A porous silicon-based nanoscale enzyme complex, and a preparation method and application thereof

Abstract

This invention discloses a porous silica-based nanozyme complex, its preparation method, and its applications, belonging to the field of biomedical technology. The preparation method includes the following steps: mixing porous silica particles with a noble metal salt in a solution, followed by shaking to obtain porous silica loaded with a noble metal-based nanozyme; mixing the porous silica loaded with the noble metal-based nanozyme with a carbon-based nanozyme in a solution, followed by shaking to obtain a solution of the porous silica-based nanozyme complex. The porous silica-based nanozyme complex of this invention exhibits excellent nanozyme activity. On one hand, it can act as a POD-like compound, catalyzing the decomposition of overexpressed H2O2 in the tumor microenzyme (TME) to generate ·OH, which kills tumor cells. On the other hand, it can also act as a GSHOx-like compound, oxidizing excess GSH in the TME and reducing the antioxidant stress capacity of cancer cells. Furthermore, near-infrared photothermal stimulation can enhance the POD-like and GSHOx-like activities of the porous silica-based nanozyme complex. This invention further utilizes the porous silica-based nanozyme complex to prepare microneedles, which can provide an effective nanocatalytic strategy for the treatment of skin diseases such as melanoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a porous silicon-based nanoenzyme complex, its preparation method, and its application. Background Technology

[0002] More than one million new cases of skin cancer are diagnosed worldwide each year, and the incidence rate has been rising over the past few decades. Cutaneous melanoma (CM) is a highly malignant tumor, accounting for 80% of skin cancer deaths. A major reason for the high mortality rate of cutaneous melanoma is the lack of reliable and effective treatments. Surgical excision of the epidermal lesion and adjacent skin tissue has been the primary standard of care for melanoma. Immunotherapy is a novel treatment approach, but it requires addressing the complexity of treatment and the long-term survival of reprogrammed cells. Furthermore, the low degree of DNA damage and high rate of DNA repair in melanoma make it resistant to radiotherapy. Therefore, developing innovative and effective treatments for melanoma is crucial for improving patient survival rates.

[0003] Microneedles (MNs) are a promising transdermal drug delivery technology. They can penetrate the epidermal barrier to form reversible microchannels, facilitating drug delivery to the dermis, where melanoma is primarily distributed. Furthermore, MN delivery is a painless administration method, avoiding contact with pain receptors in subcutaneous tissue. Soluble MNs, after dissolving in tissue fluid, can gradually deliver drug molecules to specific tumor sites, facilitating subsequent treatment and avoiding secondary pain for patients. Therefore, microneedle delivery has significant potential applications in the treatment of melanoma.

[0004] In recent years, researchers have proposed loading nanoformulations onto microneedles to develop prospective melanoma therapies. Nanozymes are a special type of nanoformulation that exhibits enzyme kinetics similar to natural enzymes under physiological conditions. The tumor microenvironment (TME) differs from normal tissues in terms of nutrient deficiency, hypoxia, weakly acidic pH, immunosuppression, and overexpression of hydrogen peroxide (H2O2) and glutathione (GSH). While other nanoformulations often employ active or passive targeting for tumor therapy, nanozymes target and regulate characteristic metabolites within the TME, thereby inducing catalytic therapy. Under endogenous stimulation of the TME, nanozymes can induce the transformation or decomposition of endogenous substances, thereby attacking cancer cells or disrupting the internal homeostasis of the TME, ultimately inhibiting tumor development. For example, peroxidase-like enzymes (PODs) can catalyze the decomposition of excess H2O2 in the TME to produce hydroxyl radicals (·OH), thereby inducing DNA damage and protein denaturation in tumor cells. Glutathione oxidase-like enzymes (GSHOs) can also catalyze this process. xCarbon nanomaterials typically contain highly oxidized metal elements (Cu(II), Mn(IV), and V(V)), which can convert overexpressed GSH in the tumor microenzyme (TME) into GSSG (oxidized glutathione) through redox reactions, thereby reducing the oxidative stress resistance of cancer cells. For example, copper(II)-doped carbon nanomaterials have been reported to possess both POD-like and GSHOx-like effects. Furthermore, nanozymes capable of responding to external stimuli (light, heat, ultrasound, etc.) have been used in multimodal tumor therapy. Exogenous stimuli can endow nanozymes with additional photodynamic, photothermal, and acoustic dynamic responses, further enhancing therapeutic performance. For example, palladium nanoparticles (PdNPs) are noble metal nanoparticles with POD-like and photothermal properties, and can be used for bimodal tumor therapy. Currently, there are few studies on loading nanozymes into microneedles for melanoma treatment.

[0005] Furthermore, loading multiple nanozymes onto carriers offers a novel approach to constructing multimodal tumor therapeutic platforms targeting complex tumor microenzymes (TMEs). To date, various materials have been developed as nanozyme carriers, including graphene, transition metal disulfide (TMD), and mesoporous silica nanospheres (MSN). Compared to these nanocarriers, electrochemically etched porous silicon (PSi) possesses tunable mesoporous channels, rich surface chemistry, and does not affect the activity of the loaded nanomaterials and drugs. Moreover, compared to MSN carriers, PSi nanocarriers exhibit drug protection, better biocompatibility, and biodegradability. The degradation product of PSi in vivo is orthosilicic acid (Si(OH)4), which is harmless to the body and can be rapidly metabolized and cleared by the kidneys. Therefore, PSi holds promise as a novel nanozyme carrier for developing nanozyme complexes for highly effective melanoma treatment. Summary of the Invention

[0006] To address at least one of the aforementioned technical problems, this invention aims to provide a technical means for treating skin diseases such as melanoma by utilizing porous silicon as a carrier to load nanozymes and combining nanozymes with a microneedle system. Therefore, the technical solution adopted by this invention is as follows:

[0007] The first aspect of this invention provides a method for preparing a porous silicon-based nanoenzyme complex, comprising the following steps:

[0008] S1, porous silicon particles are mixed with noble metal salts in solution and shaken to obtain porous silicon loaded with noble metal-based nanozymes.

[0009] S2, the porous silicon loaded with the noble metal-based nanozyme obtained in step S1 is mixed with the carbon-based nanozyme in solution, and the mixture is shaken to obtain a solution of the porous silicon-based nanozyme complex.

