A kind of scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles and preparation method and application thereof

Abstract

The invention discloses a co-loaded nanoparticle of scutellaria baicalensis self-assembled nanoparticles and terbinafine, and a preparation method and application thereof, belonging to the technical field of drug nanoparticles. The preparation method comprises two steps: preparing scutellaria baicalensis self-assembled nanoparticles and preparing co-loaded nanoparticles of scutellaria baicalensis self-assembled nanoparticles and terbinafine. The co-loaded nanoparticles prepared by the preparation method. The application of the co-loaded nanoparticles in treating dermatophytosis. The invention has the following advantages: the saturated solubility of baicalin (BCN) and terbinafine (TBF) is increased; the dissolution of terbinafine (TBF) is significantly improved; the antibacterial effect of TBF is improved; and the hyphae growth, biomass production and spore germination of three dermatophytes, namely, Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis, can be inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of drug nanoparticles (medical preparations characterized by special physical shapes), and specifically relates to co-loaded nanoparticles of scutellaria baicalensis self-assembled nanoparticles and terbinafine, and a preparation method and application thereof. Background Art

[0002] Dermatophytosis, also known as psoriasis for short, is a superficial fungal infection of the skin, hair, nail plate, etc. caused by pathogenic dermatophytes. It is the infectious skin disease with the highest incidence rate in the population. According to the different sites of invasion, psoriasis can be divided into tinea capitis, tinea manuum, tinea pedis, tinea corporis, tinea cruris, tinea unguium, etc. The main symptoms of psoriasis include rash, scaling, itching, etc. It is difficult to cure and is prone to recurrence and reinfection, which seriously affects the quality of life of patients. In recent years, the incidence of psoriasis has increased year by year. About 20-25% of people worldwide suffer from this disease. Because of its wide prevalence area and high recurrence rate, it has become a common and frequently-occurring clinical disease.

[0003] Terbinafine (TBF) is currently the first-line treatment for dermatophyte infections. TBF is an allylamine antifungal drug that selectively inhibits squalene epoxidase in fungal cell membranes, interferes with the biosynthesis of ergosterol, the main component of fungal cell membranes, and accumulates precursor squalene, destroying fungal cell membranes, thereby playing a dual role in inhibiting and killing fungi. TBF is a unique antifungal drug in the allylamine class that has both topical and oral clinical administration routes. However, long-term oral TBF treatment causes drug accumulation in the body, which can easily cause systemic adverse reactions such as nausea, vomiting, diarrhea, stomach pain and liver damage. Although topical application of TBF (creams, liniments, sprays, etc.) can avoid systemic side effects, TBF is a water-insoluble drug with poor skin permeability and retention, which prolongs the treatment time and easily causes drug resistance. In addition, a large amount of drug crystals will inevitably be produced during the storage process of the preparation, which reduces the efficacy.

[0004] In recent years, new topical delivery systems for TBF have emerged one after another, including solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, liposomes, ethosomes, etc., in order to increase the water solubility, skin permeability and retention of TBF, thereby improving the local skin bioavailability of TBF and enhancing its efficacy. However, these preparations have certain limitations, such as low drug loading, drug leakage and potential excipient toxicity, which limit the further development and application of TBF. Self-assembled nanoparticles from natural sources of traditional Chinese medicine have solubility-enhancing, absorption-promoting effects and pharmacological activity, and are highly safe, providing new ideas for the development of new TBF delivery systems. Summary of the invention

[0005] The inventors of the present application have found in their previous studies that self-assembled nanoparticles of Huangqin (HQ-SAN) can promote the dissolution of baicalin (BCN), baicalein and other main antibacterial ingredients of Huangqin, have good anti-dermatophyte effects themselves, and have synergistic antifungal effects with TBF. HQ-SAN may be a promising TBF carrier for anti-dermatophyte use.

[0006] The purpose of the invention is to disclose a pair of scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles.

[0007] The second object of the present invention is to disclose a method for preparing the above-mentioned co-loaded nanoparticles.

[0008] The third object of the present invention is to disclose the application of the above-mentioned co-loaded nanoparticles.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] A method for preparing co-loaded nanoparticles of scutellaria baicalensis self-assembled nanoparticles and terbinafine comprises the following steps:

[0011] (1) Preparation of Scutellaria baicalensis self-assembled nanoparticles (HQ-SAN);

[0012] (2) Weigh scutellaria baicalensis self-assembled nanoparticles (HQ-SAN) and terbinafine (TBF) into a 10 mL vial, add 4 mL of distilled water, sonicate for 30 min, add a magnetic stirrer, place on a magnetic stirrer, stir at 400-800 rpm for 0.5-2 h, and filter the sample through a 0.8 μm filter membrane to obtain scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles.

[0013] The preparation method described in the above technical scheme, wherein the specific method of step (1) is: weigh the scutellaria baicalensis slices, add 10 times the amount of water, decoct for 1 hour, filter while hot, add 8 times the amount of water to the residue, decoct for 1 hour, filter while hot, combine the two filtrates, concentrate to 0.5-1g crude drug / mL, centrifuge at 13000rpm for 30min, take the supernatant, put it in a dialysis bag, stir magnetically at 200rpm for 30min at room temperature, take out the sample in the dialysis bag, centrifuge at 13000rpm for 30min, repeat the dialysis-centrifugation operation twice, and the sample in the dialysis bag is the scutellaria baicalensis self-assembled nanoparticles (HQ-SAN).

[0014] The preparation method described in the above technical solution, wherein: the weight ratio of the scutellaria baicalensis self-assembled nanoparticles to terbinafine is (16-24):5.

[0015] The preparation method described in the above technical solution, wherein: the weight ratio of the Scutellaria baicalensis self-assembled nanoparticles to terbinafine is 23.4:5.

