A partitioned delivery environment-responsive hydrogel microneedle, a preparation method thereof and application thereof in oral squamous carcinoma treatment

Abstract

The application discloses a kind of partition delivery environment response type hydrogel microneedle and its preparation method and application in oral squamous carcinoma treatment.The hydrogel microneedle includes backing layer and the needle tip layer being arranged on the surface of the backing layer, the needle tip layer includes multiple array distribution microneedle tip body, each the needle tip body is connected with the backing layer respectively;Wherein, the backing layer has enzyme response, and the needle tip layer has acidic pH response.The microneedle uses innovative "integration partition design", and two hydrogels with different functions are integrated in single microneedle array: backing layer responds oral flora imbalance microenvironment, needle tip layer responds tumor weak acidic microenvironment.The design can realize partition, accurate, intelligentized synergistic treatment to oral mucosa surface and deep tumor tissue, so as to overcome the technical bottleneck of traditional dosage form in space selectivity, single drug type, release behavior uncontrollable and great systemic toxicity etc.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a zoned delivery environmentally responsive hydrogel microneedle, its preparation method, and its application in the treatment of oral squamous cell carcinoma. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the oral and maxillofacial region. Its clinical treatment currently relies mainly on traditional methods such as surgical resection, radiotherapy, and systemic chemotherapy. However, applying these treatment strategies to the unique anatomical location of the oral cavity presents numerous significant challenges.

[0003] The oral cavity has a moist, dynamic, and highly complex microenvironment. Continuous saliva secretion, tongue movement, and swallowing actions cause traditional topical medications, such as mouthwash and oral gels, to be easily diluted and washed away rapidly. This results in a very short retention time of the drug at the lesion site, low bioavailability, and difficulty in maintaining effective therapeutic concentrations.

[0004] Meanwhile, the occurrence and development of OSCC are closely related to the imbalance of the oral microecology. In cancerous areas, abnormal enrichment of opportunistic pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum can often be observed. These harmful bacteria not only directly promote tumor progression, but their metabolic activities, such as the secretion of proteases and lipopolysaccharides (LPS), also exacerbate local immunosuppression and inflammatory states, thereby forming a complex "microbiota-tumor" co-evolutionary microenvironment.

[0005] Existing treatment systems have significant limitations in addressing the aforementioned complexities. Specifically: First, there is a lack of ability to segment and precisely intervene in oral microbiota regions and deep tumor tissues. Existing drug formulations cannot spatially distinguish between drugs that act on the surface microbiota and drugs that penetrate into the tumor, resulting in a lack of targeted treatment.

[0006] Secondly, traditional drug carriers struggle to efficiently load drugs with significantly different properties simultaneously. For example, they can handle hydrophobic small-molecule chemotherapy drugs (such as docetaxel, DTX, a chemotherapy drug that induces immunogenic cell death (ICD) in tumor cells) and hydrophilic large-molecule biological agents (such as antibodies), which limits the effective implementation of multi-mechanism synergistic treatment strategies.

[0007] Third, drug release behavior generally lacks intelligent responsiveness. It cannot achieve on-demand drug release "at the required time and in the required place" based on pathological characteristics such as oral flora imbalance (e.g., upregulation of specific enzyme expression) or tumor microenvironment (e.g., weak acidity), thus failing to achieve the goal of maximizing efficacy and minimizing side effects.

[0008] Fourth, while systemic administration (such as intravenous injection) can ensure drug distribution in the body, it can cause serious toxic side effects, such as nephrotoxicity and neurotoxicity. Moreover, the actual concentration of the drug at oral lesions is often limited, resulting in an unfavorable situation of "high systemic toxicity and low local efficacy".

[0009] In summary, developing a novel drug delivery platform that can achieve zoned delivery, multi-drug co-loading, environmentally responsive release, and combines local high efficiency with systemic safety has become a critical technological bottleneck that urgently needs to be overcome in the treatment of OSCC. Summary of the Invention

[0010] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a hydrogel microneedle that enables zoned delivery and is environmentally responsive.

[0011] The present invention also proposes a method for preparing the above-mentioned hydrogel microneedles.

[0012] This invention also proposes the application of the above-mentioned hydrogel microneedles.

[0013] According to one aspect of the present invention, a hydrogel microneedle is provided, comprising a backing layer and a tip layer disposed on the surface of the backing layer, the tip layer comprising a plurality of arrayed micron-sized tips, each tip being respectively associated with the backing layer; wherein the backing layer is enzyme-responsive and the tip layer is acidic pH-responsive.

[0014] The hydrogel microneedles according to embodiments of the present invention have at least the following beneficial effects: The present invention provides an environmentally responsive hydrogel microneedle capable of zoned delivery. This microneedle employs an innovative "integrated zoned design," integrating two functionally different hydrogel layers into a single microneedle array: the backing layer responds to the dysbiotic microenvironment of the oral microbiota, and the tip layer responds to the weakly acidic microenvironment of tumors. This design enables zoned, precise, and intelligent synergistic treatment of oral mucosal surface and deep tumor tissues, thereby overcoming the technical bottlenecks of traditional dosage forms such as poor spatial selectivity, single drug loading type, uncontrollable release behavior, and high systemic toxicity. Compared with existing technologies, this solution has several advantages, including: 1) Improved precision in spatial treatment: Existing formulations struggle to distinguish between drugs that act on the surface microbiota and those that need to penetrate deep into tumor tissue within the complex oral environment. This invention utilizes a microneedle structure to achieve physical partitioning and precise drug delivery between the "oral microbiota region" and the "tumor tissue region," enabling different drugs to reach their respective optimal sites of action and avoiding reduced efficacy or local side effects caused by non-targeted release.

[0015] 2) Enhancing the versatility of drug delivery systems: Traditional nanocarriers or gel matrices struggle to simultaneously achieve efficient loading and stable coexistence of hydrophobic small molecule drugs and hydrophilic macromolecular biological agents (such as antibodies). This invention provides a universal and efficient technical platform for combination drug delivery by constructing a nanocarrier-hydrogel composite system capable of simultaneously loading hydrophobic and hydrophilic drugs.

[0016] 3) Achieving intelligent drug release: The release behavior of conventional formulations in the dynamic oral environment is uncontrollable. This invention introduces a microenvironment-responsive release mechanism, enabling the backing layer to degrade in response to sialidase secreted by dysbiotic flora, while the needle tip layer can release drugs in response to the weakly acidic microenvironment of tumor tissue, thereby achieving on-demand and intelligent controlled drug release in the target area.