[0010] Nanozymes are a class of nanomaterials with biocatalytic functions. They can catalyze the substrates of natural enzymes based on specific nanostructures and serve as enzyme substitutes. As a novel material, nanozymes possess both the physicochemical properties of nanomaterials and enzyme-like catalytic functions, combining the advantages of both natural and artificial enzymes. Furthermore, the nanostructure not only endows nanozymes with highly efficient catalytic function but also makes them more stable than natural enzymes and easier to mass-produce.

[0011] Nanozymes can be broadly classified into four categories based on their composition: metal-based nanozymes (gold, silver, platinum, etc.), metal oxide-based nanozymes (iron oxides, cerium oxides, and cobalt oxides, etc.), carbon-based nanozymes (such as carbon nanotubes, fullerenes, and carbon dots, etc.), and nanomaterial-based nanozymes (such as metal-organic frameworks, Prussian blue analogs, and metal sulfides, etc.). In this invention, the metal in the metal-based nanozyme is a noble metal, selected from one or more alloys of gold, silver, and platinum group metals (ruthenium, rhodium, palladium, osmium, iridium, platinum) (such as Pd-Pt, Pd-Au, Pd-Ag). This invention co-loads noble metal-based nanozymes and carbon-based nanozymes in a porous silica carrier to form a nanozyme complex. The noble metal-based nanozyme functions as a POD-like agent and a photothermal agent, while the carbon-based nanozyme functions as a GSHO (gold-organic framework). x It plays a role.

[0012] Metal nanoparticles (MNPs) refer to metallic particles with a size less than 100 nm in at least one dimension. Due to their small size, high specific surface area, high reactivity, and unique optical, electrical, and thermodynamic properties, they have become a hot research topic in many fields such as catalysis, sensors, clinical diagnostics, medical treatment, antibacterial agents, and environmental remediation. In this invention, noble metal nanoparticles can be synthesized simply by mixing a noble metal ion precursor with porous silicon and utilizing the silicon-hydrogen bonds on the surface of the porous silicon channels to reduce or chemically transform the noble metal ions. In some embodiments of this invention, the noble metal ions are derived from noble metal salts, such as hydrochlorides.

[0013] In some specific embodiments of the present invention, the noble metal is palladium, and the silicon-hydrogen bonds on the surface of the porous silicon channels reduce Pd(II) to PdNPs in situ, thereby obtaining porous silicon supported by palladium nanoparticles.

[0014] Carbon-based nanozymes with enzyme-like activity are known to include fullerenes, graphene, carbon nanotubes, and their derivatives. Quantum dots (QDs) are quasi-zero-dimensional semiconductor nanocrystals with dimensions smaller than or close to the exciton Bohr radius. Those skilled in the art know that engineering materials to the quantum dot size not only retains the inherent advantages of the materials but also imparts new properties, such as fluorescence, quantum effects, and edge effects. Furthermore, quantum dots exhibit low toxicity, good biocompatibility, and a larger specific surface area, more defects, and more exposed active sites compared to the original materials. Several quantum dots have been found to possess peroxidase-like activity, such as nitrogen-doped graphene quantum dots (N-GQDS), tungsten oxide quantum dots (WOx QDs), and molybdenum disulfide quantum dots.

[0015] In some embodiments of the present invention, the carbon-based nanozyme is selected from one of graphene quantum dots, nitrogen-doped graphene quantum dots, copper-doped graphene quantum dots, polyethyleneimine-modified carbon quantum dots, and single-atom iron-modified carbon quantum dots.

[0016] In some preferred embodiments of the present invention, the carbon-based nanozyme is a graphene quantum dot, more preferably, a copper-doped graphene quantum dot. In some embodiments of the present invention, the carbon-based nanozyme is a graphene quantum dot doped with a high-valence transition metal. In some specific embodiments of the present invention, the preparation method of the copper-doped graphene quantum dot is as follows:

[0017] p-phenylenediamine was dissolved in a solution containing Cu 2+ The solution was transferred to a polytetrafluoroethylene-lined autoclave for hydrothermal treatment, and then freeze-dried after microporous membrane filtration to obtain copper-doped graphene quantum dots.

[0018] Nanozymes can be broadly classified into two categories based on their catalytic reaction type: redox reactions and hydrolysis reactions. Redox reactions include four types: peroxidase-like enzymes, oxidase-like enzymes, catalase-like enzymes, and superoxide dismutase-like enzymes. Hydrolysis reactions mainly include hydrolases. This invention demonstrates that palladium nanoparticles possess peroxidase-like (POD) properties and photothermal conversion properties, and can function as both POD-like and photothermal agents. Copper-doped graphene quantum dots exhibit peroxidase-like (POD) properties and glutathione oxidase-like (GSHO) properties. x )performance.

[0019] In therapeutic applications, nanozymes primarily rely on regulating the body's reactive oxygen species (ROS) levels to treat various diseases, such as neurological disorders, inflammation, stroke, and cancer. In this invention, the skin diseases include infections and cancers; the cancers include squamous cell carcinoma, adenocarcinoma, and melanoma.

[0020] This invention loads two nanozymes onto porous silicon. Unlike most existing microneedle-based melanoma treatment systems that load chemotherapy drugs, this invention uses a nanocatalytic treatment approach, avoiding drug resistance and drug inactivation caused by chemotherapy drugs.

[0021] In this invention, the porous silica particles serve as a carrier for the nanoenzyme complex, loading the nanoenzyme complex as a tiny functional unit. The size of this unit can be determined according to the specific disease, typically at the nanometer scale, thus effectively targeting the lesion. Porous silica (PSi) possesses tunable mesoporous channels and rich surface chemical properties. Compared to MSN carriers, PSi carriers can effectively protect the activity of the nanoenzyme complex and exhibit higher biocompatibility and biodegradability. The degradation product of PSi in vivo is orthosilicic acid, which is harmless to the body and can be rapidly metabolized and eliminated by the kidneys.

[0022] In this invention, porous silicon loaded with only palladium nanoparticles does not possess GSHOX-like properties; porous silicon loaded with only copper-doped graphene quantum dots does not possess good photothermal properties. Therefore, whether it is porous silicon loaded with only metal nanoparticles or porous silicon loaded with only copper-doped graphene quantum dots, both the nanoenzyme activity and the in vitro therapeutic effect are lower than those of porous silicon loaded with both palladium nanoparticles and copper-doped graphene quantum dots.

[0023] Furthermore, due to the confined pore characteristics of porous silicon, the catalytic activity of the nanozyme complex loaded therein can be further enhanced. Based on this, the present invention also discloses the following technical solutions:

[0024] Porous silicon loaded with at least one nanozyme.

