Sulforaphanin-chitosan nanoparticle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to sulforaphane-chitosan nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: by adopting an ionic cross-linking method, taking chitosan as a carrier, dropwise adding an anionic cross-linking agent with negative electricity, and carrying out electrostatic interaction, so that the sulforaphane is loaded on the chitosan to form the nanoparticles. The sulforaphane-chitosan nanoparticles prepared by the preparation method disclosed by the invention are uniform in particle size distribution, good in stability, high in drug loading capacity and good in encapsulation effect; the preparation process is simple and feasible, the repeatability is high, and the bioavailability of the insoluble medicine is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a sulforaphane-chitosan nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Sulforaphane (SFN) is D,L-1-isothiocyanato-4-(methylsulfinyl) butane, which is a secondary metabolite after hydrolysis of glucosinolates contained in cruciferous plants. SFN has strong antioxidant, anti-inflammatory, anticancer, and neuron protection activities. It has a potential auxiliary therapeutic effect on diseases such as schizophrenia.

[0003] SFN, like most natural compounds, has poor stability and short half-life, which limits its actual clinical application. The main purpose of the present application is to develop a nano preparation of SFN and promote the popularization and application of SFN.

[0004] At present, the transdermal drug delivery system of sulforaphane includes loading sulforaphane into novel drug delivery systems such as nanoemulsion, microemulsion, liposome or nanoparticle. Ethosomes, liposomes and nanoparticles are some common nano dosage forms. Different nano carrier systems can provide different drug release and delivery characteristics, and thus affect their performance in vivo. In early studies, sulforaphane was used for anticancer, antioxidant and other studies due to its significant biological activity. As a natural biodegradable material, chitosan has become a popular material in drug delivery systems due to its biocompatibility, biodegradability and non-toxicity. The initial research mainly focused on the pharmacology of sulforaphane, such as its antioxidant, anticancer and other functions. With the deepening of the understanding of nano drug delivery systems, the combination of sulforaphane and chitosan has begun to rise, mainly focusing on the improvement of the preparation technology of sulforaphane nanoparticles, such as copolymer technology, solvent evaporation method, nanoprecipitation method, etc., aiming to improve the encapsulation efficiency and release control ability of sulforaphane. It also focuses on the evaluation of biocompatibility and biodegradability. The biodegradability and biocompatibility of chitosan nanoparticles have been evaluated in many aspects to ensure their safe release in vivo and reduce toxic side effects. At present, the research of sulforaphane-chitosan nanoparticles mainly focuses on targeted drug delivery and antioxidant and antibacterial. By loading sulforaphane on chitosan nanoparticles, tumor-targeted delivery can be achieved to enhance the anticancer effect of drugs. Chitosan nanoparticles have good stability and biocompatibility, and can be used to prepare antioxidant and antibacterial materials containing sulforaphane, and are widely used in skin repair, food preservation and other fields.

[0005] Although some progress has been made in the study of sulforaphane-chitosan nanoparticles, there are still some challenges and problems: 1. Complexity of preparation method: Although there are currently various preparation methods, how to ensure the consistency, stability and effective drug loading of the particles in large-scale production is still a challenge. 2. Control of drug release: Sulforaphane has poor solubility, how to ensure its stable release in the body and improve its bioavailability still needs further optimization. 3. Long-term safety evaluation: Although chitosan has good biocompatibility and biodegradability, the safety evaluation of long-term application in the human body is still insufficient, and more preclinical studies and clinical trials are needed. 4. Individual differences in treatment effect: Due to the strong biological activity of sulforaphane, there may be differences in its absorption and metabolism among different individuals.

[0006] Therefore, at the present stage, the combination of the two needs to be further studied to achieve better results and to realize large-scale production. SUMMARY

[0007] The purpose of the present application is to provide a sulforaphane-chitosan nanoparticle and its preparation method and application.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0009] A preparation method of sulforaphane-chitosan nanoparticles, using ion crosslinking method, taking chitosan as carrier, by adding negative anion crosslinking agent dropwise, electrostatic interaction occurs, sulforaphane is loaded on chitosan to form nanoparticles.