[0017] 4) Reducing the risk of systemic toxicity: The toxic side effects caused by systemic administration are a major bottleneck limiting the clinical application of effective drugs such as polymyxin B and docetaxel. This invention uses a local microneedle delivery method, utilizing the bioadhesive properties of microneedles to penetrate the mucosal barrier and hydrogels, significantly increasing the drug's retention time and local concentration at the lesion site, thereby enhancing efficacy while minimizing systemic exposure and related toxic side effects.

[0018] According to some embodiments of the present invention, the backing layer includes a gel matrix I and nanoparticles I distributed in the gel matrix I, wherein the gel matrix I is prepared from an enzyme-responsive material, and the surface of the nanoparticles I is modified with components that regulate oral flora.

[0019] According to some embodiments of the present invention, the needle tip layer comprises a gel matrix II and nanoparticles II distributed in the gel matrix II, wherein the gel matrix II is prepared from an acidic pH-responsive material, and the nanoparticles II are loaded with a hydrophobic chemotherapeutic drug and a hydrophilic immune checkpoint inhibitor.

[0020] According to some embodiments of the present invention, the gel matrix I and the nanoparticles I form a dynamic covalent bond through hydrazone bonds I.

[0021] According to some embodiments of the present invention, the enzyme-responsive material contains an amino group. The material may be a component that inherently contains an amino group or one that has been modified with an amino group.

[0022] According to some embodiments of the present invention, the nanoparticle I contains aldehyde groups on its surface and is formed by the self-assembly of an amphiphilic polymer. The aldehyde groups undergo a covalent reaction with amino groups in molecules that regulate oral flora, such as polymyxin B (PMB), thereby modifying the surface of the particles with drugs such as PMB. The remaining aldehyde groups form dynamic covalent bonds with amino groups in the gel substrate material through hydrazone bonds, constructing a three-dimensional network hydrogel.

[0023] According to some embodiments of the present invention, the raw materials for preparing the nanoparticle I include aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid (CHO-HPG-PLA).

[0024] According to some embodiments of the present invention, the oral flora regulating components include antibiotics, such as polymyxin B (PMB).

[0025] According to some embodiments of the present invention, the enzyme-responsive material is an oral enzyme-degradable component.

[0026] According to some embodiments of the present invention, the enzyme includes at least one of sialidase, gingival protease, or hyaluronidase. The enzyme-responsive material primarily responds to enzymes such as sialidase, gingival protease, or hyaluronidase, which are present in the oral cavity and specifically secreted by dysbiotic flora. The matrix layer component employs a component capable of being degraded by enzymes specifically secreted by dysbiotic flora, and can be selected according to changes in the target pathogenic bacteria environment. Sialidase-degradable components can be used, or biomaterials degradable by gingival protease or hyaluronidase can be employed. These enzyme components include sialidase secreted by oral pathogenic bacteria in oral squamous cell carcinoma tumor models; if the target pathogenic bacteria environment changes, the material can also target the gingival protease or hyaluronidase primarily secreted by the bacteria.

[0027] According to some embodiments of the present invention, the material used to prepare the gel matrix I comprises at least one of gelatin, collagen, amino-modified polysialic acid, or amino-modified hyaluronic acid. The responsive hydrogel matrix is ​​prepared using oral enzyme-degradable components, the specific type depending on the target organism. Gelatin and collagen can utilize their existing amino groups without additional amino modification to connect with the aldehyde groups on the surface of nanoparticles to form a hydrogel network.

[0028] According to some embodiments of the present invention, the gel matrix I is prepared by an amino-modified polysialic acid, and the nanoparticles I are prepared by an aldehyde-modified hyperbranched polyglyglycerol-polylactic acid (CHO-HPG-PLA). The surface of the nanoparticles I is modified with polymyxin B. The backing layer is a hydrogel formed by cross-linking amino-modified polysialic acid and polymyxin B-modified aldehyde-modified hyperbranched polyglyglycerol-polymyxin B nanoparticles (CHO-HPG-PMB) via hydrazone bonds.

[0029] Enzyme-responsive release: When harmful bacteria (such as Porphyromonas gingivalis) proliferate excessively in the oral cavity, it leads to the accumulation of sialidase in the oral cavity, which specifically degrades the polysialic acid (pSA) backbone, causing the hydrogel network to disintegrate and thus precisely releasing the internal CHO-HPG-PMB nanoparticles.

[0030] Selective antibacterial and detoxification: The released nanoparticles selectively kill Gram-negative harmful bacteria through PMB on their surface; at the same time, PMB can efficiently adsorb and neutralize pathogen-associated molecular patterns such as LPS released by harmful bacteria, which helps reduce chronic inflammation in the tumor microenvironment and alleviate immunosuppression.

[0031] Localized long-lasting retention: The abundant aldehyde groups on the surface of nanoparticles can form Schiff base bonds with amino groups on the surface of oral mucosa, producing a bioadhesion effect, significantly prolonging the retention time of drugs in the oral cavity, improving bioavailability, and helping to reduce antibiotic abuse and protect gut microbiota health.

[0032] According to some embodiments of the present invention, the composition of gel matrix II may be the same as or different from that of gel matrix I.

[0033] According to some embodiments of the present invention, the gel matrix II comprises at least one of gelatin, collagen, amino-modified polysialic acid, or amino-modified hyaluronic acid.

[0034] According to some embodiments of the present invention, the gel matrix II and the nanoparticles II form a dynamic covalent bond through hydrazone bonds II. The degradation of the needle tip is primarily driven by a weakly acidic pH. When the microneedle penetrates the tumor tissue, the microneedle tip responds to the acidic tumor microenvironment, directly catalyzing the breakage of the hydrazone bonds (C=N), leading to the dissociation of the hydrogel network and the release of the drug. Although the needle tip layer also contains pSA, the concentration of sialidase in the tumor tissue is extremely low; therefore, enzymatic degradation is not the main pathway for release from the needle tip layer, and the needle tip layer portion does not degrade synchronously with the degradation of the backing layer.

