Multifunctional two-section microneedle containing intelligent nano-carrier as well as preparation method and application of multifunctional two-section microneedle

Abstract

The invention discloses a multifunctional two-section microneedle containing an intelligent nano-carrier and a preparation method and application of the multifunctional two-section microneedle, and belongs to the technical field of medical instrument preparation. The preparation method specifically comprises the following steps: firstly, constructing a pH-responsive drug carrier chitosan nanoparticle by using an ionic crosslinking assembly method, then modifying gelatin and chitosan by using methacrylic anhydride, mixing the gelatin, the chitosan and the methacrylic anhydride to prepare a microneedle tip precursor solution, dropwise adding the microneedle tip precursor solution onto a microneedle mold, exhausting air in a vacuum environment, and performing vacuum drying to obtain the pH-responsive drug carrier chitosan nanoparticle micro-needle. The preparation method comprises the following steps: adding a cross-linking agent into a mold, cross-linking under ultraviolet light, drying to obtain a needle tip part, adding a gelatin-polydopamine solution above the needle tip part to form a substrate part, putting the mold into a drying box, drying overnight, and demolding to obtain the two-section microneedle. The two-section microneedle prepared by the invention has excellent capability of resisting common bacteria and promoting healing of postoperative infected wounds. The technical problem that in the prior art, a microneedle lacks intelligent drug response release and wound self-repairing promoting capacity is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical device preparation, and specifically relates to a multifunctional two-stage microneedle containing an intelligent nano-carrier, and a preparation method and application thereof. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide. It is worth noting that surgical resection remains the standard treatment for primary breast cancer. However, the risk of breast cancer recurrence after surgery is as high as 10-40%. In addition, in postoperative patients, severe local immunosuppression and significant wound infection and inflammation promote cancer cell invasion and inhibit cell activity, further increasing the incidence of breast cancer recurrence and metastasis, and ultimately leading to increased breast cancer-related mortality. To prevent postoperative recurrence, breast cancer patients usually receive adjuvant chemotherapy, a systemic intervention that can greatly reduce local recurrence and mortality. Unfortunately, multidrug resistance has become one of the major challenges in chemotherapy, resulting in treatment failure in more than 90% of patients.

[0003] In addition, traditional chemotherapy drugs are cytotoxic and may harm normal cells, leading to side effects such as bone marrow suppression, nausea, vomiting, and hair loss. Chemotherapy may also cause premature menopause in women, potentially increasing the risk of osteoporosis and impaired fertility. To date, despite great advances in chemotherapy technology, complete cure and eradication of cancer remain elusive. The potential risk of surgical infection and increased susceptibility to bacterial infection due to decreased immunity after tumor surgery may lead to life-threatening complications. Given the diversity of postoperative clinical effects, traditional tumor treatment methods may have considerable limitations in meeting the multiple treatment requirements after complex tumor surgery. Therefore, it is imperative to develop intelligent auxiliary treatment strategies to prevent tumor recurrence and potential wound infection after tumor resection.

[0004] In recent years, with the rapid development of nanotechnology and microneedle technology, new ideas have been provided for the treatment of breast cancer. Nanotechnology can achieve precise delivery and controlled release of drugs, improve the bioavailability and therapeutic effect of drugs. Microneedle technology has the advantages of minimally invasive, painless, and easy to operate. It can achieve transdermal delivery of drugs and reduce systemic distribution and side effects of drugs.

[0005] However, the existing nanocarrier and microneedle technology still has some defects. On the one hand, the preparation process of nanocarriers is complicated, and the drug loading is limited, which is difficult to meet clinical needs; on the other hand, the existing microneedle technology is mostly prepared by a single material, with limited mechanical properties and biocompatibility, and it is difficult to achieve intelligent response release of drugs. Chinese patent CN115671528A discloses a methacrylic chitosan hydrogel microneedle patch and its preparation method and application, using methacrylic anhydride MA and chitosan acetic acid solution as raw materials to prepare a hydrogel microneedle patch. However, the microneedle patch is mainly based on methacrylic chitosan hydrogel. Although it has certain drug loading and release capabilities, it cannot achieve intelligent response release of drugs. In addition, it only uses methacrylic anhydride and high-density chitosan as the main components of the microneedle matrix, resulting in the microneedle patch. The mechanical properties, flexibility and biocompatibility are not strong, and it does not have the ability to promote wound self-repair, which will be unfavorable to improve the patient's comfort and treatment effect. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a multifunctional two-stage microneedle containing intelligent nanocarriers and a preparation method and application thereof, aiming to solve the technical problems that the microneedles in the prior art lack the ability to intelligently respond to drug release and promote wound self-repair.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention also provides a method for preparing the multifunctional two-stage microneedle containing the above-mentioned intelligent nano-carrier, comprising: firstly, constructing drug-loaded chitosan nanoparticles by using an ionic crosslinking assembly method for a carboxymethyl-β-cyclodextrin-chitosan solution, an anionic crosslinking agent and a doxorubicin hydrochloride drug solution, then performing acryloyl modification on gelatin and chitosan to obtain acryloyl gelatin and acryloyl chitosan, mixing the acryloyl gelatin, acryloyl chitosan, a photoinitiator solution and the drug-loaded chitosan nanoparticles to prepare an acryloyl gelatin-chitosan composite hydrogel, dropping the composite hydrogel onto a microneedle mold, crosslinking under ultraviolet light, and drying to obtain a needle tip portion, then adding dopamine hydrochloride, a gelatin solution and a fibroblast growth factor above the needle tip portion to form a base portion, then drying the mold, and demolding to obtain a two-stage microneedle.

[0008] More preferably, methacrylic anhydride is used to perform acrylation modification on gelatin and chitosan.

[0009] Chitosan is acrylated because chitosan is extremely difficult to dissolve in water, alkaline and neutral solvents. After acrylation, it is modified to form acrylated chitosan, which improves its water solubility and has the characteristics of bonding and photocrosslinking. This substance has good biocompatibility, biodegradability, and non-toxicity, which is conducive to improving the biocompatibility of microneedles; Acryloyl modified gelatin: First, acryloyl functional groups can be introduced into the molecular structure of gelatin, making acryloyl gelatin chemically reactive. This activity allows acryloyl gelatin to form a three-dimensional network structure through photopolymerization or chemical cross-linking reaction, thereby enhancing the stability and mechanical properties of the microneedle tip; second, acryloyl gelatin has photocuring ability, which means that it can be quickly cured under ultraviolet light. This feature allows acryloyl gelatin to be tightly combined with other components through photocrosslinking technology when preparing microneedles to form a needle tip with a specific shape and structure; third, acryloyl modified gelatin retains the good biocompatibility, biodegradability and bioplasticity of gelatin. These characteristics enable microneedles to reduce rejection reactions and improve therapeutic effects when used in the human body.