[0010] The mass ratio of chitosan, negative anion crosslinking agent and sulforaphane is (1-5):1, (1-5):1 respectively; wherein the negative anion crosslinking agent is one or several of sodium hexametaphosphate, sodium tripolyphosphate and sodium dihydrogen phosphate.

[0011] Specifically:

[0012] (1) Dissolve chitosan powder in 1% acetic acid aqueous solution to prepare an acidic chitosan solution, and adjust the pH of the solution to 4.5-6.0, filter and wait for use;

[0013] (2) Take sulforaphane and add it to the chitosan acetic acid solution of step (1), so that the mass ratio of sulforaphane to chitosan acetic acid solution in the final solution is 1:(1-5);

[0014] (3) Under room temperature and magnetic stirring, sodium tripolyphosphate is added dropwise into the mixed solution of step (2) at a speed of 1d / s;

[0015] (4) The mixed solution of step (3) is dispersed with an ultrasonic cell disruptor to prepare nanoparticles.

[0016] The deacetylation degree of the step (1) chitosan powder is greater than or equal to 95%;

[0017] The concentration of the step (1) acidic chitosan solution is 1.4-1.6 mg·mL -1 ;

[0018] The step (3) is magnetic stirring at room temperature for 14-16 min.

[0019] The step (4) is ultrasonic treatment at an ultrasonic power of 90-110 W for 9-11 min.

[0020] The radish sulforaphane-chitosan nanoparticles obtained according to the preparation method are in a regular spherical shape, have a particle size of 180-220 nm, and have good particle size uniformity.

[0021] The radish sulforaphane-chitosan nanoparticles are applied as a nanodrug delivery system.

[0022] The radish sulforaphane-chitosan nanoparticles are applied in the preparation of functional cosmetics.

[0023] The radish sulforaphane-chitosan nanoparticles have the following advantages:

[0024] The process for preparing the radish sulforaphane-chitosan nanoparticles has the advantages of mild crosslinking conditions, good biocompatibility, simple and efficient operation, adjustable size and surface properties, good stability, degradability, low cost, environmental protection, and the like, and realizes the advantages of a nanodrug delivery system, specifically:

[0025] (1) The radish sulforaphane-chitosan nanoparticles use chitosan as a carrier and use a negatively charged anion as a crosslinking agent to prepare drug-loaded nanoparticles by an ionic crosslinking method.

[0026] (2) The prepared nanoparticles have uniform particle size distribution, good stability, high drug loading, and good encapsulation effect, and greatly improve the bioavailability of unstable drugs.

[0027] (3) The radish sulforaphane-chitosan nanoparticles have a long drug release time, uniform drug release, and good sustained release performance. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The single-factor ultrasonic time of the chitosan nanoparticles provided for the embodiments of the present application is investigated.

[0029] Figure 2 The single-factor ultrasonic power of the chitosan nanoparticles provided for the embodiments of the present application is investigated.

[0030] Figure 3 The single-factor stirring time of the chitosan nanoparticles provided by the embodiment of the present application is investigated.

[0031] Figure 4 The single-factor pH of the chitosan nanoparticles provided by the embodiment of the present application is investigated.

[0032] Figure 5 The single-factor chitosan concentration of the chitosan nanoparticles provided by the embodiment of the present application is investigated.

[0033] Figure 6 The single-factor mass ratio of chitosan to sulforaphane of the chitosan nanoparticles provided by the embodiment of the present application is investigated.

[0034] Figure 7 The particle size diagram and Zeta potential diagram of the chitosan nanoparticles provided by the embodiment of the present application are measured by a Malvern particle size analyzer.

[0035] Figure 8 The transmission electron microscope diagram of the chitosan nanoparticles provided by the embodiment of the present application is provided.

[0036] Figure 9 The stability of the chitosan nanoparticles, liposomes and ethosomes loaded with sulforaphane provided by the embodiment of the present application is investigated.

[0037] Figure 10 The appearance photos of the chitosan nanoparticles prepared by different negatively charged anion exchangers provided by the embodiment of the present application are provided.

[0038] Figure 11 The observation diagram of the chitosan nanoparticles treating the mouse model of irritant contact dermatitis provided by the embodiment of the present application is provided.

[0039] Figure 12 The spleen effect diagram of the chitosan nanoparticles treating the mouse model of irritant contact dermatitis provided by the embodiment of the present application is provided.

