A method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials

Abstract

This invention discloses a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, comprising: providing carbon nanotube particles loaded with calcium peroxide; grafting ferric tannate; grafting dihydroporphyrin E6 to obtain carbon nanotube particles loaded with calcium peroxide and grafted with dihydroporphyrin E6 and ferric tannate; dispersing the carbon nanotube particles loaded with calcium peroxide and grafted with dihydroporphyrin E6 and ferric tannate in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain a microneedle patch tip; providing sodium hyaluronate grafted with 3-aminophenylboronic acid; stirring the sodium hyaluronate grafted with 3-aminophenylboronic acid and polyvinyl alcohol solution on the microneedle patch tip to form a gel, thereby obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. This bilayer hydrogel microneedle patch loaded with nanomaterials can effectively achieve anti-tumor effects by directly delivering nanomaterials to the tumor site, promoting self-oxygenation within the tumor, and promoting tumor ablation.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials. Background Technology

[0002] Cancer is considered one of the major threats to human health, and melanoma is one of the most aggressive types of skin cancer, with high incidence and mortality rates, and it occurs most frequently on the skin. Currently, traditional treatments mainly include radiotherapy, chemotherapy, and surgical excision, but these can cause damage to normal tissues and organs and increase the risk of infection. Therefore, there is a need to develop a new treatment strategy.

[0003] Photothermal therapy (PTT) is an important and widely studied method for treating melanoma. PTT can destroy tumor cell membranes through heat therapy, causing protein denaturation and inducing tumor cell apoptosis. Photodynamic therapy (PDT) is considered a promising method for treating cancer. By injecting a photosensitizer into the tumor site, after it is excited by a laser of a specific wavelength, the excited state energy can be transferred to singlet molecular oxygen, thereby generating reactive oxygen species (ROS). The excessive accumulation of ROS in tumor cells can destroy cell compartments, leading to tumor cell apoptosis or necrosis. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials. This method involves dispersing carbon nanotubes loaded with calcium peroxide and grafted with dihydroporphyrin E6 and ferric tannin in a mixed solution of polyvinyl alcohol and polyvinylpyrrolidone to obtain microneedle tips. Then, a solution of sodium hyaluronate grafted with 3-aminophenylboronic acid is stirred on the microneedle tips to form a gel, resulting in a bilayer hydrogel microneedle patch loaded with nanomaterials. This bilayer hydrogel microneedle patch loaded with nanomaterials can effectively achieve anti-tumor effects by directly delivering the nanomaterials to the tumor site, promoting internal oxygen supply to the tumor, and facilitating tumor ablation.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, characterized in that it includes:

[0006] Provides carbon nanotube particles loaded with calcium peroxide;

[0007] Iron tannate was grafted onto the carbon nanotube particles loaded with calcium peroxide to obtain carbon nanotube particles loaded with calcium peroxide and grafted with iron tannate.

[0008] Dihydroporphyrin E6 was grafted onto the calcium peroxide-loaded carbon nanotube particles grafted with iron tannate to obtain calcium peroxide-loaded carbon nanotube particles grafted with both iron tannate and dihydroporphyrin E6.

[0009] The carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin and loaded with calcium peroxide were dispersed in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain microneedle patch tips.

[0010] Sodium hyaluronate grafted with 3-aminophenylboronic acid;

[0011] Sodium hyaluronate grafted with 3-aminophenylboronic acid was mixed with a polyvinyl alcohol solution and gelled onto the tip of the microneedle patch to obtain a bilayer hydrogel microneedle patch loaded with nanomaterials.

[0012] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized by providing carbon nanotube particles loaded with calcium peroxide, specifically including:

[0013] Carbon nanotubes were added to anhydrous ethanol and sonicated until uniformly dispersed to obtain a carbon nanotube dispersion. Anhydrous calcium chloride and polyvinylpyrrolidone were added to the carbon nanotube dispersion, and the mixture was sonicated and stirred for 20–120 min. Ammonia water and hydrogen peroxide solution were added, and the reaction was continued for 10–25 min. The mixture was then centrifuged and dried to obtain carbon nanotube particles loaded with calcium peroxide.

[0014] The method for preparing the above-mentioned bilayer hydrogel microneedle patch loaded with nanomaterials is characterized in that: the concentration of carbon nanotubes in the carbon nanotube dispersion is 0.25–1 mg / mL; the mass of anhydrous calcium chloride is 13–40 times the mass of carbon nanotubes; the mass of polyvinylpyrrolidone is 46–140 times the mass of carbon nanotubes; the volume of ammonia is 0.07–0.1 times the mass of carbon nanotubes, where the volume of ammonia is in mL and the mass of carbon nanotubes is in mg; the volume of hydrogen peroxide solution is 0.02–0.04 times the mass of carbon nanotubes, where the volume of hydrogen peroxide solution is in mL and the mass of carbon nanotubes is in mg.

[0015] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized by grafting iron tannate onto the carbon nanotube particles loaded with calcium peroxide to obtain carbon nanotube particles loaded with calcium peroxide and grafted with iron tannate, specifically including:

[0016] The carbon nanotube particles loaded with calcium peroxide were ultrasonically dispersed in water to obtain system A. Tannic acid was dissolved in water to obtain a tannic acid solution, and ferric chloride hexahydrate was dissolved in water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution were added to system A, and the mixture was stirred for 5–30 min. Sodium hydroxide solution was added to adjust the pH to 7.5, and the reaction was continued for 10–30 min. The mixture was then centrifuged and dried to obtain carbon nanotube particles loaded with calcium peroxide and grafted with iron tannate.

[0017] The preparation method of the above-mentioned bilayer hydrogel microneedle patch loaded with nanomaterials is characterized in that the concentration of tannic acid in the tannic acid solution is 20-40 mg / mL; the concentration of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 5-10 mg / mL; the mass of tannic acid is 0.4-1.1 times the mass of the carbon nanotube particles loaded with calcium peroxide; and the mass of ferric chloride hexahydrate is 0.1-0.3 times the mass of the carbon nanotube particles loaded with calcium peroxide.

[0018] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized by grafting dihydroporphyrin E6 onto the calcium peroxide-loaded carbon nanotube particles grafted with iron tannate, thereby obtaining calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannate, specifically including:

[0019] Dihydroporphyrin E6 was added to a PBS solution with a pH of 7.4 and sonicated until uniformly dispersed. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added and activated for 15–240 min to obtain system B. The calcium peroxide-loaded carbon nanotube particles grafted with iron tannate were added to system B and reacted in an ice bath for 18–48 h. After centrifugation and drying, calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannate were obtained.

