Ionic liquid and application thereof

Abstract

The invention discloses an ionic liquid and application thereof, the ionic liquid comprises organic acid and matrine, and the organic acid and the matrine react to form the ionic liquid; the citric acid and the matrine are combined to form the ionic liquid, so that the ionic liquid has certain safety on cells formed by human keratinization, the skin permeation effect is remarkably enhanced, a new thought is provided for subsequent percutaneous treatment, and the ionic liquid has important clinical significance.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to an ionic liquid and application thereof. Background Art

[0002] Transdermal administration refers to the delivery of drugs to the site of action through skin absorption to achieve systemic or local therapeutic effects for the prevention or treatment of diseases. Commonly used dosage forms include patches, coatings, ointments, plasters, powders, and cataplasms. Compared with other methods, transdermal absorption has many advantages, such as increasing patient compliance, continuous drug release to increase and maintain blood drug concentrations, and avoiding first-pass metabolism, protecting sensitive drugs from adverse gastrointestinal conditions and reducing gastrointestinal irritation. For example, compared with subcutaneous injection, this painless, low infection possibility, non-deep invasive and manageable approach shows higher compliance in people. The main obstacle to transdermal drug delivery is the skin itself, whose outer most dense stratum corneum (SC) is composed of keratin-rich cells embedded in multiple lipid bilayers, and its low permeability severely limits the delivery of most drug molecules. Therefore, there are obstacles that need to be solved urgently to enable local administration, such as enhancing the permeability of drugs through the skin, improving the stability of drugs, and increasing the safety of drug carriers.

[0003] There are several strategies that can be used to promote transdermal penetration: (1) physical penetration enhancers, such as iontophoresis. Iontophoresis is the process of ionic drugs passing through the skin under the action of an electric field. This method is mostly used in the study of drug delivery systems for biomacromolecules such as proteins and proteins; electroporation is a method of using an instantaneous high-voltage pulse electric field to form temporary, reversible hydrophilic channels in lipid bilayers such as cell membranes to increase permeability; ultrasound introduction is a method of using ultrasonic energy to promote drug transdermal penetration; microneedle technology forms a micron-scale hole array in the stratum corneum of the skin through the puncture of microneedles, thereby achieving drug introduction; needle-free injection drug delivery, laser-promoted transdermal drug delivery, thermal energy-promoted transdermal drug delivery, etc. However, physical penetration enhancement methods also have certain advantages and disadvantages. Taking patches as an example, it is found that the active ingredients of the drugs in the patches are not only released when applied, but also continue to be released after the patches are removed. This is a great factor affecting skin irritation. Therefore, in order to reduce irritation, it is best to find a way to remove the residual drug after the patches are removed. (2) Chemical penetration enhancers (CPEs), including water, azone, sulfoxide and its analogs, surfactants, ethanol, fatty alcohols, pyrrolidines, natural transdermal penetration enhancers, etc. The mechanism of action of traditional chemical penetration enhancers is to change the skin properties, disrupt the arrangement of lipids, form gaps in tight connections, and thus increase the water content of the stratum corneum. Its penetration enhancement process is non-specific, and high concentrations or long-term use can cause adverse reactions such as skin irritation and allergic reactions. CPEs have defects such as low biocompatibility and high irritation. (3) Biological penetration enhancers mainly include the following categories: penetration peptides, metabolic regulators, amino acid derivatives and ceramide and its analogs. However, before they are widely used, at least the following problems need to be solved: establishment of in vitro and in vivo correlation, drugability, establishment of dose-effect relationship, safety issues, etc. (4) Pharmaceutical methods such as microemulsions, liposomes, nanoparticles, etc. This method has certain advantages and disadvantages. The advantages are to improve drug permeability, adapt to specific treatment needs, and enhance drug stability. The disadvantages are that it may cause skin irritation, cost issues, safety issues, etc.