[0010] More preferably, gelatin is mixed with water in a dosage ratio of 0.75 g:4.5 ml, heated in a 50° C. water bath, and stirred evenly to obtain a gelatin solution.

[0011] Preferably, the method for preparing the above-mentioned drug-loaded chitosan nanoparticles comprises: mixing and stirring a doxorubicin hydrochloride drug solution, a carboxymethyl-β-cyclodextrin-chitosan solution and an anionic crosslinking agent in a volume ratio of (10-25): (1-6): 1, and centrifuging and washing the resulting suspension at room temperature to obtain pH-responsive drug-loaded chitosan nanoparticles.

[0012] Cross-linked chitosan / sodium tripolyphosphate constitute the core of the nanoparticles, while the hydrophilic CMCD groups (carboxymethyl-β-cyclodextrin) are arranged side by side on the periphery to form a shell structure, so that the chitosan nanoparticles loaded with drugs can remain stable under neutral conditions, resulting in slow drug release, and quickly disassemble in a weakly acidic environment. It has good pH response performance and can be used to construct pH-responsive drug carriers; Further preferably, after mixing acryloyl gelatin, acryloyl chitosan and a photoinitiator solution, a suspension of drug-loaded chitosan nanoparticles after ultrasonic treatment is added to prepare an acryloyl gelatin-chitosan composite hydrogel; the amount ratio of the acryloyl gelatin, acryloyl chitosan, photoinitiator solution and drug-loaded chitosan nanoparticles is (0.4-0.6) g: 0.09 g: 3 ml: (0.5-4) mg / ml; in the above cross-linking reaction, the ultraviolet light irradiation time is 10-20 min.

[0013] Among them, the cross-linking degree was adjusted by adjusting the mass ratio of acryloyl chitosan and acryloyl gelatin and the time of ultraviolet irradiation to obtain a microneedle tip with the best mechanical properties, and the microneedle was prepared by the template method; gelatin contains aromatic amino acids such as tyrosine, phenylalanine, and tryptophan, which can enter the B-cyclodextrin cavity to form a host-guest inclusion complex. On this basis, this study prepared a supramolecular hydrogel composed of acryloyl gelatin and chitosan nanoparticles. The aromatic residues of tyrosine, phenylalanine, and tryptophan in the gelatin molecules combined with the carboxymethyl B-cyclodextrin groups on the outside of the chitosan nanoparticles through host-guest interactions to form a supramolecular network; Preferably, the dosage ratio of dopamine hydrochloride, gelatin solution and fibroblast growth factor is 0.225 g:0.75 ml:(25-100) IU / ml.

[0014] More preferably, dopamine hydrochloride and gelatin solution are mixed and magnetically stirred to obtain gelatin-polydopamine hydrogel.

[0015] Preferably, polymyxin, sodium periodate and sodium hydroxide solution are added to the substrate part in a dosage ratio of (25-100) IU / ml: (0.1-0.15) g: (0.05-0.15) ml to prepare a microneedle substrate precursor solution as the substrate part.

[0016] Further preferably, the acryloyl gelatin-chitosan composite hydrogel is poured onto the surface of a polydimethylsiloxane mold for a cross-linking reaction, and after repeated vacuuming and drying, a microneedle tip portion is obtained; a microneedle base precursor liquid is coated on the polydimethylsiloxane mold with the microneedle tip portion to form a microneedle base portion, and after drying, a multifunctional two-stage microneedle is obtained.

[0017] The two-stage microneedle exists in the PDMS mold in the form of a microneedle patch (MN). The microneedle patch can be gently peeled off from the PDMS mold and dried and stored at low temperature before use.

[0018] Further preferably, the method for preparing the doxorubicin hydrochloride drug solution comprises: mixing doxorubicin hydrochloride, dimethylformamide and triethylamine in a dosage ratio of 25 mg:2 mL:2 ml, and dissolving by ultrasonic dispersion in an ultrasonic instrument for 20 minutes to obtain the doxorubicin hydrochloride drug solution.

[0019] Further preferably, the preparation method of the carboxymethyl-β-cyclodextrin-chitosan comprises: mixing a 1% chitosan (w / v) acetic acid aqueous solution and a carboxymethyl-β-cyclodextrin carboxyl activation solution in a volume ratio of 1:1, stirring continuously at room temperature for 24 hours to undergo an amidation reaction, followed by dialysis, and centrifuging at room temperature, taking the supernatant and freeze-drying it to remove moisture to obtain a white floc, which is carboxymethyl-β-cyclodextrin-chitosan.

[0020] Further preferably, the preparation method of the carboxymethyl-β-cyclodextrin carboxyl activation solution comprises: mixing carboxymethyl-β-cyclodextrin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt, N-hydroxysuccinimide and deionized water in a dosage ratio of 1g:0.9g:0.55g:20ml, and activating at room temperature for 1h to obtain the carboxymethyl-β-cyclodextrin carboxyl activation solution.

[0021] Preferably, the preparation method of the acryloyl gelatin comprises: weighing gelatin with a ratio of (8-10) g: 10 ml and mixing with a solvent, heating in a water bath at 40° C., stirring evenly to obtain a gelatin solution, slowly dropping 3-5 ml of methacrylic anhydride solution into the gelatin solution, stirring and fully reacting, dialyzing in deionized water for one week, freeze-drying to remove moisture, and obtaining the acryloyl gelatin.

[0022] Further preferably, in the above-mentioned method for preparing acryloyl gelatin, the solvent may be water or PBS solution.

[0023] Preferably, the photoinitiator is Ig2959 with a concentration percentage of 0.5-1%.

[0024] Further preferably, the preparation method of the acryloyl chitosan comprises: weighing chitosan in a ratio of 1 g:40 ml and mixing it with an acetic acid aqueous solution, heating it in a water bath at 40°C, ensuring that it is fully dissolved under magnetic stirring, and then dropwise adding 3-5 ml of methacrylic anhydride solution to the above-mentioned mixture, stirring and fully reacting, dialyzing it in deionized water for one week, and freeze-drying to remove moisture to obtain acryloyl chitosan.

