Anti-skin inflammation zwitterionic nano-drug and preparation method thereof

Abstract

The invention relates to an anti-scytitis zwitterionic nano-drug and a preparation method thereof, and belongs to the technical field of nano-drug preparation. The zwitterionic nano-drug provided by the invention comprises a zwitterionic polymer OPDEA-PGED as a carrier, and baicalein and curcumin which are loaded on the carrier, and the expression of the zwitterionic nano-drug is OPDEA-PGED (at) BC. The zwitterionic polymer OPDEA-PGED in the zwitterionic nano-drug has good hydrophilicity and is extremely easy to dissolve in water, the disadvantage that baicalein and curcumin are poor in solubility can be improved when the zwitterionic polymer OPDEA-PGED serves as a carrier, baicalein and curcumin have wide anti-inflammatory and anti-oxidation effects, formed OPDEA-PGED-coated BC can be slowly released, the OPDEA-PGED-coated BC has the advantages of baicalein and curcumin, and the zwitterionic nano-drug has the advantages of baicalein and curcumin. Meanwhile, the biological safety is high, the effects of resisting inflammation, resisting oxidation and relieving skin inflammation are achieved, and the toxicity is low.

Description

Technical Field

[0001] The invention relates to the technical field of nano drug preparation, and in particular to a zwitterionic nano drug for resisting skin inflammation and a preparation method thereof. Background Art

[0002] The treatment of skin inflammation currently relies mainly on topical medications, oral medications and physical therapy. Among them, although topical medications such as topical corticosteroids (TCS) are the first-line drugs for the treatment of atopic dermatitis, long-term use may cause skin atrophy, pigmentation and other adverse reactions. They are often refused for fear of side effects, affecting treatment compliance; oral medications such as oral immunosuppressants may cause a decline in immune system function and increase the risk of infection, including serious bacterial and viral infections; physical therapy such as phototherapy is an effective treatment method, but the effect is slow to show, and multiple treatments are required to observe significant improvement, which is not ideal for some patients with acute conditions.

[0003] Baicalein is a flavonoid compound derived from Scutellaria baicalensis, and curcumin is an active ingredient extracted from turmeric. Both have significant anti-inflammatory and antioxidant effects, and can inhibit inflammatory mediators produced by macrophages, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and can also inhibit a variety of inflammation-related phosphatase, cyclooxygenase and nitric oxide synthase, thereby reducing inflammatory response and tissue damage. In addition, they also have strong antioxidant capacity, which can remove free radicals in the body, thereby reducing the damage of oxidative stress to cells.

[0004] However, baicalein and curcumin have poor permeability on the skin surface, and existing topical preparations often lack sufficient stability and find it difficult to maintain the active ingredients of baicalein and curcumin, so their low bioavailability limits their effectiveness in local treatment. Summary of the invention

[0005] The present invention aims to solve the technical problem that the external preparations of baicalin and curcumin in the prior art have poor permeability on the skin surface, often lack sufficient stability, and are difficult to maintain the active ingredients of baicalin and curcumin, and provide a zwitterionic nanomedicine for resisting skin inflammation and a preparation method thereof. The zwitterionic polymer ethylenediamine-opened poly [2-(N-oxide-N, N-diethylamino) ethyl methacrylate-glycidyl methacrylate] (OPDEA-PGED) in the zwitterionic nanomedicine of the present invention has good hydrophilicity and is highly soluble in water. As a carrier, it can improve the disadvantage of poor solubility of baicalin and curcumin. The formed zwitterionic nanomedicine OPDEA-PGED@BC can be slowly released, has high biological safety, and has the effects of anti-inflammatory, anti-oxidation, and reducing skin inflammation.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A zwitterionic nanomedicine for anti-skin inflammation, comprising zwitterionic polymer ethylenediamine ring-opened poly [2-(N-oxide-N, N-diethylamino)ethyl methacrylate-glycidyl methacrylate] (OPDEA-PGED) as a carrier, and baicalein and curcumin loaded on the carrier;

[0008] The expression of the zwitterionic nanodrug is: OPDEA-PGED@BC.

