Drug-loaded silk fibroin emulsion, preparation method and application thereof

Abstract

The present application relates to a kind of drug-loaded silk fibroin emulsion and its preparation method and application, belong to biological medicine technical field.The drug-loaded silk fibroin emulsion of the present application includes silk fibroin emulsion and the oil phase material encapsulated in the inside of silk fibroin emulsion;Oil phase material has oil-soluble drug molecule dispersed in it.The silk fibroin emulsion of the present application presents strong positive electric property under gastric acid environment, improves its with gastric mucosa cell, the ability of combination of the protein of negative electricity exposed in ulcer affected area.In addition, the design of nanoscale size makes it have higher surface area and the " amphiphilic multi-block " characteristics of silk fibroin, further improve its with gastric mucosa and ulcer affected area's combination ability and cell permeability, further improve the passive targeting effect and cell permeability of emulsion.Because silk fibroin has good membrane forming capacity, it can form protective layer around drug, prolong the residence time of drug in gastrointestinal tract, improve the absorption efficiency of drug, reduce the frequency of administration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a drug-loaded silk fibroin emulsion as well as a preparation method and application thereof. BACKGROUND

[0002] Gastrointestinal diseases are a class of diseases that widely affect the digestive system of human beings, including esophagus, stomach, small intestine, large intestine and related digestive organs. There are various types of such diseases, including gastritis, gastric ulcer, irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, enteritis and various indigestion conditions. The treatment strategy for gastrointestinal diseases depends on the specific cause and condition, and the treatment methods include drug treatment, lifestyle adjustment, surgical treatment, etc. Although traditional drug treatment is effective, it is often accompanied by side effects, such as antibiotics that can cause intestinal flora imbalance, long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) that can cause gastrointestinal bleeding, etc. In addition, many drugs have low bioavailability and cannot accurately reach the lesion site, resulting in poor treatment effect.

[0003] Oral administration is the main route for treating gastrointestinal diseases, but most drugs have high lipophilicity and short gastric retention time, resulting in low bioavailability and difficulty in meeting the treatment needs. Developing a drug carrier that can deliver hydrophobic drugs, prolong the gastric retention time of drugs and have a targeted protective effect on the wound is an urgent problem in the field of gastric ulcer treatment. SUMMARY

[0004] To solve the above technical problems, the present application provides a drug-loaded silk fibroin emulsion as well as a preparation method and application thereof.

[0005] The first object of the present application is to provide a drug-loaded silk fibroin emulsion, which comprises a silk fibroin emulsion and an oil phase material encapsulated inside the silk fibroin emulsion; the oil phase material has dispersed therein oil-soluble drug molecules.

[0006] In an embodiment of the present application, the mass concentration of silk fibroin in the drug-loaded silk fibroin emulsion is 0.1%-5%, the volume fraction of the oil phase material is 0.1%-60%, and the concentration of the oil-soluble drug molecules is 0.2mg / mL-50mg / mL.

[0007] In an embodiment of the present application, the oil phase material is selected from one or more of soybean oil, corn oil, paraffin oil, ethyl oleate, oleic acid, olive oil, sunflower oil, rapeseed oil, clove oil, jojoba oil, white pool seed oil, avocado oil, wheat germ oil, perilla oil, squalane, squalene, isopropyl palmitate and caprylic / capric triglyceride.

[0008] In an embodiment of the present application, the oil-soluble drug molecules are selected from one or more of famotidine, roxatidine, lafutidine, nizatidine, esomeprazole, omeprazole, lansoprazole, pantoprazole, rabeprazole, ilaprazole, esomeprazole, sulfasalazine, olsalazine, mesalazine, balsalazide, dexamethasone and curcumin.

[0009] In an embodiment of the present application, the size of the drug-loaded silk fibroin emulsion is less than 500 nm.

[0010] In an embodiment of the present application, the silk fibroin not only has the material advantages of excellent biocompatibility, degradability, low inflammatory response and amphiphilicity, but also has the advantage of inhibiting inflammation, and has great potential as a drug carrier.

[0011] A second object of the present application is to provide a preparation method of the drug-loaded silk fibroin emulsion, comprising the following steps:

[0012] S1, dissolving the oil-soluble drug molecules in the oil phase material to obtain a drug-loaded oil phase solution;

[0013] S2, mixing the silk fibroin solution and the drug-loaded oil phase solution of S1, and stirring by a high-speed homogenizer to obtain a drug-loaded silk fibroin primary emulsion;

[0014] S3, performing stage-by-stage treatment on the drug-loaded silk fibroin primary emulsion of S2 by a high-pressure homogenizer to obtain the drug-loaded silk fibroin emulsion.