[0025] Further preferably, in the above acetic acid aqueous solution, the volume ratio of acetic acid to water is 3:100.

[0026] Basic fibroblast growth factor can stimulate the endogenous expression of fibroblasts, significantly promote cell proliferation, and effectively promote wound self-repair ability.

[0027] Preferably, the anionic crosslinking agent includes any one of sodium tripolyphosphate, sodium metaphosphate or sodium dodecyl sulfate.

[0028] Further preferably, the anionic crosslinking agent is sodium tripolyphosphate. The anionic crosslinking agent is sodium tripolyphosphate (TPP), which has the ability of rapid crosslinking and is safe and non-toxic, does not require the use of organic solvents, can form nanoparticles with uniform particle size under mild conditions, and has a positive surface charge that is easy to adjust.

[0029] The present invention also provides a multifunctional two-section microneedle containing an intelligent nano-carrier, comprising a needle tip portion and a base portion, wherein the needle tip portion is distributed on the base portion in an array form.

[0030] Preferably, the multifunctional two-stage microneedle is composed of 10×10 circular microneedles, the needle tip portion is conical, and the needle tip spacing is 400-600 microns; the needle tip portion is 600-800 microns high, and the base portion is 20-300 microns thick.

[0031] The present invention also provides the use of a multifunctional two-segment microneedle containing an intelligent nanocarrier in the treatment of breast cancer metastasis, postoperative tissue repair treatment and drug delivery.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing a multifunctional two-stage microneedle containing an intelligent nanocarrier, wherein a carboxymethyl-β-cyclodextrin-chitosan solution, an anionic crosslinking agent and a doxorubicin hydrochloride drug solution are tightly combined to form stable nanostructured drug-loaded chitosan nanoparticles. This structure can protect drug molecules from damage such as enzymolysis and oxidation in the external environment, thereby improving the stability of the drug. The introduction of the carboxymethyl-β-cyclodextrin-chitosan solution makes the drug-loaded chitosan nanoparticles pH-responsive and can adjust the release rate of the drug under different pH environments. This characteristic of precisely controlling release can reduce the side effects of the drug and improve the therapeutic effect. The drug-loaded chitosan nanoparticles are uniform in size and can be used to construct pH-responsive drug carriers because of their good pH responsiveness. They remain stable under neutral or alkaline conditions, reducing the nonspecific release of drugs, and quickly disassemble in a weakly acidic environment. The nanoparticles will disintegrate and release drugs. This is because under acidic conditions, the anionic crosslinker is protonated and loses its negative charge, causing the anionic crosslinker and the chitosan crosslink in the carboxymethyl-β-cyclodextrin-chitosan solution to dissolve, thereby causing the nanoparticles to disintegrate into macromolecular chains, thereby achieving rapid and intelligent drug release, and being able to deliver drugs to the lesion tissue in a targeted manner, reducing the side effects of sudden drug release, and solving the technical problem that the microneedle technology in the prior art cannot achieve intelligent responsive drug release. The use of acryloyl-modified gelatin and chitosan is because under the irradiation of ultraviolet light, the carbon-carbon double bonds in the structures of the two can be cross-linked with each other under the action of a photoinitiator, thereby forming a photo-crosslinked acryloyl-modified gelatin-chitosan composite hydrogel to improve the mechanical properties of the microneedles. The base also uses dopamine hydrochloride, gelatin solution and fibroblast growth factor. On the one hand, due to the presence of arginine-glycine-aspartic acid polypeptide sequence on the gelatin macromolecular chain, it can promote cell adhesion, proliferation and differentiation, so that the microneedle has the ability to promote wound self-repair. Fibroblast growth factor can stimulate the endogenous expression of fibroblasts and significantly promote cell proliferation. On the other hand, the micro-nano fibers of the gelatin-polydopamine hydrogel formed by dopamine hydrochloride and gelatin solution maintain a complex three-dimensional mesh structure, which can absorb wound exudate, increase the permeability of the wound, promote the formation of new tissue, and help maintain the normal physiological state of the wound and repair damaged tissue. Its internal porous structure can accommodate drugs, promote the release of drugs and control the release rate and dosage of drugs, thereby effectively promoting wound healing. By adding dopamine hydrochloride, gelatin solution and fibroblast growth factor, cell adhesion, proliferation and differentiation can be promoted. Fibroblast growth factor can stimulate the endogenous expression of fibroblasts, significantly promote cell proliferation, and effectively promote the self-repair ability of wounds.

[0033] Furthermore, the present invention also provides a method for preparing drug-loaded chitosan nanoparticles, wherein the drug-loaded chitosan nanoparticles are prepared by adding a doxorubicin hydrochloride drug solution to a carboxymethyl-β-cyclodextrin-chitosan solution and cross-linking with an anionic cross-linking agent. This method can effectively encapsulate drugs, and chitosan, as a material with good biocompatibility and biodegradability, helps to achieve controlled release of drugs.

[0034] Furthermore, any one of sodium tripolyphosphate, sodium metaphosphate or sodium dodecyl sulfate is selected as a cross-linking agent to undergo a cross-linking reaction with the carboxymethyl-β-cyclodextrin-chitosan solution to form a stable nanostructure.

[0035] Furthermore, the amount ratio of acryloyl gelatin, acryloyl chitosan, photoinitiator and drug-loaded chitosan nanoparticles was (0.4-0.6) g: 0.09 g: 3 ml: (0.5-4) mg / ml, and the microneedle tip was formed on the polydimethylsiloxane mold by photocrosslinking. This method not only achieves drug encapsulation, but also improves the mechanical strength and stability of the microneedle through photocrosslinking technology, while maintaining good biocompatibility; the crosslinking degree was adjusted by adjusting the mass ratio of acryloyl chitosan and acryloyl gelatin and the time of ultraviolet light irradiation to obtain a microneedle tip with the best mechanical properties.

[0036] Furthermore, by using a dopamine hydrochloride, gelatin solution and fibroblast growth factor in a dosage ratio of 0.225 g:0.75 ml:(25-100) IU / ml, dopamine hydrochloride has good adhesion, which helps the microneedle patch to adhere firmly to the skin; while gelatin provides good biocompatibility and degradability.

