Boron nitride nanoscale enzyme medicine, preparation method, application and transdermal preparation

Abstract

The application provides a boron nitride nanoscale enzyme medicine, a preparation method, application and transdermal drug preparation. The preparation method of the boron nitride nanoscale enzyme comprises the following steps: making dicyandiamide undergo thermal polycondensation reaction to synthesize graphite phase carbon nitride; mixing the graphite phase carbon nitride and boric acid with water as a solvent, then dehydrating and grinding the mixture to obtain powder graphite phase carbon nitride-boric acid precursor; first, the graphite phase carbon nitride-boric acid precursor is subjected to first calcination treatment to obtain hydroxyl boron nitride, then the hydroxyl boron nitride and iron salt are uniformly mixed, and the mixture is subjected to second calcination treatment under the condition of nitrogen to obtain the boron nitride nanoscale enzyme loaded with iron. The boron nitride nanoscale enzyme prepared by the application is loaded with iron, has small average particle size, high selectivity and inhibition activity, excellent transdermal performance, and can be used as an active agent of the transdermal drug preparation to treat superficial tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a boron nitride nanoscale enzyme drug, a preparation method and application thereof, and a transdermal drug delivery preparation. BACKGROUND

[0002] Advanced melanoma has strong invasiveness and mortality, which is a great threat to human health. For many years, the incidence of melanoma has been rising, especially in areas with strong ultraviolet light. The current treatment methods for melanoma mainly include Mohs surgery and chemotherapy. Mohs surgery is relatively effective for early-stage melanoma, but the treatment effect is poor for patients who have metastasis, and chemotherapy and other means must be used to completely remove the residual tumor tissue and cancer cells as much as possible to improve the survival rate of patients. On the other hand, most clinical chemotherapy drugs cannot target cancer cells and tissues, and often have problems such as toxic side effects and drug resistance. In view of the characteristics of melanoma as a superficial cancer, more effective drugs and drug delivery methods can be developed to target tumor sites to minimize systemic side effects.

[0003] Nanoscale enzymes 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 features and advantages of nanoscale enzymes are their ability to selectively kill tumor cells by catalyzing the high levels of endogenous hydrogen peroxide in tumors to produce reactive oxygen species. This highly selective treatment method significantly reduces damage to normal tissues and improves treatment safety. In addition, nanoscale enzymes exhibit high stability, tunability, and potential for combination therapy. However, nanoscale enzymes are generally large in size, making it difficult to deliver them 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 local drug delivery method. This method reduces systemic exposure and related side effects while enhancing the therapeutic effect at the tumor site. SUMMARY

[0005] The first object of the present application is to provide a preparation method of iron-loaded boron nitride nanoscale enzymes, which enables the preparation of boron nitride nanoscale enzymes loaded with a small amount of iron and having a small average particle size, high selectivity and inhibitory activity against tumor cells, and excellent transdermal performance due to the small size.

[0006] The second object of the present application is to provide an iron-loaded boron nitride nanoscale enzyme drug that can be used as an active agent for transdermal drug delivery preparations to treat superficial tumors with fewer side effects.

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

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

[0009] The fifth object of the present application is the use of the transdermal drug delivery preparation in the preparation of a melanoma treatment product.

[0010] To achieve the above object, in a first aspect, the present application provides a preparation method of iron-loaded boron nitride nanoscale enzyme, comprising the following steps: step 1, synthesizing graphitic carbon nitride by thermal polycondensation reaction of dicyandiamide; step 2, mixing the graphitic carbon nitride and boric acid in water as a solvent, then dehydrating and grinding the mixture to obtain a powdered graphitic carbon nitride-boric acid precursor; step 3, first performing first calcination treatment on the graphitic carbon nitride-boric acid precursor to obtain hydroxyl boron nitride, then uniformly mixing the hydroxyl boron nitride and an iron salt, and performing second calcination treatment under nitrogen to obtain the iron-loaded boron nitride nanoscale enzyme. In a specific embodiment, the thermal polycondensation reaction conditions in step 1 include a reaction temperature of 300-600℃ and a reaction time of at least 1h.

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

[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, the loading amount of iron is 0.2wt%-2wt%, preferably 0.2wt%-1wt%, based on 100% of the mass of the iron-loaded boron nitride nanoscale enzyme.