[0016] The preparation method described in the above technical scheme obtains the co-loaded nanoparticles of scutellaria baicalensis self-assembled nanoparticles and terbinafine.

[0017] The co-loaded nanoparticles described in the above technical solution, wherein: the particle size of the co-loaded nanoparticles is (254±6) nm; the PDI is (0.2154±0.0121); and the Zeta potential is (-19.89±1.21) mV.

[0018] The co-loaded nanoparticles described in the above technical solution, wherein: the baicalin encapsulation rate in the co-loaded nanoparticles is (64.53±1.12)%, and the drug loading amount is (12.89±0.16)%; the terbinafine encapsulation rate is 100%, and the drug loading amount is (5.25±0.21)%.

[0019] The co-loaded nanoparticles described in the above technical solution, wherein: the baicalin in the co-loaded nanoparticles is dissolved to 91% within 15 minutes and 100% within 30 minutes; the terbinafine in the co-loaded nanoparticles is dissolved to 83% within 120 minutes.

[0020] The use of the co-loaded nanoparticles described in the above technical solution in the treatment of dermatophytosis.

[0021] The application described in the above technical solution, wherein: the skin moss fungus is Trichophyton rubrum, Trichophyton mentagrophytes or Microsporum canis.

[0022] The present invention has the following beneficial effects:

[0023] 1. The co-loaded nanoparticles of the present invention greatly increase the saturation solubility of baicalin (BCN) and terbinafine (TBF).

[0024] 2. The dissolution rate of terbinafine (TBF) in the co-loaded nanoparticles of the present invention is significantly improved.

[0025] 3. The co-loaded nanoparticles of the present invention significantly improve the antibacterial effect of TBF.

[0026] 4. The co-loaded nanoparticles of the present invention can significantly inhibit the hyphae growth of three types of dermatophytes, namely Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis, can significantly inhibit the biomass production of the three types of dermatophytes, and can significantly inhibit the spore germination of the three types of dermatophytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 1. Figure 1 The figure shows the effect of HQ-SAN concentration and magnetic stirring speed on the TBF drug loading of co-loaded nanoparticles when the magnetic stirring time is set to 1.25 h.

[0028] 2. Figure 2 The effect of HQ-SAN concentration and magnetic stirring speed on the BCN drug loading capacity of co-loaded nanoparticles when the magnetic stirring time was set to 1.25 h.

[0029] 3. Figure 3 The figure shows the effect of HQ-SAN concentration and magnetic stirring time on the TBF drug loading of co-loaded nanoparticles when the magnetic stirring speed is set to 600 rpm.

[0030] 4. Figure 4 The effect of HQ-SAN concentration and magnetic stirring time on the BCN drug loading capacity of co-loaded nanoparticles when the magnetic stirring speed is set to 600 rpm.

[0031] 5. Figure 5 The effect of magnetic stirring speed and time on the TBF drug loading of co-loaded nanoparticles when the HQ-SAN concentration is set to 5 mg / mL.

[0032] 6. Figure 6 The effect of magnetic stirring speed and time on the BCN drug loading capacity of co-loaded nanoparticles when the HQ-SAN concentration was set to 5 mg / mL.

[0033] 7. Figure 7 This is a scanning electron microscope image of TBF raw material.

[0034] 8. Figure 8 The scanning electron micrograph of the co-loaded nanoparticles.

[0035] 9. Fig. 9 These are the XRD patterns of BCN API, TBF API, HQ-SAN, and co-loaded nanoparticles.

[0036] 10. Fig.10 This is the dissolution curve of TBF from co-loaded nanoparticles.

[0037] 11. Fig.11 This is the dissolution curve of BCN from co-loaded nanoparticles.

[0038] 12. Fig.12 Effect of co-loaded nanoparticles on hyphae growth of Trichophyton rubrum (compared with negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01).

[0039] 13. Fig.13The effect of co-loaded nanoparticles on the hyphal growth of Trichophyton mentagrophytes (compared with the negative control group, *P<0.05, **P<0.01; compared with the TBF control group, #P<0.05, ##P<0.01).

[0040] 14. Fig.14 The effect of co-loaded nanoparticles on the hyphal growth of Microsporum canis (compared with the negative control group, *P<0.05, **P<0.01; compared with the TBF control group, #P<0.05, ##P<0.01).

[0041] 15. Fig.15 The effect of co-loaded nanoparticles on the biomass of Trichophyton rubrum (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01).

[0042] 16. Fig.16 Effect of co-loaded nanoparticles on the biomass of Trichophyton mentagrophytes (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01).

[0043] 17. Fig.17 The effect of co-loaded nanoparticles on the biomass of Microsporum canis (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01).

[0044] 18. Fig.18 The effect of nanoparticles on the spore germination of Trichophyton rubrum (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ##P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01; compared with the M-co-loaded nanoparticle group, Δ P<0.05, ΔΔ P<0.01).

[0045] 19. Fig.19 for Fig.18 The effect of nanoparticles on the spore germination of Trichophyton mentagrophytes (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01; compared with the M-co-loaded nanoparticle group, Δ P<0.05, ΔΔ P<0.01).

[0046] 20. Fig. 20 for Fig.18 The effect of nanoparticles on the spore germination of Microsporum canis (compared with the negative control group, * P<0.05, ** P<0.01; compared with the TBF control group, # P<0.05, ## P<0.01; compared with the H-co-loaded nanoparticle group, & P<0.05, && P<0.01; compared with the M-co-loaded nanoparticle group, Δ P<0.05, ΔΔ P<0.01). DETAILED DESCRIPTION

[0047] To facilitate the understanding of the technical solution of the present invention, a kind of Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles and a preparation method and application thereof are further described below in conjunction with specific test examples.

