Self-assembled in-situ nanogel, preparation method and application thereof

Abstract

The application discloses a kind of self-assembly in-situ nanogel, preparation method and application, belong to the field of pharmaceutical preparation;Active ingredient of self-assembly in-situ nanogel disclosed in the application is prepared by short-chain fatty acid and natural flavonoids through chemical synthesis, resistant to gastric acid, and the gel matrix prepared in-situ forms nanogel under the condition of pH=6.5 and 37 DEG C.Self-assembly in-situ nanogel solves the problem of drug degradation caused by strong acidity of gastric juice, not only solves the palatability problem of short-chain fatty acid and avoids its large absorption in stomach, but also gives active ingredient target adhesion at inflammatory site of intestine, improves the action time and action efficiency in intestinal tract.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to a self-assembled in-situ nanogel, a preparation method and application thereof. BACKGROUND

[0002] Intestinal mucosal barrier is the most important and key defense barrier in the intestinal barrier. It is composed of intestinal mucosal epithelial cells, intercellular tight junctions and bacterial membranes, which can effectively prevent bacteria from penetrating the mucosa into the deep tissues of the body, and is the structural basis of the intestinal mucosal barrier. It is an important defense mechanism against the hostile environment in the intestinal lumen. An intact intestinal barrier not only allows specific solutes beneficial to the host to pass through, but also effectively prevents the passage of antigens, bacterial toxins and pathogens. The intestinal barrier is damaged, characterized by increased intestinal permeability, which usually increases the risk of intestinal infection, promotes the translocation of intestinal bacteria, toxins and antigens to subepithelial tissues, and ultimately leads to inflammatory reactions and gastrointestinal diseases. For a long time, antibiotics, glucocorticoids and mesalazine have been used to treat intestinal inflammation, and the long-term use of antibiotics has brought problems such as bacterial resistance and drug residues. The adverse reactions caused by long-term use of glucocorticoids cannot be ignored, and they can also severely weaken the body's resistance. Mesalazine treatment is prone to relapse. Therefore, short-chain fatty acids and natural compounds that have inhibitory effects on different links of the classical inflammatory response pathway NF-κB signaling pathway are linked by organic synthesis to form a complex with dual therapeutic effects, and nanoparticles are formed by means of self-assembly technology. The synergistic anti-inflammatory mechanism enhances its anti-inflammatory activity, repairs the intestinal barrier, and solves the treatment problem of intestinal inflammation in the environment of reducing antibiotics and hormone drugs. Therefore, finding natural drugs as substitutes is the trend of the times.

[0003] Short-chain fatty acids play an important role in maintaining intestinal homeostasis. Short-chain fatty acids are produced by the fermentation of dietary carbohydrates by large intestinal microorganisms in mammals, and are mainly metabolized in the epithelial mucosa. It is rapidly absorbed and can be used as the main energy source for the distal ileum and large intestinal epithelial cells, and stimulates the growth of small intestinal epithelium. Short-chain fatty acids inhibit the phosphorylation of p65, one of the NF-κB dimer subunits. Reduce p65 nuclear accumulation, leading to a decrease in the transcription of a series of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β, and even NF-κB itself.

[0004] Flavonoids are natural food antioxidants, and have antibacterial, antiviral, anti-inflammatory and antiallergic effects in vivo. So far, there has been no objection to their safety. However, flavonoids are almost insoluble in water, have little intestinal absorption, and thus limit their application to a certain extent due to their low oral bioavailability.

[0005] In situ gels are composed of environmentally sensitive polymers whose structure changes in response to small changes in specific conditions such as pH, temperature and ionic strength in the environment. The in situ formed gel is a liquid during the administration process, and then quickly gels at the target site to form a viscoelastic gel in response to environmental changes. Finally, the drug is slowly released under physiological conditions. Therefore, the residence time of the in situ gel will be extended, and the drug will be released in a sustained manner, thereby improving the bioavailability, minimizing systemic absorption and reducing the frequent administration regimen, thereby improving patient compliance. Natural polymers are not only widely used in the food industry, but also widely used in pharmaceutical technology because of their lower toxicity, biocompatibility and biodegradability. Incorporating therapeutic agents into a natural polymer matrix can protect active compounds from degradation, enhance absorption, improve therapeutic effect and reduce the frequency of administration.

