Boron nitride nano-enzyme drug, preparation method and application thereof, and transdermal drug delivery preparation

Abstract

The invention provides a boron nitride nano-enzyme drug as well as a preparation method, application and a transdermal delivery preparation thereof. The preparation method of the boron nitride nano-enzyme comprises the following steps: performing thermal polycondensation reaction on dicyandiamide to synthesize graphite-phase carbon nitride; taking water as a solvent, firstly mixing the graphite phase carbon nitride and boric acid, and then dehydrating and grinding the mixture to obtain a powdery graphite phase carbon nitride-boric acid precursor; the preparation method comprises the following steps: carrying out first calcination treatment on a graphite phase carbon nitride-boric acid precursor to obtain hydroxyl boron nitride, uniformly mixing hydroxyl boron nitride and iron salt, and carrying out second calcination treatment under a nitrogen condition to obtain the iron-loaded boron nitride nano-enzyme. The boron nitride nano-enzyme loaded iron prepared by the preparation method disclosed by the invention is small in average particle size, high in selectivity and inhibition activity and excellent in transdermal performance, and can be used as an active agent of a transdermal drug delivery preparation for treating superficial tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and in particular relates to a boron nitride nanoenzyme drug and a preparation method, application and transdermal drug delivery preparation thereof. Background Art

[0002] Middle and late stage melanoma is highly invasive and lethal, and is a major threat to human health. Over the years, the incidence of melanoma has continued to rise, especially in areas with strong ultraviolet rays. Currently, the main treatments for melanoma include Mohs surgery and chemotherapy. Mohs surgery is more effective for early melanoma, but the treatment effect is poor for patients with metastasis. Chemotherapy and other means must be used at the same time to completely remove residual tumor tissue and cancer cells as much as possible to improve the patient's survival rate. On the other hand, most clinical chemotherapy drugs themselves cannot target cancer cells and tissues, and often produce toxic side effects and drug resistance. Given the characteristic of melanoma as a superficial cancer, more effective drugs and drug administration methods can be developed to target tumor sites to minimize systemic side effects.

[0003] Nanozymes are a class of artificial enzymes that have attracted attention due to their unique catalytic properties and potential in cancer treatment. Compared with traditional chemotherapy, the significant feature and advantage of nanozymes is that they can selectively kill tumor cells by catalyzing high levels of endogenous hydrogen peroxide in tumors to produce reactive oxygen species. This highly selective treatment approach significantly reduces damage to normal tissues and improves treatment safety. In addition, nanozymes exhibit high stability, adjustability, and potential for combined therapy. However, nanozymes tend to be large in size and are generally difficult to deliver transdermally, which limits their application in the treatment of superficial tumors and diseases.

[0004] The advantage of transdermal drug delivery is that it provides a non-invasive, patient-friendly way to administer local medications, which reduces systemic exposure and associated toxicities while enhancing the therapeutic effect at the tumor site. Summary of the invention

[0005] The first purpose of the present invention is to provide a method for preparing iron-loaded boron nitride nanozymes, so that the prepared boron nitride nanozymes are loaded with a small amount of iron and have a small average particle size, have high selectivity and inhibitory activity against tumor cells, and also have excellent transdermal performance due to their small size.

[0006] The second object of the present invention is to provide an iron-loaded boron nitride nanozyme drug, which can be used as an active agent in a transdermal drug preparation for the treatment of superficial tumors with few side effects.

[0007] The third object of the present invention is to provide the use of iron-loaded boron nitride nanozymes in the preparation of transdermal drug delivery preparations.

[0008] The fourth object of the present invention is to provide a transdermal drug delivery preparation to solve the damage to other tissues of the whole body caused by the traditional treatment methods for superficial tumors.

[0009] The fifth object of the present invention is the application of the transdermal drug delivery preparation in the preparation of products for treating melanoma.

[0010] To achieve the above object, in the first aspect, the present invention provides a preparation method of iron-loaded boron nitride nanozyme, and the preparation method includes the following steps: Step 1, subject dicyandiamide to thermal polycondensation reaction to synthesize graphitic carbon nitride; Step 2, using water as a solvent, first mix the graphitic carbon nitride and boric acid, and then dehydrate and grind the mixture to obtain a powdery graphitic carbon nitride-boric acid precursor; Step 3, first perform a first calcination treatment on the graphitic carbon nitride-boric acid precursor to obtain hydroxyboron nitride, and then mix the hydroxyboron nitride and iron salt evenly, and perform a second calcination treatment under nitrogen conditions to obtain iron-loaded boron nitride nanozyme. In a specific embodiment, the thermal polycondensation reaction conditions in Step 1 include a reaction temperature of 300-600 °C and a reaction time of at least 1 h.

[0011] In a specific embodiment, the conditions of the first calcination treatment in Step 3 include a calcination temperature of 700-1000 °C and a calcination time of 0.1 h-3 h; the conditions of the second calcination treatment include a calcination temperature of 600-1000 °C and a calcination time of 0.1 h-2 h.

[0012] In a specific embodiment, the mass ratio of the graphitic carbon nitride to boric acid in Step 2 is 1:(0.1-0.5).