[0040] Figure 13 The H&E staining skin section diagram of the chitosan nanoparticles treating the mouse model of irritant contact dermatitis provided by the embodiment of the present application is provided.

[0041] Figure 14 The Masson staining skin section diagram of the chitosan nanoparticles treating the mouse model of irritant contact dermatitis provided by the embodiment of the present application is provided. DETAILED DESCRIPTION

[0042] The present application is further illustrated by the following examples, but the present application is not limited in the scope of the examples.

[0043] The application takes chitosan as a carrier, loads radish sulfur to prepare chitosan nanoparticles, so as to provide sustained drug release, improve the stability of radish sulfur, improve the bioavailability of the drug, and provide an experimental basis for further research on radish sulfur transdermal drug preparation.

[0044] Example 1

[0045] Precisely take chitosan (CS) and dissolve it in a 1% acetic acid solution to obtain a chitosan solution, and the final concentration of chitosan in the solution is 1.5 mg·mL -1 Obtain the acetic acid solution of CS, and slowly add 1 mol·L -1 of NaOH solution to the acetic acid solution of CS at a slow drop rate until the pH of the solution is 5, and then add it to the glass container containing radish sulfur to make the mass ratio of CS / SFN 5:1. Then, drop by drop, add a 1 mg·mL -1 aqueous solution of sodium tripolyphosphate (TPP) to the system to make the mass ratio of CS / TPP 5:1, and then stir at room temperature for 15 min, place it in a test tube, and change the ultrasonic time: 5 min, 10 min, 15 min, 20 min, and 25 min, respectively, to obtain SFN chitosan nanoparticle solutions. Measure the particle size and calculate the encapsulation efficiency (see Figure 1 ). The results are shown in Figure 1 , with the increase of the ultrasonic time, the particle size shows a trend of first decreasing and then slowly increasing, and the extension of the ultrasonic time helps to reduce the particle size of the chitosan nanoparticles. Ultrasonic waves can cause local high temperature and high pressure through cavitation effect, which can promote large particles to break into smaller particles. However, too long ultrasonic time may lead to excessive particle aggregation, which may increase the particle size. Appropriate ultrasonic time can improve the dispersibility of chitosan nanoparticles, promote uniform dispersion, and prevent particle aggregation. Too short ultrasonic time may lead to incomplete dispersion of particles, and too long ultrasonic time may lead to particle breakage. The overall particle size in the figure is not very obvious, and the encapsulation efficiency shows a trend of first increasing and then decreasing. Therefore, the ultrasonic time is selected as 10 min.

[0046] Example 2

[0047] Precisely take chitosan (CS) and dissolve it in a 1% acetic acid solution to obtain a chitosan solution, and the final concentration of chitosan in the solution is 1.5 mg·mL -1 , and slowly add 1 mol·L -1 of NaOH solution to the acetic acid solution of CS at a slow drop rate until the pH of the solution is 5, and then add it to the glass container containing radish sulfur to make the mass ratio of CS / SFN 5:1. Then, drop by drop, add a 1 mg·mL -1The aqueous solution of sodium tripolyphosphate (TPP) was added to the CS solution to obtain a CS / TPP mass ratio of 5:1, and then stirred at room temperature for 15 min. The solution was placed in a flask and subjected to ultrasonic treatment at a power of 100 W, 150 W, or 200 W for 10 min to obtain SFN chitosan nanoparticle solutions. The particle size was measured and the encapsulation efficiency was calculated (see Figure 2 ). The results are shown in Figure 2 The ultrasonic power had little effect on the particle size and encapsulation efficiency. Ultrasonic waves can break up large particles and promote the formation of nanoparticles. When the ultrasonic power is high, the particle size can become more uniform. Under the action of ultrasonic waves, the bubbles in the liquid will rapidly expand and collapse, generating local high temperature and high pressure, thereby improving the dispersibility and uniformity of the particles. However, too high an ultrasonic power can cause the particles to aggregate, which is not conducive to the stability of the nanoparticles. To reduce energy consumption and equipment wear, the ultrasonic power was selected to be 100 W.

