Cryptotanshinone manganese dioxide nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses cryptotanshinone manganese dioxide nanoparticles and a preparation method and application thereof, and belongs to the technical field of medicines.The cryptotanshinone manganese dioxide nanoparticles comprise carrier nano-manganese dioxide and cryptotanshinone loaded on the carrier nano-manganese dioxide, the average particle size is 100-300 nm, the drug loading capacity is 5%-20%, and the particle size is 100-300 nm. The preparation method comprises the following steps: dropwise adding an organic solution of cryptotanshinone into a nano-manganese dioxide solution, stirring for 12-24 hours to obtain a mixed solution, dialyzing the mixed solution, dialyzing for 12-24 hours by adopting a lauryl sodium sulfate solution as a dialysis external solution, and freeze-drying to obtain the cryptotanshinone manganese dioxide nanoparticles. On one hand, the problem that the cryptotanshinone is poor in water solubility is solved, and on the other hand, the synergistic effect of the nano-manganese dioxide and the cryptotanshinone in the aspect of acne treatment is exerted.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to cryptotanshinone manganese dioxide nanoparticles and a preparation method and application thereof. Background Art

[0002] Acne is a chronic skin disease involving the sebaceous glands of the hair follicles. Its main pathological feature is the appearance of inflammatory papules with pustules or itching symptoms on the face, back, and chest of patients, which are specifically manifested as blackheads, comedones, papules, erythema, abscesses, etc. Acne is mainly caused by abnormal desquamation in the sebaceous gland follicles that leads to obstruction of the sebaceous gland ducts of the hair follicles, excessive sebum secretion driven by androgen, pro-inflammatory stimulation caused by the proliferation of Propionibacterium acnes, and changes in immune system activity and inflammation.

[0003] Acne is a common disease, with an incidence rate of about 9% in the population. At present, the main treatments for acne include topical medication, systemic medication, phototherapy, phage therapy, etc. Topical medication refers to the use of topical preparations such as benzoyl peroxide and minocycline to inhibit the growth of Propionibacterium acnes in the sebaceous glands. Systemic medication refers to the treatment of acne through oral antibiotics, retinoic acid, hormone drugs, etc. Hormone drugs mainly include anti-androgen drugs and glucocorticoid drugs. Anti-androgen drugs are suitable for female patients with high androgen levels, polycystic ovary syndrome or significantly aggravated skin lesions before menstruation. Small doses of glucocorticoids are suitable for the inflammatory stage of acne. Intralesional injection is suitable for patients with severe conditions, often in combination with lidocaine and procaine. Phototherapy refers to the use of red and blue light, laser, photodynamic method, etc. to directly destroy the cell membrane of cells and cause cell apoptosis to achieve acne treatment. With the deepening of research on acne pathology, new therapies such as hyaluronic acid, cannabidiol, skin microbiota regulators, insulin-like growth factor, vaccines, bacteriophages, probiotics and antimicrobial peptides have been developed for the treatment of acne.

[0004] However, the above-mentioned acne treatment methods have certain drawbacks. For example, in clinical practice, topical drug treatment is prone to drug resistance and some adverse reactions; systemic drug treatment has low targeting and will affect other normal tissue cells in the body, and oral hormone drugs will also affect the body's endocrine system; light therapy is likely to cause adverse reactions such as erythema, swelling, and epidermal exfoliation, and is likely to be accompanied by severe pain reactions; new therapies such as phage therapy have shown great potential in the treatment of acne, but there are currently few clinical studies and they are still a long way from practical application.