[0013] In a specific embodiment, based on the mass of the iron-loaded boron nitride nanozyme being 100%, the iron loading amount is 0.2 wt%-2 wt%, preferably, the iron loading amount is 0.2 wt%-1 wt%.

[0014] In a specific embodiment, the iron salt in Step 3 is a divalent iron salt or a trivalent iron salt.

[0015] In a specific embodiment, the iron salt in Step 3 is ferrous dichloride tetrahydrate, and the mass ratio of the hydroxyboron nitride to ferrous dichloride tetrahydrate is: 1:(0.01-0.5).

[0016] In the second aspect, the present invention further provides an iron-loaded boron nitride nanozyme drug, and the iron-loaded boron nitride nanozyme drug is prepared by using the preparation method described above.

[0017] In a third aspect, the present invention also provides the use of the iron-loaded boron nitride nanozyme prepared by the preparation method described above in the preparation of a transdermal drug delivery preparation.

[0018] In a fourth aspect, the present invention also provides a transdermal drug delivery preparation, which includes an active agent and a transdermal enhancer coating the active agent. The active agent is the iron-loaded boron nitride nanozyme drug described above, and the average particle size of the iron-loaded boron nitride nanozyme is less than 25 nm.

[0019] In a specific embodiment, based on the total weight of the solute of the transdermal drug delivery preparation being 100%, the mass percentage content of the active agent is 1.0 wt% to 20.0 wt%, and the mass percentage content of the transdermal enhancer is 80.0 wt% to 99.0 wt%.

[0020] In a specific embodiment, based on the mass of the iron-loaded boron nitride nanozyme drug being 100%, the iron loading amount is 0.2 wt% to 2 wt%, and preferably, the iron loading amount is 0.2 wt% to 1 wt%.

[0021] In a specific embodiment, the transdermal enhancer includes pararosaniline hydrochloride and polyethyleneimine. The molecular weight range of the polyethyleneimine is 0.6 to 25 kDa. Among them, the mass ratio of the active agent to pararosaniline hydrochloride is 1:(0.05 to 0.15), and the mass ratio of the sum of the mass of the active agent and pararosaniline hydrochloride to the mass of polyethyleneimine is 1:(1 to 10).

[0022] In a specific embodiment, the transdermal enhancer further includes polyglutamic acid and protamine. The molecular weight range of polyglutamic acid is 1 to 100 kDa, and the molecular weight range of protamine is 4 to 10 kDa. Among them, the mass ratio of the sum of the mass of the active agent and pararosaniline hydrochloride to polyglutamic acid and protamine is 1:(2 to 10):(4 to 20).

[0023] In a fifth aspect, the present invention also provides the use of the transdermal drug delivery preparation described above in the preparation of a product for treating melanoma.

[0024] The beneficial effects of the present invention at least include:

[0025] I. The present invention provides a method for preparing iron-loaded boron nitride nanozyme, which comprises the following steps: Step 1, subjecting dicyandiamide to thermal polycondensation reaction to synthesize graphitic carbon nitride; Step 2, using water as a solvent, under stirring and heating conditions, first mixing the graphitic carbon nitride and boric acid, and then dehydrating and grinding the mixture to obtain a powdery graphitic carbon nitride-boric acid precursor; Step 3, first performing a first calcination treatment on the graphitic carbon nitride-boric acid precursor to obtain hydroxyboron nitride, and then uniformly mixing the hydroxyboron nitride and an iron salt, and performing a second calcination treatment under nitrogen conditions to obtain an iron-loaded boron nitride nanozyme; The boron nitride nanozyme prepared by this preparation method loads a small amount of iron. On the one hand, the iron-loaded boron nitride nanozyme has excellent catalytic activity and can selectively catalyze the peroxidation reaction in tumor cells through peroxidase-like activity to generate reactive oxygen species, thereby inducing tumor cells to die through oxidative stress and can be used as a drug to treat tumors; On the other hand, the iron-loaded boron nitride nanozyme has high-biocompatible boron nitride as the main component and loads a small amount of iron, which can minimize the damage to normal tissues and significantly improve the selectivity and safety of treatment.

[0026] II. The materials used in the method for preparing iron-loaded boron nitride nanozyme provided by the present invention are all healthy and friendly materials. The preparation process is simple and can be produced on a large scale, with high economy and sustainability.

[0027] III. The present invention provides a transdermal delivery preparation, which comprises an active agent and a transdermal enhancer coating the active agent. The active agent is an iron-loaded boron nitride nanozyme drug and has an average particle size of less than 25 nm; In this way, the small size characteristic of the Fe-BN nanozyme enables it to effectively penetrate the skin barrier and reach the tumor site directly. Transdermal administration by direct skin application not only avoids the problems of systemic toxicity and low bioavailability of traditional chemotherapy, but also can directly deliver the drug to the melanoma lesion, significantly increasing the local drug concentration and enhancing the therapeutic effect.