[0048] Example 3

[0049] The CS was precisely weighed and dissolved in a 1% acetic acid solution to obtain a CS acetic acid solution. The final concentration of chitosan in the solution was 1.5 mg·mL -1 A 1 mol·L -1 solution of NaOH was slowly added to the CS acetic acid solution at a slow drop rate until the pH of the solution was 5, and then added to a glass container containing sulforaphane to obtain a CS / SFN mass ratio of 5:1. A 1 mg·mL -1 The aqueous solution of sodium tripolyphosphate (TPP) was added to the CS solution to obtain a CS / TPP mass ratio of 5:1, and then stirred at room temperature for 15 min. The solution was placed in a flask and subjected to ultrasonic treatment at a power of 100 W, 150 W, or 200 W for 10 min to obtain SFN chitosan nanoparticle solutions. The particle size was measured and the encapsulation efficiency was calculated (see Figure 3 ). The results are shown in Figure 3 The stirring time affected the uniform dispersion and embedding of the drug molecules. Prolonged stirring time can sometimes affect the surface properties of the nanoparticles, such as changes in hydrophilicity or surface charge, which have important effects on drug loading efficiency and biocompatibility. Sufficient stirring can help improve the stability of chitosan nanoparticles and prevent the aggregation or precipitation of nanoparticles. Since the stirring time had little effect on the particle size and encapsulation efficiency, although sufficient stirring was beneficial to the formation of nanoparticles, prolonged stirring time could damage the cross-linking of chitosan and sodium tripolyphosphate. Therefore, the stirring time was selected to be 15 min.

[0050] Example 4

[0051] The CS was precisely weighed and dissolved in a 1% acetic acid solution to obtain a CS acetic acid solution. The final concentration of chitosan in the solution was 1.5 mg·mL-1 1 mol·L -1 of NaOH solution was slowly added to the acetic acid solution of CS at a slow drop rate to change the pH of the CS solution: 3, 4, 5, 6, and then added to a glass container containing sulforaphane to make the CS / SFN mass ratio 5:1. 1 mg·mL -1 of an aqueous solution of sodium tripolyphosphate (TPP) was added dropwise to the system to make the CS / TPP mass ratio 5:1, stirred at room temperature for 15 min, placed in a test tube, and subjected to ultrasonic treatment at an ultrasonic power of 100 W for 10 min to obtain SFN-chitosan nanoparticle solutions. The particle size was measured and the encapsulation rate was calculated (see Figure 4 ). The results are shown in Figure 4 With the increase of the pH value, the particle size gradually increased, while the encapsulation rate first increased and then decreased. When the pH was 5, the encapsulation rate reached the maximum. According to Coulomb's law, it can be judged that the attraction between the particles is the strongest when the pH is 5, which is most conducive to the formation and stability of the nanoparticles and the ionic crosslinking between sulforaphane and chitosan. Therefore, the pH value is selected as 5.

[0052] Example 5

[0053] CS was precisely weighed and dissolved in an acetic acid solution with a concentration of 1% to obtain acetic acid solutions containing different concentrations of CS, and the final concentration of chitosan in the solution was changed: 0.5, 1.0, 1.5, 2.0, 2.5 mg·mL -1 1 mol·L -1 of NaOH solution was slowly added to the acetic acid solution of CS at a slow drop rate until the pH of the solution was 5, and then added to a glass container containing sulforaphane to make the CS / SFN mass ratio 5:1. 1 mg·mL -1 of an aqueous solution of sodium tripolyphosphate (TPP) was added dropwise to the system to make the CS / TPP mass ratio 5:1, stirred at room temperature for 15 min, placed in a test tube, and subjected to ultrasonic treatment at an ultrasonic power of 100 W for 10 min to obtain SFN-chitosan nanoparticle solutions. The particle size was measured and the encapsulation rate was calculated (see Figure 5 ). The results are shown in Figure 5 When the final concentration of chitosan increased, the particle size of the nanoparticles also increased with the increase of the concentration of chitosan. The reason for this phenomenon is that when the concentration of chitosan increases, the polymer of chitosan that does not participate in the reaction in the solution also gradually increases. Therefore, it can be concluded that the increase of the particle size is caused by the chitosan polymer participating in the reaction. With the increase of the concentration of chitosan, the encapsulation rate of the drug first increased and then decreased, and the maximum encapsulation rate was obtained when the concentration of chitosan was 1.5 mg·mL -1This is likely to be the CS concentration increases will increase its cross-linking with sodium tripolyphosphate, but the CS concentration increases to a certain extent will gather to destroy the nano structure, thus making the drug leakage, so that the encapsulation rate is reduced. Therefore, the concentration of chitosan is 1.5mg·mL -1 .