[0048] Embodiment 1: Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles

[0049] (1) Weigh an appropriate amount of Radix Scutellariae Radix slices, add 10 times the amount of water, decoct for 1 hour, filter while hot, add 8 times the amount of water to the residue, decoct for 1 hour, filter while hot, combine the two filtrates, concentrate to 0.5-1 g crude drug / mL, centrifuge at 13000 rpm for 30 minutes, take the supernatant, put it in a dialysis bag, stir it with a magnetic force at 200 rpm for 30 minutes at room temperature, take out the sample in the dialysis bag, centrifuge at 13000 rpm for 30 minutes, repeat the dialysis-centrifugation operation twice, the sample in the dialysis bag is HQ-SAN; vacuum freeze-dry for 24 hours to obtain freeze-dried powder, store it for future use;

[0050] (2) Weigh a fixed volume of 4 mL of HQ-SAN 5.85 mg / mL and 5 mg of TBF into a 10 mL vial, add 4 mL of distilled water, ultrasonicate for 30 min, add a magnetic stirrer, place on a magnetic stirrer, stir at 700 rpm for 1.9 h, and filter the sample through a 0.8 μm filter membrane to obtain Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles.

[0051] Embodiment 2: Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles

[0052] The operation steps of this example are the same as those of Example 1, except that: in step (2), a fixed volume of 4 mL of HQ-SAN 4 mg / mL and 5 mg of TBF are weighed into a 10 mL vial, 4 mL of distilled water is added, and the mixture is ultrasonicated for 30 min. A magnetic stirrer is added, and the mixture is placed on a magnetic stirrer and stirred at 400 rpm for 0.5 h. The sample is filtered through a 0.8 μm filter membrane to obtain Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles.

[0053] Embodiment 3: Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles

[0054] The operation steps of this example are the same as those of Example 1, except that: in step (2), a fixed volume of 4 mL of HQ-SAN 6 mg / mL and 5 mg of TBF are weighed into a 10 mL vial, 4 mL of distilled water is added, and the mixture is ultrasonicated for 30 min. A magnetic stirrer is added, and the mixture is placed on a magnetic stirrer and stirred at 800 rpm for 2 h. The sample is filtered through a 0.8 μm filter membrane to obtain Scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles.

[0055] The beneficial effects of the present invention are described below through specific test examples:

[0056] Test Example 1: 1. Preparation and characterization of Scutellaria baicalensis self-assembled nanoparticles (HQ-SAN):

[0057] 1.1 Preparation of Scutellaria baicalensis self-assembled nanoparticles (HQ-SAN):

[0058] Weigh an appropriate amount of Scutellaria baicalensis slices, add 10 times the amount of water, boil for 1 hour, filter while hot, add 8 times the amount of water to the residue, boil for 1 hour, filter while hot, combine the two filtrates, concentrate to 0.5-1g crude drug / mL, centrifuge at 13000rpm for 30min, take the supernatant, put it in a dialysis bag, stir it magnetically at 200rpm for 30min at room temperature, take out the sample in the dialysis bag, centrifuge it at 13000rpm for 30min, repeat the dialysis-centrifugation operation twice, the sample in the dialysis bag is HQ-SAN; vacuum freeze-dry for 24h to obtain lyophilized powder, store it for standby use.

[0059] 1.2 Particle size analysis and morphology:

[0060] Take an appropriate amount of HQ-SAN freeze-dried powder, add distilled water to dissolve it, and use a laser particle size analyzer to measure the particle size and Zeta potential. The results show that the particle size of HQ-SAN is 172.6nm, the polydispersity index (PDI) is 0.1563, and the Zeta potential is -15.76mV, indicating that the particle size distribution of the nanoparticles is narrow and the size is uniform. Apply an appropriate amount of HQ-SAN freeze-dried powder on a copper sample table, spray gold on the surface under reduced pressure, and observe its morphology under a scanning electron microscope. The results show that the HQ-SAN freeze-dried powder is irregular granular, with uniform particle size and a particle size of about 100 to 200nm.

[0061] 1.3 Material composition analysis:

[0062] 1.3.1 Determination of protein content in HQ-SAN by Bicinchoninic Acid (BCA) method. Take 2 mg / mL bovine serum albumin (BSA) solution in the kit and dilute it to 1500, 1000, 750, 500, 250, 125, 25 μg / mL in sequence; accurately weigh 3 portions of HQ-SAN lyophilized powder (6.07, 6.10, 6.15 mg), place them in 10 mL volumetric flasks, dissolve them in distilled water and make up to volume as sample solutions. Accurately pipette 10 μL of each reference solution and sample solution, add them to a 96-well plate, two replicate wells for each sample, add 250 μL of BCA working solution to each well, mix thoroughly, cover, incubate at 37°C for 30 minutes, cool to room temperature, measure the OD value at 562 nm with an enzyme reader, and calculate the protein content in the sample according to the standard curve. The protein content of HQ-SAN is (60.23±0.82)%.

[0063] 1.3.2 Determine the polysaccharide content in HQ-SAN by anthrone-sulfuric acid method. Weigh 0.2g and dissolve anthrone reagent in 100mL 78% sulfuric acid to a concentration of 2g / L. Weigh an appropriate amount of glucose reference substance, accurately weigh it, put it into a volumetric flask, add distilled water to dissolve it ultrasonically and make up to volume, and obtain a glucose reference substance solution with a concentration of 100μg / mL. Take 0.2, 0.4, 0.6, 0.8, and 1mL of the reference substance solution to a 15mL test tube, add distilled water to make up to 1mL, and then add 5mL of the prepared anthrone reagent, shake it well, place it under boiling water for 10min, and cool it with cold water and then measure it by ultraviolet. Accurately weigh 3 portions of HQ-SAN lyophilized powder (5.38, 5.14, and 5.73mg), put them in 25mL respectively, add ultrapure water to dissolve and make up to volume, and shake them well as samples. Accurately aspirate 1mL of the sample, and determine and calculate the polysaccharide content by the same method. The polysaccharide content of HQ-SAN was (31.72±1.22)%.