[0025] This technical solution uses porous silicon as a carrier, which can enhance the catalytic activity of nanozymes.

[0026] In some embodiments of the present invention, the porous silicon particles are prepared as follows:

[0027] Using 90%–99% ethanol and hydrofluoric acid as electrolytes, boron-doped silicon wafers are anodized at a constant current density and then etched with a hydrofluoric acid-ethanol solution to obtain a porous silicon layer. Finally, the porous silicon layer is broken up by ultrasound to obtain the porous silicon particles.

[0028] The second aspect of the present invention provides a porous silicon-based nanoenzyme complex prepared by any of the preparation methods described in the first aspect of the present invention.

[0029] The third aspect of the present invention provides the application of the porous silicon-based nanoenzyme complex described in the second aspect of the present invention in the preparation of microneedles for treating skin diseases.

[0030] Microneedles contain a large number of micro-needle tip structures. Microneedles can dissolve in the human body and can directly penetrate the stratum corneum of the skin or other tissues to deliver drugs, allowing the drugs to reach the lesion directly and avoiding the first-pass effect of the liver.

[0031] Microneedles (MN) are a novel physical epidermal penetration technology, consisting of multiple micron-sized needle tips connected in an array on a base. The needles are typically 10–2000 μm high and 10–50 μm wide. The length, size, and shape of the microneedles can be individually designed according to treatment needs. In some embodiments of this invention, the height of the microneedles can be 400–800 μm, preferably 650 μm.

[0032] Microneedles can penetrate the stratum corneum in a targeted manner, creating micron-sized mechanical channels that deliver drugs directly to the dermis to participate in microcirculation and exert pharmacological responses. When delivering drugs via microneedles, the active ingredient is loaded into a microneedle array. The concentration gradient between the drug and subcutaneous tissue fluid creates a driving force, causing the drug to be slowly released into the body. Alternatively, the microneedle tip may dissolve directly in the dermis, releasing the loaded drug. This offers the advantages of transdermal delivery systems.

[0033] (1) Avoid the first-pass effect;

[0034] (2) No needles are needed to administer medication, there is no pain, medication is convenient, and patient compliance is improved;

[0035] (3) Reduce the probability of drug enzymatic degradation and maintain the drug's high activity;

[0036] (4) It can maintain a stable blood drug concentration for a long time, ensure stable efficacy, and improve safety.

[0037] In addition, it has the following advantages:

[0038] (1) Stable transdermal absorption rate;

[0039] (2) Controlling the length of the microneedle can avoid touching capillaries and nerve endings, reducing or eliminating pain.

[0040] (3) Due to the elasticity of the skin, the microchannels formed on the skin surface will close on their own after a period of time when the microneedle is administered, making it suitable for repeated administration.

[0041] (4) The administration method is convenient and can be used by oneself.

[0042] In some embodiments of the present invention, the microneedles are used in combination with photothermal stimulation to treat skin diseases. The porous silicon-based nanoenzyme complex prepared by the present invention has photothermal conversion properties, and under photothermal stimulation, it can improve the therapeutic performance for skin diseases.

[0043] In some embodiments of the present invention, the photothermal stimulation refers to irradiation with near-infrared light. Near-infrared light has the characteristics of high tissue penetration and minimal phototoxicity. Using near-infrared light, not only can drugs be released, but heat can also be generated at the drug release device, using high heat to kill lesions. In some specific embodiments of the present invention, the wavelength of the near-infrared light is 780–820 nm. In some embodiments of the present invention, when the maximum absorption wavelength of the porous silicon-based nanoenzyme complex is 808 nm, the following parameters are recommended for treatment: 808 nm near-infrared light irradiation power is 0–5 W / cm². 2 The temperature change of the microneedle after irradiation with 808nm near-infrared light is 30-80℃; the irradiation time with 808nm near-infrared light is 1-60 minutes.

[0044] In this invention, the porous silicon-based nanoenzyme complex or the microneedle performs treatment through the following functions:

[0045] (1) As a POD-like substance, it catalyzes the decomposition of H2O2 overexpressed in TME to generate ·OH that kills tumor cells;

[0046] (2) As a GSH-like substance, it oxidizes excess GSH in TME and reduces the antioxidant stress capacity of tumor cells.

[0047] (3) Near-infrared photothermal stimulation can enhance the POD-like and GSHOx-like activities of porous silicon-based nanoenzyme complexes, thereby improving the therapeutic effect.

[0048] The fourth aspect of the present invention provides a microneedle for treating skin diseases, wherein the microneedle is loaded with the porous silicon-based nanoenzyme complex described in the second aspect of the present invention.

[0049] In some embodiments of the present invention, polymer materials and the porous silicon-based nanoenzyme complex are prepared into microneedles using casting, hot pressing, injection molding or micromolding methods.

[0050] In some specific embodiments of the present invention, the microneedles are prepared by casting. Specifically, a mixture of polymer material and porous silicon-based nanoenzyme composite is filled into the top of a microneedle mold, and then a polymer material solution is dripped into the mold to form a microneedle backing layer. After drying, the microneedles are separated from the mold to obtain microneedles loaded with the porous silicon-based nanoenzyme composite.

[0051] In some embodiments of the present invention, the polymer material is a soluble material selected from at least one of the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, hyaluronic acid, dextran, chitosan, sodium alginate, polylactic acid, and polylactic acid-glycolic acid copolymer.

[0052] Soluble microneedles are the mainstream in the entire microneedle industry. Their main advantages are: they can achieve efficient drug delivery, and precise control of drug loading and microneedle morphology can achieve controlled drug release; the production process is mild, ensuring the stability of the drug during the production process; and the use of biodegradable and biocompatible materials avoids wound infection, resulting in high safety.

[0053] Beneficial effects of the present invention

[0054] Compared with the prior art, the present invention has the following technical effects:

[0055] 1. Melanoma is the deadliest type of skin cancer, and currently there is no ideal treatment option in clinical practice. Nanozymes are artificial nanomaterials with physicochemical properties and enzyme catalytic characteristics, and have attracted much attention in anti-tumor therapy. However, the therapeutic effect of nanozymes is easily affected by the tumor microenvironment (TME) and the delivery process. This invention provides a microneedle patch integrating a porous silica-based nanozyme complex for the efficient treatment of melanoma. Based on a porous silica carrier, a confined loading system of composite nanozymes with synergistic catalytic function was constructed. The prepared porous silica-based nanozyme complex has good nanozyme activity. On the one hand, it can act as a POD-like substance, catalyzing the decomposition of overexpressed H2O2 in the TME to generate ·OH, which kills tumor cells. On the other hand, it can also act as a GSHOx-like substance, oxidizing excess GSH in the TME and reducing the antioxidant stress capacity of cancer cells. In addition, the activities of both nanozymes in the porous silica-based nanozyme complex can be significantly enhanced under near-infrared light stimulation. Therefore, the porous silica-based nanozyme complex provided by this invention simultaneously possesses the intrinsic characteristics of POD-like and GSHOx-like substances as well as the photothermal conversion characteristics of exogenous stimulation.