[0006] Among the different routes of drug administration, oral administration has the advantages of convenience, painlessness, good compliance, etc. and is considered to be the most widely used administration route at present, and is the focus of drug delivery system development. The drugs administered by this route are inevitably exposed to the complex gastrointestinal microenvironment (pH = 1-3 and a large number of functional enzyme degradation), which seriously affects the absorption and utilization of the drug. Short-chain fatty acids can be easily absorbed in the stomach, which greatly limits their possibility of reaching the intestine, and their unpleasant odor is an important factor limiting their application, so there are almost no oral preparations of short-chain fatty acids approved for marketing at home and abroad. Therefore, new strategies are urgently needed to improve the ability of these drugs to pass through the harsh stomach environment and their stability in the gastrointestinal physiological environment to promote the oral absorption of drugs. SUMMARY

[0007] The purpose of the present application is to provide a self-assembled in situ nanogel, a preparation method and its application, to solve the problem of drug degradation caused by the strong acidity of gastric juice, and to impart the active ingredient with targeted adhesion at the inflammatory site of the intestinal segment, thereby improving its action time and efficiency in the intestine. The degradation of the active ingredient in the intestinal segment allows short-chain fatty acids and flavonoids to play a role in different aspects, synergistically anti-inflammatory to enhance their anti-inflammatory activity, while repairing the intestinal barrier, providing a new model for the treatment of digestive tract inflammation.

[0008] To achieve the above-mentioned purpose, the present application provides a self-assembled in situ nanogel, comprising an active ingredient and a gel matrix,

[0009] The active ingredient is formed by esterification of short-chain fatty acids and flavonoids.

[0010] Preferably, the self-assembled in situ nanogel comprises, by mass percentage: 0.1-1% of the active ingredient, 22-26% of the gel matrix, and the balance being water.

[0011] Preferably, the self-assembled in-situ nanogel comprises, in percentage by mass: 0.2% of active ingredient, 24% of gel matrix, and the balance being sterilized water.

[0012] Preferably, the short-chain fatty acid is one of acetic acid, propionic acid and butyric acid, and more preferably propionic acid;

[0013] The flavonoid compound is one of quercetin, apigenin and myricetin, and more preferably apigenin;

[0014] The mass ratio of the short-chain fatty acid to the flavonoid compound is 1:1.2.

[0015] Preferably, the active ingredient is prepared as follows:

[0016] The short-chain fatty acid is added to the organic solvent during stirring, and a solution A is obtained by reacting at 80-90°C for 2-3h, with a stirring speed of 500-2000r / min and a titration speed of 0.2-1mL / min;

[0017] The solution A is cooled to 30-60°C, and the flavonoid compound is added to obtain an active ingredient initial product;

[0018] The active ingredient initial product is extracted with ethyl acetate, and column chromatography is used for separation and purification to obtain the active ingredient.

[0019] More preferably, the active ingredient is prepared as follows:

[0020] The short-chain fatty acid is added to the organic solvent during stirring, and a solution A is obtained by reacting at 80°C for 2h, with a stirring speed of 1000r / min and a titration speed of 1mL / min;

[0021] The solution A is cooled to 50°C, and the flavonoid compound is added to obtain an active ingredient initial product;

[0022] The active ingredient initial product is extracted with ethyl acetate, and column chromatography is used for separation and purification to obtain the active ingredient.

[0023] More preferably, the active ingredient is prepared as follows:

[0024] The propionic acid is added to the organic solvent thionyl chloride during stirring, and a solution A is obtained by reacting at 80°C for 2h, with a stirring speed of 1000r / min and a titration speed of 1mL / min;

[0025] The solution A is cooled to 50°C, and the apigenin is added to obtain an active ingredient initial product;

[0026] The active ingredient initial product is extracted with ethyl acetate, and column chromatography is used for separation and purification to obtain the active ingredient.

[0027] Preferably, the organic solvent is oxalyl chloride or thionyl chloride.

[0028] Preferably, the gel matrix comprises one or more of polyacrylic acid, poloxamer, chitosan (CS) and polyvinyl alcohol.

[0029] The present application provides a preparation method of the self-assembled in-situ nanogel, comprising the following steps:

[0030] The active ingredient is dissolved in anhydrous ethanol, the gel matrix is added under stirring, and the self-assembled in-situ nanogel is obtained by continuing stirring.