[0064] 1.3.3 The content of BCN (baicalin) and other main components was determined by HPLC.

[0065] Chromatographic conditions: Shim-pack GIS C18 (4.6×250 mm, 5 μm) was used as the chromatographic column, acetonitrile (A)-0.1% phosphoric acid solution (B) was used as the mobile phase for gradient elution (0-20 min, 25% A; 20-40 min, 25% A→40% A; 40-50 min, 40% A→48% A; 50-60 min, 48% A→25% A), the flow rate was 1 mL / min, the column temperature was 30°C, the detection wavelength was 276 nm, and the injection volume was 10 μL.

[0066] The results showed that the content of BCN in HQ-SAN was (21.37±0.11)%, the content of melaleuca A glycoside was (2.67±0.00)%, the content of wogonin was (5.00±0.06)%, the content of baicalin was (0.73±0.02)%, the content of wogonin was (0.22±0.02)%, and the content of melaleuca A was (0.11±0.00)%.

[0067] Test Example 2: Preparation and optimization of HQ-SAN (self-assembled nanoparticles from Scutellaria baicalensis) and TBF (terbinafine) co-loaded nanoparticles (hereinafter referred to as: co-loaded nanoparticles):

[0068] 2.1 Preparation process of co-loaded nanoparticles:

[0069] Weigh appropriate amounts of HQ-SAN and TBF into a 10 mL vial, add 4 mL of distilled water, ultrasonicate for 30 min, add a magnetic stirrer, place on a magnetic stirrer, stir at a certain speed for a certain period of time, and pass the sample through a 0.8 μm filter membrane.

[0070] 2.2 Methods for determining indicators:

[0071] 2.2.1 Determination of particle size and Zeta potential of co-loaded nanoparticles:

[0072] The co-loaded nanoparticles were taken, diluted appropriately, and the particle size distribution and Zeta potential were determined using a laser particle size analyzer.

[0073] 2.2.2 Determination of encapsulation efficiency and drug loading of TBF and BCN in co-loaded nanoparticles:

[0074] The encapsulation efficiency and drug loading of TBF and BCN in the co-loaded nanoparticles were determined by ultrafiltration centrifugation. 1 mL of the co-loaded nanoparticles was taken into a 5 mL volumetric flask, and methanol was added to dissolve it by ultrasonication and the volume was fixed to the mark. The 0.45 μm microporous filter membrane was used for filtration, and the HPLC sample was analyzed after filtration, which was used as the total TBF and total BCN content (W 总 ); 0.4 mL of the co-loaded nanoparticles was added to the upper layer of an ultrafiltration centrifuge tube (0.5 mL, molecular weight cutoff 3000), centrifuged at 8000 rpm for 10 min, 100 μL of the lower layer solution was taken out and diluted 2 times, filtered through a 0.45 μm microporous filter membrane, and analyzed by HPLC injection, which was used as the amount of TBF and BCN not encapsulated in the preparation (W 未 HPLC chromatographic conditions are as follows: chromatographic column: Shim-pack GIS C18 column (250 mm × 4.6 mm, 5 μm), column temperature of 40 ° C, volume flow rate of 1 mL / min, injection volume of 10 μL, mobile phase of acetonitrile (A)-0.1% phosphoric acid (B) = 43:57 (v / v), detection wavelength of 276 nm (BCN), 222 nm (TBF). Drug loading % = (W 总 -W 未 ) / co-loaded nanoparticle freeze-dried powder×100%; encapsulation efficiency %=(W 总 -W 未 ) / W 总 ×100%.

[0075] 2.3 Single factor optimization:

[0076] Magnetic stirring time: The HQ-SAN concentration was fixed at 6 mg / mL, the volume was 4 mL, and the TBF was 5 mg. The magnetic stirring speed was 800 rpm. The effects of magnetic stirring time of 0.5, 1, 2, and 3 h on the particle size distribution, Zeta potential, encapsulation efficiency, and drug loading of the co-loaded nanoparticles were investigated. The results are shown in Tables 1 and 2. The results showed that different magnetic stirring times had no significant effect on the particle size distribution and Zeta potential of the co-loaded nanoparticles; with the extension of magnetic stirring time, the TBF drug loading increased first and then decreased. When the magnetic stirring was 2 h, TBF was the highest, but the BCN encapsulation efficiency and drug loading gradually decreased. Therefore, the magnetic stirring time was selected to be 0.5 to 2 h.

[0077] Table 1 Effect of different magnetic stirring times on the particle size distribution and Zeta potential of co-loaded nanoparticles

[0078]

[0079] Table 2 Effects of different magnetic stirring times on the encapsulation efficiency and drug loading of TBF and BCN in co-loaded nanoparticles

[0080]

[0081] Magnetic stirring speed: HQ-SAN 6mg / mL, volume 4mL and TBF 5mg were fixed, and the magnetic stirring time was 2h. The effects of magnetic stirring speeds of 400, 600, 800, and 1000rpm on the particle size distribution, Zeta potential, encapsulation efficiency, and drug loading of co-loaded nanoparticles were investigated. The results are shown in Tables 3 and 4. The results showed that different magnetic stirring speeds had no significant effect on the particle size distribution and Zeta potential of co-loaded nanoparticles; with the increase of magnetic stirring speed, the TBF drug loading increased first and then decreased. When the magnetic stirring speed was 800rpm, the TBF drug loading was the highest, but the BCN encapsulation efficiency and drug loading gradually decreased. Therefore, the magnetic stirring speed was selected to be 400-800rpm.