[0037] Furthermore, by adding polymyxin, sodium periodate and sodium hydroxide solution in a dosage ratio of (25-100) IU / ml: (0.1-0.15) g: (0.05-0.15) ml to the base part, polymyxin makes the microneedle have better antibacterial and melanoma wound recovery effects, thereby making the microneedle have the property of promoting wound self-repair; the addition of sodium hydroxide solution helps to adjust the pH value of the gelatin-polydopamine solution and maintain the alkaline environment of the solution, thereby promoting the oxidation and polymerization reaction of dopamine to form a stable hydrogel base.

[0038] Furthermore, the UV irradiation time in the cross-linking reaction is controlled to 10-20 minutes to ensure that the composite hydrogel is fully cross-linked on the microneedle mold and that the microneedle has sufficient mechanical strength and stability while avoiding performance degradation due to excessive cross-linking.

[0039] Furthermore, using Ig2959 at a concentration of 0.5-1% as a photoinitiator ensures that the composite hydrogel is quickly and effectively cross-linked under ultraviolet light. As a highly efficient photoinitiator, Ig2959 can significantly improve the rate and efficiency of the cross-linking reaction.

[0040] The present invention also provides a multifunctional two-stage microneedle containing intelligent nanocarriers prepared by the above method, wherein the microneedle comprises a drug-carrying needle tip portion and a base portion having antibacterial and wound healing functions. This design enables the microneedle to provide additional antibacterial and tissue repair-promoting therapeutic effects while achieving drug delivery.

[0041] Furthermore, the multifunctional two-stage microneedle is composed of 10×10 circular microneedles, the needle tip part is conical, and the needle tip spacing is 400-600 microns; the needle tip part is 600-800 microns high, and the base thickness is 20-300 microns. The advantage of this structure is that the needle tip part can responsively release anticancer drugs for the treatment of breast cancer recurrence. The base part can provide certain mechanical properties for the needle tip, so that the MN needle tip penetrates the epidermis and reaches the vicinity of the lesion to release drugs, while the base can release growth factors and polymyxins for a long time, inhibit inflammation, and promote angiogenesis and cell proliferation.

[0042] The present invention also provides the use of a multifunctional two-segment microneedle containing an intelligent nanocarrier in the treatment of breast cancer metastasis, postoperative tissue repair treatment and drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the infrared spectrum of chitosan nanoparticles; Figure 2 is the X-ray diffraction pattern of chitosan nanoparticles; Figure 3 is a scanning electron microscope image of chitosan nanoparticles; Figure 4 is the particle size distribution diagram of chitosan nanoparticles; Figure 5 The drug release performance diagram of chitosan nanoparticles and two-stage microneedles (MN); Figure 6 is a scanning electron microscope image of a two-segment microneedle (MN); Figure 7 This is an ultra-depth-of-field microscopy image of a two-stage microneedle (MN); Figure 8 It is the mechanical properties diagram of the two-stage microneedle (MN); Fig. 9 Cell viability graph and OD value bar graph of biocompatibility test of gelatin, methacrylated gelatin, microneedle tip part, two-stage microneedle (MN) of the present invention, and control group (Control); Fig.10 Fluorescence microscopy images of live / dead cells of gelatin, methacrylated gelatin, microneedle tip, two-stage microneedle (MN) of the present invention, and control group (Control); Fig.11 Schematic diagram of the preparation of multifunctional two-segment microneedles according to the present invention. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0045] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0046] The present invention provides a multifunctional two-stage microneedle containing an intelligent nano-carrier, which is used for minimally invasive treatment of breast cancer recurrence and postoperative wound healing, and comprises the following steps: a microneedle patch loaded with doxorubicin hydrochloride and chitosan nanoparticles, wherein the microneedle patch comprises a base portion and a needle array arranged on the base portion; the base portion is formed by a gelatin-polydopamine solution, and the needle array is formed by mixing methacryloyl gelatin and methacryloyl chitosan to which doxorubicin hydrochloride and chitosan nanoparticles are added.

[0047] The present invention provides a method for preparing a multifunctional two-stage microneedle containing an intelligent nanocarrier, which is specifically implemented according to the following steps: Step 1, preparation of responsive chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 4ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 4ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, dialyzed, and centrifuged at room temperature. The supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan.

[0048] Weigh 25 mg DOX·HCl (doxorubicin hydrochloride) and place it in 2 mL dimethylformamide, add excess triethylamine, and dissolve it in an ultrasonic instrument for 20 min to prepare a doxorubicin hydrochloride drug solution. Weigh 10-25 ml of the obtained doxorubicin hydrochloride drug solution and add it to 50 mL of 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution to obtain a carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution, then use a syringe pump to add 50 mL of 0.4% (w / v) sodium tripolyphosphate solution dropwise, stir continuously to make it fully contact, and the resulting suspension is centrifuged at 5000-8000 rpm for 5 min at room temperature, and the precipitate is gently blown and washed three times with deionized water to obtain drug-loaded chitosan nanoparticles.

[0049] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: weigh 10g gelatin and dissolve it in 100ml PBS solution, heat it in a water bath at 40℃, and stir evenly. Slowly add 5ml of methacrylic anhydride solution to the gelatin solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl gelatin. Weigh 5g chitosan and dissolve it in 200ml 3% acetic acid aqueous solution, heat it in a water bath at 40℃, and ensure that it is fully dissolved under magnetic stirring. Then add 5ml of methacrylic anhydride solution dropwise to the above mixed solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl chitosan.

[0050] Dissolve 0.6-0.4g of acryloyl gelatin and 0.09g of acryloyl chitosan in 3ml of 0.5-1% Ig2959 aqueous solution, and mix the two. Ultrasonic treatment is performed on the drug-loaded chitosan nanoparticles to ensure that they are evenly dispersed in deionized water, and a 1.0mg / ml suspension is obtained and added to the above mixed solution and stirred thoroughly. Acryloyl gelatin and acryloyl chitosan precursor solutions are obtained.

[0051] Step 3: Prepare the backing precursor solution Specifically, 0.75g gelatin was dissolved in 4.5ml water, heated in a water bath at 50℃, and 0.225g dopamine hydrochloride was added to the gelatin solution after uniform stirring, and magnetic stirring was performed. Polymyxin and fibroblast growth factor were added to the solution, and 0.127g sodium periodate was added, and finally a drop of sodium hydroxide was added to make a mixed solution, which was the precursor solution of the microneedle backing.