[0009] In the above technical solution, preferably, the mass ratio of OPDEA-PGED to baicalein and curcumin is 10-12:4-8:4-8.

[0010] In the above technical solution, preferably, the average particle size of the zwitterionic nanomedicine is 97.19±0.41 nm.

[0011] In the above technical solution, preferably, the drug loading amount of baicalein in the zwitterionic nanomedicine is 6-12%, and the drug loading amount of curcumin is 10-20%.

[0012] In the above technical solution, preferably, the encapsulation rate of baicalein in the zwitterionic nanomedicine is 35-50%, and the encapsulation rate of curcumin is 80-90%.

[0013] A method for preparing zwitterionic nanomedicine for resisting skin inflammation comprises the following steps:

[0014] Step 1, weigh OPDEA-PGED, add tetrahydrofuran to completely dissolve it, and obtain a first solution;

[0015] Step 2, weighing baicalein and curcumin, adding methanol to completely dissolve them, and obtaining a second solution;

[0016] Step 3, adding 4-dimethylaminopyridine (DMAP) and the second solution obtained in step 2 to the first solution obtained in step 1, then adding pyridine, reacting the reaction system in an oil bath, and after the reaction is completed, dripping the obtained mixed solution into an ultrasonic ice-water bath to obtain a light yellow emulsion; then dialyzing the obtained light yellow emulsion to obtain a micellar solution; finally, filtering the obtained micellar solution to obtain the zwitterionic nanomedicine.

[0017] In the above technical solution, preferably, the reaction conditions of the reaction system in step 3 in an oil bath are: stirring at 37° C. for 18-24 h.

[0018] In the above technical solution, preferably, after the reaction in step 3 is completed, the obtained mixed solution is added dropwise into an ultrasonic ice-water bath at a dropping speed of 8-10 L / min.

[0019] In the above technical solution, preferably, in step 3, dialyzing the obtained light yellow emulsion is performed using a dialysis bag with a molecular weight cutoff of 3000-3500 Da for 8-10 hours.

[0020] In the above technical solution, preferably, in step 3, the micellar solution obtained is finally filtered using a filter with a pore size of 250 nm or 450 nm.

[0021] The beneficial effects of the present invention are:

[0022] The zwitterionic polymer OPDEA-PGED in the zwitterionic nanomedicine of the present invention has good hydrophilicity and is very soluble in water. As a carrier, it can improve the disadvantage of poor solubility of baicalein and curcumin. The formed zwitterionic nanomedicine OPDEA-PGED@BC can be slowly released, has high biosafety, and has the effects of anti-inflammatory, anti-oxidation, and reducing skin inflammation.

[0023] The zwitterionic nanomedicine prepared by the present invention, baicalein and curcumin have extensive anti-inflammatory and antioxidant effects, OPDEA-PGED has the characteristics of low zwitterionic toxicity and strong cell permeability, and the formed zwitterionic nanomedicine OPDEA-PGED@BC has the advantages of both, has good anti-inflammatory and antioxidant effects, can improve bioavailability, has a good effect on the treatment of skin inflammation, and has low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0025] Figure 1 This is the standard curve of baicalein.

[0026] Figure 2 This is the standard curve of curcumin.

[0027] Figure 3 This is a particle size diagram of the zwitterionic nanodrug OPDEA-PGED@BC prepared in the present invention.

[0028] Figure 4The cytotoxicity graphs of the zwitterionic nanodrug OPDEA-PGED@BC prepared by the present invention and the control substance; wherein, (a) is a graph showing the cytotoxic effect of different concentrations of the zwitterionic nanodrug OPDEA-PGED@BC on RAW264.7 cells; (b) is a graph showing the cytotoxic effect of different concentrations of baicalein and curcumin mixture BC on RAW264.7 cells; (c) is a graph showing the cytotoxic effect of different concentrations of the zwitterionic polymer OPDEA-PGED on RAW264.7 cells.