[0056] 2. Currently, the application of integrating nanozymes or nanozyme complexes into microneedles in melanoma treatment is limited. Furthermore, most microneedle systems used for melanoma treatment incorporate chemotherapy drugs. Chemotherapy drugs suffer from poor solubility and selectivity, leading to complex preparation processes that may result in drug inactivation and poor storage stability. The microneedle system of this invention uses nanozyme complexes as the therapeutic agent, avoiding the use of chemotherapy drugs. Moreover, the microneedles loaded with porous silica-based nanozyme complexes exhibit good storage stability, lasting for at least three months under dry conditions at room temperature. Under near-infrared light irradiation, the porous silica-based nanozyme complex achieved a TGI of 98.8% for melanoma within 14 days.

[0057] 3. Compared with existing materials, porous silicon (PSi) possesses tunable mesoporous channels and rich surface chemical properties beneficial for drug loading efficiency and protection of drug activity. Furthermore, the PSi carrier exhibits high biocompatibility and biodegradability, enabling porous silicon-based nanozyme complexes to possess self-degradation capabilities. When applied to microneedles, this offers advantages such as high therapeutic efficacy, stable storage, safe and convenient use, and harmlessness to the human body. Therefore, microneedles encapsulated with porous silicon-based nanozyme complexes could provide an effective nanocatalytic strategy for the treatment of melanoma. Attached Figure Description

[0058] Figure 1 The following are shown: (a) photothermal response curves of the control group, porous silicon, and porous silicon-based nanoenzyme complex; (b) temperature change curve of the porous silicon-based nanoenzyme complex after five cycles of exposure to near-infrared light.

[0059] Figure 2 The following are shown: (a) the absorption spectra of porous silicon and porous silicon-based nanoenzyme complex after reacting with H2O2 and TMB at 37 °C for 5 minutes; (b) the steady-state kinetic analysis of porous silicon and porous silicon-based nanocomplex as a POD-like material under near-infrared irradiation or without near-infrared irradiation; and (c) the double reciprocal curves of porous silicon and porous silicon-based nanocomplex as a POD-like material under near-infrared irradiation or without near-infrared irradiation.

[0060] Figure 3 The following are shown: (a) the absorption spectra of porous silicon and porous silicon-based nanoenzyme complex after reacting with GSH and DTNB at 37 °C for 5 minutes; (b) the steady-state kinetic analysis of porous silicon and porous silicon-based nanoenzymes as GSH-like enzymes under near-infrared irradiation or without near-infrared irradiation; and (c) the double reciprocal curves of porous silicon and porous silicon-based nanoenzymes as GSH-like enzymes under near-infrared irradiation or without near-infrared irradiation.

[0061] Figure 4 The following are shown: steady-state kinetic analysis (a) and double reciprocal plot (b) of nanozyme complex with porous silica support removed as a POD-like product; steady-state kinetic analysis (c) and double reciprocal plot (d) of nanozyme complex with porous silica support removed as a GSHOx-like product.

[0062] Figure 5 The cell viability of B16-F10 cells after different treatments under near-infrared light irradiation is shown.

[0063] Figure 6 The use of DCFH-DA as a fluorescent probe to detect hydroxyl radicals in B16-F10 cells after different treatments is shown.

[0064] Figure 7The ratio of glutathione to oxidized glutathione in B16-F10 cells after different treatments is shown.

[0065] Figure 8 A stereomicroscopic image of microneedles loaded with a porous silicon-based nanoenzyme complex is shown.

[0066] Figure 9 The following are shown: (a) Photothermal response curves of freshly prepared porous silica-based nanozyme complex microneedles and porous silica-based nanozyme complex microneedles stored at room temperature and dried for three months. (b) Viability of B16-F10 cells after applying freshly prepared porous silica-based nanozyme complex microneedles or porous silica-based nanozyme complex microneedles stored at room temperature and dried for three months under near-infrared light irradiation.

[0067] Figure 10 The curves showing the changes in tumor volume in each group after different treatments are shown.

[0068] Figure 11 The results of H&E staining, Tunel staining, and Ki-67 staining of tumors in each group of tumor-bearing mice after different treatments are shown. Detailed Implementation

[0069] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0070] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0071] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0072] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0073] Example

[0074] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0076] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0078] Example 1: Preparation of nanoscale porous silicon (PSi)

[0079] In an electrolyte composed of hydrofluoric acid and 95% ethanol (volume ratio 4:1), a boron-doped silicon wafer was subjected to an electric shock at 77 mA / cm². 2The PSi layer on the silicon substrate was removed by anodizing at a constant current density for 600 seconds, followed by etching with a 3.3% hydrofluoric acid-ethanol solution for 180 seconds. The removed PSi layer was then ultrasonically broken up for 30 minutes (500W) to obtain nanoscale PSi.

[0080] Example 2: Preparation of porous silicon-based nanoenzyme complex

[0081] After adding 1.0 mM PdCl2 solution to the PSi solution (5 mg / mL) and shaking at room temperature for 10 minutes, Pd(II) was reduced in situ to PdNPs using the silanol groups on the surface of the PSi channels. After centrifugation at 10000 rpm for 10 minutes to remove residual PdCl2, palladium nanoparticle-supported porous silicon (PdNPs@PSi) was obtained.

[0082] Subsequently, copper-doped graphene quantum dots (CuGQD) were synthesized via a one-step hydrothermal method. In short, 10 mg / mL of p-phenylenediamine was dissolved in a 0.1 M copper chloride (CuCl2) solution, and the mixed solution was then transferred to a polytetrafluoroethylene-lined autoclave and hydrothermally treated at 180 °C for 6 hours. After filtration through a 0.22 μm filter membrane, CuGQD powder was obtained by freeze drying.