[0054] Example 6

[0055] Precisely weigh CS dissolved in a 1% acetic acid solution to obtain a CS acetic acid solution, the final concentration of chitosan in the solution is 1.5mg·mL -1 , 1mol·L -1 of NaOH solution is slowly added to the CS acetic acid solution at a slow drop rate until the pH of the solution is 5, and then added to the glass container containing SFN to make the CS / SFN mass ratio: 6:1, 5:1, 4:1, 3:1. Then 1mg·mL -1 of an aqueous solution of sodium tripolyphosphate (TPP) is added dropwise to the system to make the CS / TPP mass ratio 5:1, stirred at room temperature for 15min, placed in a test tube, and ultrasonic power is 100W, ultrasonic time is 10min, respectively, to obtain SFN chitosan nanoparticle solution. Measure the particle size and calculate the encapsulation rate (see Figure 6 ). The results are shown in Figure 6 , when the mass ratio increases, the drug encapsulation rate of SFN chitosan nanoparticles changes from high to low, and when the mass ratio of chitosan and SFN reaches 5:1, the encapsulation rate of SFN reaches the maximum. Because when the mass ratio of chitosan and SFN is 5:1, the maximum limit of chitosan-TPP nanoparticles for loading drugs is reached. If the amount of SFN is increased after the ratio reaches 5:1, the encapsulation rate will not increase, but will continue to decrease. The reason for this phenomenon is that the remaining drug molecules in the solution are not wrapped and loaded by the chitosan nanoparticle carrier, so that the remaining SFN in water continues to aggregate. The aggregated SFN drug molecules will destroy the stability of the entire reaction system, resulting in a decrease in drug loading rate and encapsulation rate. When the mass ratio increases, the particle size changes little. When the SFN content is too large, the excessive SFN that is not encapsulated in the chitosan nanoparticles will reduce the utilization rate of SFN. Therefore, the mass ratio of chitosan and SFN is 5:1.

[0056] Example 7

[0057] Precisely weigh CS dissolved in a 1% acetic acid solution to obtain a CS acetic acid solution, the final concentration of chitosan in the solution is 1.5mg·mL -1 , 1mol·L -1NaOH solution was added to the acetic acid solution of CS at a slow drop rate until the pH of the solution was 5, and then added to a glass container containing sulforaphane to give a CS / SFN mass ratio of 5:1. 1 mg·mL -1 A sodium tripolyphosphate (TPP) aqueous solution was added to the CS / TPP mass ratio of 5:1, and then stirred at room temperature for 15 min, placed in a test tube, and subjected to ultrasonic treatment at an ultrasonic power of 100 W for 10 min to obtain a SFN-chitosan nanoparticle solution (see Figure 7 ). The results are shown in Figure 7 Under the optimal process conditions, the nanoparticles prepared were regular circular particles with a uniform particle size distribution of about 200 nm, a Zeta potential of about 24.6 mV, and were relatively stable. The optimal encapsulation efficiency of the loaded sulforaphane was 93.16%, and the optimal drug loading was 16.72%.

[0058] Then, the SFN-chitosan nanoparticles obtained above were analyzed by transmission electron microscopy (TEM) for their morphology. An appropriate amount of SFN-chitosan nanoparticle (SFN-CSNPs) solution was diluted with pure water to 50 μg / mL, mixed uniformly after dilution, and then aspirated with a pipette gun and dropped on a carbon film copper mesh. 1-2 drops of 2% phosphotungstic acid were used for negative staining, and then air-dried. The structure and morphological distribution of the SFN-CSNPs were observed by transmission electron microscopy. The results are shown in Figure 8 The SFN-CSNPs prepared had a particle size of about 100 nm, and their morphology was spherical vesicles. There was a small amount of aggregation between the nanoparticles.