[0035] According to some embodiments of the present invention, the hydrazone II bond can be broken in a weakly acidic environment with a pH of 6.5-6.8. When the microneedle is inserted into the tumor tissue, the weakly acidic environment causes the hydrazone bond to break, the hydrogel to degrade, and the chemotherapeutic drugs to be slowly released from the nanoparticles; while the immune checkpoint inhibitor is linked by a Schiff base bond formed by its own amino group and the aldehyde group of the nanoparticle, which breaks in the weakly acidic environment, allowing the immune checkpoint inhibitor to be released.

[0036] According to some embodiments of the present invention, the material used to prepare the gel matrix II is an amino-containing component or an amino-modified component.

[0037] According to some embodiments of the present invention, the surface of the nanoparticles II contains aldehyde groups.

[0038] According to some embodiments of the present invention, the surface of the immune checkpoint inhibitor contains an amino group, which is linked to an aldehyde group on the surface of the nanoparticle II via a Schiff base bond. The nanoparticle II is linked to the immune checkpoint inhibitor via a Schiff base bond, thereby adsorbing antibodies.

[0039] According to some embodiments of the present invention, the hydrophobic drug is encapsulated inside nanoparticle II, and the immune checkpoint inhibitor is connected to the outer surface of nanoparticle II.

[0040] According to some embodiments of the present invention, the gel matrix II is prepared by an amino-modified polysialic acid, and the nanoparticle II is prepared by an aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid (CHO-HPG-PLA). The nanoparticle II contains a hydrophobic drug internally and an immune checkpoint inhibitor is attached to its outer surface. This allows for the efficient loading and synergistic delivery of both hydrophobic small molecule drugs (encapsulated in a hydrophobic core) and hydrophilic macromolecule drugs such as immune checkpoint inhibitors (coupled to a hydrophilic shell via Schiff base bonds) using the same nanocarrier (CHO-HPG-PLA).

[0041] pH-triggered deep delivery: Utilizing the unique weak acidity of the tumor microenvironment to trigger drug release from the needle tip layer, ensuring efficient drug release only within the tumor tissue, without interfering with the release mechanism of the backing layer, achieving true regional and targeted delivery.

[0042] According to some embodiments of the present invention, the gel matrix II is prepared by an amino-modified polysialic acid, and the nanoparticle II is prepared by an aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid (CHO-HPG-PLA). The nanoparticle II is internally loaded with docetaxel (DTX) and externally connected with an anti-Tim-3 antibody (aTim-3). The needle tip layer is composed of an amino-modified polysialic acid (pSA) and aldehyde-modified hyperbranched polyglycidyl ether nanoparticles (Docetaxel and anti-Tim-3 antibody loaded CHO-HPG Nanoparticles, HPG-DTX / aTim-3) cross-linked by hydrazone bonds to form a hydrogel.

[0043] The hydrophobic chemotherapy drug docetaxel (DTX) was encapsulated in the polylactic acid hydrophobic core of CHO-HPG-PLA nanoparticles. Subsequently, the aldehyde group on the hydrophilic shell of the nanoparticles was linked to the amino group of the anti-Tim-3 antibody (aTim-3) via a Schiff base bond, thereby adsorbing the antibody.

[0044] Hydrogel Crosslinking and Release: HPG-DTX / aTim-3 nanoparticles were mixed with amino-modified pSA to form a hydrazone-crosslinked hydrogel. When microneedles were inserted into tumor tissue (pH ~6.5-6.8), the acidic environment caused the hydrazone bonds to break, the hydrogel to degrade, and the chemotherapeutic drugs to be slowly released after translation from the nanoparticles. Immune checkpoint inhibitors, on the other hand, are formed by Schiff base bonds between their own amino groups and the aldehyde groups of the nanoparticles. These bonds break in a weakly acidic environment, thereby releasing the immune checkpoint inhibitors.

[0045] According to another aspect of the present invention, a method for preparing the above-mentioned hydrogel microneedles is provided, comprising the following steps: S1. Inject the prepolymer liquid II containing the raw materials for preparing the needle tip layer into the conical needle tip cavity of the mold; after the needle tip layer has been initially shaped, inject the prepolymer liquid I containing the raw materials for preparing the backing layer into the mold to cover the needle tip layer. S2. Dry and demold to obtain the hydrogel microneedles.

[0046] The preparation method according to the embodiments of the present invention has at least the following beneficial effects: it is prepared step by step by using a mold method, and then dried and demolded to obtain the product, which is simple to operate. At the same time, the method ensures the integration of the physical structure of the backing layer and the needle tip layer and the independence of their functional release. The final product is an innovative drug delivery system that combines minimally invasive, local, dual-response, regional targeting and long-term retention.

[0047] According to some embodiments of the present invention, step S1 further includes the preparation of prepolymer liquid II, comprising the following steps: Hydrophobic chemotherapeutic drugs are encapsulated inside nanoparticles II using raw materials, and then mixed with raw materials for the preparation of immune checkpoint inhibitors and gel matrix II to obtain the final product.

[0048] According to some embodiments of the present invention, the prepolymer II comprises HPG-DTX, aTim-3 and amino-modified pSA.

[0049] According to some embodiments of the present invention, step S1 further includes the preparation of prepolymer liquid I, comprising the following steps: Nanoparticle I is prepared by self-assembly of raw materials in water. Then, the surface of the nanoparticle I is modified with components that regulate oral flora. Finally, it is mixed with the raw materials for preparing gel matrix I to obtain the final product.

[0050] According to some embodiments of the present invention, the prepolymer liquid I comprises CHO-HPG-PMB and amino-modified pSA.

[0051] According to some embodiments of the present invention, the prepolymer liquid I or prepolymer liquid II uses water as a solvent (or dispersion medium).

[0052] According to some embodiments of the present invention, step S1 further includes the step of adding the needle tip layer prepolymer or the backing layer prepolymer to the mold and then treating it by centrifugation. Centrifugation is used to fill and remove air bubbles.

[0053] According to some embodiments of the present invention, the drying temperature in step S2 is 10~25°C. Drying at room temperature or low temperature is acceptable, and the conditions are mild. After drying, the microneedles are peeled off from the mold to obtain a structurally complete partitioned delivery environmentally responsive hydrogel microneedle array.

[0054] According to another aspect of the present invention, the application of the above-described hydrogel microneedles in the preparation of oral squamous cell carcinoma treatment drugs is provided.

[0055] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0056] Figure 1 This is a schematic diagram illustrating the design, preparation principle, and mechanism of action of the hydrogel microneedle structure according to an embodiment of the present invention.