[0005] Cryptotanshinone (CPT) is a class of diterpenoid compounds extracted from the traditional Chinese medicine Salvia miltiorrhiza. It has antioxidant, anti-inflammatory, and anti-fibrosis effects and is widely used in clinical practice to eliminate inflammation, fight viruses, and regulate hormone secretion. The ancient book "Da Ming Ben Cao" records that Salvia miltiorrhiza can treat acne, "discharging pus and relieving pain, regenerating muscles and flesh, eliminating blood evil and irritation, malignant sores, scabies, and thin swelling and poison". Clinical studies have found that cryptotanshinone can inhibit the disorder of multiple metabolic pathways in the body, especially glycolysis / gluconeogenesis, galactose metabolism, and histidine metabolism, and can inhibit sebum secretion through antibacterial, anti-inflammatory, and anti-androgen. Cryptotanshinone does not produce drug resistance and has little irritation to the skin. It is a safe and potential drug for the treatment of acne. However, cryptotanshinone has low water solubility and strong fat solubility, which limits its clinical application.

[0006] A Chinese patent document with publication number CN111202769A discloses a composition, preparation and preparation method for rosacea, which selects Chinese herbal extracts 4-terpineol, salvia miltiorrhiza root extract, lilac extract and Porphyridium cruentum extract, and forms a composite preparation with ceramide and cholesterol, which work together to balance the skin microecology, reduce skin sensitivity, alleviate skin inflammation, improve facial red papules, and repair the skin barrier, thereby achieving acne treatment.

[0007] The Chinese patent document with publication number CN112587593A discloses a composition for treating acne and its preparation method. The composition for treating acne in the invention is composed of 0.01-0.1 parts of cryptotanshinone, 0.1-0.9 parts of tea polyphenols, and 10-15 parts of ionic liquid; wherein the ionic liquid is composed of choline hydroxide and salicylic acid in a mass ratio of 1:1.14-2.28. The composition of the invention can effectively treat acne caused by various pathogenic mechanisms, and the therapeutic effect is better than that of a single component drug, showing obvious synergistic effect.

[0008] However, the composition and preparation method of the above composition are relatively complicated, and cannot improve the water solubility and stability of cryptotanshinone, nor can it achieve the effect of slow release of cryptotanshinone. Therefore, it is necessary to develop a new preparation for treating acne that has a simple preparation method, high safety, and can achieve slow release of drugs. Summary of the invention

[0009] The present invention provides cryptotanshinone manganese dioxide nanoparticles, comprising a carrier nano manganese dioxide and cryptotanshinone loaded thereon, which can solve the problem of poor water solubility of cryptotanshinone, improve the stability of the drug, and achieve slow release of the drug. The nano manganese dioxide and cryptotanshinone play a synergistic role in the treatment of acne.

[0010] The specific technical solutions adopted are as follows:

[0011] A cryptotanshinone manganese dioxide nanoparticle comprises a carrier nano manganese dioxide and cryptotanshinone loaded thereon. The average particle size of the cryptotanshinone manganese dioxide nanoparticle is 100-300nm, and the drug loading amount of the cryptotanshinone manganese dioxide nanoparticle is 5%-20%.

[0012] Nano manganese dioxide has a strong ability to adsorb drugs and can adsorb cryptotanshinone to solve the problem of poor water solubility of cryptotanshinone and improve the stability of the drug; and nano manganese dioxide can degrade reactive oxygen, relieve skin damage on the surface of acne, and regulate the low pH environment on the surface of acne; in addition, nano manganese dioxide has strong surface energy and binding activity in the acidic environment of acne, while cryptotanshinone, under corresponding conditions, mainly exists in a quinone structure, has strong reducibility, and can participate in the formation of the nano manganese dioxide coordination network framework as an active molecule. This process promotes drug adsorption and nanoparticle aggregation, slows down the release rate of the drug, prolongs the drug action time, and at the same time reduces the dispersibility of the nanoparticles and reduces the risk of nanoparticles entering the blood; that is, nano manganese dioxide and cryptotanshinone play a synergistic role in the treatment of acne.

[0013] The cryptotanshinone manganese dioxide nanoparticles can slowly release the drug cryptotanshinone in the skin acne environment and avoid entering the blood.