[0031] The preparation method of the self-assembled in-situ nanogel preferably comprises the following steps:

[0032] The active ingredient is dissolved in 5 mL of anhydrous ethanol, the gel matrix is added under stirring at 500-2000 r / min, and the self-assembled in-situ nanogel is obtained by continuing stirring for 2-12 h.

[0033] The preparation method of the self-assembled in-situ nanogel more preferably comprises the following steps:

[0034] The active ingredient is dissolved in 5 mL of anhydrous ethanol, the gel matrix is added under stirring at 1000 r / min, and the self-assembled in-situ nanogel is obtained by continuing stirring for 4 h.

[0035] The self-assembled in-situ nanogel is used for preparing an anti-inflammatory drug.

[0036] The self-assembled in-situ nanogel is used for preparing an anti-digestive tract inflammation drug.

[0037] Preferably, the self-assembled in-situ nanogel can be used for preparing an anti-intestinal inflammation drug.

[0038] The self-assembled in-situ nanogel has synergistic anti-inflammatory activity, can stably pass through the stomach, and can form a gel in-situ at an intestinal inflammation site to promote recovery of intestinal inflammation. Moreover, the self-assembled in-situ nanogel has temperature and pH response performance, can improve stability of the intestinal tract, and realizes intestinal tract directional delivery. When the temperature increases, the hydrophobic polyoxypropylene (structural unit of poloxamer) block dehydrates, and the poloxamer chain forms a micellar structure. Chitosan forms a cationic hydrogel network in water due to its primary amine groups, and it shows pH response behavior by swelling at an acidic pH value (pH < pKa) and shrinking at an alkaline pH value (pH > pKa). The ester bond formed by the carboxyl group of the short-chain fatty acid and the phenolic hydroxyl group of the flavonoids in the self-assembled in-situ nanogel well solves the problem of instability in an acidic stomach environment.

[0039] Compared with the prior art, the present application has the following advantages and technical effects:

[0040] The active ingredient in the self-assembled in-situ nanogel of the present application is prepared by chemical synthesis from short-chain fatty acids and natural flavonoids, has anti-inflammatory activity, and is resistant to gastric acid. The active ingredient forms a nanogel in-situ with the prepared gel matrix at pH 6.5 and 37℃. The self-assembled in-situ nanogel solves the problem of drug degradation caused by the strong acidity of gastric juice, and endows the active ingredient with targeted adhesion at the inflammatory site of the intestinal segment, thereby prolonging its action time and improving its action efficiency in the intestinal tract. The degradation of the active ingredient in the intestinal segment enables the short-chain fatty acids and flavonoids to exert their effects from different aspects, thereby synergistically enhancing their anti-inflammatory activity and repairing the intestinal barrier, thus providing a new mode for the treatment of digestive tract inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0041] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the application, and serve as an explanation of the illustrative embodiments of the present application, and are not intended to impose any undue limitations on the present application. In the drawings:

[0042] Figure 1 It is a physical picture of the self-assembled in-situ nanogel in Test Example 1;

[0043] Figure 2 It is a thin-layer chromatogram of the active ingredient in Test Example 1;

[0044] Figure 3 It is the peak time of the active ingredient under different conditions in Test Example 1, wherein (a) is the peak time of the active ingredient under the simulated gastric environment of pH = 2, (b) is the peak time of the active ingredient under the simulated intestinal fluid environment of pH = 5, (c) is the peak time of the active ingredient under the simulated intestinal fluid environment of pH = 6, (d) is the peak time of the active ingredient under the simulated intestinal fluid environment of pH = 7, and (e) is the peak time of apigenin butyrate dissolved directly in methanol;

[0045] Figure 4 It is the intestinal wall adhesion performance result of the gel group and the solution group after simultaneous oral administration in Test Example 2;

[0046] Figure 5 It is the inhibitory effect of the in-situ nanogel on the pro-inflammatory factors secreted by RAW264.7 in Test Example 3;

[0047] Figure 6 It is a scanning electron microscope image of the self-assembled nanoparticles without the gel matrix in the in-situ nanogel in Test Example 2;

[0048] Figure 7 It is the rheological property of the poloxamer and chitosan gel matrix of different concentrations in Test Example 2. DETAILED DESCRIPTION

[0049] The following detailed description of various exemplary embodiments of the application will not be considered limiting of the application, but rather a description of certain aspects, features, and embodiments of the application.