[0057] Figure 2 This is a transmission electron microscope image of the HPG-PMB nanoparticles prepared according to an embodiment of the present invention.

[0058] Figure 3 This is a scanning electron microscope image of the backing layer hydrogel prepared according to an embodiment of the present invention.

[0059] Figure 4 This is a scanning electron microscope image of the hydrogel microneedles prepared according to an embodiment of the present invention.

[0060] Figure 5 The following figures show the in vitro selective antibacterial effect evaluation results of HPG-PMB nanoparticles prepared in the embodiments of the present invention: (a) Antibacterial activity of HPG-PMB against Fusobacterium nucleatum; (b) Antibacterial activity of HPG-PMB against Porphyromonas gingivalis; (c) Antibacterial activity of HPG-PMB against P. anaerobius.

[0061] Figure 6These are macroscopic images of the enzyme-responsive degradation of the backing layer hydrogel prepared according to embodiments of the present invention, and transmission electron microscopy images of HPG-PMB nanoparticles after release.

[0062] Figure 7 This is a SEM image of the hydrogel backing layer prepared in the embodiments of the present invention after incubation in PBS with or without sialidase (0.5 mg / mL) for 24 hours.

[0063] Figure 8 This is a graph showing the cumulative release of HNP-PMB in simulated oral fluid after enzyme-responsive degradation of the hydrogel backing layer prepared in the embodiments of the present invention.

[0064] Figure 9 This is a stereomicroscopic image of the acid-responsive degradation of the hydrogel tip prepared in an embodiment of the present invention.

[0065] Figure 10 This describes the cumulative release kinetics of aTim3 prepared by DTX@HNP-aTim3 in the embodiments of the present invention under pH 6.8 and pH 7.4 conditions.

[0066] Figure 11 This refers to the cell survival rate of HOEC cells and HIEC-6 cells after treatment with different concentrations of the backing layer hydrogel prepared in the embodiments of the present invention.

[0067] Figure 12 The hydrogel microneedles prepared in this embodiment of the invention are used to treat tumor growth curves in a mouse oral squamous cell carcinoma in situ model.

[0068] Figure 13 The hydrogel microneedles prepared in this embodiment of the invention were used to analyze the relative abundance of oral microbiota at the genus level in mice after treatment of an orthotopic squamous cell carcinoma model in mice. Detailed Implementation

[0069] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0070] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0071] In the description of this invention, the use of I, II, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or the order of the technical features indicated.

[0072] Unless otherwise specified, "room temperature" in this invention means 25±5℃.

[0073] Unless otherwise specified, "about" in this invention means that the allowable error is within ±2%.

[0074] In the description of this invention, the following terms or abbreviations are explained as follows: OSCC: Oral Squamous Cell Carcinoma, the most common malignant tumor of the oral and maxillofacial region.

[0075] PMB: Polymyxin B, a polypeptide antibiotic that selectively kills Gram-negative bacteria and can adsorb lipopolysaccharide (LPS).

[0076] DTX: Docetaxel, a chemotherapy drug that induces immunogenic cell death (ICD) in tumor cells.

[0077] HPG: Hyperbranched Polyglycerol, a polymer material with a hyperbranched structure and good biocompatibility, which can be oxidized to introduce a large number of aldehyde groups (CHO-HPG).

[0078] CHO-HPG-PMB: Aldehyde-modified Hyperbranched Polyglycerol-Polymyxin B Nanoparticles.

[0079] pSA: Polysialic Acid, a natural polysaccharide that, after amino modification, can form hydrazone bonds with aldehyde groups to construct responsive hydrogels.

[0080] Microneedles: A micron-scale needle array that can penetrate superficial barriers such as the oral mucosa to achieve localized, minimally invasive drug delivery. In some embodiments of the present invention, a partitioned delivery hydrogel microneedle is provided, the hydrogel being composed of a backing layer responsive to sialidase and a tip layer responsive to weakly acidic pH.

[0081] (1) Backing layer – used for oral flora regulation Functional Zoning Design: For the first time, a function for regulating oral flora is integrated into the backing layer of the microneedle, complementing the tumor treatment function of the needle tip layer in both space and function. Enzyme-Responsive Drug Release: Utilizing sialidase specifically secreted by harmful bacteria as a trigger signal, antibiotics are released "on demand," avoiding overuse. Dual-Effect Mechanism of "Bactericidal-Detoxifying": Not only does it kill harmful bacteria, but it also removes their pathogenic factors by adsorbing lipopolysaccharides, comprehensively reshaping the healthy microecology.

[0082] Composition: The backing layer is composed of a hydrogel formed by cross-linking amino-modified polysialic acid and aldehyde-modified hyperbranched polyglycerol-polymyxin B nanoparticles (CHO-HPG-PMB) through hydrazone bonds.

[0083] Nanoparticle preparation: First, an amphiphilic copolymer, aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid (CHO-HPG-PLA), was synthesized and self-assembled into nanoparticles in water. Subsequently, PMB was modified onto the particle surface by covalently reacting the aldehyde groups on the nanoparticle surface with the amino groups in the polymyxin B (PMB) molecule, forming CHO-HPG-PMB nanoparticles.

[0084] Hydrogel crosslinking: The above CHO-HPG-PMB nanoparticles were mixed with an amino-modified pSA solution. The remaining aldehyde groups on the surface of the nanoparticles and the amino groups on the pSA chains formed dynamic covalent bonds through hydrazone bonds, thus constructing a three-dimensional network hydrogel.

[0085] Function implementation mechanism: Enzyme-responsive release: When harmful bacteria in the oral cavity (such as Porphyromonas gingivalis) proliferate excessively, the sialidase secreted by them will specifically degrade the pSA backbone, causing the hydrogel network to disintegrate, thereby precisely releasing the internal CHO-HPG-PMB nanoparticles.

[0086] Selective antibacterial and detoxification: The released nanoparticles selectively kill Gram-negative harmful bacteria through PMB on their surface; at the same time, PMB can efficiently adsorb and neutralize pathogen-related molecular patterns such as LPS released by harmful bacteria.

[0087] Localized long-lasting retention: The abundant aldehyde groups on the surface of nanoparticles can form Schiff base bonds with amino groups on the surface of oral mucosa, producing a bioadhesion effect, significantly prolonging the retention time of drugs in the oral cavity and improving bioavailability.