[0082] Table 3 Effect of different magnetic stirring speeds on the particle size distribution and Zeta potential of co-loaded nanoparticles

[0083]

[0084] Table 4 Effects of different magnetic stirring speeds on the encapsulation efficiency and drug loading of TBF and BCN in co-loaded nanoparticles

[0085]

[0086] HQ-SAN concentration range: The volume of HQ-SAN was fixed at 4 mL and TBF was 5 mg, and the magnetic stirring speed and time were 600 rpm and 2 h, respectively, to investigate the effect of HQ-SAN concentration on the particle size distribution, Zeta potential, encapsulation efficiency and drug loading of co-loaded nanoparticles. The results are shown in Tables 5 and 6. The results showed that different concentrations of HQ-SAN had no significant effect on the particle size distribution and Zeta potential of co-loaded nanoparticles; with the increase of HQ-SAN concentration, the TBF drug loading increased first and then decreased. When the HQ-SAN concentration was 5 mg / mL, the TBF drug loading was the highest, and the BCN encapsulation efficiency and drug loading were gradually increased. Therefore, the HQ-SAN concentration range was selected to be 4 to 6 mg / mL.

[0087] Table 5 Effect of HQ-SAN concentration on the particle size distribution and Zeta potential of co-loaded nanoparticles

[0088]

[0089]

[0090] Table 6 Effect of HQ-SAN concentration on TBF and BCN encapsulation efficiency and drug loading in co-loaded nanoparticles

[0091]

[0092] 2.4Box-Behnken response surface method optimization:

[0093] On the basis of single factor experiment, HQ-SAN concentration (X1), magnetic stirring speed (X2) and magnetic stirring time (X3) were selected as the factors to be investigated, and TBF loading (Y1) and BCN loading (Y2) were used as indicators. The Box-Behnken design with 3 factors and 3 levels was used to optimize the prescription and process of HQ-SAN and TBF co-loaded nanoparticles. The design included a central store for repeated experiments and a series of multidimensional test points. The experimental arrangement and results are shown in Tables 7 and 8, where Table 7 is the factor and level design; Table 8 is the Box-Behnken experimental arrangement and results. Y1 (%) and Y2 (%) in the table are obtained according to the method 2.2.2.

[0094] Table 7 Factor and level design

[0095]

[0096] Table 8 Box-Behnken test arrangement and results

[0097]

[0098] 2.4.1 Establishment of quadratic regression equation:

[0099] The Box-Behnken design data were processed using Design-Expert 13 software, and multiple linear regression and binomial equation fitting were performed using Y1 and Y2 as indicators. Both Y1 and Y2 were optimally fitted using binomial regression, and their quadratic multiple regression equations were Y1 = 5.84-0.2175X1 + 0.1975X2 + 0.3625X3 - 0.2425X1X2 - 0.0325X1X3 + 0.0225X2X3 - 0.8098X1 2 -0.5598X2 2 -0.0898X3 2 (R 2 =0.9896, corrected R 2 =0.9763, predicted R 2=0.9172); Y2=9.08+1.51X1-0.7912X2-1.16X3+2.04X1X2-0.2625X1X3-0.2950X2X3+3.55X1 2 +2.06X2 2 +0.36X3 2 (R 2 =0.9875, corrected R 2 =0.9715, predicted R 2 =0.9136).

[0100] 2.4.2 Analysis of variance and significance test:

[0101] The correlation coefficients of the two fitting equations indicate that the design model has a good degree of fit, and this model can be used to analyze and predict the prescription and process of HQ-SAN and TBF co-loaded nanoparticles. From the significance test of the regression coefficients in Tables 9 and 10, it can be seen that the linear and quadratic terms of HQ-SAN concentration, the linear and quadratic terms of stirring speed, the linear term of stirring time, and the interaction term between HQ-SAN concentration and stirring speed in models Y1 and Y2 all reached a significant level (P<0.05), and the other terms were not significant. After deleting the insignificant terms, the simplified equation obtained is: Y1=5.84-0.2175X1+0.1975X2+0.3625X3-0.2425X1X2-0.8098X1 2 -0.5598X2 2 ;Y2=9.08+1.51X1-0.7912X2-1.16X3+2.04X1X2+3.55X1 2 +2.06X2 2 .

[0102] Table 9 Significance test of binomial regression coefficients of Y1 model

[0103]

[0104] Table 10 Significance test of binomial regression coefficients of Y2 model

[0105]

[0106]

[0107] 2.4.3 Response surface analysis and optimization:

[0108] Using Design-Expert 13 software, according to the regression equation analysis results, two factors with significant impact on each index were selected, and the other factor was set as the center point value, and the corresponding surface graph was made ( Figures 1 to 6 ). Figure 1 and Figure 2 The results show that when the magnetic stirring time is set to 1.25 h, the effect of HQ-SAN concentration and magnetic stirring speed on the drug loading of TBF and BCN in co-loaded nanoparticles. Figure 1 It shows that the TBF drug loading first increases and then decreases with the increase of HQ-SAN concentration and magnetic stirring speed; Figure 2 It shows that the drug loading of BCN first decreases and then increases with the increase of HQ-SAN concentration and magnetic stirring speed. Figure 3 and Figure 4 The results show the effect of HQ-SAN concentration and magnetic stirring time on the drug loading of TBF and BCN in co-loaded nanoparticles when the magnetic stirring speed is set to 600 rpm. Figure 3 It showed that the TBF drug loading first increased and then decreased with the increase of HQ-SAN concentration, and increased with the extension of magnetic stirring time; Figure 4 It shows that the drug loading of BCN first decreases and then increases with the increase of HQ-SAN concentration, and gradually decreases with the extension of magnetic stirring time. Figure 5 and Figure 6 The results show the effect of magnetic stirring speed and time on the drug loading of TBF and BCN in co-loaded nanoparticles when the HQ-SAN concentration is set to 5 mg / mL. Figure 5 It shows that the TBF drug loading increases first and then decreases with the increase of magnetic stirring speed, and increases with the extension of magnetic stirring time; Figure 6 It shows that the BCN drug loading first decreases and then increases with the increase of magnetic stirring speed, and gradually decreases with the extension of magnetic stirring time.