[0004] In summary, the penetration enhancers in the prior art have low safety, are prone to cause skin irritation, and the penetration enhancing effect needs to be improved. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide an ionic liquid that is safe and has a good penetration-enhancing effect; the second purpose of the present invention is to provide the use of the ionic liquid as a penetration enhancer in transdermal penetration enhancement.

[0006] Technical solution: The ionic liquid described in the present invention includes an organic acid and matrine, which react to form an ionic liquid.

[0007] Preferably, the molar ratio of the organic acid to matrine is (0.5-2): 1. A uniform ionic liquid can be formed within the ratio range, and the ionic liquid is unstable below or above this ratio, and crystals will precipitate over a long period of time.

[0008] Further preferably, the molar ratio of the organic acid to matrine is 2:1. The structural formula of matrine is shown below:

[0009]

[0010] Preferably, the organic acid is citric acid, and the structural formula of citric acid is as follows:

[0011]

[0012] The carboxyl group of the organic acid reacts with the amine group of matrine to form a quaternary ammonium group to obtain an ionic liquid. Taking citric acid as an example, the reaction process is as follows:

[0013]

[0014] The method for forming the ionic liquid by reaction is: dissolving the organic acid and matrine in a solvent for reaction, and then removing the solvent to obtain the ionic liquid.

[0015] Preferably, the solvent is water, the reaction temperature is 20-30° C., and the reaction time is 8-10 h.

[0016] Preferably, the solvent is removed by first rotary evaporation and then drying in a drying oven.

[0017] The invention discloses an application of the ionic liquid as a penetration enhancer in promoting transdermal penetration.

[0018] Preferably, the penetration enhancer is used in a concentration of 2 to 10% (w / v) in transdermal penetration enhancement; more preferably, the concentration is 5 to 10% (w / v).

[0019] Invention mechanism:

[0020] Citric acid is an organic acid extracted from black plum, and has multiple effects such as antioxidant, anti-inflammatory, antibacterial and anti-thrombotic.

[0021] Matrine is an alkaloid extracted from the dried roots, plants and fruits of the leguminous plant Sophora flavescens through organic solvents such as ethanol. As a medicinal plant, Sophora flavescens has a history of more than 2,000 years in my country according to written records. Its main functions include clearing away heat, diuresis, killing insects, and removing dampness. It also has multiple effects such as antiviral, anti-tumor, and anti-allergic effects. It is the main active ingredient in Sophora flavescens.

[0022] Ionic liquids are salts that are liquid at or near room temperature and are composed entirely of anions and cations, also known as low-temperature molten salts. In general, ionic liquids are composed of organic cations and inorganic anions. ILs can promote transcellular and intercellular transport of drugs by disrupting the integrity of skin cells, fluidizing SCs, establishing skin diffusion channels, and extracting lipid components in SCs.

[0023] The present invention adopts citric acid and matrine to form ionic liquids (ILs). The combined effect of the two has a significant effect on enhancing skin penetration and has a certain safety for human keratinocytes (HaCaT). Compared with other transdermal technologies, the ionic liquid also greatly reduces damage to the epidermis and various toxic side effects.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: by combining citric acid and matrine to form an ionic liquid, it has a certain safety for human keratinocytes (HaCaT), significantly enhances the skin penetration effect, provides a new idea for subsequent transdermal treatment, and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the appearance of the ionic liquid (IL) prepared by using citric acid and matrine in Examples 1 to 3;

[0026] Figure 2 is the nuclear magnetic resonance hydrogen spectrum of citric acid, matrine and the ionic liquid prepared in Example 1 ( 1 H NMR);

[0027] Figure 3 is the infrared spectra (IR) of citric acid, matrine and the ionic liquid prepared in Example 1;

[0028] Figure 4 It is the graph obtained by isothermal titration calorimeter (ITC) of citric acid and matrine;

[0029] Figure 5 The particle size diagram of the ionic liquids of Examples 1 to 3 was measured by a dynamic light scattering instrument (DLS);