[0050] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only exemplary of the various preferred embodiments and are not intended to be limiting of the application. Other embodiments will occur to those skilled in the art upon consideration of this disclosure. Additionally, other combinations, permutations, applications and modifications of the application will occur to those skilled in the art upon consideration of this disclosure. The application is not to be limited, however, to the specific embodiments disclosed.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference for the disclosure and

[0052] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of this application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary only and are not intended to be limiting.

[0053] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0054] The active ingredient in the self-assembled in-situ nanogel of the present application is prepared by chemical synthesis from short-chain fatty acids and natural flavonoids, has anti-inflammatory activity, and is resistant to gastric acid. The self-assembled in-situ nanogel solves the problem of drug degradation caused by the strong acidity of gastric juice, not only solves the palatability problem of short-chain fatty acids and avoids their massive absorption in the stomach, but also endows the active ingredient with targeted adhesion at the inflammatory site in the intestinal segment, thereby improving the action time and efficiency in the intestinal tract. The self-assembled in-situ nanogel has a synergistic anti-inflammatory mechanism to enhance the anti-inflammatory activity of the active ingredient, repairs the intestinal barrier, and provides a new mode for the treatment of digestive tract inflammation.

[0055] Quercetin, apigenin and myricetin in the embodiments of the present application are purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.; acetic acid, propionic acid and butyric acid are purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; polyacrylic acid, poloxamer, chitosan and polyvinyl alcohol are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0056] Anti-inflammatory effect of active ingredients in Example 1

[0057] 1.1 Active ingredients and preparation

[0058] The composition of the active ingredients is shown in Table 1.

[0059] Table 1 Composition of active ingredients

[0060]

[0061] Preparation steps:

[0062] After 10 mL of thionyl chloride and 0.015 mol of short-chain fatty acid are reacted at 80°C for 2 h, 0.005 mol of flavonoid compound is added, and the initial product of ester is generated after 9 h of reaction at 50°C. Ethyl acetate is used for extraction, and silica gel column is used for column chromatography separation and purification. Rotary evaporation is performed at 50°C for 30 min (when there is no obvious flow, the same below), and active ingredients are obtained after vacuum drying.

[0063] 1.2 Anti-inflammatory effect of active ingredients

[0064] Table 2 Anti-inflammatory effect of active ingredients

[0065]

[0066] As shown in Table 2, propionic acid and apigenin organic synthesis propionic acid-apigenin complex have the best inhibitory effect on pro-inflammatory factor TNF-α.

[0067] Example 2 Self-assembled in-situ nanogel and determination of gelation temperature

[0068] 2.1 Composition 1 and preparation of self-assembled in-situ nanogel

[0069] The raw materials of the self-assembled in-situ nanogel composition 1 are shown in Tables 3-1 to 3-3.

[0070] Table 3-1 Composition 1 Group 1 of self-assembled in-situ nanogel

[0071]

[0072] Table 3-2 Composition 1 Group 2 of self-assembled in-situ nanogel

[0073]

[0074]

[0075] Preparation steps:

[0076] (1) 10 mL of thionyl chloride was reacted with 0.015 mol of propionic acid at 80°C for 2 h, then 0.005 mol of apigenin was added, and the mixture was reacted at 50°C for 9 h to form an initial product of apigenin propionate, which was extracted with ethyl acetate and separated and purified by column chromatography, and then rotary evaporation was performed.

[0077] After vacuum drying, the active ingredient was obtained;

[0078] (2) The active ingredient was dissolved in ethanol to form an active ingredient solution, which was stirred at 25°C for 8 h at a stirring speed of 1000 r / min to form active ingredient self-assembled nanoscale particles.

[0079] (3) According to the cold-sol method, chitosan was dissolved in 1 wt% acetic acid water and poloxamer to form a uniform gel matrix, and the active ingredient self-assembled nanoscale particles prepared in (2) were added to the gel matrix and stirred at a stirring speed of 1000 r / min to form self-assembled in-situ nanogel.

[0080] 2.2 Gelling temperature of the self-assembled in-situ nanogel at pH 1.5 and pH 6.5:

[0081] Table 4 Gelling temperature of the self-assembled in-situ nanogel composition 1

[0082]

[0083] As can be seen from Table 4, the gelling temperature at different pH is greater than 40°C, and no gelling occurs at physiological temperature 37°C.

[0084] Example 3 Self-assembled in-situ nanogel composition 2 and determination of its gelling temperature

[0085] 3.1 Self-assembled in-situ nanogel composition 2 and preparation

[0086] The raw materials of the self-assembled in-situ nanogel composition 2 are shown in Tables 5-1 to 5-3.