[0014] The present invention also provides a method for preparing the cryptotanshinone manganese dioxide nanoparticles, which specifically comprises the following steps: dripping an organic solution of cryptotanshinone into a nano manganese dioxide solution drop by drop, stirring in the dark for 12-24 hours to obtain a mixed solution, dialyzing the mixed solution, using a sodium dodecyl sulfate solution as the dialysis external solution, and lyophilizing after dialysis for 8-12 hours to obtain the cryptotanshinone manganese dioxide nanoparticles.

[0015] Preferably, the concentration range of the cryptotanshinone organic solution is 5-30 mg / mL, and the solvent is acetone, methanol, ethanol or ether; the concentration range of the nano manganese dioxide solution is 5-30 mg / mL.

[0016] The preparation method of the nano manganese dioxide is preferably as follows: 1-5wt% hydrogen peroxide aqueous solution is added to 20-80mg / mL methylammonium hydroxide aqueous solution to obtain a mixed solution, and then 25-60mg / mL manganese chloride aqueous solution is dripped into the mixed solution drop by drop, stirred for reaction, and washed with water and centrifuged after the reaction is completed to obtain nano manganese dioxide.

[0017] Preferably, during the dialysis process, a dialysis bag of 7000Da is used, and the concentration of the dialysis external liquid sodium dodecyl sulfate solution is 0.5-5.0wt%.

[0018] The present invention also provides use of the cryptotanshinone manganese dioxide nanoparticles in preparing a drug for treating acne.

[0019] The present invention also provides a medicine for treating acne, comprising the cryptotanshinone manganese dioxide nanoparticles.

[0020] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0021] The medicine is an external medicine or a local medicine, and the dosage forms include gel, ointment, cream and the like.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The cryptotanshinone manganese dioxide nanoparticles provided by the present invention introduce cryptotanshinone as the main drug for acne treatment, which has high safety and wide sources, and avoids problems such as adverse reactions, pain reactions, and drug resistance. At the same time, nano manganese dioxide is introduced into the field of acne treatment and used as an active oxygen nanozyme to generate active oxygen for antibacterial purposes. In addition, the nano manganese dioxide and cryptotanshinone interact with each other, increasing the interaction repulsion between the drug-loaded nanoparticles, improving their dispersibility, reducing the risk of entering the blood, and increasing safety.

[0024] (2) The cryptotanshinone manganese dioxide nanoparticles provided by the present invention have a simple preparation process, a high drug loading capacity, high safety, and are easy to mass produce. The nano manganese dioxide preparation method adopted avoids the use of a large amount of toxic and volatile solvents, has mild and non-harsh conditions, high yield, and low cost, thereby improving the safety of the industrial production of acne preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the particle size diagram of nano manganese dioxide and cryptotanshinone manganese dioxide nanoparticles.

[0026] Figure 2 The transmission electron microscope images (×100000) of nano manganese dioxide and cryptotanshinone manganese dioxide nanoparticles, wherein a is nano manganese dioxide and b is cryptotanshinone manganese dioxide nanoparticles.

[0027] Figure 3 This is a statistical chart of the encapsulation efficiency and drug loading of cryptotanshinone manganese dioxide nanoparticles.

[0028] Figure 4 This is the XPS analysis diagram of cryptotanshinone manganese dioxide nanoparticles.

[0029] Figure 5 This is the in vitro release diagram of cryptotanshinone manganese dioxide nanoparticles.

[0030] Figure 6 The morphological diagrams of the right ears of model mice, among which a is an acne model mouse, b is a normal mouse, and c is a model mouse treated with cryptotanshinone manganese dioxide nanoparticles.

[0031] Figure 7 These are HE staining images of the right ears of model mice, where a is a normal mouse, b is an acne model mouse, c is a model mouse treated with cryptotanshinone, d is a model mouse treated with nano-manganese dioxide, and e is a model mouse treated with cryptotanshinone manganese dioxide nanoparticles. DETAILED DESCRIPTION

[0032] The present invention is further illustrated below in conjunction with the examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0033] Example 1 Preparation of Nano-Manganese Dioxide