[0020] The method for preparing the above-mentioned bilayer hydrogel microneedle patch loaded with nanomaterials is characterized in that the mass of the dihydroporphyrin E6 is 1 to 2 times the mass of the calcium peroxide-loaded carbon nanotube particles grafted with iron tannin; the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 47 to 95 times the mass of the dihydroporphyrin E6; the mass of the N-hydroxysuccinimide is 28 to 57 times the mass of the dihydroporphyrin E6; the volume of the PBS solution is 1 to 2 times the mass of the dihydroporphyrin E6, the volume of the PBS solution is in mL, and the mass of the dihydroporphyrin E6 is in mg.

[0021] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized in that the carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin and loaded with calcium peroxide are dispersed in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain the microneedle patch tip, specifically including:

[0022] The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin were ultrasonically dispersed in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain system C. System C was placed in a PDMS mold, vacuumed, and then allowed to dry naturally at room temperature to obtain microneedle patch tips. The mass of polyvinyl alcohol was 25 to 75 times the mass of dihydroporphyrin E6, and the mass of polyvinylpyrrolidone was 0.6 to 3 times the mass of polyvinyl alcohol.

[0023] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized by providing sodium hyaluronate grafted with 3-aminophenylboronic acid, specifically including:

[0024] Sodium hyaluronate was completely dissolved in water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The mixture was stirred and activated for 20–60 min. 3-aminophenylboronic acid powder was then added, and the reaction was carried out in the dark for 16–48 h. After dialyzing with deionized water and lyophilizing, 3-aminophenylboronic acid-grafted sodium hyaluronate was obtained. The mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 0.4–1.1 times the mass of sodium hyaluronate, and the mass of N-hydroxysuccinimide was 0.2–0.7 times the mass of sodium hyaluronate. The volume of water was 50–200 times the mass of sodium hyaluronate, and the volume of water was in mL. The mass of sodium hyaluronate was in g. The mass of sodium hyaluronate was 50–100 times the mass of dihydroporphyrin E6.

[0025] The above-mentioned method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials is characterized in that the sodium hyaluronate grafted with 3-aminophenylboronic acid and a polyvinyl alcohol solution are stirred on the tip of the microneedle patch to form a gel, thereby obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials, specifically including:

[0026] The hyaluronic acid-grafted sodium hyaluronate is dissolved to obtain system D. System D is placed on the upper layer of the microneedle patch tip. A 5%–10% polyvinyl alcohol solution is added to the upper layer of system D, and the mixture is stirred to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 2%–4%. The volume of the polyvinyl alcohol solution is 15–30 times the mass of the sodium hyaluronate. The volume of the polyvinyl alcohol solution is in μL, and the mass of the sodium hyaluronate is in g.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. The method of the present invention, by constructing a bilayer hydrogel containing a needle tip and a backing, wherein the needle tip portion is sequentially incorporating calcium peroxide, ferric tannate, and dihydroporphyrin E6, can specifically address the problem of tumor ablation. The carbon nanotube particles grafted with dihydroporphyrin E6 and ferric tannate obtained by this preparation method and loaded with calcium peroxide, allow the calcium peroxide to generate H2O2 within the tumor microenvironment, and the ferric tannate to convert the H2O2 within the tumor microenvironment into O2, alleviating tumor hypoxia. Under 660nm laser irradiation, dihydroporphyrin E6 converts O2 into ROS, achieving synergistic treatment of melanoma with combined mild photothermal therapy, thereby ablating the tumor.

[0029] 2. The method of the present invention, by using hyaluronic acid-grafted sodium hyaluronate and polyvinyl alcohol as a backing, fully utilizes the good moisturizing and biocompatibility of hyaluronic acid, and the excellent self-healing, antibacterial and anti-inflammatory properties of the boron ester bonds after the hyaluronic acid modified with 3-aminophenylboronic acid and polyvinyl alcohol are cross-linked by boron ester bonds, thereby promoting the regeneration of skin tissue at the tumor site.

[0030] 3. The preparation method of the nanomaterial-loaded bilayer hydrogel microneedle patch of the present invention involves dispersing carbon nanotube particles loaded with calcium peroxide and grafted with dihydroporphyrin E6 and iron tannin in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain microneedle patch tips. Then, sodium hyaluronate grafted with 3-aminophenylboronic acid and polyvinyl alcohol solution are stirred on the microneedle patch tips to form a gel, thereby obtaining the nanomaterial-loaded bilayer hydrogel microneedle patch. This nanomaterial-loaded bilayer hydrogel microneedle patch can effectively achieve anti-tumor effects by directly delivering nanomaterials to the tumor site, promoting self-oxygenation within the tumor, and facilitating tumor ablation.

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the preparation of calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin in Example 1, and the preparation of bilayer hydrogel microneedles loaded with nanomaterials.

[0033] Figure 2 The electron microscope image and elemental distribution map of the calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannate in Example 1 are shown.

[0034] Figure 3Morphology and gelation diagram of the bilayer hydrogel microneedle patch loaded with nanomaterials in Example 1, and NMR and IR spectra of sodium hyaluronate grafted with 3-aminophenylboronic acid.

[0035] Figure 4 This is a schematic diagram characterizing the intracellular and extracellular antioxidant, antibacterial, L929 cell proliferation and migration-promoting abilities of sodium hyaluronate grafted with 3-aminophenylboronic acid in Example 1.

[0036] Figure 5 This is a schematic diagram showing the results of tests on the intracellular melanoma cell killing ability, ROS production capacity, and influence on mitochondrial membrane potential of carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannate and loaded with calcium peroxide, as well as the effects on mitochondrial membrane potential in Example 1. Detailed Implementation

[0037] This invention provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, comprising:

[0038] Step 1: Add carbon nanotubes to anhydrous ethanol and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add anhydrous calcium chloride and polyvinylpyrrolidone to the carbon nanotube dispersion, sonicate and stir for 20-120 min. Add ammonia and hydrogen peroxide solution, continue the reaction for 10-25 min, centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide. The mass percentage concentration of the ammonia is 25%; the mass percentage concentration of the hydrogen peroxide solution is 30%; the stirring temperature is 25°C; and the drying temperature is 37°C. The concentration of carbon nanotubes in the nanotube dispersion is 0.25–1 mg / mL; the mass of the anhydrous calcium chloride is 13–40 times the mass of the carbon nanotubes; the mass of the polyvinylpyrrolidone is 46–140 times the mass of the carbon nanotubes; the volume of the ammonia solution is 0.07–0.1 times the mass of the carbon nanotubes, where the volume of the ammonia solution is in mL and the mass of the carbon nanotubes is in mg; the volume of the hydrogen peroxide solution is 0.02–0.04 times the mass of the carbon nanotubes, where the volume of the hydrogen peroxide solution is in mL and the mass of the carbon nanotubes is in mg.