[0028] IV. When the transdermal delivery preparation provided by the present invention is used to treat melanoma, since it is applied by topical application to the affected skin, compared with traditional chemotherapy and surgery, it has the characteristics of non-invasive, painless, convenient, etc., significantly reducing the side effects and discomfort during the treatment process and improving the compliance and quality of life of patients; In addition, topical administration reduces systemic exposure and further reduces the risk of systemic side effects.

[0029] V. The transdermal delivery preparation prepared by using iron-loaded boron nitride nanozyme has the advantages of high efficiency, safety and convenience, can significantly improve the therapeutic effect of superficial tumors such as melanoma, reduce side effects, and has the prospect of clinical transformation and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Transmission electron microscopy image of the objective spherical aberration corrected Fe-BN nanozyme prepared in Example 1;

[0031] Figure 2 Particle size distribution diagram of the Fe-BN nanozyme prepared in Example 1;

[0032] Figure 3 Condenser spherical aberration corrected transmission electron microscopy analysis diagram of the Fe-BN nanozyme prepared in Example 1;

[0033] Figure 4 Detection results of peroxidase-like activity of the Fe-BN nanozyme prepared in Example 1 by TMB method;

[0034] Figure 5 Infrared spectrum diagram of the Fe-BN nanozymes prepared in Examples 1 to 4;

[0035] Figure 6 XRD diagram (before dialysis) of the Fe-BN nanozymes prepared in Examples 1 to 4;

[0036] Figure 7 XRD diagram (after dialysis) of the Fe-BN nanozymes prepared in Examples 1 to 4;

[0037] Figure 8 In vivo imaging diagrams of biological tissues at different times of the Fe-BN transdermal drug delivery preparation prepared in Example 1;

[0038] Figure 9 Therapeutic effect diagram of the Fe-BN transdermal drug delivery preparation prepared in Example 1 on melanoma in a mouse model. Detailed implementation manners

[0039] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0040] In the present invention, the iron-loaded boron nitride nanozyme is simply referred to as Fe-BN nanozyme, and pararosaniline hydrochloride is simply referred to as BF, and its full English name is Basic Fuchsin.

[0041] The "including" described in the present invention means that in addition to the components described, other components may also be included. In addition, the "including" described in the present invention can also be replaced by the closed "consisting of" or "composed of...".

[0042] According to the first aspect of the present invention, a preparation method of a boron nitride nanozyme is provided, and the preparation method includes the following steps:

[0043] Step 1: Subject dicyandiamide to a thermal polycondensation reaction to synthesize graphitic carbon nitride.

[0044] The dicyandiamide is also known as cyanamide and dicyandiamide.

[0045] Preferably, the conditions of the thermal polycondensation reaction include a reaction temperature of 300 to 600 °C, preferably 500 to 550 °C; the reaction time is at least 1 h, preferably 4 h to 6 h.

[0046] Preferably, the conditions of the thermal polycondensation reaction further include a heating rate of 2.2 °C / min.

[0047] Step 2: Using water as a solvent, first mix the graphitic carbon nitride and boric acid, and then dehydrate and grind the mixture to obtain a powdery graphitic carbon nitride-boric acid precursor.

[0048] Preferably, the mass ratio of the graphitic carbon nitride to boric acid is 1:(0.1 to 0.5).

[0049] In Step 2, the dehydration method of the mixture is evaporation dehydration.

[0050] Step 3: First perform a first calcination treatment on the graphitic carbon nitride-boric acid precursor to obtain hydroxyboron nitride, and then uniformly mix the hydroxyboron nitride and an iron salt, and perform a second calcination treatment under a nitrogen atmosphere to obtain an iron-loaded boron nitride nanozyme.

[0051] Preferably, based on the mass of the iron-loaded boron nitride nanozyme being 100%, the iron loading amount is 0.2 wt% to 2 wt%, and more preferably, the iron loading amount is 0.2 wt% to 1 wt%.

[0052] Preferably, the conditions of the first calcination treatment include a calcination temperature of 700 to 1000 °C, preferably 750 to 850 °C; the calcination time is 0.1 h to 3 h, preferably 0.1 h to 2 h.

[0053] The specific method for preparing hydroxyboron nitride in Step 3 is as follows: Spread the graphitic carbon nitride-boric acid precursor flat in a covered alumina crucible / reaction bed, and perform calcination in a nitrogen-purged tubular furnace at a heating rate of 3.3 °C / min, a calcination temperature of 700 to 1000 °C, and a calcination time of 0.1 h to 3 h to obtain a white hydroxyboron nitride powder.

[0054] Preferably, the spreading thickness of the graphitic carbon nitride-boric acid precursor does not exceed 4 mm.

[0055] Preferably, the iron salt is a divalent iron salt or a trivalent iron salt.

[0056] More preferably, the iron salt is ferrous dichloride tetrahydrate, and the mass ratio of the hydroxyboron nitride to the ferrous dichloride tetrahydrate is 1:(0.01 - 0.5).

[0057] In an alternative embodiment, typical but non-limiting mass ratios of the hydroxyboron nitride to the ferrous dichloride tetrahydrate are 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3 or 1:0.5.

[0058] Preferably, the conditions for the second calcination treatment include a calcination temperature of 600 - 1000 °C and a calcination time of 0.1 h - 2 h; more preferably, the conditions for the second calcination treatment include a calcination temperature of 900 - 1000 °C and a calcination time of 1 h - 2 h.