[0029] Figure 5 The graphs show the regulatory effects of the zwitterionic nanodrug OPDEA-PGED@BC prepared by the present invention and the control substance on the inflammatory factors of RAW264.7 cells; wherein, (a) is a graph showing the regulatory effects on the inflammatory factor IL-1β of RAW264.7 cells, (b) is a graph showing the regulatory effects on the inflammatory factor IL-6 of RAW264.7 cells, and (c) is a graph showing the regulatory effects on the inflammatory factor TNF-α of RAW264.7 cells.

[0030] Wherein: TNF-α stands for tumor necrosis factor-α, IL-1β stands for interleukin-1β, and IL-6 stands for interleukin-6;

[0031] OPDEA-PGED group represents the zwitterionic polymer group; BC group represents the baicalein and curcumin mixture group; OPDEA-PGED@BC group represents the zwitterionic nanomedicine group. DETAILED DESCRIPTION

[0032] The inventive concept of the present invention is: the present invention aims to solve the technical problem that the existing baicalein and curcumin have poor permeability on the skin surface, and the external preparations often lack sufficient stability, making it difficult to maintain the active ingredients of baicalein and curcumin, so the low bioavailability limits their effect in local treatment. Zwitterionic polymers are macromolecular compounds composed of positive and negative ions, including organic anionic macromolecular polymers and organic cationic macromolecular polymers. Zwitterionic polymers have good hydrophilicity, polarity and solubility, can be dissolved in water, have better stability and dispersibility, and are therefore widely used in various fields. In view of this, the present invention provides a zwitterionic nano drug OPDEA-PGED@BC for anti-skin inflammation and a preparation method thereof. In the invention, baicalein and curcumin have a wide range of anti-inflammatory and antioxidant effects, OPDEA-PGED has the characteristics of low zwitterionic toxicity and strong cell permeability, and the formed zwitterionic nano drug OPDEA-PGED@BC has the advantages of both, has good anti-inflammatory and antioxidant effects, can improve bioavailability, and has a good effect on the treatment of skin inflammation.

[0033] The invention discloses a zwitterionic nanomedicine for preventing skin inflammation, comprising a zwitterionic polymer ethylenediamine ring-opening poly[2-(N-oxide-N,N-diethylamino)methylacrylate-glycidyl methacrylate] (OPDEA-PGED, the full name of the English abbreviation: Poly[2-(N-oxide-N,N-diethylamino)methylacrylate-glycidyl methacrylate] derived from ethylenediamine ring-opening) as a carrier, and baicalein and curcumin loaded on the carrier; the expression of the zwitterionic nanomedicine is: OPDEA-PGED@BC.

[0034] Preferably, the mass ratio of OPDEA-PGED to baicalein and curcumin is 10-12:4-8:4-8; further preferably, the mass ratio of OPDEA-PGED to baicalein and curcumin is 10:4:4.

[0035] Preferably, the average particle size of the zwitterionic nanomedicine of the present invention is 97.19±0.41 nm.

[0036] Preferably, the drug loading of baicalein in the zwitterionic nanodrug of the present invention is 6-12%, and the drug loading of curcumin is 10-20%. The encapsulation efficiency of baicalein in the zwitterionic nanodrug is 35-50%, and the encapsulation efficiency of curcumin is 80-90%; further preferably, the drug loading of baicalein in the zwitterionic nanodrug of the present invention is 9.08%, and the encapsulation efficiency is 40.85%, and the drug loading of curcumin is 18.37%, and the encapsulation efficiency is 82.67%.

[0037] A method for preparing zwitterionic nanomedicine for resisting skin inflammation comprises the following steps:

[0038] Step 1, weigh OPDEA-PGED, add tetrahydrofuran to completely dissolve it, and obtain a first solution;

[0039] Step 2, weighing baicalein and curcumin, adding methanol to completely dissolve them, and obtaining a second solution;

[0040] Among them, preferably, the mass ratio of OPDEA-PGED to baicalein and curcumin is 10-12:4-8:4-8, and more preferably, the mass ratio of OPDEA-PGED to baicalein and curcumin is 10:4:4.