[0039] Step 2: The carbon nanotube particles loaded with calcium peroxide described in Step 1 are ultrasonically dispersed in water to obtain System A. Tannic acid is dissolved in water to obtain a tannic acid solution, and ferric chloride hexahydrate is dissolved in water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 5–30 min. Sodium hydroxide solution is added to adjust the pH to 7.5, and the reaction continues for 10–30 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate. The concentration of tannic acid in the tannic acid solution is 20–40 mg / mL; the concentration of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 5–10 mg / mL; the mass of tannic acid is 0.4–1.1 times the mass of the calcium peroxide-loaded carbon nanotube particles, and the mass of ferric chloride hexahydrate is 0.1–0.3 times the mass of the calcium peroxide-loaded carbon nanotube particles.

[0040] Step 3: Add dihydroporphyrin E6 to a PBS solution with pH 7.4, sonicate until uniformly dispersed, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, activate for 15–240 min to obtain system B. Add the calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 18–48 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin. The mass of dihydroporphyrin E6 is 1 to 2 times the mass of the calcium peroxide-loaded carbon nanotube particles grafted with iron tannin; the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 47 to 95 times the mass of dihydroporphyrin E6; the mass of N-hydroxysuccinimide is 28 to 57 times the mass of dihydroporphyrin E6; the volume of the PBS solution is 1 to 2 times the mass of dihydroporphyrin E6, the volume of the PBS solution is in mL, and the mass of dihydroporphyrin E6 is in mg.

[0041] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to dry naturally at room temperature to obtain the microneedle patch tip. The mass of the polyvinyl alcohol is 25 to 75 times the mass of dihydroporphyrin E6 in Step 3, and the mass of the polyvinylpyrrolidone is 0.6 to 3 times the mass of the polyvinyl alcohol.

[0042] Step 5: Completely dissolve sodium hyaluronate in water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate for 20-60 min, add 3-aminophenylboronic acid powder, react in the dark for 16-48 h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.4-1.1 times the mass of sodium hyaluronate, and the mass of N-hydroxysuccinimide is 0.2-0.7 times the mass of sodium hyaluronate. The volume of water is 50-200 times the mass of sodium hyaluronate, and the volume of water is mL. The mass unit of sodium hyaluronate is g. The mass of sodium hyaluronate is 50-100 times the mass of dihydroporphyrin E6.

[0043] Step Six: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add a 5%–10% polyvinyl alcohol solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 2%–4%. The volume of the polyvinyl alcohol solution is 15–30 times the mass of the sodium hyaluronate in step five. The volume of the polyvinyl alcohol solution is in μL, and the mass of the sodium hyaluronate is in g.

[0044] The following description, in conjunction with specific embodiments, illustrates the content of the present invention. However, the following description is not intended to limit the scope of the present invention.

[0045] A series of bilayer hydrogel microneedle patches were prepared according to the method of the present invention, as detailed below.

[0046] Example 1

[0047] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps:

[0048] Step 1: Add 50 mg of carbon nanotubes (CNTs) to 100 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 1 g of anhydrous calcium chloride (CaCl2) and 3 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 20 min, add 5 mL of ammonia water (NH3·H2O) and 1 mL of hydrogen peroxide solution (H2O2), continue the reaction for 15 min, centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2); the mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0049] Step 2: 50 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 are ultrasonically dispersed in water to obtain System A. 40 mg of tannic acid (TA) is dissolved in 1 mL of water to obtain a tannic acid solution. 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in 1 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 10 min. Sodium hydroxide (NaOH) solution is added to adjust the pH to 7.5, and the reaction continues for 15 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water is 2000 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution is 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 1%.

[0050] Step 3: Add 20 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 950 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 570 mg of N-hydroxysuccinimide, activate for 20 min to obtain system B, add 20 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 24 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin (CNT@CaO2@TA-Fe / Ce6);

[0051] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution composed of 0.5g polyvinyl alcohol (PVA) and 1.5g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0052] Step 5: Dissolve 1g of sodium hyaluronate (HA) completely in 100mL of water, add 0.96g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.5g of N-hydroxysuccinimide (NHS), stir and activate for 20min, add 0.3g of 3-aminophenylboronic acid powder, react in the dark for 18h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 100 times the mass of the sodium hyaluronate.

[0053] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of a 10% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 2%.

[0054] Example 2

[0055] This embodiment describes a method for preparing a bilayer hydrogel microneedle patch using negatively loaded nanomaterials, comprising the following steps:

[0056] Step 1: Add 75 mg of carbon nanotubes (CNTs) to 100 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 2 g of anhydrous calcium chloride (CaCl2) and 3.5 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 30 min. Add 5 mL of ammonia water (NH3·H2O) and 2 mL of hydrogen peroxide solution (H2O2), and continue the reaction for 20 min. Centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2). The mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0057] Step 2: 50 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 are ultrasonically dispersed in water to obtain System A. 40 mg of tannic acid (TA) is dissolved in 1 mL of water to obtain a tannic acid solution. 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in 1 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 20 min. Sodium hydroxide (NaOH) solution is added to adjust the pH to 7.5, and the reaction continues for 10 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water is 2000 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution is 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 1%.

[0058] Step 3: Add 20 mg of dihydroporphyrin E6 to 40 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 1.425 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.855 g of N-hydroxysuccinimide, activate for 15 min to obtain system B, add 20 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 18 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin (CNT@CaO2@TA-Fe / Ce6);

[0059] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution consisting of 1g polyvinyl alcohol (PVA) and 1g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0060] Step 5: Dissolve 2g of sodium hyaluronate (HA) completely in 100mL of water, add 1.9g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.14g of N-hydroxysuccinimide (NHS), stir and activate for 30min, add 0.4g of 3-aminophenylboronic acid powder, react in the dark for 20h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 50 times the mass of the sodium hyaluronate.

[0061] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of a 10% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 3%.