[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 iron dichloride tetrahydrate, and the mass ratio of the hydroxyl boron nitride to iron dichloride tetrahydrate is 1:(0.01-0.5).

[0016] In a second aspect, the present application also provides an iron-loaded boron nitride nanoscale enzyme drug, which is prepared by the above-mentioned preparation method.

[0017] In a third aspect, the present application further provides an application of the iron-loaded boron nitride nanoscale enzyme prepared by the preparation method in the above in the preparation of a transdermal drug delivery preparation.

[0018] In a fourth aspect, the present application further provides a transdermal drug delivery preparation, which comprises an active agent and a transdermal enhancer for coating the active agent, wherein the active agent is the iron-loaded boron nitride nanoscale enzyme drug prepared by the preparation method in the above, and the average particle size of the iron-loaded boron nitride nanoscale enzyme is less than 25 nm.

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

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

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

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

[0023] In a fifth aspect, the present application further provides an application of the transdermal drug delivery preparation in the above in the preparation of a product for treating melanoma.

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

[0025] I. The application provides a preparation method of iron-loaded boron nitride nanoscale enzyme, comprising the following steps: step 1, dicyandiamide is subjected to thermal polycondensation reaction to synthesize graphite phase carbon nitride; step 2, in a stirring and heating state, the graphite phase carbon nitride and boric acid are first mixed in water as a solvent, and then the mixture is dehydrated and ground to obtain powdered graphite phase carbon nitride-boric acid precursor; step 3, the graphite phase carbon nitride-boric acid precursor is first subjected to first calcination treatment to obtain hydroxyl boron nitride, and then the hydroxyl boron nitride and iron salt are uniformly mixed and subjected to second calcination treatment under nitrogen condition to obtain the iron-loaded boron nitride nanoscale enzyme; the boron nitride nanoscale enzyme prepared by the preparation method is loaded with a small amount of iron, on the one hand, the iron-loaded boron nitride nanoscale enzyme has excellent catalytic activity and can selectively catalyze peroxide reaction in tumor cells through peroxidase-like activity to produce reactive oxygen, 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 nanoscale enzyme mainly comprises high biocompatibility boron nitride and a small amount of iron, which can minimize damage to normal tissues and significantly improve the selectivity and safety of treatment.

[0026] II. The preparation method of the iron-loaded boron nitride nanoscale enzyme provided by the application uses healthy friendly materials, has a simple preparation process and can be produced on a large scale, and has high economic efficiency and sustainability.

[0027] III. The application provides a transdermal drug preparation, which comprises an active agent, and a transdermal enhancer for coating the active agent, wherein the active agent is an iron-loaded boron nitride nanoscale enzyme drug and has an average particle size of less than 25 nm; in this way, the small size of the Fe-BN nanoscale enzyme enables it to effectively penetrate the skin barrier and reach the tumor site, and direct application of the drug to the skin not only avoids the systemic toxicity and low bioavailability of traditional chemotherapy, but also directly delivers the drug to the melanoma lesion, significantly improves the local drug concentration, and enhances the treatment effect.

[0028] IV. When the transdermal drug preparation provided by the application is applied to treat melanoma, it has the characteristics of non-invasiveness, painlessness and convenience compared with traditional chemotherapy and surgery, significantly reduces the side effects and discomfort during treatment, and improves the patient's compliance and quality of life; in addition, local administration reduces systemic exposure, further reducing the risk of systemic side effects.

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

[0030] Figure 1 Objective spherical aberration corrected transmission electron microscopy image of Fe-BN nanoszyme prepared in Example 1;

[0031] Figure 2 Particle size distribution graph of Fe-BN nanoszyme prepared in Example 1;

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

[0033] Figure 4 Peroxidase-like activity TMB method detection result of Fe-BN nanoszyme prepared in Example 1;

[0034] Figure 5 Infrared spectrogram of Fe-BN nanoszyme prepared in Example 1 to Example 4;

[0035] Figure 6 XRD graph (before dialysis) of Fe-BN nanoszyme prepared in Example 1 to Example 4;

[0036] Figure 7 XRD graph (after dialysis) of Fe-BN nanoszyme prepared in Example 1 to Example 4;

[0037] Figure 8 Biological tissue in vivo imaging image of Fe-BN transdermal preparation prepared in Example 1 at different times;

[0038] Figure 9 Treatment effect image of Fe-BN transdermal preparation prepared in Example 1 on melanoma in a mouse model. DETAILED DESCRIPTION

[0039] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0040] In the present application, the iron-loaded boron nitride nanoszyme is referred to as Fe-BN nanoszyme, and hydrochloric acid pararosaniline is referred to as BF, and its English full name is Basic Fuchsin.