[0059] Comparative Example 1

[0060] SFN-BEs were prepared by the ethanol injection method. 104 mg of soybean lecithin and 8 mg of sulforaphane were precisely weighed to give a soybean lecithin to sulforaphane mass ratio of 13:1. 3 ml of ethanol and 0.6 ml of propylene glycol were precisely weighed to give a volume ratio of ethanol to propylene glycol of 5:1, and dissolved in a 25 mL flask as an organic phase. The ratio between the mixture and the organic solvent was 280:9. The mixture was placed in a water bath under the condition of 30±1℃ and 700 rpm magnetic stirring. 15 mg of poloxamer 188 was precisely weighed and dissolved in 1.8 ml of distilled water as an aqueous phase, which was preheated to 30±1℃. The aqueous phase was slowly injected into the organic phase at a constant rate (1.2 ml / min) to give a ratio of 2:1 between the organic phase and the aqueous phase. The temperature was maintained at 30±1℃ during the experiment. After stirring for 10 min, the SFN-BEs ethosomes with uniform particle size were obtained by passing through a 0.22 μm microporous organic filter membrane, and the obtained SFN-BEs were sealed and stored in a 4℃ refrigerator for further characterization.

[0061] SFN-Lips was prepared by thin film dispersion method. 4mg of sulforaphane in acetonitrile with a concentration of 0.8mg / ml was placed in a 25ml flask, and the organic solvent was removed under reduced pressure at 35℃. 24mg of hydrogenated soybean lecithin and 2mg of cholesterol were precisely weighed into a 25ml flask, and the mass ratio of hydrogenated soybean lecithin to cholesterol was 12:1. 5ml of chloroform was added, and the mixture was dissolved by ultrasonic or heating stirring. The organic solvent was removed under reduced pressure at 35℃ to obtain a uniform dry lipid film. The flask was removed and placed in a water bath. The water bath temperature was 40 degrees, the time was 15 minutes, and 5ml of pure water was added. The temperature was kept constant at 40 degrees during the experiment, and the stirring speed was 1200rpm. Continue stirring for 15 minutes, then take out and ice water bath ultrasonic for 10 minutes, ultrasonic power is 340W. 0.22μm microporous organic filter membrane was used to obtain SFN-Lips with uniform particle size. The obtained SFN-Lips was sealed and stored in a 4℃ refrigerator for further characterization.

[0062] The particle size and pdi of the alcoholosomes, liposomes and nanoparticles obtained in Example 7 above were measured by Malvern particle size analyzer (see Figure 9 ).

[0063] The results are shown in Figure 9 , the particle size of the chitosan nanoparticles changed less with time, and the pdi of the particle size was also smaller. It can be seen that the nanoparticles loaded with sulforaphane are more stable than the alcoholosomes and liposomes.

[0064] Comparative Example 2

[0065] CS was precisely weighed and dissolved in a 1% acetic acid solution to obtain a CS acetic acid solution. The final concentration of chitosan in the solution was 1.5mg·mL -1 A 1mol·L -1 of NaOH solution was slowly added to the CS acetic acid solution at a slow drop rate until the pH of the solution was 5, and then added to the glass container containing sulforaphane to make the mass ratio of CS / SFN 5:1. 1mg·mL -1 of a sodium alginate (SA) aqueous solution was added dropwise to the system to make the mass ratio of CS / SA 5:1, and then stirred at room temperature for 15 minutes. The SFN-CSNPs obtained were sealed and stored in a 4℃ refrigerator for further characterization.

[0066] The SFN-CSNPs obtained in the comparative example were characterized and observed as described in Example 7 above. The results are shown in Figure 10, using an anion exchanger sodium tripolyphosphate to prepare chitosan nanoparticles, the appearance showed as blue translucent liquid, slightly pale blue opalescence, using an anion exchanger sodium alginate to prepare chitosan nanoparticles, the appearance showed as white precipitate, and the crosslinking time was long, and a large amount of by-products were produced. When using sodium tripolyphosphate, usually, too many by-products were not generated as sodium alginate, sodium tripolyphosphate as a crosslinking agent had the characteristics of strong, efficient, and widely applicable, usually, this method did not need complex equipment or expensive raw materials, the preparation process was simple and the cost was low, and it was suitable for large-scale production. Therefore, sodium tripolyphosphate was selected as an anion exchanger.