[0062] Example 3

[0063] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps:

[0064] Step 1: Add 25 mg of carbon nanotubes (CNTs) to 100 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 1 g of anhydrous calcium chloride (CaCl2) and 3.5 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 60 min. Add 2.5 mL of ammonia water (NH3·H2O) and 1 mL of hydrogen peroxide solution (H2O2), and continue the reaction for 10 min. Centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2). The mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0065] Step 2: 75 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 were ultrasonically dispersed in water to obtain System A. 80 mg of tannic acid (TA) was dissolved in 2 mL of water to obtain a tannic acid solution. 21.2 mg of ferric chloride hexahydrate (FeCl3·6H2O) was dissolved in 2 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution were added to System A, and the mixture was stirred for 20 min. Sodium hydroxide (NaOH) solution was added to adjust the pH to 7.5, and the reaction was continued for 10 min. The mixture was then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water was 1333 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution was 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution was 1%.

[0066] Step 3: Add 20 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 0.95 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.57 g of N-hydroxysuccinimide, activate for 15 min to obtain system B, add 10 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannate as described in Step 2 to system B, react in an ice bath for 20 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannate (CNT@CaO2@TA-Fe / Ce6);

[0067] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution consisting of 1.5g polyvinyl alcohol (PVA) and 1g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0068] Step 5: Dissolve 1.5g of sodium hyaluronate (HA) completely in 100mL of water, add 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.5g of N-hydroxysuccinimide (NHS), stir and activate for 30min, add 0.36g of 3-aminophenylboronic acid powder, react in the dark for 16h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 67 times the mass of the sodium hyaluronate.

[0069] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of 5% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 2%.

[0070] Example 4

[0071] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps:

[0072] Step 1: Add 50 mg of carbon nanotubes (CNTs) to 50 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 1 g of anhydrous calcium chloride (CaCl2) and 3.5 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 120 min. Add 5 mL of ammonia water (NH3·H2O) and 2 mL of hydrogen peroxide solution (H2O2), and continue the reaction for 25 min. Centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2). The mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0073] Step 2: 100 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 are ultrasonically dispersed in water to obtain System A. 40 mg of tannic acid (TA) is dissolved in 1 mL of water to obtain a tannic acid solution. 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in 1 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 30 min. Sodium hydroxide (NaOH) solution is added to adjust the pH to 7.5, and the reaction continues for 10 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water is 1000 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution is 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 1%.

[0074] Step 3: Add 20 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 0.95 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.57 g of N-hydroxysuccinimide, activate for 60 min to obtain system B, add 10 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 24 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin (CNT@CaO2@TA-Fe / Ce6);

[0075] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution consisting of 1g polyvinyl alcohol (PVA) and 2g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0076] Step 5: Dissolve 1.75g ​​of sodium hyaluronate (HA) completely in 100mL of water, add 1.92g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.152g of N-hydroxysuccinimide (NHS), stir and activate for 60min, add 0.55g of 3-aminophenylboronic acid powder, react in the dark for 20h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 57 times the mass of the sodium hyaluronate.

[0077] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of 8% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 4%.

[0078] Example 5

[0079] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps:

[0080] Step 1: Add 50 mg of carbon nanotubes (CNTs) to 150 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 1 g of anhydrous calcium chloride (CaCl2) and 3.5 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 30 min. Add 5 mL of ammonia water (NH3·H2O) and 2 mL of hydrogen peroxide solution (H2O2), and continue the reaction for 10 min. Centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2). The mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0081] Step 2: 50 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 are ultrasonically dispersed in water to obtain System A. 40 mg of tannic acid (TA) is dissolved in 1 mL of water to obtain a tannic acid solution. 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in 1 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 10 min. Sodium hydroxide (NaOH) solution is added to adjust the pH to 7.5, and the reaction continues for another 10 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water is 2000 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution is 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 1%.

[0082] Step 3: Add 20 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 0.95 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.57 g of N-hydroxysuccinimide, activate for 2 h to obtain system B, add 20 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 24 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin (CNT@CaO2@TA-Fe / Ce6);

[0083] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution consisting of 1g polyvinyl alcohol (PVA) and 1.5g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0084] Step 5: Dissolve 1g of sodium hyaluronate (HA) completely in 200mL of water, add 0.96g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.57g of N-hydroxysuccinimide (NHS), stir and activate for 60min, add 0.39g of 3-aminophenylboronic acid powder, react in the dark for 24h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 200 times the mass of the sodium hyaluronate.

[0085] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of a 10% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 2%.

[0086] Example 6

[0087] This embodiment provides a method for preparing a bilayer hydrogel microneedle patch loaded with nanomaterials, including the following steps:

[0088] Step 1: Add 75 mg of carbon nanotubes (CNTs) to 150 mL of anhydrous ethanol, and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Add 1 g of anhydrous calcium chloride (CaCl2) and 3.5 g of polyvinylpyrrolidone (PVP) to the carbon nanotube dispersion, sonicate and stir for 45 min. Add 7.5 mL of ammonia water (NH3·H2O) and 3 mL of hydrogen peroxide solution (H2O2), and continue the reaction for 20 min. Centrifuge and dry to obtain carbon nanotube particles loaded with calcium peroxide (CNT@CaO2). The mass percentage concentration of the ammonia water (NH3·H2O) is 25%; the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%; the stirring temperature is 25℃; and the drying temperature is 37℃.

[0089] Step 2: 50 mg of the calcium peroxide-loaded carbon nanotube particles (CNT@CaO2) described in Step 1 are ultrasonically dispersed in water to obtain System A. 40 mg of tannic acid (TA) is dissolved in 2 mL of water to obtain a tannic acid solution. 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in 2 mL of water to obtain a ferric chloride solution. The tannic acid solution and ferric chloride solution are added to System A, and the mixture is stirred for 5 min. Sodium hydroxide (NaOH) solution is added to adjust the pH to 7.5, and the reaction continues for 30 min. The mixture is then centrifuged and dried to obtain calcium peroxide-loaded carbon nanotube particles grafted with iron tannate (CNT@CaO2@TA-Fe). The mass of the water is 2000 times the mass of the calcium peroxide-loaded carbon nanotube particles. The mass percentage of tannic acid in the tannic acid solution is 3.8%. The mass percentage of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 1%.