[0041] Step 3, adding 4-dimethylaminopyridine (DMAP) and the second solution obtained in step 2 to the first solution obtained in step 1, and then adding pyridine, stirring the reaction system in an oil bath at 37° C. for 18-24 h, and after the reaction is completed, dropping the obtained mixed solution into an ultrasonic ice-water bath at a dropping speed of 8-10 L / min to obtain a light yellow emulsion; then dialyzing the obtained light yellow emulsion using a dialysis bag (molecular weight cutoff (MWCO): 3000-3500 Da) for 8-10 h to obtain a micellar solution; finally, filtering the obtained micellar solution using a filter with a pore size of 250 nm or 450 nm to obtain the zwitterionic nanodrug OPDEA-PGED@BC.

[0042] The preparation and performance characterization of the zwitterionic nanodrug of the present invention are described in more detail below through examples. The zwitterionic polymer OPDEA-PGED used in the examples of the present invention is synthesized with reference to the existing literature (A pH-triggered N-oxide polyzwitterionic nano-drug loaded system for the anti-tumor immunity activation research, Zhao et al. Journal of Nanobiotechnology (2024) 22:420, https: / / doi.org / 10.1186 / s12951-024-02677-0), and the specific synthesis part is detailed in the supplementary information section of the literature. All other reagents used are commercially available products.

[0043] Example 1 Preparation of zwitterionic nanomedicine OPDEA-PGED@BC of the present invention

[0044] The dual-drug loaded micellar zwitterionic nanodrug OPDEA-PGED@BC was prepared with a mass ratio of OPDEA-PGED to baicalein and curcumin of 10:4:4.

[0045] Step 1. Weigh 10 mg of OPDEA-PGED in a 25 mL brown single-necked bottle, add 2 mL of tetrahydrofuran, and stir for 24 h to completely dissolve it to obtain a first solution;

[0046] Step 2: Weigh 4 mg of baicalein and 4 mg of curcumin in a 10 mL brown single-mouth bottle, add 2 mL of methanol, and stir for five minutes to completely dissolve them to obtain a second solution;

[0047] Step 3: Add 4 mg of 4-dimethylaminopyridine (DMAP) and the second solution obtained in step 2 to the first solution obtained in step 1, and then add 2 mL of pyridine, and stir the reaction solution in an oil bath at 37°C for 24 h. After the reaction, the obtained mixed solution was added dropwise to an 8 mL ultrasonic ice-water bath at a dropping speed of 8 L / min to obtain a light yellow emulsion, which was then transferred into a dialysis bag (MWCO: 3500 Da) and dialyzed for 8 h to obtain a micellar solution; finally, the obtained micellar solution was filtered through a filter with a pore size of 450 nm to obtain the zwitterionic nanodrug OPDEA-PGED@BC.

[0048] Example 2 Determination of in vitro drug loading of zwitterionic nanodrug OPDEA-PGED@BC prepared in Example 1

[0049] First, the UV absorbance of baicalin at 325nm and curcumin at 426nm was measured by UV spectrophotometer to establish a standard curve. 10 mg of baicalin and 10 mg of curcumin were accurately weighed, and the volume was adjusted to 10 mL with methanol to prepare 1 mg / mL baicalin solution and curcumin solution, respectively. The mother liquor was diluted step by step to the set mass concentration gradient. The set mass concentration gradient of baicalin solution was 1μg / mL, 2μg / mL, 5μg / mL, 6μg / mL, and 8μg / mL; the set mass concentration gradient of curcumin solution was 25μg / mL, 50μg / mL, 110μg / mL, 125μg / mL, and 160μg / mL; the absorbance of each mass concentration at 325nm and 426nm was measured, and the standard curve of baicalin was established as follows: y=0.0498x+0.0551, R 2 = 0.9994 (see Figure 1 ); The standard curve of curcumin is: y=61.628x-0.0286, R 2 = 0.9993 (see Figure 2 ). The pH value was adjusted to about 6, and the structure of the zwitterionic nanodrug OPDEA-PGED@BC was destroyed by ultrasonication with an appropriate amount of methanol, so that the baicalin and curcumin loaded in the zwitterionic nanodrug were dissolved in dichloromethane. After high-speed centrifugation, the dichloromethane layer was transferred to a cuvette, and the ultraviolet absorption peaks of baicalin and curcumin were observed at 25°C, and the drug loading and encapsulation efficiency of the zwitterionic nanodrug OPDEA-PGED@BC were obtained according to the established standard curve.