[0015] In an embodiment of the present application, in S2, the stirring speed is 200 rmp-20000 rmp and the time is more than 3 min.

[0016] Further, in S2, the stirring speed is 200 rmp-20000 rmp and the time is 3 min-60 min.

[0017] In an embodiment of the present application, in S3, the stage-by-stage treatment is specifically divided into two stages, the first stage is overpressure of 100 bar-450 bar at least once, and the second stage is overpressure of 500 bar-1000 bar at least twice.

[0018] Further, in S3, the stage-by-stage treatment is specifically divided into two stages, the first stage is overpressure of 150 bar-450 bar 1-3 times, and the second stage is overpressure of 500 bar-1000 bar 2-6 times.

[0019] A third object of the present application is to provide an application of the drug-loaded silk fibroin emulsion in preparing a drug for treating gastrointestinal diseases.

[0020] In one embodiment of the present application, the gastrointestinal disease comprises one or more of Helicobacter pylori infection, duodenal ulcer, duodenitis, gastric ulcer, acute gastritis, chronic gastritis, remnant gastritis, chronic atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, gastric cancer, colitis, intestinal cancer and irritable bowel syndrome.

[0021] The technical solution of the present application has the following advantages compared with the prior art:

[0022] (1) The drug-loaded silk fibroin emulsion of the present application dissolves oil-soluble drug molecules in the oil phase material, so that its solubility is much higher than that of the water phase system. The oil phase solution of the dissolved drug molecules is completely coated inside the silk fibroin by using the "amphiphilic multi-block" and other characteristics of silk fibroin, greatly improving the drug encapsulation efficiency, and thus improving the drug dosage, ensuring the stability of the drug, reducing the toxicity of the drug, and achieving better therapeutic effect.

[0023] (2) The silk fibroin in the drug-loaded silk fibroin emulsion of the present application is a natural protein produced by certain insects such as silkworms, spiders or transgenic expression, which not only has good biocompatibility, biodegradability, high mechanical strength and excellent chemical modification properties, but also has certain anti-inflammatory activity. As a natural oil-water complex forming material (emulsion and porous particles), silk fibroin has a great influence on the feasibility and stability of the dispersion. From the molecular chain structure, the silk fibroin molecular chain with "amphiphilic multi-block" characteristics can spontaneously aggregate on the water / air surface in the form of hydrophobic segment pointing to the air. Silk fibroin has the ability to act as a macromolecular surfactant in a mixture solution of hydrophobic liquid and water, and then generate an emulsion. The degummed silk fibroin is composed of random coil and alpha-helix, which can be converted to a densely and orderly arranged anti-parallel beta-sheet structure when stimulated by shear force, high temperature, acid and alkali, and organic solvents such as alcohol. This structure can keep stable in complex external environment. Therefore, the regenerated silk fibroin not only has relatively strong emulsifying ability, but also forms a very stable oil-in-water (O / W) emulsion. At the same time, silk fibroin has excellent biocompatibility, biodegradability, low inflammatory response and other advantages, and has great potential as a drug carrier component.

[0024] (3) The silk fibroin emulsion in the drug-loaded silk fibroin emulsion has strong positive electric property in a gastric acid environment, which improves the binding capacity of the silk fibroin emulsion to the negatively charged protein exposed on the gastric mucosa and the ulcer affected area. In addition, the nano-sized design makes the silk fibroin emulsion have a high surface area and a "amphiphilic multi-block" property, which further improves the binding capacity and cell permeability of the silk fibroin emulsion to the gastric mucosa and the ulcer affected area, and improves the passive targeting effect and cell permeability of the silk fibroin emulsion. In addition, the silk fibroin has good film forming ability, which can form a protective layer around the drug, prolong the residence time of the drug in the gastrointestinal tract, improve the absorption efficiency of the drug, and reduce the frequency of drug administration. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to the specific embodiments of the present application and in conjunction with the drawings, in which:

[0026] Figure 1 The left graph is the particle size value of the drug-loaded silk fibroin primary emulsion at different homogenization times, and the right graph is the particle size value of the drug-loaded silk fibroin emulsion at different homogenization times;

[0027] Figure 2 The graph is the sustained release graph of the present application test example 4;

[0028] Figure 3 The graph is the fluorescence imaging graph of the gastric tissue section in the present application test example 5; wherein the upper graph is the free rhodamine model, the middle graph is the silk fibroin emulsion model, and the lower graph is the drug-loaded silk fibroin emulsion model;