[0109] 2.4.4 Prediction and verification of optimal prescription and process:

[0110] Design-Expert 13 experimental design software was used to set the target according to the maximum value of Y1 and Y2, and the optimal conditions were X1 = 5.85 mg / mL, X2 = 700 rpm, and X3 = 1.9 h. The prescription and process conditions were determined as follows: 5.85 mg / mL HQ-SAN and 5 mg TBF were weighed into a 10 mL vial, 4 mL of distilled water was added, ultrasonicated for 30 min, a magnetic stirrer was added, placed on a magnetic stirrer, stirred at 700 rpm for 1.9 h, and the sample was filtered through a 0.8 μm filter membrane. The particle size distribution and Zeta potential were determined by Malvern laser particle size analyzer, and the encapsulation efficiency and drug loading were determined by HPLC. The results are shown in Table 11. The model predicted values ​​for Y1 and Y2 were 5.13% and 13.03%, respectively. The actual measured values ​​were (5.25±0.21)% and (12.89±0.16)%, respectively. The results showed that the actual measured values ​​were close to the model measured values, indicating that the model has good predictive power.

[0111] Table 11 Verification results of three batches of co-loaded nanoparticles

[0112]

[0113]

[0114] 3. Morphological observation of co-loaded nanoparticles:

[0115] TBF API and co-loaded nanoparticle freeze-dried powder were pasted onto a silicon plate, sprayed with gold, and their morphology was observed and photographed under a scanning electron microscope (SEM). Figure 7 and Figure 8 .Depend on Figure 7 It can be seen that TBF API is irregular in shape under a scanning electron microscope, and the particle size ranges from tens to hundreds of microns. Figure 8 It can be seen that the co-loaded nanoparticles are spherical, relatively uniform in size, and have a particle size of about 300 nm.

[0116] 4. Effect of co-loaded nanoparticles on the saturated solubility of TBF and BCN:

[0117] TBF API, BCN API (purchased from the market) and co-loaded nanoparticles freeze-dried powder were added in excess to an appropriate amount of distilled water, stirred at 100 rpm for 48 hours at 37 ° C, centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken and dissolved with 50 times the volume of methanol. The concentrations of BCN and TBF were determined by HPLC after passing through a 0.45 μm microporous filter membrane. The results showed that the saturated solubility of BCN API was (14.79 ± 1.13) μg / mL, the saturated solubility of TBF API was (2.85 ± 0.36) μg / mL, the saturated solubility of BCN in the co-loaded nanoparticles was (11.56 ± 0.84) mg / mL, and the saturated solubility of TBF was (192.40 ± 4.60) μg / mL. After BCN and TBF were loaded into nanoparticles, their saturated solubilities increased by 769 times and 68 times, respectively.

[0118] 5. Effect of co-loaded nanoparticles on TBF and BCN crystal forms:

[0119] Powder X-ray diffraction was used to scan and analyze BCN API, TBF API, HQ-SAN, and co-loaded nanoparticles. Working conditions: Cu target, tube voltage 40kv, tube current 60mA. Scan rate 2° / min, scanning 2θ range 5°~55°. Results are shown in Fig. 9 BCN and TBF have obvious characteristic diffraction peaks between 5 and 35°, indicating that BCN and TBF have typical crystal structure characteristics; HQ-SAN and co-loaded nanoparticles have no diffraction peaks, indicating that BCN and TBF are encapsulated in the nanoparticles in an amorphous state.

[0120] 6. Effect of co-loaded nanoparticles on the in vitro dissolution of TBF and BCN:

[0121] The dissolution was determined by the paddle method according to Part IV (General Rule 0931) of the 2020 edition of the Chinese Pharmacopoeia. Because the solubility of BCN and TBF in water is quite different, different dissolution media were used to investigate the effect of co-loaded nanoparticles on the dissolution of TBF and BCN. Dissolution conditions for BCN: At a temperature of (37±1)°C, 200mL of distilled water was used as the release medium, the stirring speed was 100rpm, and an appropriate amount of BCN bulk drug (diluted with sodium carboxymethyl cellulose) and co-loaded nanoparticles (20mg in terms of BCN) were loaded into capsules, 3 portions each; Dissolution conditions for TBF: At a temperature of (37±1)°C, 100mL of 40% ethanol water was used as the release medium, the stirring speed was 100rpm, and an appropriate amount of TBF bulk drug (diluted with sodium carboxymethyl cellulose) and co-loaded nanoparticles (2mg in terms of TBF) were loaded into capsules, 3 portions each. After the dissolution conditions are stable, the capsules are placed in the dissolution cup and the time is immediately set from the time of contact with the solvent. 1 mL of samples are taken at 5, 15, 30, 45, 60, and 120 min, and fresh dissolution medium of the same temperature and volume is added. The samples are filtered through a 0.45 μm filter membrane, and the filtrate is taken for HPLC analysis to calculate the cumulative dissolution rate. The results are shown in Fig.10 and Fig.11 ,Depend on Fig.10 and Fig.11 It can be seen that only 46% of BCN was dissolved from its raw material within 60 minutes, while 91% was dissolved from the co-loaded nanoparticles within 15 minutes and 100% was dissolved within 30 minutes, indicating that the dissolution of BCN increased significantly after being encapsulated in self-assembled nanoparticles; only 16% of TBF was dissolved from its raw material within 120 minutes, while 83% was dissolved from the co-loaded nanoparticles within 120 minutes, indicating that after TBF was loaded into the nanoparticles, its solubility was significantly improved.