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

[0042] According to a first aspect of the present application, there is provided a method for preparing a boron nitride nanoscale enzyme, the method comprising the steps of:

[0043] Step 1, synthesizing graphite phase carbon nitride by subjecting dicyandiamide to a thermal polycondensation reaction.

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

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

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

[0047] Step 2, mixing the graphite phase carbon nitride and boric acid in water as a solvent, and then dehydrating and grinding the mixture to obtain a graphite phase carbon nitride-boric acid precursor in powder form.

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

[0049] In Step 2, the method for dehydrating the mixture is steam dehydration.

[0050] Step 3, first subjecting the graphite phase carbon nitride-boric acid precursor to a first calcination treatment to obtain hydroxyl boron nitride, and then mixing the hydroxyl boron nitride and an iron salt uniformly and performing a second calcination treatment under a nitrogen atmosphere to obtain an iron-loaded boron nitride nanoscale enzyme.

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

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

[0053] The hydroxyl boron nitride obtained in Step 3 is prepared by: placing the graphite phase carbon nitride-boric acid precursor in a covered alumina crucible / reaction bed, performing calcination in a nitrogen-purged tube furnace, a heating rate of 3.3°C / min, a calcination temperature of 700-1000°C, and a calcination time of 0.1-3 h, to obtain white hydroxyl boron nitride powder.

[0054] Preferably, the graphite phase carbon nitride-boric acid precursor is laid flat to a thickness of no more than 4 mm.

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

[0056] More preferably, the iron salt is ferric chloride tetrahydrate, and the mass ratio of the hydroxyl boron nitride to the ferric chloride tetrahydrate is 1:(0.01-0.5).

[0057] In an alternative embodiment, the mass ratio of the hydroxyl boron nitride to the ferric chloride tetrahydrate is typically but not limitedly 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3 or 1:0.5.

[0058] Preferably, the second calcination treatment is performed at a temperature of 600-1000℃ for 0.1h-2h; more preferably, the second calcination treatment is performed at a temperature of 900-1000℃ for 1h-2h.

[0059] The step 3 is to prepare the iron-loaded boron nitride nanoscale enzyme by mixing and grinding the hydroxyl boron nitride powder obtained in the first calcination treatment and the ferric chloride tetrahydrate in a mortar, and then laying them in a covered alumina crucible / reaction bed, and performing calcination in a nitrogen-tube furnace at a temperature increasing rate of 6℃ / min, a reaction temperature of 600-1000℃ and a reaction time of 0.1h-2h to obtain the iron-loaded boron nitride nanoscale enzyme (Fe-BN nanoscale enzyme).

[0060] In the present application, the boron nitride in the iron-loaded boron nitride nanoscale enzyme is hexagonal boron nitride.

[0061] According to a second aspect of the present application, there is also provided an iron-loaded boron nitride nanoscale enzyme prepared by the above preparation method.

[0062] The iron-loaded boron nitride nanoscale enzyme prepared by the present application has excellent catalytic activity and can selectively catalyze peroxide reaction in tumor cells through peroxidase-like activity to produce reactive oxygen, thereby inducing tumor cells to die through oxidative stress. Since high biocompatibility boron nitride (BN) is used as the main component and a small amount of Fe is loaded, the damage to normal tissues is minimized, and the selectivity and safety of the treatment are significantly improved.

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

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

[0065] According to a fourth aspect of the present application, there is further provided a transdermal drug delivery formulation comprising an active agent, and a transdermal enhancer coated on the active agent, wherein the active agent is the iron-loaded boron nitride nanoscale enzyme drug as described above, and the average particle size of the iron-loaded boron nitride nanoscale enzyme is less than 25 nm.

[0066] When the average particle size of the iron-loaded boron nitride nanoscale enzyme is less than 25 nm, it can effectively penetrate the skin barrier and reach the tumor site, thereby serving as an active drug of the transdermal drug delivery formulation.