[0059] The preparation of the iron-loaded boron nitride nanozyme in step 3 is specifically as follows: First, the hydroxyboron nitride powder obtained by the first calcination treatment and ferrous dichloride tetrahydrate are placed in a mortar and ground evenly, then spread out flat in a covered alumina crucible / reaction bed, and calcined in a nitrogen-purged tubular furnace at a heating rate of 6 °C / min, a reaction temperature of 600 - 1000 °C, and a reaction time of 0.1 h - 2 h to obtain the iron-loaded boron nitride nanozyme (Fe-BN nanozyme).

[0060] In the present invention, the boron nitride in the iron-loaded boron nitride nanozyme is hexagonal boron nitride.

[0061] According to the second aspect of the present invention, there is also provided an iron-loaded boron nitride nanozyme drug prepared by the above-described preparation method.

[0062] The iron-loaded boron nitride nanozyme prepared in the present invention, as an anti-tumor nanozyme drug, has excellent catalytic activity, can selectively catalyze peroxidation reactions in tumor cells through peroxidase-like activity to generate reactive oxygen species, thereby inducing tumor cells to die through oxidative stress. Since it uses highly biocompatible boron nitride (BN) as the main component and loads a trace amount of Fe, it can minimize damage to normal tissues to the greatest extent and significantly improve the selectivity and safety of treatment.

[0063] According to the third aspect of the present invention, there is also provided the application of the above iron-loaded boron nitride nanozyme drug in the preparation of a transdermal delivery preparation.

[0064] In view of the fact that the iron-loaded boron nitride nanozyme drug provided by the present invention has a small size, good biocompatibility, and high catalytic activity and selectivity, it has good application prospects in transdermal delivery preparations.

[0065] According to the fourth aspect of the present invention, a transdermal drug delivery preparation is further provided. The transdermal drug delivery preparation includes an active agent and a transdermal enhancer coating the active agent. The active agent is the iron-loaded boron nitride nanozyme drug described above, and the average particle size of the iron-loaded boron nitride nanozyme is less than 25 nm.

[0066] When the average particle size of the iron-loaded boron nitride nanozyme is less than 25 nm, it can effectively penetrate the skin barrier and reach the tumor site, so it can be used as an active drug in the transdermal drug delivery preparation.

[0067] It can be understood that in the present invention, the transdermal drug delivery preparation is specifically formed by dispersing the active agent coated with the transdermal enhancer in water. Among them, the active agent coated with the transdermal enhancer is the solute, and water is the solvent.

[0068] In an optional embodiment, based on the total weight of the solute of the transdermal drug delivery preparation being 100%, the mass percentage content of the active agent is 1.0 wt% - 20.0 wt%, and the mass percentage content of the transdermal enhancer is 80.0 wt% - 99.0 wt%.

[0069] In an optional embodiment, based on the mass of the iron-loaded boron nitride nanozyme being 100%, the iron loading amount is 0.2 wt% - 2 wt%, and more preferably, the iron loading amount is 0.2 wt% - 1 wt%.

[0070] In an optional embodiment, the transdermal enhancer includes pararosaniline hydrochloride and polyethyleneimine. The molecular weight range of the polyethyleneimine is 0.6 - 25 kDa. Among them, the mass ratio of the active agent to pararosaniline hydrochloride is 1:(0.05 - 0.15), and the mass ratio of the sum of the mass of the active agent and pararosaniline hydrochloride to the mass of the polyethyleneimine is 1:(1 - 10).

[0071] In an optional embodiment, the transdermal enhancer further includes polyglutamic acid and protamine. The molecular weight range of polyglutamic acid is 1 - 100 kDa, and the molecular weight range of protamine is 4 - 10 kDa. Among them, the mass ratio of the sum of the mass of the active agent and pararosaniline hydrochloride to polyglutamic acid and protamine is 1:(2 - 10):(4 - 20).

[0072] In the present invention, the transdermal drug delivery preparation is prepared by the following method, including:

[0073] Step (1), provide iron-loaded boron nitride nanozyme Fe-BN as the active agent.

[0074] Among them, the iron-loaded boron nitride nanozyme is prepared by the following method: Step a, subject dicyandiamide to thermal polycondensation reaction to synthesize graphitic carbon nitride; Step b, using water as a solvent, under stirring and heating conditions, first mix the graphitic carbon nitride and boric acid, and then dehydrate and grind the mixture to obtain a powdery graphitic carbon nitride-boric acid precursor; Step c, first perform a first calcination treatment on the graphitic carbon nitride-boric acid precursor to obtain hydroxyboron nitride, and then uniformly mix the hydroxyboron nitride and iron salt, and perform a second calcination treatment under nitrogen conditions to obtain the iron-loaded boron nitride nanozyme.

[0075] In an optional embodiment, the conditions of the thermal polycondensation reaction include a reaction temperature of 300-600 °C and a reaction time of at least 1 h.