[0083] The above-mentioned PSi or PdNPs@PSi were mechanically shaken in CuGQD solution (1 mg / mL) for 2 hours, followed by centrifugation at 10000 rpm for 5 minutes to remove unbound CuGQD, yielding a copper-doped graphene quantum dot-loaded porous silicon or porous silicon-based nanozyme complex solution. Finally, the solution was freeze-dried to obtain copper-doped graphene quantum dot-loaded porous silicon or porous silicon-based nanozyme complex powder.

[0084] Example 3: Photothermal properties of porous silicon-based nanoenzyme complex

[0085] 500 μg / mL porous silicon, copper-doped graphene quantum dot-supported porous silicon, palladium nanoparticle-supported porous silicon, and porous silicon-based nanozyme complex were placed in 1.5 mL centrifuge tubes and analyzed under near-infrared light (808 nm, 1.0 W / cm²). 2 The solution was irradiated for 5 minutes, during which the temperature change was recorded using a thermal imager (FLIR ONE Pro, FLIR Systems, USA). Additionally, the porous silica-based nanozyme composite was placed under near-infrared light irradiation for five minutes, then removed and allowed to cool naturally to room temperature. This heating-cooling cycle was repeated five times to evaluate the photothermal stability of the porous silica-based nanozyme composite.

[0086] The results are as follows Figure 1As shown, after irradiation with an 808 nm near-infrared laser for 5 minutes, the temperature of the control group solution remained almost unchanged, the temperature of the porous silicon solution increased by only ≈3℃, and the temperature of the porous silicon solution loaded with copper-doped graphene quantum dots increased by ≈9℃, demonstrating that the thermal energy conversion property of copper-doped graphene quantum dots through absorption of near-infrared light is poor. The temperature of the porous silicon solution loaded with palladium nanoparticles increased by ≈27℃, and the temperature of the porous silicon-based nanoenzyme complex increased from 22℃ to 52℃. Figure 1 Figure a shows that the introduction of palladium nanoparticles endows porous silicon-based nanozymes with good photothermal conversion capabilities.

[0087] Furthermore, the temperature rise of the porous silicon-based nanoenzyme complex remained essentially unchanged after five cycles of near-infrared irradiation. Figure 1 Figure b shows that it has good near-infrared photothermal conversion stability.

[0088] Example 4: Determination of peroxidase (POD) activity in porous silica-based nanoenzyme complexes

[0089] In this embodiment, the inventors determined the POD-like properties of the porous silica-based nanozyme composite prepared in Example 2. Specifically, the inventors studied the POD-like properties of the porous silica-based nanozyme composite based on the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine (TMB). Generally, POD can catalyze the decomposition of H2O2 to produce ·OH, and ·OH can oxidize TMB to generate an oxidation product (oxTMB) with characteristic absorption at 652 nm.

[0090] Porous silicon, copper-doped graphene quantum dot-loaded porous silicon, palladium nanoparticle-loaded porous silicon or porous silicon-based nanozyme complex (500 μg / mL), TMB (0.5 mM) and H2O2 (3.3 mM) were added to PBS buffer (pH = 6.5). The absorbance of the mixed solution was recorded after reacting for 5 minutes under near-infrared light conditions or with near-infrared light conditions.

[0091] At normal physiological temperature (37℃), using different concentrations of H2O2 as substrates, steady-state kinetics were analyzed after adding porous silica-based nanozyme complexes (500 μg / mL) to PBS buffer (pH = 6.5) containing TMB (0.5 mM). The Michaelis equation 1 / V0 = K was used. m / V max +1 / V max The Michaelis constant (K) was calculated. m ) and maximum reaction rate (V max ), where V0 represents the initial reaction rate. The same method was used to analyze the POD-like activity of the porous silicon-based nanozyme composite when heated to 50°C under near-infrared light irradiation.

[0092] The results are as follows Figure 2 As shown, from Figure 2 As can be seen, compared with porous silicon groups, porous silicon groups supported by copper-doped graphene quantum dots, and porous silicon groups supported by palladium nanoparticles, the absorbance value of the porous silicon-based nanoenzyme complex group at 652 nm is significantly increased. Figure 2 Figure a) demonstrates that the porous silicon-based nanozyme complex can most effectively catalyze the decomposition and oxidation of TMB by H₂O₂. Steady-state kinetic analysis ( Figure 2 Figures b and c show the Michaelis constant (K), a characteristic parameter used to evaluate the performance of nanozymes. m ) and maximum reaction rate (V max K m The value represents the binding affinity of nanozymes to reaction substrates, K. m A lower value indicates that the nanozyme binds more easily to the substrate. max The value V represents the ability of nanozymes to catalyze reactions. max A higher Kc value indicates a faster rate of nanozyme catalysis. When H2O2 is used as the substrate, the Kc value of the porous silica-based nanozyme complex... m The value is 0.9735 mM, V max The value is 5.417 × 10 -8 M / s, demonstrating that the porous silicon-based nanozyme complex possesses POD-like properties. Similarly, under near-infrared irradiation-induced high-temperature conditions of 50℃, steady-state kinetic analysis yielded the K0 of the porous silicon-based nanozyme complex. m The value is 0.7726 mM, V max The value is 6.423 × 10 -8 M / s. Compared to the reaction at 37 °C, the Kc of the porous silica-based nanozyme complex induced by near-infrared irradiation at 50 °C increased. m The value decreases, V max The increase in the value proves that near-infrared light-induced high temperature of 50℃ can further enhance the POD-like performance of porous silicon-based nanoenzyme complexes.

[0093] Example 5: Glutathione oxidase (GSHOx)-like activity of porous silica-based nanoenzyme complex

[0094] The reaction product of GSH and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has a characteristic absorption peak at 412 nm. The remaining amount of GSH in the reaction system can be assessed by monitoring the absorbance value at 412 nm.

[0095] Porous silicon, copper-doped graphene quantum dot-supported porous silicon, palladium nanoparticle-supported porous silicon, or porous silicon-based nanozyme complexes (500 μg / mL) were added to PBS buffer (pH 6.5) containing GSH (0.2 mM) and 5,5'-disulfide bis(2-nitrobenzoic acid) (DTNB, 200 μg / mL). The absorbance changes of the DTNB and GSH reaction products at 412 nm were measured using a UV-Vis spectrophotometer. Steady-state kinetics were performed at 37 °C and 50 °C, respectively, using different concentrations of GSH as substrates and porous silicon-based nanozyme complexes (500 μg / mL) as catalysts in the presence of DTNB (200 μg / mL), as described in Example 4.