[0052] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0053] The present invention cleverly combines nanotechnology and tumor microenvironment-responsive microneedle technology, and designs a needle-head two-stage microneedle platform containing smart nanocarriers through a step-by-step perfusion strategy for multifunctional treatment of breast cancer postoperative recurrence and wound infection / healing. The proposed micro-nanostructure includes a photo-crosslinked hydrogel tip containing chitosan-based self-assembled nanocarriers and a gelatin-polydopamine hydrogel backing layer. Due to the two-stage structure design of the microneedle, its compression performance reaches 4.48N-needle -1 , fully meeting the stress requirements of transdermal penetration. In addition, the microneedle also has long-term pH responsiveness and specific tumor microenvironment recognition capabilities, which can be used for targeted drug release. In addition, MN also exhibits excellent biocompatibility and auxiliary multifunctional therapeutic properties, including antibacterial properties, adhesion, self-healing properties, swelling ability and biodegradability, further broadening its potential applicability as a precision therapeutic material. In addition, comprehensive in vitro and in vivo experiments confirmed the comprehensive therapeutic effects of microneedles on metastatic breast cancer and its recurrence, as well as the healing of postoperative infected wounds, which greatly promoted the development of intelligent two-stage microneedles for postoperative adjuvant treatment of breast cancer. The multifunctional two-stage microneedles containing intelligent nanocarriers prepared by the present invention have multifunctional properties such as antibacterial properties, inhibition of cancer cell growth, and the ability to induce tissue regeneration, which can effectively inhibit the metastasis of breast cancer and can be used in wound repair and reconstruction of missing skin tissue to achieve "cancer treatment-tissue repair integration".

[0054] The present invention takes into account the harm of anti-tumor drugs to the human body and the activity of anti-tumor drugs when locally administered, and when preparing microneedles with tumor healing properties, it is also necessary to load anti-tumor drugs so that they can kill tumor cells and induce and stimulate tissue repair. The microneedles of the present invention have antibacterial properties, adhesion, self-healing properties, expansion ability, biodegradability, specific release of drugs, and are conducive to cell adhesion and growth. By constructing a transdermal drug delivery system, the precise release of doxorubicin hydrochloride is achieved, the toxic and side effects are reduced, and the synergistic treatment of metastatic breast cancer and its recurrence and postoperative wound infection is achieved.

[0055] The present invention is described in further detail below in conjunction with embodiments: Embodiment 1: The present invention is based on the preparation method of paper-based leather biomass materials, which is specifically implemented according to the following steps: Step 1, preparation of responsive drug-loaded chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 4ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 4ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, dialyzed, and centrifuged at room temperature. The supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan.

[0056] Weigh 25 mg DOX·HCl, place in 2 mL dimethylformamide, add 2 ml triethylamine, and dissolve in ultrasonic dispersion in an ultrasonic instrument for 20 min to prepare doxorubicin hydrochloride drug solution. Take 10 ml of the obtained doxorubicin hydrochloride drug solution and add it to 50 mL 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution to obtain carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution. Then, 50 ml of sodium tripolyphosphate solution was added dropwise to the carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution, and stirred continuously at room temperature to make it fully contacted. The resulting suspension was centrifuged at 5000 rpm for 5 min at room temperature, and the precipitate was gently blown and washed with deionized water three times to form a milky white nanoparticle suspension, which is the drug-loaded chitosan nanoparticles, and it was collected by centrifugation.

[0057] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: weigh 10g gelatin and dissolve it in 100ml phosphate buffer solution (PBS) solution, heat it in a water bath at 40℃, stir it evenly, slowly drop 5ml of methacrylic anhydride solution into the gelatin solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl gelatin. Weigh 5g chitosan and dissolve it in 200ml 3% acetic acid aqueous solution, heat it in a water bath at 40℃, and ensure that it is fully dissolved under magnetic stirring. Then add 3ml of methacrylic anhydride solution dropwise to the above mixed solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl chitosan.

[0058] 0.6 g of acryloyl gelatin and 0.09 g of acryloyl chitosan were dissolved in 3 ml of 0.5% Ig2959 aqueous solution, and the two were mixed. Chitosan nanoparticles were ultrasonically treated to ensure that they were evenly dispersed in deionized water to obtain a 1.0 mg / ml suspension and added to the above mixed solution and stirred thoroughly to obtain acryloyl gelatin and acryloyl chitosan precursor solutions.

[0059] Step 3: Prepare the backing precursor solution Specifically: 0.75g gelatin was dissolved in 4.5ml water, heated in a water bath at 50°C, and stirred evenly to obtain a gelatin solution; 0.225g dopamine hydrochloride was added to 0.75ml gelatin solution, and a mixed solution was obtained after magnetic stirring; then 30IU / ml polymyxin and fibroblast growth factor were added respectively, and 0.14g sodium periodate was added, and finally 0.1ml sodium hydroxide solution was added to prepare a mixed solution, which was the precursor solution for the microneedle backing.

[0060] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold containing the microneedle tip part to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0061] Example 2 Step 1, preparation of responsive drug-loaded chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 5ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 5ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, then dialyzed and centrifuged at room temperature. The supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan. 25mg of DOX·HCl was weighed and placed in 2mL of dimethylformamide, 2ml of triethylamine was added, and ultrasonic dispersion and dissolution were performed in an ultrasonicator for 20min to obtain doxorubicin hydrochloride drug solution. 15 ml of the obtained doxorubicin hydrochloride drug solution was added to 150 mL of 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution to obtain a carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution. Subsequently, 50 ml of sodium metaphosphate was added dropwise to the carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution and stirred continuously at room temperature to allow full contact. The resulting suspension was centrifuged at 6000 rpm for 5 min at room temperature, and the precipitate was washed three times with deionized water to form a milky white nanoparticle suspension, which was the drug-loaded chitosan nanoparticles, and was collected by centrifugation.

[0062] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: 8g gelatin was weighed and dissolved in 100ml PBS solution, heated in a water bath at 40℃, stirred evenly, 3ml methacrylic anhydride solution was slowly added to the gelatin solution, stirred and reacted fully, dialyzed in deionized water for one week, and freeze-dried to remove moisture to obtain acryloyl gelatin. 5g chitosan was weighed and dissolved in 200ml 3% acetic acid aqueous solution, heated in a water bath at 40℃, and ensured that it was fully dissolved under magnetic stirring. Then 4ml methacrylic anhydride solution was added dropwise to the above mixture, stirred and reacted fully, dialyzed in deionized water for one week, and freeze-dried to remove moisture to obtain acryloyl chitosan.