[0076] In an optional embodiment, the mixing conditions of the graphitic carbon nitride and boric acid include a mixing temperature of 70-100 °C and a reaction time of 0.1 h-1 h.

[0077] In an optional embodiment, the conditions of the first calcination treatment include a calcination temperature of 700-1000 °C and a calcination time of 0.1 h-3 h.

[0078] In an optional embodiment, the conditions of the second calcination treatment include a calcination temperature of 600-1000 °C and a calcination time of 0.1 h-2 h.

[0079] In an optional embodiment, the mass ratio of the graphitic carbon nitride to boric acid is 1:(0.1-0.5).

[0080] In an optional embodiment, based on the mass of the iron-loaded boron nitride nanozyme being 100%, the iron loading is 0.2 wt%-2 wt%, and more preferably, the iron loading is 0.2 wt%-1 wt%.

[0081] In an optional embodiment, the iron salt is a divalent iron salt or a trivalent iron salt.

[0082] In an optional embodiment, the iron salt is ferrous dichloride tetrahydrate, and the mass ratio of the hydroxyboron nitride to ferrous dichloride tetrahydrate is 1:(0.01-0.5).

[0083] Step (2), modify the iron-loaded boron nitride nanozyme Fe-BN with pararosaniline hydrochloride BF to obtain an Fe-BN / BF solution, wherein the mass ratio of the iron-loaded boron nitride nanozyme Fe-BN to pararosaniline hydrochloride BF is 1:(0.05-0.15).

[0084] In this step, first disperse the iron-loaded boron nitride nanozyme Fe-BN in water, then add pararosaniline hydrochloride BF, and magnetically stir at a rotation speed of 250 rpm for 1 h to obtain the Fe-BN / BF solution.

[0085] Step (3): Add polyethyleneimine to the Fe-BN / BF solution to prepare the transdermal delivery preparation, where the mass ratio of Fe-BN / BF to polyethyleneimine is 1:(1-10); or Step (3): First add polyethyleneimine to the Fe-BN / BF solution for the first coating, and then add polyglutamic acid and protamine to the solution of Fe-BN / BF coated with polyethyleneimine for the second coating to prepare the transdermal delivery preparation, where the mass ratio of Fe-BN / BF to polyethyleneimine is 1:(1-10), the mass ratio of Fe-BN / BF to polyglutamic acid is 1:(2-10), and the mass ratio of Fe-BN / BF to protamine is 1:(4-20).

[0086] In this step, after adding polyethyleneimine to the Fe-BN / BF solution, magnetically stir at a rotation speed of 250 rpm for 2 h to obtain the solution of Fe-BN / BF coated with polyethyleneimine; add the mixed solution dissolved with polyglutamic acid and protamine to the solution of Fe-BN / BF coated with polyethyleneimine, and magnetically stir at a rotation speed of 250 rpm for 2 h to prepare the transdermal delivery preparation.

[0087] In the present invention, the preparation method of the mixed solution dissolved with polyglutamic acid and protamine is: dissolve polyglutamic acid and protamine in water, and ultrasonically treat for 30 min and magnetically stir for 30 min to obtain it.

[0088] Example 1

[0089] Preparation of Iron-Loaded Boron Nitride Nanozyme

[0090] Step 1.1: Weigh 10 g of dicyandiamide, add it to an alumina boat, place it in a muffle furnace, set the heating rate to 2.2 °C / min -1 , and perform thermal polycondensation at a temperature of 500 °C for 4 h to synthesize graphitic carbon nitride.

[0091] Step 1.2: First weigh 3.0 g of graphitic carbon nitride and 0.9 g of boric acid, add them to 100 mL of deionized water, ultrasonically treat for 30 min, then heat and stir until the water is completely evaporated, and then grind to obtain the graphitic carbon nitride-boric acid precursor powder.

[0092] Step 1.3: Weigh 0.5 g of the graphitic carbon nitride-boric acid precursor obtained in the second step and place it in a covered alumina crucible. Calcinate it in a tube furnace with a heating rate of 3.3 °C / min, a reaction temperature of 800 °C, and a reaction time of 1 h to obtain white hydroxyboron nitride powder.

[0093] Step 1.4: Weigh 200 mg of the hydroxyboron nitride powder obtained in the third step, and then weigh 10 mg of ferric dichloride tetrahydrate. Mix and grind them evenly in a mortar, spread them out in a covered alumina crucible, and calcinate them in a nitrogen-purged tube furnace with a heating rate of 6 °C / min, a reaction temperature of 900 °C, and a reaction time of 1 h to prepare iron-loaded boron nitride nanozyme, abbreviated as Fe-BN nanozyme. Among them, the Fe loading is 0.87 wt%.

[0094] Perform objective lens spherical aberration correction transmission electron microscopy, particle size distribution statistics, and condenser lens spherical aberration correction transmission electron microscopy analysis on the Fe-BN nanozyme prepared in Example 1. The results are shown in Figures 1 to 3 , where Figure 1 is the objective lens spherical aberration correction transmission electron microscopy image of the Fe-BN nanozyme prepared in Example 1, Figure 2 is the particle size distribution diagram of the Fe-BN nanozyme prepared in Example 1, Figure 3 is the condenser lens spherical aberration correction transmission electron microscopy analysis image of the Fe-BN nanozyme prepared in Example 1. It can be seen from Figures 1 to 3 that Fe-BN is evenly dispersed, the average particle size is 7.5 nm, and Fe is dispersed on the boron nitride carrier in the form of single atoms.