[0088] (2) Needle tip layer - used for tumor area treatment Universal drug delivery platform: Utilizing the same nanocarrier (CHO-HPG-PLA), it can simultaneously achieve efficient loading and synergistic delivery of hydrophobic small molecule drugs (encapsulated in a hydrophobic core) and hydrophilic macromolecule drugs (coupled to a hydrophilic shell via Schiff base bonds).

[0089] pH-triggered deep delivery: Utilizing the unique weak acidity of the tumor microenvironment to trigger the release of nanoparticles from the needle tip layer. After the nanoparticles are released, the drug is released slowly, ensuring that the drug is released only within the tumor tissue and does not interfere with the release mechanism of the backing layer, thus achieving true regional and targeted delivery.

[0090] Composition: The needle tip layer is a hydrogel formed by cross-linking amino-modified polysialic acid (pSA) with aldehyde-modified hyperbranched polyglycidyl ether nanoparticles (DHO-HPG Nanoparticles, HPG-DTX / aTim-3) loaded with docetaxel and anti-Tim-3 antibody through hydrazone bonds.

[0091] Nanoparticle preparation: The hydrophobic chemotherapy drug docetaxel (DTX) was encapsulated in the polylactic acid hydrophobic core of CHO-HPG-PLA nanoparticles. Subsequently, the aldehyde group on the hydrophilic shell of the nanoparticles was linked to the amino group of the anti-Tim-3 antibody (aTim-3) via a Schiff base bond, thereby adsorbing the antibody.

[0092] Hydrogel Crosslinking and Release: HPG-DTX / aTim-3 nanoparticles were mixed with amino-modified pSA to form a hydrazone-crosslinked hydrogel. When microneedles were inserted into tumor tissue (pH ~6.5-6.8), the acidic environment caused the hydrazone bonds to break, the hydrogel to degrade, and DTX and aTim-3 were released in situ and synergistically into the tumor area.

[0093] The preparation method and operation steps are as follows: Step 1: Needle tip layer filling. The needle tip layer hydrogel prepolymer (containing HPG-DTX / aTim-3 and amino-modified pSA) is injected into the conical needle tip cavity of the PDMS mold, and centrifugal force is used to fully fill it and remove air bubbles.

[0094] Step 2: Backing layer filling and integration. After the needle tip layer is initially shaped, the backing layer hydrogel prepolymer (containing CHO-HPG-PMB and amino-modified pSA) is injected into the mold to cover the needle tip layer. It is then centrifuged again to fill the space of the backing layer.

[0095] Step 3: Curing and Demolding. After drying at room temperature or low temperature, the microneedles are peeled off from the mold to obtain a structurally complete partitioned delivery environmentally responsive hydrogel microneedle array.

[0096] This invention employs a two-step centrifugal mold method for preparation, requiring mainly a polydimethylsiloxane microneedle mold and a centrifuge. This method ensures the integration of the physical structure of the backing layer and the tip layer while maintaining independence in functional release. The final product is an innovative drug delivery system that combines minimally invasive, localized, dual-response, zone-targeting, and long-lasting effects.

[0097] According to some embodiments of the present invention, the needle tip layer is a pH-responsive hydrogel formed by crosslinking amino-modified polysialic acid with aldehyde-modified hyperbranched polyglycidyl ether nanoparticles (HPG-DTX / aTim-3) loaded with docetaxel and anti-Tim-3 antibody via hydrazone bonds.

[0098] According to some embodiments of the present invention, the aldehyde-modified hyperbranched polyglycidyl ether nanoparticles are formed by the self-assembly of CHO-HPG-PLA copolymers, possessing a hydrophobic core encapsulating hydrophobic drugs and an aldehyde-modified shell connecting hydrophilic drugs via Schiff base bonds. The nanocarrier platform of the present invention has a universal drug-carrying function of "one core, one shell".

[0099] The core concept of this invention lies in constructing an integrated microneedle system capable of zoned delivery, environmentally responsive release, and synergistic treatment of oral flora and tumors. Within this core concept, those skilled in the art can foresee various alternative solutions that can achieve the same inventive objective. Firstly, regarding the selection of the responsive hydrogel matrix, this invention preferably uses polysialic acid as the enzyme-responsive framework for the backing layer, the key being its ability to be degraded by enzymes specifically secreted by dysbiotic flora. If the environment of the target pathogenic bacteria changes, for example, if it primarily secretes gingival protease or hyaluronidase, polysialic acid can be equivalently replaced with gelatin / collagen or hyaluronic acid, or other biomaterials that can be degraded by the corresponding enzymes, still achieving the intelligent drug release function of the backing layer in areas rich in pathogens.

[0100] Secondly, the platform of this invention exhibits significant versatility in the selection of therapeutic agents. The immune checkpoint inhibitor loaded on the needle tip layer functions primarily to block immunosuppressive signals to reverse T cell exhaustion in the tumor microenvironment. Therefore, the anti-Tim-3 antibody (aTim-3) is merely a preferred embodiment and can be completely replaced by other inhibitors with similar mechanisms of action, such as the currently widely used anti-PD-1 / PD-L1 antibodies, or emerging anti-LAG-3 antibodies, anti-TIGIT antibodies, etc. Similarly, the chemotherapy drug docetaxel (DTX) loaded on the needle tip layer can also be replaced with other hydrophobic chemotherapy drugs (such as paclitaxel, cisplatin) or small molecule targeted drugs, as long as they can produce a synergistic effect with immunotherapy, depending on the specific treatment needs.

[0101] The preparation method of the above-mentioned partitioned delivery microneedle system includes the following steps: (1) providing a tip layer prepolymer liquid, which contains amino-modified polysialic acid and HPG nanoparticles loaded with DTX and aTim-3; (2) providing a backing layer prepolymer liquid, which contains amino-modified polysialic acid and aldehyde-modified HPG-PMB nanoparticles; (3) injecting the tip layer prepolymer liquid into a microneedle mold, and subjecting it to a first centrifugation step to selectively fill the tip cavity of the mold and perform preliminary cross-linking; (4) injecting the backing layer prepolymer liquid into the mold, and subjecting it to a second centrifugation step to fill the backing layer space and integrate it with the tip layer formed in step (3) at the interface; (5) drying and curing, demolding, and obtaining the partitioned delivery microneedle system.