[0067] Example 8

[0068] In order to explore the anti-inflammatory effect of SFN-loaded chitosan nanoparticles, a mouse model of irritant contact dermatitis was established by using a mixture of dinitrochlorobenzene and acetone / olive oil. In this study, 20 BALB / c mice were randomly divided into 5 groups, 5 mice in each group, namely ① blank control group, ② ICD disease model group (only modeling without treatment), ③ dexamethasone+ICD group, ④ SFN+ICD group, and ⑤ SFN-CSNPs+ICD group. The anti-inflammatory effect of SFN-CSNPs and the effect on the stability of sulforaphane were evaluated.

[0069] Results are shown in Figure 11 , except for the normal control group, before starting the experiment, the exposed skin with a diameter of 2 cm was prepared by completely removing the animal's back hair. Then, on days 0, 2, 4, and 6, 50 μL of a mixture of 0.5% DNCB and acetone / olive oil (4:1) was applied to the shaved area. On days 8 and 10 of modeling, 25 μL of a mixture of 0.25% DNCB and acetone / olive oil (4:1) was applied to the back of the mouse. After each treatment, the animals were kept outside the cage for a few minutes to dry the solution. The mice were sacrificed on day 17, and samples were collected. The mice in each administration group were coated with SFN-CSNPs and dexamethasone on the back of the mouse on day 8, and the drug-loaded microneedle and the non-drug-loaded microneedle were pressed on the back of the mouse, and the medical tape was fixed for 10 minutes. The whole experiment lasted for 10 days. The mice were sacrificed on day 17, and the skin wound condition of the mice was recorded after each application for subsequent evaluation. Compared with the model group, the skin score of the SFN-CSNPs group of mice was the lowest, and the skin had only redness, no scaling, and no erosion. The decrease in body weight was not obvious. The pharmacodynamics experiment showed that the SFN-CSNPs group had the smallest skin damage and the lightest epidermal inflammation in the case of contact with the stimulating chemical. The hydrogel microneedle combined with nanotechnology strategy could promote the absorption of drugs, improve the relative bioavailability, prolong the half-life of drugs, and had good in vivo sustained release.

[0070] Example 9

[0071] Splenomegaly is a manifestation of inflammation. After the end of the experiment modeling, the mice were weighed, and the mice were killed by decapitation, and the spleen was taken out by dissection, the size of the spleen was measured with a ruler and its weight was measured. The organ index of the spleen was calculated to judge the severity of inflammation. The larger the spleen, the larger the spleen index, the more severe the inflammation on the back of the mouse.

[0072] As shown in the results Figure 12 , splenomegaly is a manifestation of inflammation. After the end of the experiment modeling, the mice were weighed, and the mice were killed by decapitation, and the spleen was taken out by dissection, the size of the spleen was measured with a ruler and its weight was measured. The organ index of the spleen was calculated to judge the severity of inflammation. The larger the spleen, the larger the spleen index, the more severe the inflammation on the back of the mouse. Spleen index = spleen weight / mouse weight x 100%. Compared with the blank control group, the ICD model group had enlarged spleen, and the spleen index was significantly increased from 0.32 ± 0.08 to 1.37 ± 0.05 (P < 0.001); the spleen index of the SFN-CSNPs group was significantly reduced to 0.56 ± 0.06 (P < 0.05).

[0073] Example 10

[0074] The morphological changes of the skin in the skin lesion area on the back of the mouse with contact dermatitis were observed by hematoxylin-eosin staining (H&E staining) to determine whether there were changes in skin histopathology.

[0075] Specifically, after the mice in the above Example 8 were killed, the skin lesion skin of about 0.5-0.8 cm2in size was cut with a medical scissors, fixed with 4% paraformaldehyde fixing solution, and then the skin lesion tissue was cut into a slice of about 5 μm thick, paraffin-embedded; hematoxylin staining solution for 15 min, eosin staining solution for 1 min, observed under a white light microscope (Nikon Eclipse C1), and the prepared slice was analyzed and evaluated by adjusting the parameters. As shown in the results Figure 13 , the H&E staining results showed that the cells in the normal control group were arranged closely, the skin tissue had a thin epidermis, the cells in the epidermis were round in shape, and the epidermis was closely connected with the dermis layer. The epidermis of the ICD model group and the ointment group was thickened with edema, and the skin damage was obvious. The structure of the basement membrane area (the connection between the epidermis and the dermis layer) was destroyed and flattened, and a small amount of vacuoles could be seen in the cytoplasm of the epidermis. As can be seen from the results of H&E staining of other groups, compared with the model group, the treatment of the SFN-CSNPs group correspondingly reduced the thickness and edema of the epidermis, the keratin layer keratinization was reduced, and the inflammatory reaction was reduced. It shows that SFN has an anti-inflammatory effect on ICD, SFN-CSNPs has good anti-inflammatory function, and has a preventive effect on contact dermatitis.