[0087] Table 5-1 Self-assembled in-situ nanogel composition 2 group 1

[0088]

[0089]

[0090] Table 5-2 Self-assembled in-situ nanogel composition 2 group 2

[0091]

[0092] Table 5-3 Self-assembled in-situ nanogel composition 2 group 3

[0093]

[0094] Preparation step:

[0095] (1) 10 mL of thionyl chloride was reacted with 0.015 mol of propionic acid at 80°C for 2 h, then 0.005 mol of apigenin was added, and the mixture was reacted at 50°C for 9 h to form an initial product of apigenin propionate, which was extracted with ethyl acetate and separated and purified by column chromatography, and then rotary evaporation was performed.

[0096] After vacuum drying, the active ingredient was obtained;

[0097] (2) The active ingredient was dissolved in ethanol to form an active ingredient solution, which was stirred at 25°C for 8 h at a stirring speed of 1000 r / min to form active ingredient self-assembled nanoscale particles;

[0098] (3) According to the cold-sol method, chitosan was dissolved in 1 wt% acetic acid water and poloxamer to form a uniform gel matrix, and the active ingredient self-assembled nanoscale particles prepared in (2) were added to the gel matrix and stirred at a stirring speed of 1000 r / min to form in-situ nanogel.

[0099] 3.2 Gelling temperature of the self-assembled in-situ nanogel at pH 1.5 and pH 6.5:

[0100] Table 6 Gelling temperature of the self-assembled in-situ nanogel composition 2

[0101]

[0102] As shown in Table 6, the gelling was tested at 40°C, and the results showed that, in the case of 3 wt% poloxamer 188, the gelling temperature of a solution of 20 wt% poloxamer 407 had a difference at pH = 1.5 and pH = 6.5. When the CS concentration was 0.5 wt%, the gelling temperature at pH = 1.5 and pH = 6.5 was not lower than 40°C; when the CS concentration was 1 wt%, the gelling temperature at pH = 1.5 was not lower than 40°C, and the gelling temperature at pH = 6.5 was about 37°C; when the CS concentration was 1 wt%, the gelling temperature at pH = 1.5 was not lower than 40°C, and the gelling temperature at pH = 6.5 was about 35°C. Therefore, the CS concentration of 1 wt% to 1.5 wt% was a more suitable concentration.

[0103] Example 4 Self-assembled in-situ nanogel composition 2 and determination of its gelling temperature

[0104] 4.1 Self-assembled in-situ nanogel composition 3 and preparation

[0105] The self-assembled in-situ nanogel composition 3 is shown in Tables 7-1 to 7-3.

[0106] Table 7-1 Composition of self-assembled in-situ nanogel 3 Group 1

[0107]

[0108] Table 7-2 Composition of self-assembled in-situ nanogel 3 Group 2

[0109]

[0110] Table 7-3 Composition of self-assembled in-situ nanogel 3 Group 3

[0111]

[0112] Preparation steps:

[0113] (1) 10 mL of thionyl chloride was reacted with 0.015 mol of propionic acid at 80°C for 2 h, then 0.005 mol of apigenin was added, and the reaction was carried out at 50°C for 9 h to generate the initial product of apigenin propionate. Ethyl acetate was used for extraction, column chromatography separation and purification, and rotary evaporation. After vacuum drying, the active ingredient was obtained;

[0114] (2) The active ingredient was dissolved in ethanol to prepare an active ingredient solution, which was stirred at 25°C for 8 h at a stirring speed of 1000 r / min to prepare active ingredient self-assembled nanoscale;

[0115] (3) According to the cold-sol method, chitosan was dissolved in 1 wt% acetic acid water and poloxamer to prepare a uniform gel matrix. The active ingredient self-assembled nanoscale prepared in (2) was added to the gel matrix and stirred at a stirring speed of 1000 r / min to prepare a self-assembled in-situ nanogel.

[0116] 4.2 Gelling temperature of self-assembled in-situ nanogel at pH 1.5 and pH 6.5:

[0117] Table 8 Gelling temperature of self-assembled in-situ nanogel composition 3

[0118]

[0119] As shown in Table 8, the gelling was tested at 37°C, and the results showed that in the case of 3 wt% poloxamer 188, the gelling temperature of 22 wt% poloxamer 407 solution at pH 1.5 and pH 6.5 was lower than 37°C, indicating that when the content of poloxamer 407 was 22 wt%, chitosan had little effect on the gelling temperature at different pH, and poloxamer 407 was the main factor affecting the gelling temperature. The content should be reduced so that chitosan has a better adjusting effect on the gelling temperature of poloxamer 407 solution at different pH.