[0063] 0.5 g of acryloyl gelatin and 0.09 g of acryloyl chitosan were dissolved in 3 ml of 1% Ig2959 aqueous solution, and the two were mixed. Chitosan nanoparticles were ultrasonically treated to ensure that they were evenly dispersed in deionized water, and a 0.5 mg / ml suspension was obtained and added to the above mixed solution and stirred thoroughly. Acryloyl gelatin and acryloyl chitosan precursor solutions were obtained.

[0064] Step 3: Prepare the backing precursor solution Specifically: 0.75g gelatin was dissolved in 4.5ml water, heated in a 50°C water bath, and stirred evenly to obtain a gelatin solution; 0.225g dopamine hydrochloride was added to 0.75ml gelatin solution, and magnetic stirring was performed to obtain a mixed solution; then 25IU / ml polymyxin and fibroblast growth factor were added respectively, and 0.12g sodium periodate was added, and finally 0.15ml sodium hydroxide solution was added to prepare a mixed solution, which was the precursor solution for the microneedle backing.

[0065] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold containing the microneedle tip part to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0066] Example 3 Step 1, preparation of responsive drug-loaded chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 6ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 6ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, then dialyzed and centrifuged at room temperature. The supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan. 25mg of DOX·HCl was weighed and placed in 2mL of dimethylformamide, 2ml of triethylamine was added, and ultrasonic dispersion and dissolution were performed in an ultrasonicator for 20min to obtain doxorubicin hydrochloride drug solution. 20 ml of the obtained doxorubicin hydrochloride drug solution was added to 200 mL of 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution to obtain a carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution. Subsequently, 40 ml of sodium dodecyl sulfate was added dropwise to the carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution and stirred continuously at room temperature to allow full contact. The resulting suspension was centrifuged at 7000 rpm for 5 min at room temperature, and the precipitate was washed three times with deionized water to form a milky white nanoparticle suspension, i.e., chitosan nanoparticles, which was then collected by centrifugation.

[0067] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: weigh 10g gelatin and dissolve it in 100ml PBS solution, heat it in a water bath at 40℃, and stir evenly. Slowly add 4ml of methacrylic anhydride solution to the gelatin solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl gelatin. Weigh 5g chitosan and dissolve it in 200ml 3% acetic acid aqueous solution, heat it in a water bath at 40℃, and ensure that it is fully dissolved under magnetic stirring. Then add 5ml of methacrylic anhydride solution dropwise to the above mixed solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl chitosan.

[0068] 0.4 g of acryloyl gelatin and 0.09 g of acryloyl chitosan were dissolved in 3 ml of 0.5% Ig2959 aqueous solution, and the two were mixed. Chitosan nanoparticles were ultrasonically treated to ensure that they were evenly dispersed in deionized water to obtain a 2.0 mg / ml suspension and added to the above mixed solution and stirred thoroughly. Acryloyl gelatin and acryloyl chitosan precursor solutions were obtained.

[0069] Step 3: Prepare the backing precursor solution Specifically: 0.75g gelatin was dissolved in 4.5ml water, heated in a water bath at 50°C, and stirred evenly to obtain a gelatin solution; 0.225g dopamine hydrochloride was added to 0.75ml gelatin solution, and a mixed solution was obtained after magnetic stirring; then 35IU / ml polymyxin and fibroblast growth factor were added to the mixed solution respectively, and 0.13g sodium periodate was added, and finally 0.1ml sodium hydroxide was added to prepare a mixed solution, which was the precursor solution for the microneedle backing.

[0070] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold containing the microneedle tip part to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0071] Example 4 Step 1, preparation of responsive drug-loaded chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 7ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 7ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, then dialyzed and centrifuged at room temperature. The supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan. 25mg of DOX·HCl was weighed and placed in 2mL of dimethylformamide, 2ml of triethylamine was added, and ultrasonic dispersion and dissolution were performed in an ultrasonicator for 20min to obtain doxorubicin hydrochloride drug solution. 25 ml of the obtained doxorubicin hydrochloride drug solution was added to 250 mL of 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution to obtain a carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution. Subsequently, 50 ml of sodium tripolyphosphate was added dropwise to the carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution and stirred continuously at room temperature to allow full contact. The resulting suspension was centrifuged at 8000 rpm for 5 min at room temperature, and the precipitate was washed three times with deionized water to form a milky white nanoparticle suspension, which was the drug-loaded chitosan nanoparticles, and was collected by centrifugation.

[0072] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: weigh 8g gelatin and dissolve it in 100ml PBS solution, heat it in a water bath at 40℃, stir it evenly, slowly drop 5ml of methacrylic anhydride solution into the gelatin solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl gelatin. Weigh 5g chitosan and dissolve it in 200ml 3% acetic acid aqueous solution, heat it in a water bath at 40℃, and ensure that it is fully dissolved under magnetic stirring. Then add 5ml of methacrylic anhydride solution dropwise to the above mixed solution, stir and react fully, dialyze it in deionized water for one week, freeze-dry to remove moisture, and obtain acryloyl chitosan.

[0073] 0.4 g of acryloyl gelatin and 0.09 g of acryloyl chitosan were dissolved in 3 ml of 1% Ig2959 aqueous solution, and the two were mixed. Chitosan nanoparticles were ultrasonically treated to ensure that they were evenly dispersed in deionized water, and a 3.0 mg / ml suspension was obtained and added to the above mixed solution and stirred thoroughly. Acryloyl gelatin and acryloyl chitosan precursor solutions were obtained.

[0074] Step 3: Prepare the backing precursor solution Specifically: 0.75g gelatin was dissolved in 4.5ml water, heated in a water bath at 50°C, and stirred evenly to obtain a gelatin solution; 0.225g dopamine hydrochloride was added to 0.75ml gelatin solution, and a mixed solution was obtained after magnetic stirring; then 100IU / ml polymyxin and fibroblast growth factor were added respectively, and 0.14g sodium periodate was added, and finally 0.1ml sodium hydroxide solution was added to prepare a mixed solution, which was the precursor solution for the microneedle backing.