[0050] Drug loading ( ) and encapsulation efficiency ( ) is calculated as follows:

[0051]

[0052]

[0053] Example 3 Particle size and potential test of zwitterionic nanodrug OPDEA-PGED@BC prepared in Example 1

[0054] The particle size scattering intensity and distribution of the prepared zwitterionic nanodrug OPDEA-PGED@BC in Test Example 1 were analyzed by dynamic light scattering (DLS) to analyze the particle size of the micelles, and the PDI dispersion coefficient and zeta potential were recorded. The measurement was repeated three times, and the particle size measurement results are shown in FIG. Figure 3 , and finally expressed as mean ± standard deviation.

[0055] Take 1 mg of zwitterionic nanodrug OPDEA-PGED@BC, dilute it ten times, and measure the average particle size to be 97.19±0.41 nm, the potential: 0.184±3.93 mV, the drug loading of baicalein is 9.08%, and the encapsulation efficiency is 40.85%; the drug loading of curcumin is 18.37%, and the encapsulation efficiency is 82.67%.

[0056] Table 1 Particle size, potential, drug loading and encapsulation efficiency of OPDEA-PGED@BC

[0057] Example 4 Cytotoxicity test of zwitterionic nanodrug OPDEA-PGED@BC prepared in Example 1

[0058] The cell viability of RAW264.7 cells was detected by CCK8 assay. RAW264.7 cells (derived from male Abelson murine leukemia virus-induced macrophages) were cultured at 1×10 4Cells / well were inoculated in a 96-well plate and cultured overnight. After the cells attached to the wall, 20 μL of different concentrations of BC (the concentrations were 0 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and the mass ratio of baicalein to curcumin was 1:1), OPDEA-PGED (the concentrations were 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL), and OPDEA-PGED@BC (the concentrations were 0 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL), were added and cultured at 37 °C and 5% CO2 for 24 h. After discarding the culture medium, wash twice with PBS, add 100 μL of the prepared CCK8:MDEM=1:10 working solution, culture at 37 ℃ in the dark for 2 h, measure the absorbance of each well at 450 nm using a microplate reader, and calculate the cell survival rate using the following formula:

[0059]

[0060] in, and are the absorbance of OPDEA-PGED@BC and blank control (DMEM medium containing cells), respectively. is the absorbance of cell-free DMEM medium. Figure 4 The results of ac in the experiment showed that, compared with the control group, the viability of RAW264.7 cells decreased with the increase of drug concentration after 24 h of co-culture. When the concentration of OPDEA-PGED@BC was ≤5μg / mL, the cell survival rate was higher than 85%, indicating that OPDEA-PGED@BC had good biocompatibility when the concentration did not exceed 5μg / mL (see the results). Figure 4 (a) in the figure.

[0061] Example 5 Example 1: Regulatory effect of zwitterionic nanodrug OPDEA-PGED@BC on inflammatory factors in RAW264.7 cells