[0090] Step 3: Add 10 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicate until evenly dispersed, then add 0.95 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.57 g of N-hydroxysuccinimide, activate for 4 h to obtain system B, add 10 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, react in an ice bath for 48 h, centrifuge and dry to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin (CNT@CaO2@TA-Fe / Ce6);

[0091] Step 4: The calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannins described in Step 3 are ultrasonically dispersed in a mixed solution composed of 0.5g polyvinyl alcohol (PVA) and 1.5g polyvinylpyrrolidone (PVP) to obtain System C. System C is placed in a PDMS mold, vacuumed, and then allowed to air dry at room temperature to obtain the microneedle patch tip. The polyvinyl alcohol is MACKLIN polyvinyl alcohol 0588 low viscosity type (PVA-205); the polyvinylpyrrolidone is MACKLIN polyvinylpyrrolidone (PVP-K30).

[0092] Step 5: Dissolve 1g of sodium hyaluronate (HA) completely in 100mL of water, add 480mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 288mg of N-hydroxysuccinimide (NHS), stir and activate for 20min, add 0.24g of 3-aminophenylboronic acid powder, react in the dark for 48h, dialyze with deionized water, and freeze-dry to obtain 3-aminophenylboronic acid-grafted sodium hyaluronate. The molecular weight cutoff of the dialysis bag used for dialysis is 8k-14k. The mass of the water is 100 times the mass of the sodium hyaluronate.

[0093] Step 6: Dissolve the hyaluronic acid-grafted sodium hyaluronate to obtain system D. Place system D on the upper layer of the microneedle patch tip. Add 30 μL of a 10% polyvinyl alcohol (PVA) solution to the upper layer of system D and stir to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials. The mass percentage of hyaluronic acid-grafted sodium hyaluronate in system D is 4%.

[0094] Comparative Example 1

[0095] This comparative example is the same as Example 1, except that...

[0096] Step 1 is as follows: Add 50 mg of carbon nanotubes (CNTs) to 100 mL of deionized water and sonicate until uniformly dispersed to obtain a carbon nanotube dispersion. Dissolve 40 mg of tannic acid (TA) in 1 mL of water to obtain a tannic acid solution. Dissolve 10.6 mg of ferric chloride hexahydrate (FeCl3·6H2O) in 1 mL of water to obtain a ferric chloride solution. Add the tannic acid solution and ferric chloride solution to the carbon nanotube dispersion, and then add sodium hydroxide (NaOH) solution to adjust the pH to 7.5. Continue the reaction for 15 min, centrifuge and dry to obtain carbon nanotubes grafted with iron tannate.

[0097] Step two involves ultrasonically dispersing the iron tannate-grafted carbon nanotubes described in Step one in 100 mL of anhydrous ethanol to obtain System A. 1 g of anhydrous calcium chloride (CaCl2) and 3 g of polyvinylpyrrolidone (PVP) are added to System A, and the mixture is ultrasonically dispersed and stirred for 20 min. Then, 5 mL of ammonia water (NH3·H2O) and 1 mL of hydrogen peroxide solution (H2O2) are added, and the reaction continues for 15 min. The mixture is then centrifuged and dried to obtain iron tannate-grafted carbon nanotube particles loaded with calcium peroxide. The mass percentage concentration of the ammonia water (NH3·H2O) is 25%, the mass percentage concentration of the hydrogen peroxide solution (NH3·H2O) is 30%, the stirring temperature is 25°C, and the drying temperature is 37°C.

[0098] Step 3 involves adding 20 mg of dihydroporphyrin E6 to 20 mL of PBS solution with pH 7.4, sonicating until evenly dispersed, then adding 950 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 570 mg of N-hydroxysuccinimide, activating for 20 min to obtain system B. Then, adding 20 mg of calcium peroxide-loaded carbon nanotube particles grafted with iron tannin as described in Step 2 to system B, reacting in an ice bath for 24 h, centrifuging and drying to obtain calcium peroxide-loaded carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin.

[0099] Comparative Example 2

[0100] This comparative example is the same as Example 1, except that step five is not included, and step six is ​​as follows: 200 mg of sodium hyaluronate powder is dissolved in 10 mL of water to obtain a sodium hyaluronate solution. The sodium hyaluronate solution is placed on the upper layer of the microneedle patch tip, and 30 μL of a 10% polyvinyl alcohol (PVA) solution is added to the upper layer. The mixture is stirred to form a gel, thus obtaining a bilayer hydrogel microneedle patch loaded with nanomaterials.

[0101] The results showed that the bilayer hydrogel microneedle patch loaded with nanomaterials obtained by the method in Comparative Example 1 had a weaker tumor treatment effect. This may be because calcium peroxide is wrapped in the outer layer of ferric tannate, which causes calcium peroxide to produce H2O2 first, while ferric tannate cannot be converted into H2O2 in time. As a result, H2O2 accumulates excessively inside the tumor, and the tumor microenvironment still has the problem of hypoxia.

[0102] The bilayer hydrogel microneedle patch loaded with nanomaterials obtained by the method in Comparative Example 2 showed poor cell proliferation and migration effects as well as poor ability to promote skin tissue regeneration at the tumor site. This may be due to the lack of boronic ester bonds with antibacterial and anti-inflammatory effects.

[0103] Performance testing:

[0104] Figure 1 The diagram shows the construction of carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannin and loaded with calcium peroxide, as well as the construction of a bilayer hydrogel microneedle patch loaded with nanomaterials, as shown in Example 1. The construction process of this invention shows that by providing carbon nanotube particles loaded with calcium peroxide, grafting iron tannate onto the carbon nanotube particles loaded with calcium peroxide to obtain carbon nanotube particles loaded with calcium peroxide and grafted with iron tannate, grafting dihydroporphyrin E6 onto the carbon nanotube particles loaded with calcium peroxide and grafted with iron tannate to obtain carbon nanotube particles loaded with calcium peroxide and grafted with dihydroporphyrin E6 and iron tannate, dispersing the carbon nanotube particles loaded with calcium peroxide and grafted with dihydroporphyrin E6 and iron tannate in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain microneedle patch tips, providing sodium hyaluronate grafted with 3-aminophenylboronic acid, and stirring the sodium hyaluronate grafted with 3-aminophenylboronic acid and polyvinyl alcohol solution on the microneedle patch tips to form a gel, a bilayer hydrogel microneedle patch loaded with nanomaterials is obtained. The bilayer hydrogel microneedle patch loaded with nanomaterials obtained by this method can effectively achieve anti-tumor effects. By directly delivering nanomaterials to the tumor site, it promotes self-oxygenation within the tumor and facilitates tumor ablation.