[0075] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold containing the microneedle tip part to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0076] Example 5 Step 1, preparation of responsive drug-loaded chitosan nanoparticles: Specifically, 1g of carboxymethyl-β-cyclodextrin, 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt and 0.55g of N-hydroxysuccinimide were added to 20ml of deionized water and activated at room temperature for 1h to obtain carboxymethyl-β-cyclodextrin carboxyl activation solution. 8ml of 1% chitosan (w / v) acetic acid aqueous solution was added to 8ml of carboxymethyl-β-cyclodextrin carboxyl activation solution, stirred at room temperature for 24h to undergo amidation reaction, then dialyzed and centrifuged at room temperature, and the supernatant was freeze-dried to remove moisture to obtain a white flocculent, which was carboxymethyl-β-cyclodextrin-chitosan. 25mg of DOX·HCl was weighed, placed in 2mL of dimethylformamide, 2ml of triethylamine was added, and ultrasonic dispersion and dissolution were performed in an ultrasonicator for 20min to obtain doxorubicin hydrochloride drug solution. 25 ml of the obtained doxorubicin hydrochloride drug solution was added to 300 mL of 1% (w / v) carboxymethyl-β-cyclodextrin-chitosan solution. 50 ml of sodium tripolyphosphate was added dropwise to the carboxymethyl-β-cyclodextrin-chitosan-doxorubicin hydrochloride solution, and stirred continuously at room temperature to allow full contact. The resulting suspension was centrifuged at 5000 rpm for 5 min at room temperature, and the precipitate was washed three times with deionized water to form a milky white nanoparticle suspension, namely chitosan nanoparticles, which was collected by centrifugation.

[0077] Step 2: Preparation of acryloyl gelatin and acryloyl chitosan precursor solutions Specifically: 8g gelatin was weighed and dissolved in 100ml PBS solution, heated in a water bath at 40℃, stirred evenly, 5ml methacrylic anhydride solution was slowly added to the gelatin solution, stirred and reacted fully, dialyzed in deionized water for one week, and freeze-dried to remove moisture to obtain acryloyl gelatin. 5g chitosan was weighed and dissolved in 200ml 3% acetic acid aqueous solution, heated in a water bath at 40℃, and ensured that it was fully dissolved under magnetic stirring. Then 5ml methacrylic anhydride solution was added dropwise to the above mixture, stirred and reacted fully, dialyzed in deionized water for one week, and freeze-dried to remove moisture to obtain acryloyl chitosan.

[0078] 0.4 g of acryloyl gelatin and 0.09 g of acryloyl chitosan were dissolved in 3 ml of 1% Ig2959 aqueous solution, and the two were mixed. Chitosan nanoparticles were ultrasonically treated to ensure that they were evenly dispersed in deionized water, and a 4.0 mg / ml suspension was obtained and added to the above mixed solution and stirred thoroughly. Acryloyl gelatin and acryloyl chitosan precursor solutions were obtained.

[0079] Step 3: Prepare the backing precursor solution Specifically: 0.75g gelatin was dissolved in 4.5ml water, heated in a water bath at 50°C, and stirred evenly to obtain a gelatin solution; 0.225g dopamine hydrochloride was added to 0.75ml gelatin solution, and a mixed solution was obtained after magnetic stirring; then 60IU / ml polymyxin and fibroblast growth factor were added respectively, and 0.15g sodium periodate was added, and finally 0.15ml sodium hydroxide solution was added to prepare a mixed solution, which was the precursor solution for the microneedle backing.

[0080] Step 4, pouring the acryloyl gelatin and acryloyl chitosan precursor liquid onto the surface of the polydimethylsiloxane mold, exposing the mold to ultraviolet light for crosslinking, then vacuuming to remove bubbles and drying, repeating the steps and leaving it at room temperature until it is completely dried to obtain the microneedle tip part. The backing part precursor liquid is coated on the mold containing the microneedle tip part to form a microneedle substrate and then dried to make a two-stage microneedle. Finally, the microneedle patch is gently peeled off from the polydimethylsiloxane mold and dried and stored at low temperature before use.

[0081] Figure 1 The infrared spectrum of chitosan nanoparticles shows that compared with chitosan, the amide peak of chitosan nanoparticles has a slight red shift and the signal is significantly enhanced. This may be because the amino group of chitosan forms a new amide bond with the carboxyl group of carboxymethyl-β-cyclodextrin. In addition, chitosan nanoparticles have a strong infrared spectrum at 1416 cm -1 A carboxymethyl absorption peak appeared nearby, and the absorption peak signal originated from the carboxymethyl group in carboxymethyl-β-cyclodextrin. The results showed that chitosan nanoparticles were successfully prepared.

[0082] Figure 2 The X-ray diffraction diagram of chitosan nanoparticles shows that CS contains type I and type II crystals, and type II crystals are dominant. CMCD also contains type I and type II crystals, and type II crystals are slightly more than type I crystals. In the XRD spectrum of CS-g-CD, the diffraction peak of type II crystals is significantly broader than that of CS, almost covering the area of ​​the diffraction peak of type II crystals in CS and CMCD, while the diffraction peak of type I crystals in CS-g-CD migrates from 11° of CS to 7°, and the signal is enhanced. The results show that the introduction of carboxymethyl-β-cyclodextrin not only maintains the original ordered structure of chitosan to a certain extent, but also changes the orderliness of part of the chitosan structure and generates a new ordered structure.

[0083] Figure 3 This is a scanning electron microscope image of chitosan nanoparticles. The chitosan nanoparticles are spherical particles with a diameter of about 450 nm and a relatively uniform size distribution.

[0084] Figure 4 The particle size distribution diagram of chitosan nanoparticles shows that as the amount of sodium tripolyphosphate added increases, the average particle size of the particles gradually increases. If the amount of sodium tripolyphosphate added is too small, the resulting nanoparticles are too small and the drug loading is insufficient. When the proportion increases, fine particles can be seen precipitating. At this time, the drug cannot be delivered in the tumor environment because the carrier is too large. When the mixing ratio is 4:1, the particle size is 443.9nm, which can meet the standard of drug delivery and can encapsulate an appropriate amount of drug.

[0085] Figure 5 The results show that chitosan nanoparticles and two-stage microneedles have obvious pH responsiveness and remain stable in neutral and alkaline environments, but decompose rapidly in weakly acidic environments, indicating that chitosan nanoparticles disintegrate under pH 5.5 conditions, and the nanoparticles decompose into carboxymethyl-β-cyclodextrin macromolecular chains. This is because under the acidic conditions, sodium tripolyphosphate is protonated and loses its negative charge, causing the sodium tripolyphosphate / chitosan cross-linking to dissolve, thereby causing the nanoparticles to disintegrate into macromolecular chains. Therefore, it is expected to be widely used in nano drug delivery systems in the acidic microenvironment of tumors.