[0096] The results are as follows Figure 3 As shown, compared with the control group and the PSi group, the absorption peak at 412 nm of the reaction system containing copper-doped graphene quantum dots loaded porous silicon or porous silicon-based nanoenzyme complex was significantly reduced, while the absorption value of the reaction system containing palladium nanoparticles loaded porous silicon was almost unchanged, proving that copper-doped graphene quantum dots have GSHOx-like activity. Figure 3 Figure a). Through steady-state dynamic analysis ( Figure 3 Figures b and c show the Ki of the porous silica-based nanozyme complex when GSH is used as a substrate. m The value is 0.4662 mM, V max The value is 5.079 × 10 -7 M / s, demonstrating that the porous silica-based nanozyme complex possesses GSHOx-like properties. Furthermore, by inducing the system temperature to 50℃ using near-infrared light irradiation, followed by steady-state kinetic analysis, the K0 of the porous silica-based nanozyme complex for simulating GSHOx was obtained. m The value is 0.3327 mM, V max The value is 6.184 × 10 -7 M / s. Compared to the reaction at 37 °C, the Kc of the porous silica-based nanozyme complex induced by near-infrared irradiation at 50 °C increased. m The value decreases, V max The increase in the value proves that the high heat caused by near-infrared light irradiation can further enhance the GSHOx-like performance of porous silicon-based nanoenzyme complexes.

[0097] Example 6: Investigation into the Enhancement of Nanozyme Catalytic Activity on Porous Silica Support

[0098] To investigate the effect of porous silica support on nanozyme activity, the PSi support was oxidized using a weakly alkaline PBS solution (pH = 7.4) to disrupt the PSi pore structure, resulting in a nanozyme complex without the porous silica support. As described in Examples 4 and 5 above, the POD-like activity and GSHOx-like activity of the nanozyme complex without the porous silica support were tested.

[0099] The results are as follows Figure 4 As shown, through steady-state dynamic analysis ( Figure 4 Figures a and b show the Ka of the nanozyme complex with the porous silica support removed when H2O2 is used as the substrate. m The value is 1.109 mM, V max The value is 3.416 × 10 -8 M / s. Compared to porous silicon-based nanozyme complexes, the Kz of nanozyme complexes without porous silicon supports is significantly lower. m The value increased significantly, V max The decrease in the value indicates that the POD-like activity of the nanozyme decreased after the porous silica support was removed. Further analysis using steady-state kinetics... Figure 4 Figures c and d show the Ki of the nanozyme complex with the porous silica support removed when using GSH as a substrate. m The value is 0.9785 mM, V max The value is 4.143 × 10 -7 M / s. Compared to porous silicon-based nanozyme complexes, the Kz of nanozyme complexes without porous silicon supports is significantly lower. m Value increases, V max The decrease in the value indicates that the GSHOx-like activity of the nanozyme also decreased after the porous silica support was removed. The above steady-state kinetic results demonstrate that the pore confinement characteristics of the PSi nanosupport enable the dual nanozyme to exhibit synergistic POD-like and GSHOx-like activities.

[0100] Example 7: Effects of porous silicon-based nanoenzyme complex on melanoma cells

[0101] 1. Cell Culture

[0102] Mouse melanoma cells (B16-F10) were purchased from ProSino Biotechnology Co., Ltd. and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin (100 units / mL) and streptomycin (100 μg / mL) at 37°C with 5% CO2.

[0103] 2. Cytotoxicity assay

[0104] In vitro cytotoxicity was assessed using the CellTiter-Lumin kit (Beyotime Biotechnology Co., Ltd.). First, 8 × 10⁸ cells were injected into each well. 3 B16-F10 cells were seeded into 96-well plates at a density of [number] cells / well. Then, they were treated with an 808nm laser (1.0W / cm²). 2B16-F10 cells were irradiated for 5 minutes per well with different concentrations (0, 100, 200, 300, 400, 500 μg / mL) of porous silica, copper-doped graphene quantum dot-loaded porous silica, palladium nanoparticle-loaded porous silica, or porous silica-based nanozyme complexes. After 24 hours of incubation, 100 μL of Celltiter Lumin solution was added to each well and the cells were shaken for 10 minutes to stabilize the chemiluminescent signal. Cell viability was detected using a microplate reader (Thermo Scientific 5250040, USA).

[0105] To evaluate the effect of near-infrared irradiation on the ability of porous silicon-based nanozyme complexes to inhibit tumor cell growth, B16-F10 cells (8 × 10⁶ cells per well) were used. 3 Cells were seeded and cultured in 96-well plates, and then different concentrations of porous silicon, copper-doped graphene quantum dot-loaded porous silicon, palladium nanoparticle-loaded porous silicon, or porous silicon-based nanoenzyme complex solutions (0, 100, 200, 300, 400, 500 μg / mL) were added. After irradiation with near-infrared light, cell viability was detected using CellTiter-Lumin.

[0106] The results are as follows Figure 5 As shown, under near-infrared light irradiation, the viability of B16-F10 cells remained essentially unchanged (above 93%) with increasing porous silicon concentration in the reaction system. The cell viability of the porous silicon group loaded with 500 μg / mL copper-doped graphene quantum dots was 51%, while the cell viability of the porous silicon group loaded with 500 μg / mL palladium nanoparticles was 35%. The cell viability of B16-F10 cells showed a significant concentration dependence when the porous silicon-based nanozyme complex was added; when 500 μg / mL of the porous silicon-based nanozyme complex was added, the B16-F10 cell viability was only 10%, demonstrating that the porous silicon-based nanozyme complex significantly outperformed the copper-doped graphene quantum dot-loaded and palladium nanoparticle-loaded porous silicon groups in vitro.

[0107] 3. Intracellular ROS detection

[0108] B16-F10 cells were treated as follows:

[0109] Control group: Irradiated only with 808nm laser (1.0W / cm²). 2 (5 minutes per hole).

[0110] Porous silicon array: Porous silicon was used for treatment. After adding 500 μg / mL PSi to the well plate and incubating for 12 hours, irradiation with or without 808 nm laser (1.0 W / cm²) was applied. 2 (5 minutes per hole).

[0111] Porous silicon arrays supported by copper-doped graphene quantum dots: Porous silicon was fabricated using copper-doped graphene quantum dots. After adding 500 μg / mL CuGQD@PSi to the well plates and incubating for 12 hours, irradiation with or without 808 nm laser (1.0 W / cm²) was applied. 2 (5 minutes per hole).