[0122] 7. Evaluation of the antibacterial effect of co-loaded nanoparticles on dermatophytes:

[0123] 7.1 Drug sensitivity test:

[0124] The final concentration of the bacterial suspension was prepared with reference to the "Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi" (M38-3rd) of the Clinical and Laboratory Standards Institute (CLSI) to be (1-5)×104 CFU / mL for later use. A 96-well plate was taken, 100 μL of RPMI 1640 medium was added to each well, and then 100 μL of 4 μg / mL TBF and 0.5 mg / mL co-loaded nanoparticles (prepared in Example 1) were added in sequence in the first column, mixed with a pipette, and 100 μL was drawn to the second well, and then 2-fold dilution was performed in sequence to the 10th well. The final concentrations of TBF were 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008, and 0.004 μg / mL; the final concentrations of co-loaded nanoparticles were 0.25, 0.125, 0.0625, 0.0313, 0.0156, 0.0078, 0.0039, 0.0020, 0.0010, and 0.0005 mg / mL. 100 μL of the prepared bacterial suspension was added to wells 1 to 10 and well 12, well 11 was used as a negative control, and well 12 was used as a positive control. The prepared 96-well drug sensitivity plate was placed in a biochemical incubator, Trichophyton rubrum was cultured for 7 days, Trichophyton mentagrophytes and Microsporum canis were cultured for 5 days, and the growth of fungi in each test well was observed with the naked eye and compared with the positive and negative control wells. The lowest drug mass concentration at which no fungal growth was observed with the naked eye was the MIC value. The experimental results are shown in Table 12. The results showed that the MIC values ​​of TBF against the three bacteria were in the range of 0.25-0.5 μg / mL, which had an inhibitory effect, indicating that the experimental operation was feasible; the MIC values ​​of the co-loaded nanoparticles against the three bacteria were in the range of 3.9-7.8 μg / mL, which was calculated according to the TBF drug loading of 5.25%, equivalent to TBF 0.2028-0.4056 μg / mL. Because TBF is a poorly soluble component, the TBF control group was diluted with culture medium after being solubilized with DMSO, while the co-loaded nanoparticles were directly dispersed with culture medium, indicating that loading TBF into the nanoparticles significantly improved the antibacterial effect of TBF.

[0125] Table 12 Drug sensitivity results

[0126]

[0127] 7.2 Effect of co-loaded nanoparticles on the growth of dermatophyte hyphae:

[0128] The experiment was divided into 5 groups, including negative control group, TBF control group (1×MIC), high-dose co-loaded nanoparticles group (H-co-loaded nanoparticles, 2×MIC), medium-dose co-loaded nanoparticles group (M-co-loaded nanoparticles, 1×MIC), and low-dose co-loaded nanoparticles group (L-co-loaded nanoparticles, 0.5×MIC). Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis were inoculated on SDA culture plates, respectively. After a period of culture, a 9mm diameter puncher was used to take out the bacterial blocks as bacterial cakes for standby use. The drug was mixed with the preheated SDA culture medium and poured into a 9cm diameter sterile culture dish to prepare a drug-containing culture medium. In this culture dish, a 9mm puncher was used to remove the intermediate culture medium and inoculate a 9mm bacterial cake. The culture dish was sealed with a sealing film and placed in a 28℃ incubator for incubation. Trichophyton rubrum was cultured for 12 days, Trichophyton mentagrophytes and Microsporum canis were cultured for 6 days, and the mycelial growth diameter was measured by the cross method every 24 hours. Mycelium length (cm) = (average mycelium length measured - 0.9) / 2, mycelium growth inhibition rate % = (mycelium length of negative control group - mycelium length of drug treatment group) / mycelium length of negative control group × 100. Colony diameter measurement results are shown in Figure 12 to Figure 14 Compared with the negative control group, the TBF control group and the different doses of co-loaded nanoparticles groups could significantly inhibit the hyphae growth of the three dermatophytes (P<0.05) (the hyphae length of Trichophyton rubrum is shown in Fig.12 , the length of hyphae of Trichophyton mentagrophytes is shown in Fig.13 , the hypha length of Microsporum canis is shown in Fig.14 ), the mycelial growth inhibition rate was 50.08-73.95%; compared with the TBF control group, on the last day of the test, the high-dose co-loaded nanoparticles group had a significantly higher inhibitory effect on mycelial growth (P<0.05), while there was no significant difference in the medium-dose co-loaded nanoparticles group (P>0.05).

[0129] 7.3 Effect of co-loaded nanoparticles on the biomass synthesis of dermatophytes:

[0130] The experiment was divided into 5 groups: negative control group, TBF control group (1×MIC), high-dose co-loaded nanoparticles group (H-co-loaded nanoparticles, 2×MIC), medium-dose co-loaded nanoparticles group (M-co-loaded nanoparticles, 1×MIC), and low-dose co-loaded nanoparticles group (L-co-loaded nanoparticles, 0.5×MIC). Take a 12mL sterile shaking tube, add 5mL SDB culture medium and 1mL containing 5.0×10 5 CFU / mL bacterial suspension, seal the tube with sealing film, shake and culture at 120rpm and 28℃ for 6 days, collect the mycelium by filtration, dry and weigh. Biomass inhibition rate % = (biomass of negative control group - biomass of drug treatment group) / biomass of negative control group × 100. Results are shown in Figure 15 to Figure 17Compared with the negative control group, the TBF control group and the different doses of co-loaded nanoparticles groups could significantly inhibit the biomass production of the three dermatophytes (P<0.05) (the biomass of Trichophyton rubrum is shown in Fig.15 , Trichophyton mentagrophytes biomass Fig.16 , Microsporum canis biomass Fig.17 ); The inhibition rates of the TBF control group on the biomass production of Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis were 71.22%, 76.16%, and 72.62%, respectively; compared with the TBF control group, the high and medium doses of co-loaded nanoparticles had no significant effect on the biomass production of the three dermatophytes (P>0.05), and the inhibition rates were 63.83-81.20%. This shows that the high and medium doses of co-loaded nanoparticles and the positive drugs have the same effect on inhibiting the biomass synthesis of dermatophytes.