[0120] Table 9 Gelling temperature of different compositions of self-assembled in-situ nanogel

[0121]

[0122] As shown in Table 9, in the 18wt%, 20wt% and 22wt% poloxamer 407 solutions, the gelling temperature of the 18wt% poloxamer 407 solution under the conditions of pH 1.5 and pH 6.5 is greater than 40℃, the gelling temperature of the 20wt% poloxamer 407 solution under the conditions of pH 1.5 and pH 6.5 is different, and the gelling temperature is about 37℃, and the gelling temperature of the 22wt% poloxamer 407 solution under the conditions of pH 1.5 and pH 6.5 is not different, and is less than 37℃. Therefore, the 20wt% poloxamer 407 solution is the best concentration.

[0123] Three concentrations of 3wt%, 5wt% and 10wt% poloxamer 188 are prepared into different concentrations of solutions with 18wt%, 20wt% and 22wt% poloxamer 407 solutions respectively. The gelling conditions are tested at 40℃, and the results show that the gelling temperature of the 18wt% poloxamer 407 solution with 3wt% poloxamer 188 is higher than 40℃, the 20wt% and 22wt% poloxamer 407 solutions with 3wt% poloxamer 188 are solidified at 40℃, the 22wt% poloxamer 407 solution with 5wt% is solidified at 40℃, and the 18wt%, 20wt% and 22wt% poloxamer 407 solutions with 10wt% are not solidified at 40℃. Therefore, 3wt% poloxamer 188 solution is selected. In addition, according to Table 4, 1wt% CS solution is the best concentration. Therefore, the final gel matrix is 20wt% poloxamer 407, 3wt% poloxamer 188 and 1wt% CS.

[0124] Test Example 1 Preparation of self-assembled in-situ nanogel and stability test in simulated gastric environment

[0125] 1.1 Preparation of self-assembled in-situ nanogel

[0126] Table 10 Composition of self-assembled in-situ nanogel

[0127]

[0128] Preparation method of self-assembled in-situ nanogel:

[0129] (1) 10mL thionyl chloride was reacted with 0.015mol propionic acid at 80℃ for 2h, then 0.005mol apigenin was added, and the initial product apigenin propionate was generated by reacting at 50℃ for 9h, extracted with ethyl acetate, separated and purified by column chromatography, and rotary evaporated. After vacuum drying, the active ingredient was obtained;

[0130] (2) The active ingredient was dissolved in ethanol to prepare an active ingredient solution, which was stirred at 25°C for 8 h at a stirring speed of 1000 r / min to prepare active ingredient self-assembled nano;

[0131] (3) According to the cold dissolution method, chitosan was dissolved in 1wt% acetic acid water and poloxamer to prepare a uniform gel matrix, and the active ingredient self-assembled nano prepared in (2) was added to the gel matrix and stirred at a stirring speed of 1000 r / min to prepare self-assembled in-situ nano gel.

[0132] 1.2 Stability test of active ingredient self-assembled nano and self-assembled in-situ nano gel in gastric juice and intestinal juice environment

[0133] 1.2.1 Materials and methods

[0134] (1) Drug: self-assembled in-situ nano gel prepared in 1.1 (referred to as gel group) and active ingredient self-assembled nano obtained in step (2) of 1.1 (referred to as nano group).

[0135] (2) Test reagents: NaCl, pepsin, HCl, bile extract, lipase.

[0136] (3) Instrument: high performance liquid chromatography.

[0137] (4) Test steps:

[0138] The ester bond rupture was detected. The ester formed by short-chain fatty acid and flavonoids was dispersed in simulated gastric juice (20 mg NaCl and 5 mg pepsin were dissolved in HCl solution to adjust pH to 2.0, and ultrapure water was added to 10 mL), and stirred at 37°C for 4 h. The ester formed by short-chain fatty acid and flavonoids was dispersed in simulated intestinal juice (5 mg bile extract and 16 mg lipase were weighed and dissolved in a neutral solution, and adjusted to pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, respectively, and then ultrapure water was added to 10 mL), and stirred at 37°C for 4 h. After being prepared with methanol, high performance liquid chromatography analysis was performed, and each group was repeated 3 times.