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

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

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

[0070] In an alternative embodiment, the transdermal enhancer comprises pararosaniline hydrochloride and polyethyleneimine, the molecular weight of the polyethyleneimine ranges from 0.6 to 25 kDa, wherein the mass ratio of the active agent to the pararosaniline hydrochloride is 1:(0.05 to 0.15), and the mass ratio of the sum of the active agent and the pararosaniline hydrochloride to the polyethyleneimine is 1:(1 to 10).

[0071] In an alternative embodiment, the transdermal enhancer further comprises polyglutamic acid and protamine, the molecular weight of the polyglutamic acid ranges from 1 to 100 kDa, and the molecular weight of the protamine ranges from 4 to 10 kDa, wherein the mass ratio of the sum of the active agent and the pararosaniline hydrochloride to the polyglutamic acid and the protamine is 1:(2 to 10):(4 to 20).

[0072] In the present application, the transdermal drug delivery formulation is prepared by the following method, comprising:

[0073] Step (1), providing an active agent of iron-loaded boron nitride nanoscale enzyme Fe-BN.

[0074] The load iron boron nitride nanoscale enzyme is prepared by the following method: step a, dicyandiamide is subjected to a thermal polycondensation reaction to synthesize graphite phase carbon nitride; step b, in a stirring and heating state, the graphite phase carbon nitride and boric acid are mixed first, and then the mixture is dehydrated and ground to obtain a powder-shaped graphite phase carbon nitride-boric acid precursor; step c, the graphite phase carbon nitride-boric acid precursor is subjected to a first calcination treatment to obtain hydroxyl boron nitride, and then the hydroxyl boron nitride and an iron salt are uniformly mixed and subjected to a second calcination treatment under a nitrogen condition to obtain the load iron boron nitride nanoscale enzyme.

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

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

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

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

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

[0080] In an alternative embodiment, the load amount of iron is 0.2wt%-2wt% based on 100% of the mass of the load iron boron nitride nanoscale enzyme, and more preferably, the load amount of iron is 0.2wt%-1wt%.

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

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

[0083] Step (2), the load iron boron nitride nanoscale enzyme Fe-BN is subjected to pararosaniline BF modification to obtain a Fe-BN / BF solution, wherein the mass ratio of the load iron boron nitride nanoscale enzyme Fe-BN to pararosaniline BF is 1:(0.05-0.15).

[0084] In this step, the iron-loaded boron nitride nanoscale enzyme Fe-BN is first dispersed in water, then paraformaldehyde BF is added, and magnetic stirring is carried out at a speed of 250 rpm for 1 h to obtain a Fe-BN / BF solution.

[0085] Step (3), polyethyleneimine is added to the Fe-BN / BF solution to prepare the transdermal drug delivery preparation, wherein the mass ratio of Fe-BN / BF to polyethyleneimine is 1:(1-10), or step (3), polyethyleneimine is first added to the Fe-BN / BF solution to perform the first coating, and then polyglutamic acid and protamine are added to the Fe-BN / BF solution coated with polyethyleneimine to perform the second coating, to prepare the transdermal drug delivery preparation, wherein 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 polyethyleneimine is added to the Fe-BN / BF solution, magnetic stirring is carried out at a speed of 250 rpm for 2 h to obtain a Fe-BN / BF solution coated with polyethyleneimine; a mixed solution containing dissolved polyglutamic acid and protamine is added to the Fe-BN / BF solution coated with polyethyleneimine, and magnetic stirring is carried out at a speed of 250 rpm for 2 h to prepare the transdermal drug delivery preparation.

[0087] In the present application, the preparation method of the mixed solution containing dissolved polyglutamic acid and protamine is as follows: polyglutamic acid and protamine are dissolved in water, ultrasonic treatment is carried out for 30 min, and magnetic stirring is carried out for 30 min.

[0088] Example 1

[0089] Preparation of iron-loaded boron nitride nanoscale enzyme

[0090] Step 1.1, weigh 10 g of dicyandiamide into an alumina porcelain boat, and place it in a muffle furnace, with a heating rate setting of 2.2℃ / min -1 At a temperature of 500℃, heat polycondensation is carried out for 4 h to synthesize graphite phase carbon nitride.