[0095] Perform catalytic activity analysis on the Fe-BN nanozyme prepared in Example 1. The results are shown in Figure 4 , Figure 4 is the detection result of the peroxidase-like activity of the Fe-BN nanozyme prepared in Example 1 by the TMB method. It can be seen from Figure 4 that it can effectively catalyze the decomposition of hydrogen peroxide to produce hydroxyl radicals and has peroxidase-like nanozyme characteristics.

[0096] Examples 2 to 4

[0097] Same as Example 1, except that the calcination temperature in the tube furnace in Step 1.4 is different. The calcination temperature in Example 1 is 900 °C, the calcination temperature in Example 2 is 600 °C, the calcination temperature in Example 3 is 700 °C, and the calcination temperature in Example 4 is 800 °C.

[0098] Perform characterization analysis on the Fe-BN nanozymes prepared in Examples 1 to 4. The results are shown in detail in Figures 5 to 7 , where Figure 5Infrared spectra of the Fe-BN nanozymes prepared in Examples 1 to 4. In this figure, the curve indicated by I represents the Fe-BN calcined at 600 °C, the curve indicated by II represents the Fe-BN calcined at 700 °C, the curve indicated by III represents the Fe-BN calcined at 800 °C, and the curve indicated by IV represents the Fe-BN calcined at 900 °C; Figure 6 XRD pattern (before dialysis) of the Fe-BN nanozymes prepared in Examples 1 to 4. Among them, 600 represents the calcination temperature of 600 °C, 700 represents the calcination temperature of 700 °C, 800 represents the calcination temperature of 800 °C, and 900 represents the calcination temperature of 900 °C; Figure 7 XRD pattern (after dialysis) of the Fe-BN nanozymes prepared in Examples 1 to 4. Among them, 600 represents the calcination temperature of 600 °C, 700 represents the calcination temperature of 700 °C, 800 represents the calcination temperature of 800 °C, and 900 represents the calcination temperature of 900 °C; It can be seen that Figures 5 to 7 the product framework is BN.

[0099] Examples 5 - 6

[0100] Preparation of boron nitride nanozymes with different iron loadings

[0101] Example 5

[0102] Same as Example 1, except that the mass of ferrous dichloride tetrahydrate added in step 1.4 is different. The mass of ferrous dichloride tetrahydrate added in step 1.4 of Example 1 is 10 mg, and the mass of ferrous dichloride tetrahydrate added in step 1.4 of Example 5 is 2 mg. The iron loading in the prepared Fe-BN nanozyme is 0.27 wt%.

[0103] Example 6

[0104] Same as Example 1, except that the mass of ferrous dichloride tetrahydrate added in step 1.4 is different. The mass of ferrous dichloride tetrahydrate added in step 1.4 of Example 1 is 10 mg, and the mass of ferrous dichloride tetrahydrate added in step 1.4 of Example 6 is 15 mg. The iron loading in the prepared Fe-BN nanozyme is 0.9 wt%.

[0105] It should be noted that the iron loadings in Examples 1, 5, and 6 are calculated based on the total mass of the Fe-BN nanozyme being 100%.

[0106] Example 7

[0107] Investigate the effect of the solid-phase reaction temperature (calcination temperature) in step 1.4 on the average particle size of Fe-BN

[0108] Example 7

[0109] Same as Example 1, except that the calcination temperature in the tubular furnace in Step 1.4 is different. The calcination temperature in Example 1 is 900 °C, and the calcination temperature in Example 7 is 1000 °C. The average particle size of the prepared Fe-BN is 7.6 nm.

[0110] When the calcination temperature in Example 1 is 900 °C, the average particle size of the prepared Fe-BN is 7.5 nm. When the calcination temperature in Example 4 is 800 °C, the average particle size of the prepared Fe-BN is 15.2 nm. When the calcination temperature in Example 7 is 1000 °C, the average particle size of the prepared Fe-BN is 7.6 nm. Comparing the experimental results of Example 1, Example 4, and Example 7, it can be seen that when the calcination temperature is 800-1000 °C, the average particle size of the prepared Fe-BN nanozyme is less than 25 nm, and when the calcination temperature is 900-1000 °C, the average particle size of the prepared Fe-BN is less than 10 nm; at the same time, when the calcination temperature is increased from 900 °C to 1000 °C, the average particle size of the Fe-BN nanozyme varies little. Considering the calcination cost, the optimal calcination temperature is 900 °C.

[0111] Examples 8-9

[0112] Investigate the effect of the solid-phase reaction time in Step 1.4 on the average particle size of Fe-BN

[0113] Example 8

[0114] Same as Example 1, except that the calcination time in the tubular furnace in Step 1.4 is different. The calcination time in Example 1 is 1 h, and the calcination time in Example 8 is 0.1 h. The average particle size of the prepared Fe-BN is 18.5 nm.