[0034] Take 2.2g of tetramethylammonium hydroxide pentahydrate, add 20mL of deionized water, stir evenly, and prepare a tetramethylammonium hydroxide solution with a concentration of 55mg / mL; add 2mL of 3wt% hydrogen peroxide aqueous solution to obtain a mixed solution; dissolve 1.2g of manganese chloride tetrahydrate in 10mL of deionized water to prepare a manganese chloride aqueous solution with a concentration of 44mg / mL, add it dropwise to the mixed solution, stir at 500rpm and react overnight; after the reaction is completed, resuspend the precipitate with deionized water, centrifuge at 14000rpm for 10min, repeat three times, pre-freeze the precipitate in an ultra-low temperature refrigerator for 4h, and freeze-dry in a freeze dryer for 24h to obtain nano manganese dioxide freeze-dried powder.

[0035] Example 2 Determination of particle size and Zeta potential of nano manganese dioxide

[0036] 10 mg of the nano manganese dioxide freeze-dried powder prepared in Example 1 was accurately weighed and placed in a 25 mL beaker. 10 mL of deionized water was added and the mixture was evenly dispersed by water bath ultrasound for 5 min. The particle size and surface potential were tested using a particle size analyzer.

[0037] The test results show that the particle size of the nano manganese dioxide is 170.96±5.82nm ( Figure 1 The potential is -11.40±1.77 mV and the polydispersity index PDI is 0.252.

[0038] Example 3 Preparation of Cryptotanshinone Manganese Dioxide Nanoparticles

[0039] Take 450 mg of the nano manganese dioxide prepared in Example 1, add 30 mL of deionized water, and perform water bath ultrasound for 5 min to prepare a nano manganese dioxide solution with a concentration of 15 mg / mL; add 60 mg of cryptotanshinone to 4 mL of acetone and shake well; use a dropper to drip the cryptotanshinone acetone solution drop by drop into the nano manganese dioxide solution, protect from light, and magnetically stir at 500 rpm for 24 h to obtain a mixed solution, dialyze the mixed solution for 8 h using a dialysis bag (7000 Da), and the dialysis external liquid is 250 mL of a 1.5 wt% sodium dodecyl sulfate solution. The sample obtained after the dialysis is pre-frozen in an ultra-low temperature refrigerator for 4 h, and freeze-dried in a freeze dryer for 12 h to obtain the cryptotanshinone manganese dioxide nanoparticle freeze-dried powder.

[0040] Example 4 Determination of Particle Size and Zeta Potential of Cryptotanshinone Manganese Dioxide Nanoparticles

[0041] Accurately weigh 10 mg of the lyophilized powder of cryptotanshinone manganese dioxide nanoparticles prepared in Example 3, put it in a 25 mL beaker, add 10 mL of deionized water, and disperse it evenly by water bath ultrasound for 5 min. Use a particle size analyzer to test the particle size and surface potential.

[0042] The test results show that the particle size of the cryptotanshinone manganese dioxide nanoparticles is 157.49±2.10nm ( Figure 1 The particle size did not change much before and after drug loading, the potential was -52.36±2.24mV, and the polydispersity index PDI was 0.163.

[0043] After loading with cryptotanshinone, the potential of the nanoparticles changed from -11.40±1.77mV to -52.36±2.24mV, and the intermolecular repulsion increased, which is beneficial to increase its stability.

[0044] Example 5 Transmission electron microscopy characterization of cryptotanshinone manganese dioxide nanoparticles

[0045] The nano manganese dioxide and cryptotanshinone manganese dioxide nanoparticle samples prepared in Example 1 and Example 3 were dropped on the carbon film, the excess sample liquid was absorbed by filter paper, and pre-dried under an infrared baking lamp. The samples were placed on a transmission electron microscope, observed at an appropriate magnification, and photographed and recorded. The results are shown in Figure 2 .from Figure 2 As can be seen from a and b, the nanoparticles are regular spherical before and after drug loading, and the particle size is consistent with the particle size measurement results.