[0131] 7.4 Effect of co-loaded nanoparticles on germination of dermatophyte spores:

[0132] The experiment was divided into 5 groups: negative control group, TBF control group (1×MIC), high-dose co-loaded nanoparticles group (H-co-loaded nanoparticles, 2×MIC), medium-dose co-loaded nanoparticles group (M-co-loaded nanoparticles, 1×MIC), and low-dose co-loaded nanoparticles group (L-co-loaded nanoparticles, 0.5×MIC). 5 CFU / mL) was fully shaken and mixed, then inoculated on a concave glass slide containing SDA medium, placed in a humidified box, and cultured at 28°C for 24 hours. The spore germination was examined under a microscope. The number of germinated spores per 200 spores was calculated based on the extraction of dental tubes and the growth of hyphae. The spore germination rate (%) = number of germinated spores / total number of examined spores × 100. The results are shown in Figure 18 to Figure 20 Compared with the negative control group, the TBF control group and the different doses of co-loaded nanoparticles groups could significantly inhibit the spore germination of the three types of dermatophytes (P<0.05) (the spore germination rate of Trichophyton rubrum is shown in Fig.18 , Trichophyton mentagrophytes spore germination rate Fig.19 , the germination rate of Microsporum canis spores Fig. 20 ); after TBF treatment, the spore germination inhibition rates of Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis were 79.53%, 76.08% and 68.58%, respectively; compared with the TBF control group, the high and medium doses of co-loaded nanoparticles had no significant effect on the spore germination inhibition rates of the three dermatophytes (P>0.05), and the inhibition rates were 61.30-79.61%, indicating that the high and medium doses of co-loaded nanoparticles had the same inhibitory effect on germination of dermatophyte spores as the positive drugs.

[0133] The above description is only a preferred embodiment of the present invention and does not impose any formal or substantial limitation on the present invention. Any technician familiar with the profession can make use of the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and any slight changes, modifications and evolutions made by equivalent changes are all equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing co-loaded nanoparticles of scutellaria baicalensis self-assembled nanoparticles and terbinafine, comprising the following steps: (1) Preparation of self-assembled nanoparticles of Scutellaria baicalensis: Weigh the decoction pieces of Scutellaria baicalensis, add 10 times the amount of water, decoct for 1 hour, filter while hot, add 8 times the amount of water to the residue, decoct for 1 hour, filter while hot, combine the two filtrates, concentrate to 0.5-1 g crude drug / mL, centrifuge at 13000 rpm for 30 min, take the supernatant, put it in a dialysis bag, stir it magnetically at 200 rpm for 30 min at room temperature, take out the sample in the dialysis bag, centrifuge it at 13000 rpm for 30 min, repeat the dialysis-centrifugation operation twice, and the sample in the dialysis bag is the self-assembled nanoparticles of Scutellaria baicalensis; (2) Weigh the scutellaria baicalensis self-assembled nanoparticles and terbinafine in a 10 mL vial, add 4 mL of distilled water, ultrasonicate for 30 min, add a magnetic stirrer, place on a magnetic stirrer, stir at 400 ~ 800 rpm for 0.5 ~ 2 h, filter the sample through a 0.8 μm filter membrane to obtain scutellaria baicalensis self-assembled nanoparticles and terbinafine co-loaded nanoparticles; the weight ratio of the scutellaria baicalensis self-assembled nanoparticles to terbinafine is (16 ~ 24):

5.

2. The preparation method according to claim 1, characterized in that: The weight ratio of the scutellaria baicalensis self-assembled nanoparticles to terbinafine is 23.4:

5.

3. The co-loaded nanoparticles of Scutellaria baicalensis self-assembled nanoparticles and terbinafine prepared by the preparation method according to claim 1 or claim 2.

4. The co-loaded nanoparticles according to claim 3, characterized in that: The particle size of the co-loaded nanoparticles was (254±6) nm; the PDI was (0.2154 ± 0.0121); and the Zeta potential was (-19.89 ± 1.21) mV.

5. The co-loaded nanoparticles according to claim 3, characterized in that: The encapsulation efficiency of baicalin in the co-loaded nanoparticles was (64.53 ± 1.12)%, and the drug loading capacity was (12.89 ± 0.16)%; the encapsulation efficiency of terbinafine was 100%, and the drug loading capacity was (5.25 ± 0.21)%.

6. The co-loaded nanoparticles according to claim 3, characterized in that: The dissolution of baicalin in the co-loaded nanoparticles reached 91% within 15 minutes and 100% within 30 minutes; the dissolution of terbinafine in the co-loaded nanoparticles reached 83% within 120 minutes.

7. Use of the co-loaded nanoparticles according to any one of claims 3 to 6 in the preparation of a medicament for treating dermatophytosis.

8. The use according to claim 7, characterized in that: The skin moss fungus is Trichophyton rubrum, Trichophyton mentagrophytes or Microsporum canis.

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