[0030] Figure 6 is a graph of pH values ​​of ionic liquids of different molar ratios in Examples 1 to 3 measured by a pH meter;

[0031] Figure 7 is the cytotoxicity graph of citric acid, matrine and the ionic liquid prepared in Example 1 to HaCaT;

[0032] Figure 8 is the standard curve between FITC-Dextran concentration and fluorescence intensity;

[0033] Fig. 9 It is a diagram of the in vitro transdermal effects of ionic liquids of different molar ratios in Examples 1 to 3;

[0034] Fig.10 This is a graph showing the in vitro transdermal effects of different concentrations of citric acid:matrine = 2:1M ionic liquid;

[0035] Fig.11 This is a graph comparing the difference in transdermal data between different work areas. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with embodiments.

[0037] Example 1

[0038] Experimental materials: citric acid, matrine, FITC-Dextran, DMEM culture medium, fetal bovine serum (FBS) and penicillin-streptomycin mixture (100×), PBS (Keygen Biotechnology), Cell Counting Kit-8 (CCK8),

[0039] Experimental instruments: 1 / 10,000 balance, 1 / 100,000 balance, heating stirrer, rotary evaporator, vacuum drying oven, isothermal titration calorimeter (ITC) (TA Company, USA), dynamic light scattering (DLS), pH meter, 96-well plate, VS-1300L-U clean bench (Suzhou Antai Air Technology Co., Ltd.), carbon dioxide incubator (Thermo Scientific), Invitrogen blood cell counting plate, inverted fluorescence microscope (Leica Company, Germany), and microplate reader.

[0040] 1.82 g of citric acid and 1.18 g of matrine were weighed in a molar ratio of 2:1, and the weighed drugs were dissolved in 30 mL of ultrapure water, and heated in a water bath at 25°C for 8 h to prepare a uniform solution of about 10% (w / v); most of the aqueous solution in the solution was further removed by rotary evaporation, with the temperature set at 70°C and the rotation speed set at 40 r / min to form a concentrated solution, and finally the concentrated solution was dried in a vacuum drying oven at a temperature of 60°C to achieve a constant weight state to obtain an ionic liquid.

[0041] Example 2

[0042] On the basis of Example 1, the molar ratio of citric acid to matrine was changed to 1:1, 1.31 g of citric acid and 1.69 g of matrine were weighed, and the other conditions remained unchanged.

[0043] Example 3

[0044] On the basis of Example 1, the molar ratio of citric acid to matrine was changed to 1:2, 0.84 g of citric acid and 2.16 g of matrine were weighed, and the other conditions remained unchanged.

[0045] 1. Characterization of ionic liquids

[0046] (1) Figure 1 As shown, the ionic liquids prepared by using citric acid and matrine in different molar ratios in Examples 1 to 3 are all light yellow and transparent and have certain fluidity.

[0047] (2) By nuclear magnetic resonance spectroscopy ( 1 H NMR), infrared (IR), isothermal titration calorimeter (ITC), dynamic light scattering (DLS) and pH meter were used to characterize the ionic liquids. Figures 2 to 6 shown.

[0048] pass Figure 2 The nuclear magnetic resonance hydrogen spectrum shows that citric acid reacts with matrine and connects an H to the No. 1 N of matrine to form a quaternary ammonium group, which is an electron-withdrawing group, causing the H on the adjacent C to shift to a low field, that is, to a high chemical shift; citric acid loses H to form an electron-donating group, causing the H on the methylene to shift to a high field, that is, to a low chemical shift.