[0086] Figure 6 It is a scanning electron microscope image of a two-section microneedle, which shows the macroscopic image of the complete microneedle and the complete microneedle under a microscope. The microneedle is composed of 10×10 circular microneedles, the needle tip part is conical, and the needle tip distance is about 500 microns.

[0087] Figure 7This is an ultra-depth microscope image of a two-stage microneedle, which can visualize the two-stage structure of the microneedle. It can be seen from the ultra-depth microscope image that the microneedle has an obvious two-stage structure, which is divided into a needle tip part (red) and a base part. The height of the microneedle tip is about 800 microns and the thickness of the base is about 200 microns. The advantage of this structure is that the needle tip part can responsively release anticancer drugs for the treatment of breast cancer recurrence. The base part can provide certain mechanical properties for the needle tip, so that the MN needle tip penetrates the epidermis and reaches the vicinity of the lesion to release drugs, while the base can release growth factors and polymyxins for a long time, inhibit inflammation, and promote angiogenesis and cell proliferation.

[0088] Figure 8 This is a diagram of the mechanical properties of a two-stage microneedle. The results in the figure show that the force applied to the microneedle gradually increases with the strain, and the maximum stress that can be achieved is 448N, which fully meets the stress required for skin penetration.

[0089] Fig. 9 The cell viability graph and OD value bar graph of the biocompatibility test of gelatin, methacrylated gelatin, microneedle needle tip, two-stage microneedle of the present invention, and control group are shown. The results show that after incubation for a uniform time, the cell viability and OD value of the experimental group are basically consistent with those of the control group, and on the fifth day, the cell activity of the microneedle group is significantly better than that of the control group, and the cell activity is more active than before, indicating that the microneedle group has a positive regulatory effect on the activity of cells and can effectively promote the growth and proliferation of cells. This is due to the presence of arginine-glycine-aspartic acid polypeptide sequences on the gelatin macromolecular chain, which can promote cell adhesion, proliferation and differentiation. On the other hand, the addition of basic fibroblast growth factor to the polydopamine hydrogel base can stimulate the endogenous expression of fibroblasts and significantly promote cell proliferation.

[0090] Fig.10 The live / dead cell fluorescence microscope images of gelatin, methacrylated gelatin, microneedle tip, two-stage microneedle of the present invention, and control group show that the introduction of each experimental group did not cause obvious cytotoxicity, and the cell growth and distribution were good. There was no significant difference compared with the control group on the 3rd day, and no large number of red dead cells appeared. It was clearly observed that the number of green cells in the microneedle group increased after 5 days, which means that the microneedle has good biocompatibility and promotes cell proliferation, which is crucial for its clinical application.

[0091] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a multifunctional two-stage microneedle containing an intelligent nanocarrier, characterized in that: First, carboxymethyl-β-cyclodextrin-chitosan solution, anionic crosslinking agent and doxorubicin hydrochloride drug solution are used to construct drug-loaded chitosan nanoparticles, then gelatin and chitosan are acrylated to obtain acrylated gelatin and acrylated chitosan, acrylated gelatin, acrylated chitosan, photoinitiator solution and drug-loaded chitosan nanoparticles are mixed to prepare acrylated gelatin-chitosan composite hydrogel, the composite hydrogel is dropped onto a microneedle mold, cross-linked under ultraviolet light, and dried to obtain a needle tip part, dopamine hydrochloride, gelatin solution and fibroblast growth factor are added to the needle tip part to form a base part, the mold is dried and demolded to obtain a two-stage microneedle.

2. The method for preparing a multifunctional two-stage microneedle containing a smart nanocarrier according to claim 1, characterized in that: The preparation method of drug-loaded chitosan nanoparticles comprises: mixing and stirring a doxorubicin hydrochloride drug solution, a carboxymethyl-β-cyclodextrin-chitosan solution and an anionic crosslinking agent, and centrifuging and washing the obtained suspension at room temperature; the volume ratio of the doxorubicin hydrochloride drug solution, the carboxymethyl-β-cyclodextrin-chitosan solution and the anionic crosslinking agent is (10-25): (1-6):

1.

3. The method for preparing the multifunctional two-stage microneedle containing smart nanocarriers according to claim 1, characterized in that: The anionic crosslinking agent includes any one of sodium tripolyphosphate, sodium metaphosphate or sodium dodecyl sulfate.

4. The method for preparing the multifunctional two-stage microneedle containing smart nanocarriers according to claim 1, characterized in that: The usage ratio of the acryloyl gelatin, acryloyl chitosan, photoinitiator solution and drug-loaded chitosan nanoparticles is (0.4-0.6) g: 0.09 g: 3 ml: (0.5-4) mg / ml; in the above cross-linking reaction, the ultraviolet light irradiation time is 10-20 min.

5. The method for preparing the multifunctional two-stage microneedle containing smart nanocarriers according to claim 1, characterized in that: The dosage ratio of dopamine hydrochloride, gelatin solution and fibroblast growth factor is 0.225g:0.75ml:(25-100)IU / ml.

6. The method for preparing the multifunctional two-stage microneedle containing smart nanocarriers according to claim 1, characterized in that: Polymyxin, sodium periodate and sodium hydroxide solution in a dosage ratio of (25-100) IU / ml: (0.1-0.15) g: (0.05-0.15) ml are added to the substrate part to prepare a microneedle substrate precursor solution as the substrate part.

7. The method for preparing a multifunctional two-stage microneedle containing smart nanocarriers according to claim 1, characterized in that: The photoinitiator used was Ig2959 with a concentration of 0.5-1%.

8. The multifunctional two-segment microneedle prepared by the method for preparing a multifunctional two-segment microneedle containing smart nanocarriers according to any one of claims 1 to 7, characterized in that: It comprises a needle tip part and a base part, wherein the needle tip part is distributed on the base part in an array form.

9. The multifunctional two-stage microneedle containing smart nanocarriers according to claim 8, characterized in that: The multifunctional two-stage microneedle is composed of 10×10 circular microneedles, the needle tip part is conical, and the needle tip spacing is 400-600 microns; the needle tip part is 600-800 microns high, and the base part is 20-300 microns thick.

10. Use of the multifunctional two-segment microneedle containing smart nanocarriers according to any one of claims 8 to 9 in drug delivery.

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