[0112] Palladium nanoparticle-supported porous silicon: Porous silicon was treated using palladium nanoparticle-supported porous silicon. 500 μg / mL of PdNPs@PSi was added to the well plates and incubated for 12 hours, followed by irradiation with or without 808 nm laser (1.0 W / cm²). 2 (5 minutes per hole).

[0113] Porous silica-based nanozyme complex group: Treatment was performed using a porous silica-based nanozyme complex. After adding 500 μg / mL of the porous silica-based nanozyme complex to the well plates and incubating for 12 hours, irradiation with or without 808 nm laser (1.0 W / cm²) was applied. 2 (5 minutes per hole)

[0114] After different treatments, the cells were incubated with reactive oxygen species fluorescent probe (DCFH-DA) dye for 30 minutes. After washing away residual DCFH-DA with PBS, the POD-like performance of the porous silica-based nanozyme complex in B16-F10 cells was evaluated. Specifically, fluorescent staining images were obtained by fluorescence microscopy to estimate the level of intracellular reactive oxygen species (ROS).

[0115] First, the production of ·OH in B16-F10 cells after the addition of different materials was investigated under conditions without near-infrared light irradiation. For example... Figure 6 As shown, compared with porous silicon, copper-doped graphene quantum dot-loaded porous silicon, and palladium nanoparticle-loaded porous silicon, the porous silicon-based nanozyme complex group exhibited stronger green fluorescence, demonstrating that the porous silicon-based nanozyme complex can also be used at the cellular level as a POD-like nanozyme to catalyze the decomposition of H2O2 to generate a large amount of ·OH for killing melanoma cells. Furthermore, after near-infrared light irradiation, the green fluorescence of the porous silicon-based nanozyme complex group was further enhanced, indicating that photothermal stimulation can enhance the POD-like performance of the porous silicon-based nanozyme complex in B16-F10 cells.

[0116] 4. Detection of intracellular glutathione levels

[0117] B16-F10 cells were planted at a rate of 1×10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates and divided into 6 groups for assay: a control group without any treatment, a group treated with 500 μg / mL porous silica, a group treated with 500 μg / mL copper-doped graphene quantum dot-loaded porous silica, a group treated with 500 μg / mL palladium nanoparticle-loaded porous silica, a group treated with 500 μg / mL porous silica-based nanozyme complex, and a group treated with both 500 μg / mL porous silica-based nanozyme complex and near-infrared light irradiation (808 nm, 5 min). After each treatment, the cells were centrifuged at 3000 rpm for 5 min at 4 °C to remove the supernatant. Protein removal reagent M (30 μL) was mixed with the cells and vortexed thoroughly. Subsequently, the cell samples were alternately frozen and thawed twice using liquid nitrogen and a 37 °C water bath. The supernatant after centrifugation (4 °C, 10000 rpm) was collected, and the total glutathione content in the cells was determined according to the protocol in the glutathione / oxidized glutathione assay kit (Beyotime Biotechnology Co., Ltd.).

[0118] The results are as follows Figure 7 As shown, compared with the control group (no treatment), the ratio of glutathione to oxidized glutathione in B16-F10 cells remained essentially unchanged in both the porous silicon group and the porous silicon group loaded with palladium nanoparticles. However, after adding porous silicon loaded with copper-doped graphene quantum dots, the ratio of glutathione to oxidized glutathione in B16-F10 cells decreased. The ratio of glutathione to oxidized glutathione in the porous silicon-based nanozyme complex system further decreased, demonstrating that the porous silicon-based nanozyme complex can effectively oxidize intracellular glutathione, exhibiting GSHOx-like performance. After near-infrared light irradiation, the ratio of glutathione to oxidized glutathione in the cells of the porous silicon-based nanozyme complex group further decreased, indicating that near-infrared light irradiation can promote the oxidation of intracellular glutathione by the porous silicon-based nanozyme complex, enhancing the GSHOx-like performance of the porous silicon-based nanozyme complex.

[0119] Example 8: Microneedles loaded with porous silicon-based nanoenzyme complex

[0120] 1. Preparation of microneedles loaded with porous silica-based nanoenzyme complexes

[0121] The inventors further integrated porous silicon-based nanoenzyme complexes into microneedles, developing a dissolvable microneedle patch specifically for melanoma treatment. The microneedles loaded with the porous silicon-based nanoenzyme complex were prepared using a stepwise casting method.

[0122] Using a polydimethylsiloxane (PDMS) mold (Singapore, Micropoint Technologies Pte Ltd, 5mm × 5mm), 10% hyaluronic acid (HA, molecular weight less than 5000 Da) hydrogel containing a porous silica-based nanozyme complex solution (500 μg / mL) was dropped into the PDMS mold. The mixture was then centrifuged at 3000 rpm for 20 minutes to fill the mold cavity. After removing excess mixture, the mold was filled with 20% HA hydrogel to obtain a backing layer. The mold was then placed in a drying tower and dried for at least 12 hours. After drying, the microneedles were carefully separated from the mold by holding the edge region with tweezers, yielding microneedles loaded with the porous silica-based nanozyme complex. The preparation methods for porous silica-loaded microneedles, porous silica microneedles loaded with copper-doped graphene quantum dots, and porous silica microneedles loaded with palladium nanoparticles were the same as described above.

[0123] 2. Microscopic observation of microneedles loaded with porous silica-based nanoenzyme complexes

[0124] The morphology of the prepared microneedles was observed using a stereomicroscope (Nikon, SMZ745T, Japan). The results are as follows: Figure 8 As shown, the microneedles loaded with the porous silicon-based nanoenzyme complex were observed to be arranged in a neat and orderly manner, with a complete pyramid shape, a tip height of 650 μm, and an array size of 10 × 10.

[0125] 3. Stability of microneedles supported on porous silica-based nanoenzyme complexes

[0126] After being stored at room temperature and under dry conditions for three months, the photothermal response performance was tested in the same manner as in the above examples. Furthermore, freshly prepared microneedles or microneedles stored for three months were co-incubated with B16-F10 cells for 1 hour before being stimulated with 808 nm light (1.0 W / cm²). 2 (5 minutes), and then cell viability was measured as described in the above embodiments.