[0091] Step 1.2, first weigh 3.0 g of graphite phase carbon nitride and 0.9 g of boric acid into 100 mL of deionized water, ultrasonic treatment is carried out for 30 min, then heating and stirring are carried out to completely volatilize the water, and then grinding is carried out to obtain graphite phase carbon nitride-boric acid precursor powder.

[0092] Step 1.3, 0.5 g of the second step obtained graphite phase carbon nitride-boric acid precursor was weighed into a covered alumina crucible, and calcined in a tube furnace, with a heating rate of 3.3 ℃ / min, a reaction temperature of 800 ℃, and a reaction time of 1 h, to obtain white hydroxyl boron nitride powder.

[0093] Step 1.4, 200 mg of the third step obtained hydroxyl boron nitride powder was weighed, and 10 mg of iron dichloride tetrahydrate was weighed, and the two were mixed and ground in a mortar, and then spread in a covered alumina crucible, and calcined in a nitrogen gas tube furnace, with a heating rate of 6 ℃ / min, a reaction temperature of 900 ℃, and a reaction time of 1 h, to obtain iron-loaded boron nitride nanoscale enzyme, referred to as Fe-BN nanoscale enzyme, wherein the Fe loading amount is 0.87 wt%.

[0094] The Fe-BN nanoscale enzyme prepared in Example 1 was subjected to objective spherical aberration correction transmission electron microscopy, particle size distribution statistics, and condenser spherical aberration correction transmission electron microscopy analysis, and the results are shown in Figures 1 to 3 , wherein, Figure 1 is the objective spherical aberration correction transmission electron microscopy image of the Fe-BN nanoscale enzyme prepared in Example 1, Figure 2 is the particle size distribution graph of the Fe-BN nanoscale enzyme prepared in Example 1, Figure 3 is the condenser spherical aberration correction transmission electron microscopy analysis graph of the Fe-BN nanoscale enzyme prepared in Example 1. From Figures 1 to 3 it can be seen that the Fe-BN is uniformly dispersed, with an average particle size of 7.5 nm, and Fe is dispersed in the form of a single atom on the boron nitride carrier.

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

[0096] Examples 2-4

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

[0098] The Fe-BN nanoscale enzymes prepared in Examples 1-4 were subjected to characterization analysis, and the results are shown in Figures 5 to 7 , wherein, Figure 5The infrared spectra of Fe-BN nanoscale enzymes prepared in Examples 1 to 4 are shown in the figure, wherein curve I represents Fe-BN calcined at 600℃, curve II represents Fe-BN calcined at 700℃, curve III represents Fe-BN calcined at 800℃, and curve IV represents Fe-BN calcined at 900℃; Figure 6 The XRD patterns of Fe-BN nanoscale enzymes prepared in Examples 1 to 4 (before dialysis) are shown in the figure, wherein 600 represents a calcination temperature of 600℃, 700 represents a calcination temperature of 700℃, 800 represents a calcination temperature of 800℃, and 900 represents a calcination temperature of 900℃; Figure 7 The XRD patterns of Fe-BN nanoscale enzymes prepared in Examples 1 to 4 (after dialysis) are shown in the figure, wherein 600 represents a calcination temperature of 600℃, 700 represents a calcination temperature of 700℃, 800 represents a calcination temperature of 800℃, and 900 represents a calcination temperature of 900℃; Figures 5 to 7 It can be seen that the product framework is BN.

[0099] Examples 5-6

[0100] Preparation of boron nitride nanoscale enzymes with different iron loadings

[0101] Example 5

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

[0103] Example 6

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

[0105] It should be noted that the iron loadings of Example 1, Example 5 and Example 6 are all based on the total mass of the Fe-BN nanoscale enzyme being 100%.

[0106] Example 7

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

[0108] Example 7

[0109] The same as example 1, except that the calcination temperature in step 1.4 in the tube furnace is not the same, the calcination temperature in example 1 is 900℃, and the calcination temperature in example 7 is 1000℃, and the average particle size of the prepared Fe-BN is 7.6nm.