[0115] Example 9

[0116] Same as Example 1, except that the calcination time in the tubular furnace in Step 1.4 is different. The calcination time in Example 1 is 1 h, and the calcination time in Example 9 is 2 h. The average particle size of the prepared Fe-BN is 7.3 nm.

[0117] Comparing the experimental data of Example 1, Example 8, and Example 9, it can be seen that there is a correlation between the calcination time and the average particle size of Fe-BN. When the calcination time is less than 1 h, as the calcination time increases, the average particle size of the prepared Fe-BN becomes smaller. When the calcination time is greater than 1 h, as the calcination time increases, the average particle size of the prepared Fe-BN varies little, indicating that the optimal calcination time is 1 h.

[0118] Example 10

[0119] Preparation of transdermal drug delivery preparation

[0120] Step 2.1: Provide the iron-loaded boron nitride nanozyme prepared in Example 1.

[0121] Step 2.2: Weigh 20 mg of the Fe-BN nanozyme and place it in 2 mL of water. Sonicate for 30 min to disperse it evenly. Then weigh 2 mg of pararosaniline hydrochloride (BF) and add it thereto. Stir magnetically at 250 rpm for 1 h to obtain the Fe-BN / BF solution.

[0122] Step 2.3: Add 20 mg of polyethyleneimine to the Fe-BN / BF solution obtained in Step 2.2. Stir magnetically at 250 rpm for 2 h to obtain the Fe-BN / BF / polyethyleneimine solution.

[0123] Step 2.4: Weigh 40 mg of polyglutamic acid and 80 mg of protamine and dissolve them in 1 mL of water. Sonicate for 30 min and stir magnetically for 30 min to obtain a mixed solution.

[0124] Step 2.5: Add the mixed solution obtained in Step 2.4 to the Fe-BN / BF / polyethyleneimine solution. Stir magnetically at 250 rpm for 2 h to prepare the transdermal delivery preparation.

[0125] Apply the transdermal delivery preparation prepared in Example 10 to the skin of the tumor-bearing mice at the tumor site. Take the ex vivo tissues of the mice at different time periods for fluorescence imaging analysis. The results are shown in Figure 8 , and it can be seen from Figure 8 that the Fe-BN nanozyme can effectively penetrate the skin and reach the melanoma lesion site.

[0126] Animal experiments were carried out on the anti-tumor activity of Fe-BN. A control group (PBS), an injection group (intratumoral injection), and a transdermal group (transdermal administration) were set up respectively. In the transdermal group, the transdermal delivery preparation prepared in Example 10 was applied to the skin of the tumor site of the mice three times a day; in the injection group, the mice were intratumorally injected with 20 mg / kg of the nanozyme on day 0 and day 7 respectively. The results are shown in Figure 9 , and the drug in the transdermal group has a significant inhibition rate on the growth of melanoma (the inhibition rate at 2 weeks is 93%), and there is no significant difference statistically compared with the intratumoral injection method.

[0127] Examples 11 to 25

[0128] Investigate the influence of different formulations of transdermal enhancers on the skin penetration performance

[0129] The active agents used in Examples 11 to 25 are all the Fe-BN nanozymes prepared in Example 1. The main difference is that the transdermal enhancers are not completely the same. The masses of the transdermal enhancers added in each example are shown in Table 1 in detail.

[0130] Table 1 Influence of Transdermal Enhancers with Different Formulations on Skin Penetration Performance

[0131]

[0132]

[0133] Examples 26 to 31, and Comparative Example 1 and Comparative Example 2

[0134] Investigate the influence of Fe-BN nanozymes with different iron loadings and different particle sizes on skin penetration performance

[0135] The transdermal enhancers used in Examples 26 to 31 are exactly the same as those in Example 10 (2 mg of pararosaniline hydrochloride, 20 mg of polyethyleneimine, 40 mg of polyglutamic acid, 80 mg of protamine), and the active agents used are the Fe-BN nanozymes prepared in Examples 4 to 9. The iron loadings and average particle sizes of the Fe-BN nanozymes added in each example are shown in detail in Table 2.

[0136] The transdermal enhancers used in Comparative Example 1 and Comparative Example 2 are exactly the same as those in Example 10 (2 mg of pararosaniline hydrochloride, 20 mg of polyethyleneimine, 40 mg of polyglutamic acid, 80 mg of protamine), and the active agents used are boron nitride nanoparticles with other particle sizes. The specific particle size dimensions are shown in detail in Table 2.

[0137] Table 2 Influence of Fe-BN Nanozymes with Different Iron Loadings and Different Particle Sizes on Skin Penetration Performance

[0138]

[0139]

[0140] As can be seen from Table 2, the Fe-BN nanozymes prepared by using the preparation method of the iron-loaded boron nitride nanozymes provided by the present invention have an average particle size of less than 20 nm and have good transdermal performance when used to prepare transdermal drug delivery preparations; the skin permeability of the transdermal drug delivery preparation prepared with 25-nm boron nitride nanoparticles as the raw material in Comparative Example 1 is general, and only a small amount can penetrate, and the skin permeability of the transdermal drug delivery preparation prepared with 100-nm boron nitride nanoparticles as the raw material in Comparative Example 2 is poor and cannot penetrate the skin at all.