[0046] Example 6 Determination of drug loading and encapsulation efficiency of cryptotanshinone manganese dioxide nanoparticles

[0047] Accurately weigh 1 mg of the lyophilized powder of cryptotanshinone manganese dioxide nanoparticles prepared in Example 3 into a 15 mL centrifuge tube, add 8 mL of 1.5% sodium dodecyl sulfate solution, 1 mL of 10% HCl, and 1 mL of 3 wt% hydrogen peroxide, sonicate in a water bath for 5 min, and then dilute it 10 times. Use a UV-visible spectrophotometer to measure the absorbance at 264 nm to calculate the drug loading and encapsulation efficiency of the cryptotanshinone manganese dioxide nanoparticles.

[0048] The drug loading and encapsulation efficiency of cryptotanshinone manganese dioxide nanoparticles were 11.38±0.43% and 92.96±3.52%, respectively. Figure 3 As shown, it indicates that cryptotanshinone is almost completely loaded into the nano manganese dioxide particles, with a high drug loading, which solves the problem of low solubility of cryptotanshinone.

[0049] Example 7 XPS Study of Cryptotanshinone Manganese Dioxide Nanoparticles

[0050] The prepared cryptotanshinone manganese dioxide nanoparticles were analyzed using X-ray photoelectron spectroscopy. The results of X-ray photoelectron spectroscopy analysis are shown in Figure 4 As shown, cryptotanshinone manganese dioxide nanoparticles contain elements such as C, Mn, and O, indicating that cryptotanshinone is successfully loaded in nano manganese dioxide.

[0051] Example 8 In vitro release of cryptotanshinone manganese dioxide nanoparticles

[0052] 36 mg of the cryptotanshinone manganese dioxide nanoparticle freeze-dried powder prepared in Example 3 was taken, and 2 mL of 1.5 wt% sodium dodecyl sulfate solution was added for dispersion, and 30 mL of 1.5 wt% sodium dodecyl sulfate at pH = 4.0 and pH = 5.5 were used as release media, respectively, to conduct an in vitro drug release test, pH = 4.0 and pH = 5.5 were the pH values ​​of the simulated acne surface and skin surface. 2 mL of the release medium was taken at each time point (0, 0.5, 1, 2, 4, 8, 12, 24, 48 h), and the cryptotanshinone concentration was measured at 264 nm using an ultraviolet-visible spectrophotometer, and 2 mL of 1.5 wt% sodium dodecyl sulfate solution at each pH was added to draw an in vitro release curve.

[0053] In vitro release results Figure 5 As shown, there is basically no difference in drug release rate under the conditions of pH = 4.0 and pH = 5.5. At 48h, the cumulative release rate of cryptotanshinone manganese dioxide nanoparticles is about 50%, indicating that cryptotanshinone manganese dioxide nanoparticles can significantly achieve a sustained-release effect of the drug.

[0054] Example 9 Animal efficacy evaluation

[0055] A mouse right ear acne model was constructed with xylene for pharmacodynamic experiments. 15 mice were divided into 5 groups, 3 mice in each group, including normal mice, model group mice, cryptotanshinone treatment group mice, nano manganese dioxide treatment group mice, and cryptotanshinone manganese dioxide nanoparticles treatment group mice. The five groups of mice were treated with normal saline, normal saline, cryptotanshinone, nano manganese dioxide, and cryptotanshinone manganese dioxide nanoparticles, respectively, and applied twice a day, 50 μL each time, for 5 consecutive days. One hour after the last administration, except for the normal mouse group, the rest of the mice were treated with 50 μL of xylene in the right ear. The mice were killed 3 hours later and HE staining of tissue sections was performed.

[0056] After treatment, the morphological observations of the model group mice, normal mice, and cryptotanshinone manganese dioxide nanoparticles treated mice showed significant differences, such as Figure 6 The right ear of the mice in the model group was obviously red and swollen, and there were more wrinkles, as shown in Figure 2. Figure 6 a in the figure, while the right ear of a normal mouse is smooth and has no redness or swelling. Figure 6 In b, the ear auricle of the mice in the cryptotanshinone manganese dioxide nanoparticles treatment group was no longer obviously red and swollen, and the auricle was relatively smooth, but there was still a small amount of blood in the inner ear, such as Figure 6 c in the figure. This indicates that the mouse right ear acne model was successfully established, and the cryptotanshinone manganese dioxide nanoparticles treatment group had a significant therapeutic effect on inflammatory edema.