[0102] Example This example provides a hydrogel microneedle composed of a tip layer and a backing layer. The tip layer consists of multiple arrayed micron-sized needle tips, each of which is connected to the backing layer; wherein the backing layer is enzyme-responsive, and the tip layer is acid-pH-responsive.

[0103] Its preparation process is as follows Figure 1 As shown, the details are as follows: 1) Preparation of the backing layer prepolymer solution: a) Preparation of HPG-PMB nanoparticles Synthesis and oxidation of CHO-HPG-PLA copolymer: Take 1 g of HPG-PLA copolymer and dissolve it in 26 mL of DMSO.

[0104] Under magnetic stirring, the above DMSO solution was slowly injected into 450 mL of deionized water to form a nanoparticle suspension.

[0105] Centrifuge at 4500 rpm for 15 minutes using an ultrafiltration centrifuge tube (molecular cutoff: 100 kDa), discard the filtrate, and wash twice with deionized water to remove DMSO.

[0106] The washed nanoparticles were redispersed in 20 mL of deionized water, and sodium periodate (NaIO4) was added. The reaction was carried out overnight at room temperature in the dark.

[0107] After the reaction was completed, excess sodium sulfite (Na2SO3) was added to quench the reaction. The product was washed three times again by ultrafiltration centrifuge tube, redispersed in deionized water, and freeze-dried to obtain a white solid CHO-HPG-PLA copolymer.

[0108] 22.5 mg of the CHO-HPG-PLA copolymer obtained in the above operation was dissolved in 0.85 mL of DMSO and slowly added dropwise to 10 mL of deionized water under stirring, forming CHO-HPG nanoparticles through self-assembly. 5 mL of an aqueous solution containing 10 mg of polymyxin B (PMB) was added to the nanoparticle suspension, and the mixture was incubated at room temperature in the dark for 6 hours. After incubation, the nanoparticles were washed three times by centrifugation using an ultrafiltration centrifuge tube (molecular cutoff: 100 kDa) to remove unreacted PMB, yielding the HPG-PMB nanoparticle suspension. The microstructure of the prepared HPG-PMB nanoparticles was characterized, and the transmission electron microscopy (TEM) images are shown below. Figure 2 As shown in the figure (the scale bar is 50 nm). It can be seen from the figure that the HPG-PMB nanoparticles exhibit a uniform spherical shape.

[0109] b) Synthesis of amino-modified pSA: 100 mg of polysialic acid (pSA) was dissolved in deionized water to prepare a 3 mg / mL solution, and the pH of the reaction mixture was adjusted to 6.8. 30 times the volume of polysialic acid, adipic acid dihydrazide (ADH), 192 mg EDC, and 135 mg NHS were added, and the reaction was carried out at room temperature for 12 hours. Subsequently, the pSA was adjusted to 7.0 with 0.1 M NaOH, and the reaction solution was transferred to a dialysis bag (molecular cutoff: 3500 Da) and dialyzed against deionized water for 48 hours. The product was purified by sodium chloride-ethanol precipitation and lyophilized to obtain a white solid amino-modified pSA.

[0110] c) Construction of pSA / HPG-PMB hydrogel prepolymer: 400 mg of amino-modified pSA was dissolved in 1 mL of deionized water to obtain solution A. A suspension containing HPG-PMB nanoparticles was taken as solution B. Equal volumes of solutions A and B were mixed, vortexed, and allowed to stand for about 30 seconds to obtain the pSA / HPG-PMB hydrogel prepolymer.

[0111] 2) Preparation of needle tip layer prepolymer solution: a) Preparation of HPG-DTX / aTim-3 nanoparticles: 22.5 mg of CHO-HPG-PLA copolymer and 2.5 mg of docetaxel (DTX) were dissolved together in 0.85 mL of DMSO. This solution was injected into 10 mL of stirred deionized water using a syringe to form HPG-DTX nanoparticles. The nanoparticles were purified using ultrafiltration to remove free DTX and DMSO. The purified HPG-DTX nanoparticles were mixed with 1 mL of PBS solution containing 200 μg of anti-Tim-3 antibody (aTim-3) and incubated at room temperature in the dark for 4 hours. The nanoparticles were then purified again by ultrafiltration to obtain HPG-DTX / aTim-3 nanoparticles.

[0112] An amino-modified pSA solution (prepared according to the preparation process of solution A in the previous step) was mixed with an equal volume of HPG-DTX / aTim-3 nanoparticle suspension to form a DTX / aTim3@pSA / HNP hydrogel prepolymer.

[0113] 3) Assembly of microneedles Assembly was performed using a PDMS microneedle mold (needle height 650 μm, bottom width 300 μm).

[0114] The DTX / aTim3@pSA / HNP hydrogel prepolymer (i.e., needle tip layer prepolymer) prepared by the above operation was dropped onto the surface of the mold and centrifuged at 4500 rpm for 10 minutes to fill the needle tip cavity.

[0115] After removing the excess prepolymer, the prepared pSA / HPG-PMB hydrogel prepolymer (i.e., the backing layer prepolymer) was dropped onto the mold and centrifuged at 4500 rpm for 10 minutes to fill the backing layer.

[0116] Place the mold in a desiccator and let it stand at room temperature overnight to allow it to fully cure and dry.

[0117] Peeling it off from the mold yields the DT@S / HP bilayer hydrogel microneedles.

[0118] The microstructure of the prepared hydrogel microneedles was characterized, and the scanning electron microscope (SEM) image of the backing layer is shown below. Figure 3 As shown in the image (scale bar is 100 μm), the pSA / HPG-PMB hydrogel exhibits a porous microstructure, as seen in the scanning electron microscope (SEM) image. Figure 4 As shown in the figure (the scale bar is 100 μm), SEM reveals the sharp cone morphology of a single microneedle and the seamless, complete interface between the drug injection needle tip and the backing layer.

[0119] The effectiveness of the system was then verified at the cellular and animal levels.

[0120] 1) Evaluation of selective antibacterial activity of HPG-PMB: The selective antibacterial activity of HPG-PMB against three bacteria, including *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Peptostreptococcus anaerobicans*, was further evaluated. The bacterial suspension was adjusted to 1 × 10⁻⁶. 6 CFU / mL, bacteria were exposed to HPG-PMB at anaerobic conditions at 37°C for 24 hours. After incubation, bacterial viability was assessed by serial dilution, blood agar plate application, and 24-hour anaerobic culture. CFU were counted by colony counting, and the counts in the treated groups were standardized with those in the untreated control group (0 μg / mL PMB) to calculate the relative bacterial viability (%). Figure 5 As shown, the released HPG-PMB nanoparticles exhibit selective, dose-dependent bactericidal activity against pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and are non-toxic to symbiotic bacteria such as Streptococcus anaerobicans.