[0141] It should be noted that in the present invention, the method for evaluating skin permeability is specifically as follows: a fluorescent substance (such as a fluorescent dye or a fluorescently labeled nanomaterial) is used as a tracer, and the distribution, migration, and metabolic processes of the target substance in vivo or in vitro are traced by detecting the fluorescence signal emitted by it. The basic steps of this method are as follows: First, in an aqueous solution, the Fe-BN nanomaterial and rhodamine B dye are stirred and mixed for 6 hours to ensure sufficient binding between the two. Subsequently, the unbound rhodamine B is removed by centrifugation, and the precipitate is redispersed in deionized water. Then, a transdermal penetration enhancer is added to this dispersion and stirred overnight at room temperature to ensure uniform mixing. Next, an appropriate amount of the rhodamine B-labeled Fe-BN nanozyme transdermal preparation solution is evenly applied to a specific skin site of a tumor-bearing mouse. After application, for a certain period of time (such as 3 to 12 hours), the drug attached to the surface is washed off with water. Subsequently, the mouse is dissected, and the skin and subcutaneous tissue (tumor or muscle) treated with the drug are removed and analyzed by a small animal fluorescence imaging system. By observing the intensity of the fluorescence signal appearing in the subcutaneous tissue, the skin permeability of the Fe-BN nanozyme is judged. A strong fluorescence signal indicates good skin permeability, and vice versa indicates poor permeability.

[0142] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions and substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for preparing an iron-loaded boron nitride nanozyme, characterized in that: The preparation method comprises the following steps: Step 1, subjecting dicyandiamide to a thermal polycondensation reaction to synthesize graphite phase carbon nitride; Step 2: Using water as a solvent, firstly mixing the graphite phase carbon nitride and boric acid, and then dehydrating and grinding the mixture to obtain a powdered graphite phase carbon nitride-boric acid precursor; Step 3: firstly subject the graphite phase carbon nitride-boric acid precursor to a first calcination treatment to obtain hydroxy boron nitride, then evenly mix the hydroxy boron nitride and iron salt, and then subject the mixture to a second calcination treatment under nitrogen conditions to obtain an iron-loaded boron nitride nanozyme.

2. The method for preparing the iron-loaded boron nitride nanozyme according to claim 1, characterized in that: The thermal polycondensation reaction conditions in step 1 include a reaction temperature of 300-600° C. and a reaction time of at least 1 hour; and / or, The conditions of the first calcination treatment include a calcination temperature of 700 to 1000° C. and a calcination time of 0.1 to 3 hours; and / or, The conditions of the second calcination treatment include a calcination temperature of 600 to 1000° C. and a calcination time of 0.1 h to 2 h.

3. The method for preparing the iron-loaded boron nitride nanozyme according to claim 1 or 2, characterized in that: The mass ratio of the graphite phase carbon nitride to boric acid is 1:(0.1-0.5); and / or, Taking the mass of the iron-loaded boron nitride nanozyme as 100%, the iron loading amount is 0.2wt% to 2wt%.

4. A boron nitride nanozyme drug loaded with iron, characterized in that: The iron-loaded boron nitride nanozyme drug is prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the iron-loaded boron nitride nanozyme prepared by the preparation method according to any one of claims 1 to 3 in the preparation of a transdermal drug delivery preparation.

6. A transdermal drug delivery preparation, characterized in that: The transdermal drug delivery preparation comprises an active agent and a transdermal enhancer coating the active agent, wherein the active agent is the iron-loaded boron nitride nanozyme drug according to claim 4, and the average particle size of the iron-loaded boron nitride nanozyme is less than 25 nm.

7. The transdermal drug delivery preparation according to claim 6, characterized in that: Based on the total weight of the solute of the transdermal drug delivery preparation being 100%, the mass percentage of the active agent is 1.0wt% to 20.0wt%, and the mass percentage of the transdermal enhancer is 80.0wt% to 99.0wt%.

8. The transdermal drug delivery preparation according to claim 7, characterized in that: The transdermal enhancer comprises para-mercury hydrochloride and polyethyleneimine, wherein the molecular weight of the polyethyleneimine is in the range of 0.6 to 25 kDa, wherein the mass ratio of the active agent to the para-mercury hydrochloride is 1:(0.05 to 0.15), and the mass ratio of the sum of the mass of the active agent and the para-mercury hydrochloride to the mass of the polyethyleneimine is 1:(1 to 10).

9. The transdermal drug delivery preparation according to claim 8, characterized in that The transdermal enhancer also includes polyglutamic acid and protamine, the molecular weight range of polyglutamic acid is: 1-100kDa, and the molecular weight range of protamine is: 4-10kDa, wherein the mass ratio of the sum of the mass of the active agent and the parafuchsin hydrochloride to the mass of the polyglutamic acid and protamine is 1:(2-10):(4-20).

10. Use of the transdermal drug delivery preparation according to any one of claims 6 to 9 in the preparation of a product for treating melanoma.

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