[0139] 1.2.2 Results

[0140] The test results of self-assembled in-situ nano gel are shown in Figure 1 , and the thin layer chromatogram of the active ingredient is shown in Figure 2 .

[0141] The peak time of the active ingredient under different conditions is shown in Figure 3 , as shown in Figure 3 (a), in the simulated gastric environment of pH=2, the peak time of the active ingredient was 17.4 min (the peak time of the ester). As shown in Figure 3As shown in (b) to (d), under simulated intestinal fluid conditions at pH 5–7, the peak elution time was 5.9 min (the peak elution time of apigenin), with no or very little elution at 17.4 min. Figure 3 As shown in (e), apigenin butyrate, without any treatment, was dissolved in methanol and analyzed. Its peak time was consistent with that under simulated gastric conditions at pH 2. This indicates that the active ingredient can maintain its ester form in acidic gastric juice, with the ester bond remaining intact; however, in weakly acidic, neutral, and weakly alkaline intestinal juices, the ester bond breaks, decomposing into apigenin.

[0142] Experimental Example 2: Intestinal Wall Adhesion Performance Test of Self-Assembled In-situ Nanogels

[0143] 2.1 Test Methods

[0144] According to experimental requirements, mice were inoculated with CY7 fluorescently labeled self-assembled in situ nanogels via gavage. After different time intervals following gavage, mice were injected intraperitoneally with chloral hydrate and then placed in an imaging darkroom platform. The platform was raised and lowered to a suitable field of view using software control, and images were taken. Each treatment group had three replicates.

[0145] 2.2 Results

[0146] like Figure 4 As shown, after simultaneous gavage administration of the gel group (in situ gel) and the solution group (solution), the gel moved more slowly in the intestines compared to the solution group within 1-8 hours. At 8 hours, almost no fluorescence signal was detected in the intestines of the solution group, while a strong fluorescence signal was still detected in the gel group at 8 hours, indicating that the gel still retained drug in the intestines at 8 hours. In contrast, the drug in the intestines of the solution group was completely emptied by 8 hours. This demonstrates the adhesion performance of self-assembled in situ nanogels to the intestines and also enhances the sustained-release performance of the drug.

[0147] The scanning electron microscope image of the self-assembled nanoparticles without a gel matrix in the in-situ nanogel in Experimental Example 2 is shown below. Figure 6 ,Depend on Figure 6 It can be seen that the self-assembled nanoparticles exhibit a relatively uniform spherical shape with a particle size of 500-600 nm.

[0148] The rheological properties of different concentrations of poloxamer and chitosan gel matrices in Experiment Example 2 are shown in [reference needed]. Figure 7 (where F127 is Poloxamer 407), by Figure 7 It is known that when the poloxamer content is 20wt% and the chitosan content is 1wt% or 1.5wt%, the gelation temperature differs greatly under the conditions of pH=1.5 and pH=6.5. It can be in a solution state at pH=1.5 and in a gel state at pH=6.5 at the same temperature.

[0149] In vitro anti-inflammatory test of active ingredient self-assembled nano and self-assembled in situ nano-hydrogel of test example 3

[0150] The key importance of macrophages as the main gatekeepers of tissue homeostasis has been well established. Macrophages rapidly adapt their functions by sensing the surrounding microenvironment and acquiring specific phenotypes according to the microanatomic niche they are in. This is particularly true for complex heterogeneous organs such as the gastrointestinal (GI) tract. Therefore, macrophage RAW264.7 cells were chosen as model cells to establish an inflammation model and detect inflammatory cytokines. Intestinal epithelial cells IPEC-J2 cells and Caco-2 cells are an important barrier to protect the intestinal tract. Damage to epithelial cells and subsequent damage to barrier function can ultimately lead to the occurrence of inflammatory reactions. Therefore, self-damage repair of epithelial cells is of great significance to the reconstruction of the intestinal barrier and the homeostatic regulation of tissues. Healing of mucosal wounds depends on the proliferation and migration of epithelial cells. Unlike immune cells and fibroblasts, the migration of epithelial cells is carried out in a sheet-like structure. Directional migration depends on the polar distribution of cells and the reconstruction of the cytoskeleton. Therefore, intestinal epithelial cells IPEC-J2 cells and Caco-2 cells were chosen as model cells to establish an inflammation model.