[0076] Example 11

[0077] The mice obtained in Example 8 above were further subjected to Masson staining, which is also used to observe the histomorphology of the inflammatory mouse back skin lesion area. Paraffin sections were used, and xylene was used for deparaffinization, gradient alcohol was used for rehydration, and then Masson staining kit was used for staining. The pictures were observed and taken by upright microscope.

[0078] The results are shown in Table 1. Figure 14 As shown in Table 1, the Masson staining results show that collagen fibers, mucous, cartilage, and nerve fibers are blue, muscles and elastic fibers are red, and red blood cells are orange-red. Further analysis of the Masson staining results shows that the number of collagen fibers in the skin inflammation site is small, the synthesis of collagen fibers is blocked, the arrangement is disordered and loose, there are many red blood cells, and there are mucous-like substances and necrotic substances. Compared with the normal control group, the red area of the ICD model group increased, and the inflammation was most obvious. It can be seen that the collagen fibrosis of the SFN-CSNPs group is the smallest, so the inflammation is the lightest, and the anti-ICD ability is the strongest.

Claims

1. A method of preparing sulforaphane-chitosan nanoparticles, characterized by: The ion cross-linking method is adopted, and the sulforgin is loaded on the chitosan to form the nanoparticles by adding the negative anion cross-linking agent and the electrostatic interaction.

2. The method for preparing sulforaphane-chitosan nanoparticles according to claim 1, characterized in that: The mass ratio of the chitosan and the negative anion cross-linking agent and the sulforgin is (1-5):1, (1-5):1 respectively; wherein the negative anion cross-linking agent is one or several of sodium hexametaphosphate, sodium tripolyphosphate and sodium dihydrogen phosphate.

3. The preparation method of the sulforgin-chitosan nanoparticles according to claim 1 or 2, characterized in that: (1) the chitosan powder is dissolved in 1% acetic acid aqueous solution to prepare an acidic chitosan solution, and the pH of the solution is adjusted to 4.5-6.0, and the solution is filtered and used; (2) the sulforgin is added into the chitosan acetic acid solution of step (1), so that the mass ratio of the sulforgin and the chitosan acetic acid solution in the final solution is 1:(1-5); (3) under the magnetic stirring at room temperature, the sodium tripolyphosphate is added into the mixed solution of step (2) at a speed of 1d / s; (4) the mixed solution of step (3) is dispersed by the ultrasonic cell disruptor to prepare the nanoparticles.

4. The preparation method of the sulforgin-chitosan nanoparticles according to claim 3, characterized in that: the deacetylation degree of the chitosan powder of step (1) is greater than or equal to 95%; The concentration of the acidic chitosan solution in step (1) is 1.4-1.6 mg·mL -1 .

5. The preparation method of the sulforgin-chitosan nanoparticles according to claim 3, characterized in that: the magnetic stirring in step (3) is performed at room temperature for 14-16 min; the ultrasonic power in step (4) is 90-110 W, and the ultrasonic time is 9-11 min.

6. A sulforaphane-chitosan nanoparticle obtained by the preparation method of claim 1, characterized in that: The sulforgin-chitosan nanoparticles prepared by the method of claim 1 have a regular spherical shape, a particle size of 180-220 nm, and good particle size uniformity.

7. Use of the sulforaphane-chitosan nanoparticle of claim 1, wherein: The application of the sulforgin-chitosan nanoparticles in the nano-drug delivery system.

8. Use of the sulforaphane-chitosan nanoparticle of claim 1, wherein: The application of the sulforgin-chitosan nanoparticles in the preparation of functional cosmetics.

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