[0127] The results are as follows Figure 9 As shown, the photothermal response of the microneedles loaded with the porous silicon-based nanoenzyme composite can retain 99.31% of its original value after three months of storage at room temperature and under dry conditions. Figure 9 (Figure a). Furthermore, under near-infrared light stimulation, compared to freshly prepared porous silica-based nanozyme complex-loaded microneedles, microneedles stored for three months still maintained an inhibition rate of over 98.5% on the growth of B16-F10 cells. Figure 9 (Figure b in the middle)

[0128] The above results demonstrate that the microneedles loaded with porous silicon-based nanoenzyme complexes have good storage stability and potential for practical applications.

[0129] Example 9: In vivo anti-melanoma performance of porous silicon-based nanoenzyme complex loaded with microneedles

[0130] In this embodiment, the inventors verified the ability of the microneedles loaded with the porous silicon-based nanoenzyme complex prepared in Example 8 to treat melanoma in vivo.

[0131] BALB / c nude mice (5-6 weeks old, female) were provided by the Shanghai Laboratory Animal Center, China. All animal experiments were conducted in a standard barrier environment at the Laboratory Animal Center of Zhejiang University and were supervised and approved by the Animal Ethics Committee of Zhejiang University (ZJU20220361).

[0132] 50 μL of B16-F10 cells (1 × 10⁶) were injected into the right upper limb of mice. 6 A mouse model of melanoma was established using PBS solution (number of mice). When the tumor volume reached 100 mm², the model was further developed. 3 At that time, the tumor-bearing nude mice were randomly divided into five groups (n=4 per group):

[0133] (i) Near-infrared illumination group;

[0134] (ii) Porous silicon-supported microneedles + near-infrared irradiation group;

[0135] (iii) Microneedles of porous silicon containing copper-doped graphene quantum dots loaded with near-infrared light;

[0136] (iv) Microneedles of porous silicon containing palladium nanoparticles loaded with near-infrared light;

[0137] (v) Microneedles loaded with porous silicon-based nanoenzyme complex + near-infrared light irradiation group.

[0138] After puncturing the skin, the microneedles were firmly pressed onto the tumor site using a medical bandage and secured. One hour later, the microneedle patch was removed, and the tumor site was then irradiated with an 808nm laser (1.0W / cm², 5 minutes). During near-infrared irradiation, photothermal images of the tumor-bearing mice were recorded using a thermal imager. Mouse weight and tumor volume were measured and recorded every two days. Tumor volume = 0.5 × tumor length × tumor width. 2 After 14 days of different treatments, the mice were euthanized and the tumor tissue was removed for photographing and weighing. The tumor growth inhibition rate (TGI) was calculated as follows: TGI = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%.

[0139] The results are as follows Figure 10 As shown, after 14 days of treatment, the tumors in the nude mice in the near-infrared light stimulation group grew in a completely uncontrolled manner (tumor volume = 2220 mm). 3 The tumor volume of nude mice subjected to both porous silicon-loaded microneedles and near-infrared irradiation was 1900 mm².3 At that time, the tumor growth trend was slightly inhibited. Meanwhile, the tumor volume in the group using microneedles containing copper-doped graphene quantum dots loaded with porous silicon, combined with near-infrared light irradiation, reached 750 mm² on day 14. 3 The treatment effect was also unsatisfactory. In contrast, the tumor volume of the group treated with microneedles containing palladium nanoparticles loaded with porous silicon combined with near-infrared light irradiation only reached 300 mm² after 14 days of treatment. 3 The TGI value was 86.3%, indicating that photothermal-enhanced POD-like catalytic therapy can delay the development of melanoma in vivo. Surprisingly, the tumor volume of nude mice subjected to simultaneous application of porous silicon-based nanoenzyme complex-loaded microneedles and near-infrared light irradiation was significantly reduced, with a TGI value as high as 98.8%, demonstrating that the porous silicon-based nanoenzyme complex-loaded microneedles have excellent in vivo anti-melanoma growth effects.

[0140] The inventors further explored the anti-melanoma mechanism of the prepared microneedles through histological analysis. Specifically, the tumor tissue and major organs of the sacrificed mice were immersed in 4% paraformaldehyde for 24 hours, and then paraffin-embedded and sectioned for H&E, TUNEL and Ki-67 staining analysis.

[0141] The results are as follows Figure 11 As shown, in the different treatment groups, H&E staining of tumor tissue treated with microneedles loaded with porous silica nanozyme complexes revealed the highest degree of tumor cell apoptosis. Furthermore, immunofluorescence staining results from terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) and Ki-67 assays also confirmed that, among the three treatment groups, the microneedles loaded with porous silica nanozyme complexes combined with near-infrared light irradiation were the most effective at killing tumor cells and inhibiting their proliferation.

[0142] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing a porous silicon-based nanoenzyme complex, characterized in that, Includes the following steps: S1, porous silicon particles are mixed with noble metal salts in solution and shaken to obtain porous silicon loaded with noble metal-based nanozymes. S2, the porous silicon loaded with the noble metal-based nanozyme obtained in step S1 is mixed with the carbon-based nanozyme in solution, and after shaking, a solution of the porous silicon-based nanozyme composite is obtained. The method for preparing the porous silicon particles is as follows: Using 90%–99% ethanol and hydrofluoric acid as the electrolyte, boron-doped silicon wafers were anodized at a constant current density, followed by etching with a hydrofluoric acid-ethanol solution to obtain a porous silicon layer. Finally, the porous silicon layer was ultrasonically broken up to obtain porous silicon particles. The noble metal-based nanozyme is palladium nanoparticles. The carbon-based nanozyme is a graphene quantum dot.

2. The porous silicon-based nanoenzyme complex prepared by the preparation method according to claim 1.

3. The use of the porous silicon-based nanoenzyme complex according to claim 2 in the preparation of microneedles for treating skin diseases.

4. The application according to claim 3, characterized in that, The microneedles are used in combination with photothermal stimulation to treat skin diseases.

5. The application according to claim 4, characterized in that, The photothermal stimulation refers to the use of near-infrared light for irradiation.

6. A microneedle for treating skin diseases, characterized in that, The microneedles are loaded with the porous silicon-based nanoenzyme complex as described in claim 2.

7. The method for preparing microneedles according to claim 6, characterized in that, Microneedles are prepared from polymer materials and the porous silicon-based nanoenzyme complex using casting, hot pressing, injection molding, or micromolding methods.

8. The preparation method according to claim 7, characterized in that, The polymer material is selected from at least one of the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, hyaluronic acid, dextran, chitosan, sodium alginate, polylactic acid, and polylactic acid-glycolic acid copolymer.

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