[0105] Figure 2 This is an electron micrograph of carbon nanotube particles grafted with dihydroporphyrin E6 and iron tannate, supported on calcium peroxide, from Example 1. Figure 2 A and 2B are scanning electron microscope (SEM) images of CaO2 nanoparticles and pure carbon nanotubes, respectively. Figure 2 C is a lattice of calcium peroxide-supported carbon nanotubes grafted with dihydroporphyrin E6 and iron tannate. Figure 2 D is the DHAADF-STEM elemental distribution map of calcium peroxide-loaded carbon nanotubes grafted with dihydroporphyrin E6 and iron tannate. Figure 2 E is Figure 2 D is a distribution map of element N within the shown region. Figure 2 F is Figure 2 Distribution map of Ca element in region D. Figure 2 G is Figure 2 Distribution diagram of element C in region D. Figure 2 H is Figure 2 Fe element distribution map in region D. Figure 2 I am Figure 2 The distribution map of element O in the region shown in D, combined with... Figure 2 D~2I demonstrated the successful preparation of CNT@CaO2@TA-Fe / Ce6 and the uniform distribution of N, Ca, C, Fe and O elements within the nanoparticles.

[0106] Figure 3 The images show the morphology of the bilayer hydrogel microneedle patch loaded with nanomaterials in Example 1, a schematic diagram of the gelation process, and the NMR and IR spectra of sodium hyaluronate grafted with 3-aminophenylboronic acid. Based on... Figure 3 As can be seen from A, the dual-layer smart microneedle patch has a complete needle tip structure. Figure 3 B is the hydrogel backing of the bilayer smart microneedle patch obtained by cross-linking. Figure 3 C is the infrared spectrum of sodium hyaluronate grafted with 3-aminophenylboronic acid, based on Figure 3 C shows that at 1340cm -1 and 700cm -1 The formation of a new peak indicates successful grafting. Figure 3 D is the NMR spectrum of sodium hyaluronate grafted with 3-aminophenylboronic acid. A new characteristic peak appears in the range of 7.3–7.7 ppm, indicating that the grafting of 3-aminophenylboronic acid was successful.

[0107] Figure 4 This is a schematic diagram illustrating the intracellular and extracellular antioxidant, antibacterial, and L929 cell proliferation and migration promotion capabilities of sodium hyaluronate grafted with 3-aminophenylboronic acid as described in Example 1.

[0108] Methods for testing intracellular and extracellular antioxidant capacity include: O2 ·- The scavenging ability test method included: dissolving the gel microneedle patch, riboflavin, methionine, and NBT in PBS (pH=7.4), irradiating with white light at 25℃ for 15 min, collecting the supernatant into a cuvette for photographing and recording, and measuring the full-wavelength scan curve using 1 mL of the supernatant. The absorbance value at 560 nm was calculated to evaluate O2. ·- Clearance ability;

[0109] The H2O2 scavenging ability test method includes: incubating 10 mL of H2O2 and the gel microneedle patch at 37 °C for 2 h to obtain system E; dissolving 1.33 mL of titanium sulfate in 8.33 mL of sulfuric acid to obtain titanium sulfate solution; taking 50 μL of system E and 100 μL of titanium sulfate solution into 50 mL of deionized water to obtain system F; collecting the supernatant into a cuvette for photographing and recording; and taking 1 mL of the supernatant to measure the full wavelength scan curve, calculating the absorbance value at 405 nm, and evaluating the H2O2 scavenging ability.

[0110] The method for testing ·OH scavenging ability includes: adding the gel microneedle patch, FeSO4, H2O2 and salicylic acid solution to PBS (pH=7.4), incubating at 37℃ for 30 min, collecting the supernatant into a cuvette for photographing and recording, and taking 1 mL of the supernatant to measure the full wavelength scan curve, calculating the absorbance value at 510 nm, and evaluating the ·OH scavenging ability;

[0111] The antibacterial ability test method includes: using a concentration of 1x10 7 1 mL of Escherichia coli and Staphylococcus aureus at CFU / mL were co-incubated with the gel microneedle patch, and then irradiated with an 808 nm near-infrared laser before being incubated at 37°C for 12 h. The cell suspension was then diluted to 10⁻⁶ of its original concentration. -6 Then, 100 μL was spread onto an agar plate and incubated at 37°C and 120 rpm for 24 hours. The plate was then photographed and counted to calculate the antibacterial activity.

[0112] The test method for promoting L929 proliferation included: the gel microneedle patch was extracted in 1640 culture medium for 3 days, L929 cells were cultured in the extract for 24 hours, and after complete adhesion, AO / EB staining solution was added, and the images were taken and recorded under an inverted fluorescence microscope to evaluate the ability to promote L929 proliferation.

[0113] The test method for promoting L929 cell migration included: seeding L929 cells in 6-well plates and culturing them for 24 hours. After they were fully adhered, the gel microneedle patch was scratched in the 6-well plate with a 200 μL pipette tip and photographed. The patch was then extracted in 1640 medium for 3 days. The scratched cells were cultured in the extract and the scratch size was photographed after 12 hours to assess the ability to promote L929 cell migration.

[0114] in Figure 4 A is a schematic diagram illustrating the intracellular and extracellular antioxidant properties of sodium hyaluronate grafted with 3-aminophenylboronic acid. According to... Figure 4 As shown in Figure A, the sodium hyaluronate backing grafted with 3-aminophenylboronic acid of the present invention has an effective O2 scavenging effect. · - The ability. Figure 4B is a schematic diagram illustrating the H2O2 scavenging performance of sodium hyaluronate grafted with 3-aminophenylboronic acid. Figure 4 As shown in B, the sodium hyaluronate backing grafted with 3-aminophenylboronic acid of the present invention has an effective ability to remove H2O2. Figure 4 C is a schematic diagram illustrating the ·OH scavenging performance of sodium hyaluronate grafted with 3-aminophenylboronic acid. According to... Figure 4 As shown in C, the sodium hyaluronate backing grafted with 3-aminophenylboronic acid of the present invention has an effective ability to remove ·OH. Figure 4 D is a schematic diagram illustrating the antibacterial properties of sodium hyaluronate grafted with 3-aminophenylboronic acid. Figure 4 D indicates that the 3-aminophenylboronic acid-grafted sodium hyaluronate has excellent antibacterial ability and can significantly promote the proliferation and migration of L929 cells.