[0057] Acne treatment effects Figure 7 As shown in the figure, the right ear skin of normal mice has multiple layers of thin squamous cells, and there is basically no bacterial colonization in the hair follicles and sebaceous glands. Figure 7 a in the figure; the dermis of the right ear skin of mice in the model group was significantly thickened, with obvious inflammation and visible bacterial colonization, such as Figure 7 b. The lymphocyte infiltration in the dermis of the right ear of the model mice treated with cryptotanshinone was significantly alleviated, but some bacteria were still colonized, such as Figure 7 c; In the model mice treated with nano-manganese dioxide, the symptoms of lymphocyte infiltration were slightly alleviated, but keratin hyperplasia and dermal inflammation were still obvious. Figure 7 d in the figure; The inflammation of the dermis and hair follicle sebaceous glands in the right ear of the model mice treated with cryptotanshinone manganese dioxide nanoparticles was significantly alleviated, and the skin inflammation was basically restored, with only a small amount of bacterial colonization, such as Figure 7 Therefore, the mouse right ear acne model was successfully established, cryptotanshinone can significantly treat acne inflammation, nano-manganese dioxide has a slight alleviating effect on acne, and the synergistic cryptotanshinone manganese dioxide nanoparticles have the best therapeutic effect.

[0058] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cryptotanshinone manganese dioxide nanoparticle, characterized in that: The invention comprises carrier nano manganese dioxide and cryptotanshinone loaded thereon. The average particle size of the cryptotanshinone manganese dioxide nanoparticles is 100-300nm, and the drug loading amount of the cryptotanshinone manganese dioxide nanoparticles is 5%-20%.

2. The method for preparing cryptotanshinone manganese dioxide nanoparticles according to claim 1, characterized in that: The following steps are involved: The cryptotanshinone organic solution is added dropwise into the nano manganese dioxide solution, stirred in the dark for 12-24 hours to obtain a mixed solution, and the mixed solution is dialyzed with sodium dodecyl sulfate solution as the external dialysis solution. After dialysis for 8-12 hours, the solution is freeze-dried to obtain the cryptotanshinone manganese dioxide nanoparticles.

3. The method for preparing cryptotanshinone manganese dioxide nanoparticles according to claim 2, characterized in that: The concentration range of the cryptotanshinone organic solution is 5-30 mg / mL, and the solvent is acetone, methanol, ethanol or ether; the concentration range of the nano manganese dioxide solution is 5-30 mg / mL.

4. The method for preparing cryptotanshinone manganese dioxide nanoparticles according to claim 2, characterized in that: The preparation method of the nano manganese dioxide is as follows: adding a 1-5wt% hydrogen peroxide aqueous solution to a 20-80mg / mL methylammonium hydroxide aqueous solution to obtain a mixed solution, then dripping a 25-60mg / mL manganese chloride aqueous solution into the mixed solution drop by drop, stirring for reaction, washing with water and centrifuging after the reaction is completed to obtain the nano manganese dioxide.

5. The method for preparing cryptotanshinone manganese dioxide nanoparticles according to claim 2, characterized in that: During the dialysis process, a 7000Da dialysis bag was used, and the concentration of the dialysis external liquid sodium dodecyl sulfate solution was 0.5-5.0wt%.

6. Use of the cryptotanshinone manganese dioxide nanoparticles according to claim 1 in the preparation of a drug for treating acne.

7. A drug for treating acne, characterized in that: It includes the cryptotanshinone manganese dioxide nanoparticles as described in claim 1.

8. The acne treatment drug according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

9. The acne treatment drug according to claim 7, characterized in that: The medicine is an external medicine or a local medicine, and the dosage form includes a gel, an ointment or a cream.

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