[0121] 2) Assessment of the responsive degradation capacity of the backing layer and needle tip: To assess the enzymatic degradation ability of the hydrogel backing layer, pSA / HNP-PMB hydrogel samples were equilibrated in PBS and then incubated in PBS solutions with or without sialidase (0.5 mg / mL) for 0, 24, and 48 hours. Bright-field images were taken to observe macroscopic morphological changes. Samples from 0 and 24 hours were washed, lyophilized, and observed using scanning electron microscopy (SEM) to assess structural degradation. Figure 6 As shown in ab, the gradual degradation of the hydrogel over time can be observed macroscopically with the naked eye, and the released HNP-PMB nanoparticles can be observed by TEM. This indicates that the pSA / HNP-PMB hydrogel can be specifically degraded by sialidase secreted by harmful bacteria. Figure 7 As shown, after 24 hours of incubation with sialidase, a large number of pores appeared on the surface of the hydrogel, and the structure was obviously loose.

[0122] To assess the stability of HNP-PMB in the oral environment, HNP-PMB was dispersed in simulated oral fluid and placed in a 37 °C constant-temperature shaker at 100 rpm in the dark. Samples were taken at specified time points. During sampling, the samples were transferred to ultrafiltration centrifuge tubes (100 kDa) and centrifuged at 4500 rpm for 15 min at 4 °C. The PMB concentration in the filtrate was determined by HPLC, and the cumulative release rate was calculated. Results are as follows: Figure 8 As shown, the release rate of the nanoparticles in artificial saliva is only about 25% after 48 hours, and it has good stability in a slightly neutral oral environment. The stability of HNP-PMB in the oral environment endows it with the ability to achieve long-term oral adhesion and microbial regulation.

[0123] To evaluate the acid-responsive degradation ability of the hydrogel tips, DTX / aTim3@pSA / HNP hydrogel tip samples were incubated in PBS solutions at pH 7.4 and 6.8, respectively. The degradation was assessed using a stereomicroscope at 5 min, 24 h, and 48 h. Figure 9 As shown, the hydrogel tip degrades slowly in an environment of pH 7.4 and retains its original tip shape after 48 hours. However, in an acidic environment of pH 6.8, the hydrogel tip undergoes significant degradation after 24 hours and is completely degraded after 48 hours. This indicates that the DTX / aTim3@pSA / HNP hydrogel can specifically degrade in an acidic environment.

[0124] Further pH-responsive drug release capacity of DTX@HNP-aTim3 was evaluated. The drug was dispersed in buffer solutions at pH 7.4 and 6.8, respectively, and shaken at 37 °C in the dark (100 rpm). Samples were taken at specified time points. Upon sampling, the samples were transferred to ultrafiltration centrifuge tubes (100 kDa) and centrifuged at 4500 rpm for 15 min at 4 °C. The aTim3 content in the filtrate was determined using a BCA protein quantification kit, and the cumulative release rate was calculated. Figure 10 As shown, aTim3 is released slowly in a pH 7.4 environment; however, when the pH 6.8 simulates the extracellular environment of tumor cells, the release rate of aTim3 is significantly increased, with a release rate of up to 81.23% after 72 hours, demonstrating the ability of acid-responsive degradation to release aTim3.

[0125] 3) In vitro biosafety evaluation of pSA / HNP hydrogel The in vitro safety of pSA / HNP hydrogels was assessed using the CCK-8 assay. pSA / HNP hydrogels were extracted in serum-free medium at 37°C for 48 h to obtain the stock extract, which was then diluted with medium to create concentration gradients of 12.5%, 25%, 50%, and 75%. HOEC and HIEC-6 cells were seeded into 96-well plates and cultured overnight. The original medium was discarded, and 0.1 mL of the different concentrations of hydrogel extract was added to each well. After 24 h of incubation, the original medium was discarded and replaced with fresh medium containing CCK-8. After 2 h of incubation, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated for each group. The results are shown below. Figure 11 As shown, cell viability was not significantly affected after incubation with different concentrations of hydrogel extract in both cell types, indicating that pSA / HNP hydrogel has good in vitro biocompatibility.

[0126] 4) Evaluation of the inhibitory effect on a bacterial infection-complicated oral squamous cell carcinoma in situ tumor model: To establish an in situ tumor model in the oral microenvironment infected with bacteria, this study first administered a mixed bacterial suspension (containing 1×10⁶ Porphyromonas gingivalis) through three oral inoculations (once every other day). 9 CFU (dissolved in 100 μL PBS containing 2% CMC) was introduced into the oral cavity of mice. SCC-7 cells (8 × 10⁻⁶ cells) were then introduced. 5 (Number of mice) were injected submucosally into the buccal mucosa to induce tumor formation. Once the tumors reached a palpable size, the tumor-bearing mice were randomly divided into 4 groups (…). n =5), receiving oral mucosal administration of different microneedle formulations: bacterial negative control (G1), bacterial positive control (G2), S / HP microneedles (G3), and DT@S / HP microneedles (G4). All groups received a single dose on day 1, and thereafter, tumor volume was measured every two days using the formula V=0.5×L×W² (L is the longest diameter, W is the shortest vertical diameter) to assess treatment efficacy. Figure 12 As shown, compared with the uninfected group (G1), the bacterial-positive group (G2) exhibited accelerated tumor progression, highlighting the pro-tumor effect of dysbiotic oral microbiota. Compared with group G3, the intact microneedle system group (G4) achieved a more significant anti-tumor effect, indicating that the microbiota regulation capability of the backing layer HNP-PMB synergistically works with the anti-tumor drugs delivered by the needle tip, demonstrating the significance of the partitioned delivery microneedle design.