[0049] pass Figure 3 Infrared spectrum shows that 3500-3000cm -1 :N + -H stretching vibration; 2949cm -1 :Alkyl CH stretching vibration; 894cm -1 : CH out-of-plane bending vibration; 2600cm -1 : Carboxylic acid COH stretching vibration and bending vibration double frequency and combination frequency, which is the characteristic absorption peak of carboxylic acid; 1415cm -1 : Carboxylic acid and alcohol COH in-plane bending vibration; 1334 cm -1 : Carboxylic acid COH stretching vibration; 963cm -1 : Carboxylic acid COH out-of-plane bending vibration; 1725cm -1 : Carboxylic acid C=O stretching vibration; 1584cm -1 :Valerolactam C=O stretching vibration; 1584, 1415cm -1 : antisymmetric and symmetric stretching vibrations of carboxylate COO; 802 cm -1 : COO-angle vibration of carboxylate; 471cm -1 : Carboxylate COO-out-of-plane rocking vibration; 1204 cm -1 : cyclic lactam CN stretching vibration; 1113cm -1 :CC stretching vibration; 1072cm -1 : Alcohol CO stretching vibration; 679, 598cm -1: Bending vibration of NH and O=CN of valerolactam.

[0050] Depend on Figure 4 It can be obtained that the ITC reaction shows that there is an obvious heat change (exothermic) in the process of citric acid titration of matrine, indicating the existence of binding. According to Gibbs free energy, △G<0, it is calculated that the reaction can proceed spontaneously when the temperature is greater than -423.98℃.

[0051] pass Figure 5 DLS shows that when the ionic liquid is diluted in water, it has a certain particle size and is not a single solution.

[0052] Depend on Figure 6 It can be seen that due to the combination of citric acid and matrine in different ratios, the ionic liquid is diluted to a concentration of 5% (w / v), so the pH value measured by the pH meter here is acidic.

[0053] 2. Ionic liquid transdermal penetration test

[0054] (1) In vitro cytotoxicity assay

[0055] Logarithmically growing HaCaT cells were inoculated into 96-well cell culture plates (10,000 cells per well) and incubated overnight at 37°C and 5% CO2. Different concentrations of the test samples were added according to the experimental design. After 24 hours of co-culture, 10 μl of CCK-8 solution was added to each well, and the incubation continued for about 2 hours. The absorbance at 450 nm was measured by an ELISA reader. The calculation formula for cell viability is as follows:

[0056] Cell viability (%) = [(experimental well - blank well) / (control well - blank well)] x 100%.

[0057] The results of HaCaT cell proliferation inhibition by CCK-8 method were as follows Figure 7 As shown in the figure, it can be seen that citric acid, matrine, and the ionic liquids prepared in Examples 1 to 3 all have certain safety.

[0058] (2) In vitro transdermal diffusion test

[0059] Cell line: Human keratinocytes (HaCaT), purchased from Servicebio.

[0060] Animals: Female SD rats weighing 220-250 g were selected.

[0061] Establishment of standard curve: accurately weigh 1 mg FITC-Dextran and dissolve it in 1 mL purified water, vortex to mix it evenly, and prepare solutions of different concentrations by gradient dilution method, namely 10, 5, 1, 0.5, 0.1, 0.05, 0.01 μg / mL, and measure its fluorescence by microplate reader, setting the excitation wavelength to 488 nm and the emission wavelength to 521 nm. Perform linear regression, draw the standard curve with FITC-Dextran concentration as the horizontal axis and fluorescence intensity as the vertical axis, and get the regression equation Y=1902X+339.9, R 2 =0.9963, indicating a good linear relationship ( Figure 8 ).

[0062] Preparation of reagents: Weigh different molar ratios of ionic liquids and dissolve them in purified water to a concentration of 5% (w / v), then weigh a certain amount of FITC-Dextran (1 mg / mL) and dissolve it in the above solution, adjust the pH value with a 10% (w / v) NaOH solution, measure the initial fluorescence intensity to the same level using an enzyme reader, and stir at room temperature overnight using a magnetic stirrer.

[0063] Skin preparation: Select the abdominal skin of SD rats and shave the abdominal hair with an electric shaver and depilatory cream. Wash the fat and mucosal tissue remaining on the skin tissue with PBS, absorb the residual liquid on the skin surface with filter paper, and finally wrap it in tin foil and store it in a -20℃ refrigerator to ensure the freshness of the mouse skin for use the next day. The mouse skin needs to be used within 1 to 2 weeks.