[0115] Figure 5 This is a schematic diagram showing the results of tests on the intracellular melanoma cell killing ability, ROS production capacity, and influence on mitochondrial membrane potential of carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannate and loaded with calcium peroxide, as well as the results of tests on mitochondrial membrane potential of the carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) in Example 1. Figure 5 AM / PI is a schematic diagram illustrating the intracellular melanoma cell-killing performance of carbon nanotube particles grafted with dihydroporphyrin e6 and ferric tannin loaded with calcium peroxide. The testing method included: seeding B16F10 cells in 6-well plates and culturing for 24 hours until complete adhesion; then treating with CNT@CaO2@TA-Fe / Ce6 particles for 6 hours; finally, adding AM / PI staining solution; and recording the results under an inverted fluorescence microscope to assess its melanoma cell-killing ability. Figure 5 AM / PI, the carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannin and loaded with calcium peroxide (CNT@CaO2@TA-Fe / Ce6) of the present invention exhibit the best anti-tumor effect and the highest number of dead cells under the combined action of photodynamic therapy and mild photothermal therapy.

[0116] Figure 5 ROS is a schematic diagram illustrating the ROS-producing capacity of carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannin loaded with calcium peroxide. The testing method included: seeding B16F10 cells in 6-well plates and culturing for 24 hours until complete adhesion; then adding CNT@CaO2@TA-Fe / Ce6 particles for 6 hours; followed by the addition of DCFH-DA and DAPI probes; and finally, photographing and recording the results under an inverted fluorescence microscope to assess the ROS-producing capacity. Figure 5 According to ROS, the fluorescent reaction regions of the carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannin and loaded with calcium peroxide (CNT@CaO2@TA-Fe / Ce6) in this invention are significantly dense, indicating that they can significantly promote the generation of the most ROS.

[0117] Figure 5 JC-1 is a schematic diagram illustrating the effect of carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannin loaded with calcium peroxide on mitochondrial membrane potential. The testing method included: seeding B16F10 cells in 6-well plates and culturing for 24 hours until complete adhesion; then adding CNT@CaO2@TA-Fe / Ce6 particles for 6 hours; followed by adding JC-1 staining solution; and recording the results under an inverted fluorescence microscope to assess its ability to reduce mitochondrial membrane potential. Figure 5 As can be seen in JC-1, the fluorescent reaction region of the carbon nanotube particles (CNT@CaO2@TA-Fe / Ce6) grafted with dihydroporphyrin e6 and iron tannin supported on calcium peroxide (CNT@CaO2@TA-Fe / Ce6) of the present invention is significantly thin, indicating that it can reduce the mitochondrial membrane potential.

[0118] Figure 5 In this context, Control represents the blank control group;

[0119] MN is a blank microneedle, which is prepared by uniformly dropping a mixed solution of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) into a PDMS mold, evacuating the vacuum, and then allowing it to dry naturally at room temperature to obtain a blank microneedle; wherein the concentration of the mixed solution of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) is the same as in Example 1;

[0120] Ce6+660nm refers to microneedles loaded with dihydroporphyrin e6 that have been irradiated with a 660nm laser (Ce6+660). The preparation method includes: dissolving dihydroporphyrin e6 in a mixed solution composed of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), uniformly dropping the solution into a PDMS mold, evacuating the mold, and allowing it to dry naturally at room temperature to obtain microneedles loaded with dihydroporphyrin e6; wherein the amounts of the mixed solution of dihydroporphyrin e6, polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) are the same as in Example 1;

[0121] CCa@TF / Ce6 is a microneedle of carbon nanotube particles grafted with dihydroporphyrin e6 and iron tannin and loaded with calcium peroxide (CCa@TF / Ce6).

[0122] CCa@TF / Ce6+808nm laser irradiates microneedles loaded with calcium peroxide-coated carbon nanotube particles grafted with dihydroporphyrin e6 and iron tannate.

[0123] CCa@TF / Ce6+660nm laser irradiates microneedles loaded with calcium peroxide-coated carbon nanotube particles grafted with dihydroporphyrin e6 and iron tannate.

[0124] The microneedles of CNT@CaO2@TA-Fe / Ce6 nanoparticles grafted with dihydroporphyrin e6 and ferric tannin and loaded with calcium peroxide are prepared by irradiation with 660nm and 808nm lasers (CCa@TF / Ce6+660nm / 808nm laser). The preparation method includes: ultrasonically dispersing the CNT@CaO2@TA-Fe / Ce6 nanoparticles obtained in step 3 of Example 1 in a mixed solution of 1g of polyvinyl alcohol (PVA) and 1.5g of polyvinylpyrrolidone (PVP) to obtain a dispersed system; uniformly dropping the dispersed system into a PDMS mold; vacuuming; and allowing it to dry naturally at room temperature to obtain microneedles of CNT@TF / Ce6 grafted with dihydroporphyrin e6 and ferric tannin and loaded with calcium peroxide; the concentrations of the polyvinyl alcohol (PVA) solution and the polyvinylpyrrolidone (PVP) mixed solution are the same as in Example 1.

[0125] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a dual-layer hydrogel microneedle patch loaded with nanomaterials, characterized in that, The application relates to a method for preparing a microneedle patch needle tip, and a microneedle patch needle tip prepared by the method. The method comprises the following steps: Providing calcium peroxide-loaded carbon nanotube particles; Grafting iron tannic acid on the calcium peroxide-loaded carbon nanotube particles to obtain iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles; Grafting chlorin e6 on the iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles to obtain chlorin e6 and iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles; Dispersing the chlorin e6 and iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain the microneedle patch needle tip, specifically comprising the following steps: Ultrasonic dispersion of the chlorin e6 and iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles in a mixed solution composed of polyvinyl alcohol and polyvinylpyrrolidone to obtain system C, placing the system C in a PDMS mold, vacuumizing and naturally drying at room temperature to obtain the microneedle patch needle tip; the mass of the polyvinyl alcohol is 25-75 times that of chlorin e6, and the mass of the polyvinylpyrrolidone is 0.6-3 times that of the polyvinyl alcohol; Providing 3-aminobenzene boronic acid-grafted sodium hyaluronate; 2. The method of claim 1, wherein the method further comprises: Forming a gel layer of the 3-aminobenzene boronic acid-grafted sodium hyaluronate and a polyvinyl alcohol solution on the microneedle patch needle tip to obtain a nanomaterial-loaded double-layer hydrogel microneedle patch. The method comprises the following steps:

3. The method of claim 2, wherein the method further comprises: Providing calcium peroxide-loaded carbon nanotube particles, specifically comprising the following steps:

4. The method of claim 1, wherein the method further comprises: Adding carbon nanotubes into anhydrous ethanol and ultrasonically dispersing the carbon nanotubes until uniform dispersion is obtained to obtain a carbon nanotube dispersion liquid, adding anhydrous calcium chloride and polyvinylpyrrolidone into the carbon nanotube dispersion liquid, ultrasonic dispersion and stirring reaction for 20-120 min, adding ammonia water and hydrogen peroxide solution, continuing to react for 10-25 min, centrifugal drying to obtain calcium peroxide-loaded carbon nanotube particles. The concentration of the carbon nanotubes in the carbon nanotube dispersion liquid is 0.25-1 mg / mL; the mass of the anhydrous calcium chloride is 13-40 times that of the carbon nanotubes; the mass of the polyvinylpyrrolidone is 46-140 times that of the carbon nanotubes; the volume of the ammonia water is 0.07-0.1 times that of the carbon nanotubes, the unit of the volume of the ammonia water being mL and the unit of the mass of the carbon nanotubes being mg; the volume of the hydrogen peroxide solution is 0.02-0.04 times that of the carbon nanotubes, the unit of the volume of the hydrogen peroxide solution being mL and the unit of the mass of the carbon nanotubes being mg. Grafting iron tannic acid on the calcium peroxide-loaded carbon nanotube particles to obtain iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles, specifically comprising the following steps: Ultrasonic dispersion of the calcium peroxide-loaded carbon nanotube particles in water to obtain system A, dissolving tannic acid in water to obtain a tannic acid solution, dissolving ferric chloride hexahydrate in water to obtain a ferric chloride solution, adding the tannic acid solution and the ferric chloride solution into the system A, stirring reaction for 5-30 min, adding a sodium hydroxide solution to adjust the pH to 7.5, continuing to react for 10-30 min, centrifugal drying to obtain iron tannic acid-grafted calcium peroxide-loaded carbon nanotube particles.

5. The method of claim 4, wherein the method further comprises: The concentration of tannic acid in the tannic acid solution is 20-40 mg / mL; the concentration of ferric chloride hexahydrate in the ferric chloride hexahydrate solution is 5-10 mg / mL; the mass of the tannic acid is 0.4-1.1 times the mass of the calcium peroxide-loaded carbon nanotube particles loaded with calcium peroxide, and the mass of the ferric chloride hexahydrate is 0.1-0.3 times the mass of the calcium peroxide-loaded carbon nanotube particles loaded with calcium peroxide.

6. The method of claim 1, wherein the method further comprises: The grafting of chlorin e6 and tannic acid iron on the calcium peroxide-loaded carbon nanotube particles is carried out by adding chlorin e6 to the grafting tannic acid iron calcium peroxide-loaded carbon nanotube particles, and then adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to the system, and stirring for 15-240 min to obtain system B, and then adding the grafting tannic acid iron calcium peroxide-loaded carbon nanotube particles to the system B, and then carrying out ice bath reaction for 18-48 h, and then centrifuging and drying to obtain the grafting chlorin e6 and tannic acid iron calcium peroxide-loaded carbon nanotube particles. The mass of the chlorin e6 is 1-2 times the mass of the grafting tannic acid iron calcium peroxide-loaded carbon nanotube particles; the mass of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 47-95 times the mass of the chlorin e6, and the mass of the N-hydroxysuccinimide is 28-57 times the mass of the chlorin e6; the volume of the PBS solution is 1-2 times the mass of the chlorin e6, and the unit of the volume of the PBS solution is mL, and the unit of the mass of the chlorin e6 is mg.

7. The method of claim 6, wherein the method further comprises: The grafting of 3-aminobenzoic acid on the sodium hyaluronate is carried out by completely dissolving the sodium hyaluronate in water, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, stirring and activating for 20-60 min, adding 3-aminobenzoic acid powder, and then carrying out dark reaction for 16-48 h, and then dialyzing with deionized water, and then freeze-drying to obtain the grafting 3-aminobenzoic acid sodium hyaluronate; the mass of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 0.4-1.1 times the mass of the sodium hyaluronate, and the mass of the N-hydroxysuccinimide is 0.2-0.7 times the mass of the sodium hyaluronate; the volume of the water is 50-200 times the mass of the sodium hyaluronate, and the unit of the volume of the water is mL, and the unit of the mass of the sodium hyaluronate is g; the mass of the sodium hyaluronate is 50-100 times the mass of the chlorin e6.

8. The method of claim 1, wherein the method further comprises: The grafting of 3-aminobenzoic acid on the sodium hyaluronate is carried out by completely dissolving the sodium hyaluronate in water, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, stirring and activating for 20-60 min, adding 3-aminobenzoic acid powder, and then carrying out dark reaction for 16-48 h, and then dialyzing with deionized water, and then freeze-drying to obtain the grafting 3-aminobenzoic acid sodium hyaluronate; the mass of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 0.4-1.1 times the mass of the sodium hyaluronate, and the mass of the N-hydroxysuccinimide is 0.2-0.7 times the mass of the sodium hyaluronate; the volume of the water is 50-200 times the mass of the sodium hyaluronate, and the unit of the volume of the water is mL, and the unit of the mass of the sodium hyaluronate is g; the mass of the sodium hyaluronate is 50-100 times the mass of the chlorin e6. The grafting of 3-aminobenzoic acid on the sodium hyaluronate is carried out by completely dissolving the sodium hyaluronate in water, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, stirring and activating for 20-60 min, adding 3-aminobenzoic acid powder, and then carrying out dark reaction for 16-48 h, and then dialyzing with deionized water, and then freeze-drying to obtain the grafting 3-aminobenzoic acid sodium hyaluronate; the mass of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 0.4-1.1 times the mass of the sodium hyaluronate, and the mass of the N-hydroxysuccinimide is 0.2-0.7 times the mass of the sodium hyaluronate; the volume of the water is 50-200 times the mass of the sodium hyaluronate, and the unit of the volume of the water is mL, and the unit of the mass of the sodium hyaluronate is g; the mass of the sodium hyaluronate is 50-100 times the mass of the chlorin e6.

9. The method of claim 1, wherein the method further comprises, ​ ​ The mass percentage of hyaluronic acid sodium grafted with 3-aminobenzene boronic acid in the system D is 2% to 4%; The volume of the polyvinyl alcohol solution is 15 to 30 times of the mass of the hyaluronic acid sodium, the unit of the volume of the polyvinyl alcohol solution is μL, and the unit of the mass of the hyaluronic acid sodium is g.

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