[0110] The average particle size of the Fe-BN prepared when the calcination temperature in example 1 is 900℃ is 7.5nm, the average particle size of the Fe-BN prepared when the calcination temperature in example 4 is 800℃ is 15.2nm, and the average particle size of the Fe-BN prepared when the calcination temperature in example 7 is 1000℃ is 7.6nm. Comparing the experimental results of example 1, example 4 and example 7, it can be seen that when the calcination temperature is 800-1000℃, the average particle size of the Fe-BN nanometer enzyme prepared is less than 25nm, and when the calcination temperature is 900-1000℃, the average particle size of the Fe-BN prepared is less than 10nm; at the same time, when the calcination temperature increases from 900℃ to 1000℃, the average particle size of the Fe-BN nanometer enzyme is not much different. Considering the cost of calcination, the optimal calcination temperature is 900℃.

[0111] Example 8-Example 9

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

[0113] Example 8

[0114] The same as example 1, except that the calcination time in step 1.4 in the tube furnace is not the same, the calcination time in example 1 is 1h, and the calcination time in example 8 is 0.1h, and the average particle size of the prepared Fe-BN is 18.5nm.

[0115] Example 9

[0116] The same as example 1, except that the calcination time in step 1.4 in the tube furnace is not the same, the calcination time in example 1 is 1h, and the calcination time in example 9 is 2h, and the average particle size of the prepared Fe-BN is 7.3nm.

[0117] Comparing the experimental data of example 1, example 8 and example 9, it can be seen that the calcination time is related to the average particle size of Fe-BN. When the calcination time is less than 1h, the average particle size of the prepared Fe-BN becomes smaller as the calcination time increases. When the calcination time is greater than 1h, the average particle size of the prepared Fe-BN is not much related to the calcination time, indicating that the optimal calcination time is 1h.

[0118] Example 10

[0119] Preparation of transdermal drug delivery preparation

[0120] Step 2.1, providing the iron-loaded boron nitride nanosensor prepared in Example 1.

[0121] Step 2.2, weighing 20 mg of Fe-BN nanosensor into 2 mL of water, ultrasonicating for 30 min to make it uniformly dispersed, then weighing 2 mg of pararosaniline hydrochloride (BF) into it, and magnetically stirring at 250 rpm for 1 h to obtain a Fe-BN / BF solution.

[0122] Step 2.3, adding 20 mg of polyethyleneimine into the Fe-BN / BF solution obtained in Step 2.2, and magnetically stirring at 250 rpm for 2 h to obtain a Fe-BN / BF / polyethyleneimine solution.

[0123] Step 2.4, dissolving 40 mg of polyglutamic acid and 80 mg of protamine in 1 mL of water, ultrasonicating for 30 min, and magnetically stirring for 30 min to obtain a mixed solution.

[0124] Step 2.5, adding the mixed solution obtained in Step 2.4 into the Fe-BN / BF / polyethyleneimine solution, and magnetically stirring at 250 rpm for 2 h to obtain a transdermal drug delivery preparation.

[0125] The transdermal drug delivery preparation prepared in Example 10 was applied to the tumor skin of tumor-bearing mice, and the ex vivo tissues of mice at different time points were subjected to fluorescence imaging analysis, and the results are shown in Figure 8 From the above results, it can be seen that the Fe-BN nanosensor can effectively penetrate the skin to reach the melanoma lesion site. Figure 8

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

[0127] Examples 11-25

[0128] Investigation of the effect of different transdermal enhancers on skin penetration

[0129] The active agent used in Examples 11-25 is the Fe-BN nanosensor prepared in Example 1, and the main difference is that the transdermal enhancers are not completely the same. The mass of the transdermal enhancer added in each example is shown in Table 1.​

[0130] Table 1 Influence of transdermal enhancer of different formulations on skin permeability

[0131]

[0132]

[0133] Examples 26-31 and Comparative Examples 1 and 2

[0134] Influence of Fe-BN nanoszyme with different iron loadings and different particle sizes on skin permeability was investigated

[0135] The transdermal enhancer used in Examples 26-31 is exactly the same as that in Example 10 (2 mg of pararosaniline hydrochloride, 20 mg of polyethyleneimine, 40 mg of polyglutamic acid, and 80 mg of protamine), and the active agent used is the Fe-BN nanoszyme prepared in Examples 4-9, and the iron loading and average particle size of the Fe-BN nanoszyme added in each example are shown in Table 2.