[0062] The qPCR experiment was used to detect the regulatory effect of the zwitterionic nanodrug OPDEA-PGED@BC prepared in Example 1 on inflammatory factors in RAW264.7 cells. 5RAW264.7 cells were inoculated per well and cultured at 37°C and 5% CO2 for 24 h. The culture medium containing LPS (1 mg / mL), LPS (1 mg / mL) + OPDEA-PGED@BC (calculated as Cur 5 μg / mL), LPS (1 mg / mL) + OPDEA-PGED (17.4 μg / mL), LPS (1 mg / mL) + BC (5 μg / mL, the mass ratio of baicalein and curcumin was 1:1) was replaced and incubated at 37°C and 5% CO2 for 6 h. The culture medium was discarded and washed twice with PBS. Total RNA was extracted with TRIzol reagent. RNA concentration was measured using Nanodrop 2000 fiber optic spectrophotometer (NanodrodropND-1000, Thermo). cDNA was synthesized using the Hifair® Ⅲ 1st Strand cDNA Synthesis Kit according to the manufacturer's description. Quantification was performed using Hieff® qPCR SYBR Green Master Mix. The PCR reaction conditions were 95°C for 5 min, 95°C for 10 s, and 60°C for 30 s, for a total of 40 cycles. β-actin was used as the internal reference gene. −ΔΔCt The data were calculated by the method of Figure 5 ).Depend on Figure 5 It can be seen from ac that compared with the control substance, the zwitterionic nanodrug OPDEA-PGED@BC group can significantly reduce the expression of inflammatory factors IL-1β, IL-6 and TNF-α in RAW264.7 cells induced by lipopolysaccharide, suggesting that inflammation is inhibited.

[0063] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A zwitterionic nanomedicine for anti-skin inflammation, characterized in that: The invention comprises a zwitterionic polymer ethylenediamine ring-opened poly [2-(N-oxide-N, N-diethylamino)ethyl methacrylate-glycidyl methacrylate] (OPDEA-PGED) as a carrier, and baicalein and curcumin loaded on the carrier; The expression of the zwitterionic nanodrug is: OPDEA-PGED@BC.

2. The zwitterionic nanomedicine for anti-skin inflammation according to claim 1, characterized in that: The mass ratio of OPDEA-PGED to baicalein and curcumin is 10-12:4-8:4-8.

3. The zwitterionic nanomedicine for anti-skin inflammation according to claim 1, characterized in that: The average particle size of the zwitterionic nanomedicine is 97.19±0.41 nm.

4. The zwitterionic nanomedicine for anti-skin inflammation according to claim 1, characterized in that: The drug loading amount of baicalein in the zwitterionic nanomedicine is 6-12%, and the drug loading amount of curcumin is 10-20%.

5. The zwitterionic nanomedicine for preventing skin inflammation according to claim 1, characterized in that: The encapsulation rate of baicalein in the zwitterionic nanomedicine is 35-50%, and the encapsulation rate of curcumin is 80-90%.

6. A method for preparing the zwitterionic nanomedicine for preventing skin inflammation according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1, weigh OPDEA-PGED, add tetrahydrofuran to completely dissolve it, and obtain a first solution; Step 2, weighing baicalein and curcumin, adding methanol to completely dissolve them, and obtaining a second solution; Step 3, adding 4-dimethylaminopyridine (DMAP) and the second solution obtained in step 2 to the first solution obtained in step 1, then adding pyridine, reacting the reaction system in an oil bath, and after the reaction is completed, dripping the obtained mixed solution into an ultrasonic ice-water bath to obtain a light yellow emulsion; then dialyzing the obtained light yellow emulsion to obtain a micellar solution; finally, filtering the obtained micellar solution to obtain the zwitterionic nanomedicine.

7. The method for preparing the zwitterionic nanomedicine for resisting skin inflammation according to claim 6, characterized in that: The reaction conditions of the reaction system in step 3 in an oil bath are: stirring at 37° C. for 18-24 h.

8. The method for preparing the zwitterionic nanomedicine for resisting skin inflammation according to claim 6, characterized in that: After the reaction in step 3 is completed, the obtained mixed solution is added dropwise into an ultrasonic ice-water bath at a dropping speed of 8-10 L / min.

9. The method for preparing the zwitterionic nanomedicine for resisting skin inflammation according to claim 6, characterized in that: In step 3, the obtained light yellow emulsion is dialyzed using a dialysis bag with a molecular weight cutoff of 3000-3500 Da for 8-10 h.

10. The method for preparing the zwitterionic nanomedicine for resisting skin inflammation according to claim 6, characterized in that: In step 3, the micellar solution is finally filtered using a filter with a pore size of 250 nm or 450 nm.

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