[0064] Franz diffusion cells and ex vivo mouse skin were used to conduct in vitro percutaneous permeation experiments on the prepared different preparations. First, the ex vivo mouse skin that had been processed the day before was taken out and thawed at room temperature. After thawing, it was cleaned with PBS, and the surface moisture of the mouse skin was wiped dry with filter paper. Then it was fixed in the diffusion cell with the back facing up. PBS buffer solution was added to the receiving cell, and 800 μL of blank FITC-Dextran and solutions with a molar ratio of citric acid: matrine of 2:1, 1:1, and 1:2 M were injected into the donor cell. The diffusion area A of the diffusion cell was 3.14 cm 2 , the volume of the receiving pool was 8.3 mL, the stirring speed was set to 500 r / min, the temperature was 37°C, and finally the opening of the supply pool was sealed with a sealing film. The sampling times were set to 0.5, 1, 2, 4, 8, 12, and 24 h, respectively. When the sampling time was up, 0.3 mL of sample was drawn from the receiving pool with a syringe, and an equal volume of PBS was added to the receiving pool at the same time. The fluorescence intensity was measured by an ELISA instrument, and the excitation wavelength was set to 488 nm and the emission wavelength was set to 521 nm. The cumulative percentage of FITC-Dextran that penetrated through the skin (Q n). Calculated according to the following equation:

[0065]

[0066] In this formula, V is the volume of the receiving cell (mL); A is the diffusion area of ​​the diffusion cell (cm 2 );C n : Drug concentration at the nth sampling point (μg / mL). Linear regression was performed on the cumulative drug permeation per unit area Q versus time t to obtain the cumulative permeation curve.

[0067] The Franc diffusion cell was used to measure the transdermal effects of ionic liquids with different ratios. Fig. 9 As shown, it was found that the transdermal penetration enhancement effect of citric acid:matrine = 2:1M was the best.

[0068] Based on the above results, the transdermal effects of different concentrations were further tested for this ratio. The results are as follows Fig.10 As shown, it can be seen that the effect of 5% concentration is the best, which is increased by 22 times compared with the blank group.

[0069] The ionic liquid prepared in Example 1 of the present invention with a concentration of 5 wt% was used as a penetration enhancer to compare the molecular weight and penetration enhancement multiple of the existing penetration enhancers. The results are as follows: Fig.11 shown.

[0070] Depend on Fig.11 It can be seen that the present invention has certain advantages in terms of both the molecular weight of penetration enhancement and the penetration enhancement multiple.

Claims

1. An ionic liquid, characterized in that It includes an organic acid and matrine, which react to form an ionic liquid.

2. The ionic liquid according to claim 1, characterized in that The molar ratio of the organic acid to matrine is (0.5-2):

1.

3. The ionic liquid according to claim 2, characterized in that The molar ratio of the organic acid to matrine is 2:

1.

4. The ionic liquid according to claim 1, characterized in that The organic acid is citric acid.

5. The ionic liquid according to claim 1, characterized in that The method for forming the ionic liquid by reaction is: dissolving the organic acid and matrine in a solvent for reaction, and then removing the solvent to obtain the ionic liquid.

6. The ionic liquid according to claim 5, characterized in that The solvent is water, and the reaction temperature is 20-30°C.

7. The ionic liquid according to claim 5, characterized in that The solvent removal is performed by first rotary evaporation and then drying in a drying oven.

8. Use of the ionic liquid according to any one of claims 1 to 7 as a penetration enhancer in enhancing transdermal penetration.

9. The use according to claim 8, characterized in that: The penetration enhancer is used in a concentration of 2-10% (w / v) in transdermal penetration enhancement.

10. The use according to claim 9, characterized in that: The concentration is 5-10% (w / v).

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