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

[0137] Table 2 Influence of Fe-BN nanoszyme with different iron loadings and different particle sizes on skin permeability

[0138]

[0139]

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

[0141] It should be noted that in the present application, the method for evaluating skin permeability is specifically: using a fluorescent substance (such as a fluorescent dye or a fluorescently labeled nanomaterial) as a tracer, the technology for tracking the distribution, migration and metabolic process of the target substance in vivo or in vitro by detecting the fluorescent signal emitted thereby. The basic steps of the method are as follows: first, in an aqueous solution, the Fe-BN nanomaterial is stirred and mixed with rhodamine B dye for 6 hours to ensure that the two are fully combined. Subsequently, the uncombined rhodamine B is removed by centrifugal treatment, and the precipitate is redispersed in deionized water. Next, the transdermal penetration enhancer is added to the dispersion and stirred overnight at room temperature to ensure uniform mixing. Next, take an appropriate amount of rhodamine B labeled Fe-BN nanoscale enzyme transdermal preparation solution and evenly apply it to the specific skin site of the tumor-bearing mouse. After application, keep for a certain period of time (such as 3-12 hours), and wash off the surface adhering drug with water. Subsequently, the mouse is dissected, and the drug-treated skin, subcutaneous tissue (tumor or muscle) is removed, and analyzed by a small animal fluorescence imaging system. By observing the fluorescence signal intensity appearing in the subcutaneous tissue, the skin permeability of the Fe-BN nanoscale enzyme is determined. A strong fluorescence signal indicates good skin permeability, and vice versa.

[0142] The above is a further detailed description of the present application in combination with specific preferred embodiments, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the concept of the present application, a number of simple deductions and substitutions can be made, which should be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing an iron-loaded boron nitride nanoszyme, characterized in that, The average particle size of the iron-loaded boron nitride nanoscale enzyme is less than 25 nm, and the preparation method comprises the following steps: Step 1, a thermal polycondensation reaction of dicyandiamide is carried out to synthesize graphite phase carbon nitride; Step 2, graphite phase carbon nitride and boric acid are mixed in water as a solvent, and then the mixture is dehydrated and ground to obtain a powder of graphite phase carbon nitride-boric acid precursor; Step 3, the graphite phase carbon nitride-boric acid precursor is first subjected to a first calcination treatment to obtain hydroxyl boron nitride, and then the hydroxyl boron nitride and an iron salt are uniformly mixed and subjected to a second calcination treatment under a nitrogen atmosphere to obtain the iron-loaded boron nitride nanoscale enzyme, wherein the second calcination treatment is carried out at a calcination temperature of 600-1000℃ for 0.1h-2h.

2. The method for preparing iron-loaded boron nitride nanozymes according to claim 1, characterized in that, The thermal polycondensation reaction conditions in step 1 include a reaction temperature of 300-600℃ and a reaction time of at least 1h; and / or, The first calcination treatment is carried out at a calcination temperature of 700-1000℃ for 0.1h-3h.

3. The method of claim 1 or 2, wherein the method is characterized by, The mass ratio of the graphite phase carbon nitride to the boric acid is 1:(0.1-0.5); and / or, The loading amount of iron is 0.2wt%-2wt% based on 100wt% of the mass of the iron-loaded boron nitride nanoscale enzyme.

4. An iron-loaded boron nitride nanoszyme drug, characterized in that, The iron-loaded boron nitride nanoscale enzyme drug is prepared by the preparation method of any one of claims 1-3.

5. Use of the iron-loaded boron nitride nanoscale enzyme prepared by the preparation method of any one of claims 1-3 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 for coating the active agent, and the active agent is the iron-loaded boron nitride nanoscale enzyme drug of claim 4.

7. The transdermal drug delivery preparation according to claim 6, wherein The mass percentage of the active agent is 1.0wt%-20.0wt% and the mass percentage of the transdermal enhancer is 80.0wt%-99.0wt% based on 100wt% of the total weight of the solutes in the transdermal drug delivery preparation.

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

9. The transdermal drug delivery preparation according to claim 8, wherein The transdermal enhancer further comprises polyglutamic acid and protamine, the molecular weight of the polyglutamic acid ranges from 1 to 100kDa, and the molecular weight of the protamine ranges from 4 to 10kDa, and the mass ratio of the sum of the active agent and the pararosaniline hydrochloride to the polyglutamic acid and protamine is 1:(2-10):(4-20).

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

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