[0127] Oral swabs and fecal samples were collected from mice in each group on day 7 post-treatment and flash-frozen on dry ice for transport. DNA samples for bacterial 16S rDNA fragment amplification were extracted from mouse swabs and feces using a DNA extraction kit according to the product instructions. The highly variable V3-V4 region of the 16S rDNA fragment was amplified by polymerase chain reaction (PCR). Sequencing was performed at LC Biotechnology Co., Ltd. according to the company's standard procedures. Figure 13 As shown, in the tumor-bearing harmful bacteria infection group (Bacteria (+)), the abundance of the opportunistic pathogen *Porphyromonas* was significantly increased, while the abundance of the oral commensal bacteria *Streptococcus* was significantly decreased. After intervention with the complete microneedle D / T@S / HP MN group, the abundance of *Porphyromonas* decreased to near the level of the healthy control group, while the abundance of *Streptococcus* returned to normal levels. These results highlight the benefits of the synergistic integration of microbiome regulation and chemoimmunotherapy.

[0128] Compared with existing technologies, the partitioned delivery environmentally responsive hydrogel microneedle system provided by this invention represents a significant technological advancement, with its beneficial effects primarily reflected in a comprehensive improvement in precision, versatility, safety, and intelligence. Specifically, through a unique integrated "backing layer-needle tip layer" design, this system achieves, for the first time, partitioned, precise intervention and synergistic treatment of oral mucosal surface flora and deep tumor tissue in both physical space and functional release: the backing layer specifically responds to the sialidase secreted by harmful bacteria, degrading and releasing nanoparticles that selectively kill Gram-negative bacteria and adsorb lipopolysaccharides, precisely reshaping the microecology; simultaneously, the needle tip layer responds only to drug release in the weakly acidic environment of the tumor, directly delivering chemotherapy drugs and antibodies into the tumor lesion. This dual isolation in both space and mechanism ensures that different therapies can be administered simultaneously and efficiently without interference, fundamentally solving the problem of poor targeting in traditional dosage forms.

[0129] Secondly, the core nanocarrier (CHO-HPG-PLA) of this system constitutes a highly flexible and multifunctional drug delivery platform. Its hydrophobic core can efficiently encapsulate drugs with poor water solubility (such as docetaxel), while the aldehyde group of the hydrophilic shell can stably bind hydrophilic macromolecules (such as antibodies) through covalent bonds. This successfully breaks through the technical bottleneck of traditional carriers that cannot simultaneously deliver hydrophilic / hydrophobic drugs, and provides a universal solution for complex combination drug strategies.

[0130] In terms of safety, the local minimally invasive drug delivery method of microneedles, combined with the bioadhesion properties of nanoparticles, ensures that the drug can be concentrated in the lesion for a long time, greatly improving bioavailability. At the same time, because the amount of drug entering the systemic circulation is greatly reduced, drugs such as polymyxin B, which are effective but have high systemic toxicity, can be used safely, successfully achieving the unity of "highly effective local treatment" and "low systemic toxicity and side effects".

[0131] Finally, this invention introduces a dual environmental response mechanism based on enzymes and pH, enabling drug release to be intelligently triggered by specific biomarkers of the disease microenvironment (sialidase, low pH), achieving on-demand, precise controlled release of drugs—"antibacterial only when bacteria are present, chemotherapy only when the tumor is reached." This intelligent drug release mode not only maximizes treatment efficiency but also effectively avoids unnecessary exposure and damage to healthy tissues, representing a more advanced and precise new paradigm for disease treatment.

[0132] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A hydrogel microneedle, characterized in that: It includes a backing layer and a tip layer disposed on the surface of the backing layer. The tip layer contains a plurality of arrayed micron-sized tips, each of which is connected to the backing layer. The backing layer is enzyme-responsive, and the tip layer is acidic pH-responsive.

2. The hydrogel microneedles according to claim 1, characterized in that: The backing layer comprises a gel matrix I and nanoparticles I distributed in the gel matrix I, wherein the gel matrix I is prepared by a material containing an enzyme-responsive material, and the surface of the nanoparticles I is modified with components that regulate oral flora; and / or, the tip layer comprises a gel matrix II and nanoparticles II distributed in the gel matrix II, wherein the gel matrix II is prepared by a material containing an acidic pH-responsive material, and the nanoparticles II are loaded with a hydrophobic chemotherapeutic drug and a hydrophilic immune checkpoint inhibitor.

3. The hydrogel microneedles according to claim 2, characterized in that: The gel matrix I and the nanoparticles I are connected by a dynamic covalent bond through hydrazone bonds I; and / or, the enzyme-responsive material contains amino groups.

4. The hydrogel microneedles according to claim 2, characterized in that: The raw materials for preparing nanoparticle I include aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid; and / or, the oral flora regulating component includes antibiotics; and / or, the enzyme includes at least one of sialidase, gingival protease, or hyaluronidase.

5. The hydrogel microneedles according to claim 2, characterized in that: The gel matrix I is prepared by a material comprising at least one of gelatin, collagen, amino-modified polysialic acid, or amino-modified hyaluronic acid; and / or the composition of the gel matrix II is the same as or different from that of the gel matrix I.

6. The hydrogel microneedles according to claim 2, characterized in that: The gel matrix II and the nanoparticles II form a dynamic covalent bond through hydrazone bonds II; the hydrazone bonds II can be broken in a weakly acidic environment with a pH of 6.5 to 6.

8.

7. The hydrogel microneedles according to claim 2, characterized in that: The gel matrix II is prepared from an amino-containing component or an amino-modified component; and / or, the surface of the nanoparticle II contains aldehyde groups; and / or, the surface of the immune checkpoint inhibitor contains amino groups, and the amino groups are linked to the aldehyde groups on the surface of the nanoparticle II via Schiff base bonds.

8. The hydrogel microneedles according to claim 2, characterized in that: The gel matrix II is prepared by an amino-modified polysialic acid, and the nanoparticle II is prepared by an aldehyde-modified hyperbranched polyglycidyl ether-polylactic acid. The nanoparticle II is internally loaded with docetaxel and externally linked with an anti-Tim-3 antibody.

9. A method for preparing hydrogel microneedles as described in any one of claims 1 to 8, characterized in that: Includes the following steps: S1. Inject the prepolymer liquid II containing the raw materials for preparing the needle tip layer into the conical needle tip cavity of the mold; after the needle tip layer has been initially shaped, inject the prepolymer liquid I containing the raw materials for preparing the backing layer into the mold to cover the needle tip layer. S2. Dry and demold to obtain the hydrogel microneedles.

10. The use of the hydrogel microneedles as described in any one of claims 1 to 8 in the preparation of oral squamous cell carcinoma treatment drugs.

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