[0151] 3.1 Test method

[0152] First, the macrophages were co-incubated with self-assembled nanoparticles and self-assembled in situ nano-hydrogel, respectively. The specific steps are as follows: RAW264.7 cells were cultured, and when the cells entered the logarithmic growth phase, they were used for the test. The cell density was adjusted to 5×10 5 / mL, and the cells were inoculated in a 6-well plate and cultured in a 37℃, 5% CO2 incubator. When the cell density reached 60%-70%, the cells were replaced with serum-free DMEM basic culture medium. The in situ nano-hydrogel group, the nanoparticle group (nanoparticles are small molecules synthesized after short-chain fatty acids and flavonoids, which form self-assembled nano after stirring), the complex group (the complex group is a small molecule synthesized after short-chain fatty acids and flavonoids), the physical mixing group of short-chain fatty acids and flavonoids, and the short-chain fatty acid and flavonoid compound 12.5 μmol / L were incubated with the cells for 4 h, and then the drug was aspirated and lipopolysaccharide (LPS) was added for incubation for 24 h. Each treatment group was set in triplicate.

[0153] 3.2 Results

[0154] As Figure 5(Control is the negative control group without LPS and without drug, LPS is the control group with LPS incubation without drug, E gel represents the in-situ nanogel group, E NPs is the self-assembled nano group (i.e. nanoparticle group), E is the active ingredient complex solution group (i.e. complex group), PA API is the short-chain fatty acid, flavonoids without synthetic complex, single mixed solution group, i.e. short-chain fatty acid and flavonoids compound physical mixing group, PA is short-chain fatty acid, and API is flavonoids), as shown, LPS induces RAW264.7, and significantly increases the protein expression of inflammatory factor TNF-α (p<0.001), and significantly increases the protein expression of IL-6, IL-1β and IL-8 (p<0.0001). Compared with the LPS group, the gel group and the self-assembled nanoparticles significantly reduce TNF-α (p<0.05), and significantly reduce the protein expression of IL-6, IL-1β and IL-8 (p<0.001). The physical mixing group of apigenin and sodium propionate has better inhibitory effect on the protein expression of pro-inflammatory factors TNF-α, IL-6, IL-1β and IL-8 than the single group of apigenin and propionic acid. The protein expression of pro-inflammatory factors TNF-α, IL-6 and IL-1β of apigenin propionate is slightly lower than that of the physical mixing group, and it has been proved in the foregoing that apigenin propionate is reduced to apigenin and propionic acid in the simulated intestinal fluid environment, so it plays an anti-inflammatory role in the form of apigenin and propionic acid in the intestinal tract. The nano group and the apigenin propionate group have similar effects, and the in-situ gel group has better inhibitory effect on the protein expression of pro-inflammatory factors TNF-α, IL-6, IL-1β and IL-8 than the nano group and the apigenin propionate group.

[0155] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A self-assembling in situ nanogel, characterized in that, The self-assembled in-situ nano-hydrogel comprises 0.2% active ingredient, 24% gel matrix and the balance water by mass percentage; The active ingredient is obtained by esterification of short-chain fatty acid and flavonoid compound; The short-chain fatty acid is propionic acid; The flavonoid compound is apigenin; The mass ratio of the short-chain fatty acid to the flavonoid compound is 1:1.2; The gel matrix is composed of 20wt% poloxamer 407, 3wt% poloxamer 188 and 1wt% chitosan; The self-assembled in-situ nano-hydrogel forms gel in-situ at pH=6.5 and 37℃.

2. The self-assembled in situ nanogel according to claim 1, characterized in that, The preparation method of the active ingredient is as follows: The short-chain fatty acid is added into an organic solvent during stirring, and a solution A is obtained after reaction at 80-90℃ for 2-3h; The solution A is cooled to 30-60℃, and the flavonoid compound is added to obtain an initial product of the active ingredient; The initial product of the active ingredient is extracted with ethyl acetate, and column chromatography is used for separation and purification to obtain the active ingredient.

3. The self-assembled in situ nanogel of claim 2, wherein, The organic solvent is oxalyl chloride or thionyl chloride.

4. Use of the self-assembled in-situ nano-hydrogel according to any one of claims 1-3 in the preparation of an anti-inflammatory drug.

5. Use of the self-assembled in-situ nano-hydrogel according to any one of claims 1-3 in the preparation of an anti-inflammation drug for digestive tract.

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