[0029] Figure 4 The graph is the actual graph of the gastric tissue section in the present application test example 5;

[0030] Figure 5 The graph is the phagocytosis laser confocal photo of the GES-1 cell in the present application test example 6; wherein the left graph is a 20x magnification graph, and the right graph is a 40x magnification graph;

[0031] Figure 6 The graph is the ELISA detection result of the expression of inflammatory cytokines of RAW246.7 after incubation in different samples in the present application test example 7; wherein the left graph is the expression of TNF-α, and the right graph is the expression of IL-6 (*p≤0.05, **p≤0.01, ***p≤0.001);

[0032] Figure 7 The graph is the morphology of RAW246.7 after incubation for 24h under different samples in the present application test example 7;

[0033] Figure 8Figure 1 is a general diagram of rat stomach tissue for different samples in Example 8 of the present application, which were gavaged for 1 / 3 / 5 days;

[0034] Figure 9 Figure 2 is an experimental result of gastric ulcer repair for different samples in Example 8 of the present application, which were gavaged for 1 / 3 / 5 days; wherein, the left graph is a statistical diagram of the total area ratio of gastric ulcer, and the right graph is a statistical diagram of ulcer inhibition rate (**p≤0.01, ***p≤0.001);

[0035] Figure 10 Figure 3 is a H&E staining diagram of rat stomach tissue sections for different samples in Example 9 of the present application, which were gavaged for 1 / 3 / 5 days;

[0036] Figure 11 Figure 4 is a colon fluorescence imaging diagram in Example 10 of the present application;

[0037] Figure 12 Figure 5 is an ELISA detection result of mouse colon tissue homogenate for different samples in Example 11 of the present application, which were gavaged for 1 / 3 / 5 days; wherein, the left graph is the expression of TNF-α, and the right graph is the expression of IL-6 (*p≤0.05; **p≤0.01). DETAILED DESCRIPTION

[0038] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting to the present application.

[0039] In the present application, unless otherwise specified, the samples involved in the examples are adjusted based on Example 1, specifically as follows: OMP aqueous solution (0.5 mg / mL), squalene aqueous solution (110 μL / mL), famotidine aqueous solution (110 μL / mL), unloaded silk fibroin emulsion (SO), drug-loaded silk fibroin emulsion (OMP concentration is 1 / 2 / 4 / 8 / 12 mg / mL), dexamethasone aqueous solution (0.01 wt%), and all samples are subjected to high-temperature and high-pressure sterilization treatment.

[0040] Example 1

[0041] The drug-loaded silk fibroin emulsion of the present application and the preparation method thereof specifically include the following steps:

[0042] S1, dissolve the soluble silk fibroin in deionized water to obtain a silk fibroin solution with a concentration of 10 wt%;

[0043] Dissolve omeprazole (OMP) in squalene, stir at 37℃ in the dark for more than 2h, until omeprazole is completely dissolved in squalene, to obtain a drug-loaded squalene solution with a concentration of 36.36 mg / mL;

[0044] S2, the silk fibroin solution and drug-loaded squalene solution were mixed according to the volume ratio of 6:11, then purified water was added to 100 mL (the mass concentration of silk fibroin was 0.6%, the volume fraction of squalene was 11%, and the concentration of omeprazole was 4 mg / mL), and the weighed solution was stirred in a high-speed homogenizer at 3000 rpm for 5 min to obtain a drug-loaded silk fibroin primary emulsion;

[0045] S3, the drug-loaded silk fibroin primary emulsion was poured into a high-pressure homogenizer, and was treated under the conditions of 400 bar overpressure once and 700 bar overpressure four times to obtain a drug-loaded silk fibroin emulsion with a particle size of less than 500 nm.

[0046] Example 2

[0047] The same as example 1, except that omeprazole was replaced by curcumin.

[0048] Example 3

[0049] The same as example 1, except that omeprazole was replaced by dexamethasone.

[0050] Example 4

[0051] The same as example 1, except that omeprazole was replaced by famotidine (FM).

[0052] Example 5

[0053] The same as example 1, except that squalene was replaced by soybean oil.

[0054] Example 6

[0055] The same as example 1, except that the volume ratio of the silk fibroin solution and the drug-loaded squalene solution was adjusted so that the mass concentration of silk fibroin in the drug-loaded silk fibroin emulsion was 1.5%, the volume fraction of squalene was 30%, and the concentration of omeprazole was 30 mg / mL.

[0056] Example 7

[0057] The same as example 1, except that the volume ratio of the silk fibroin solution and the drug-loaded squalene solution was adjusted so that the mass concentration of silk fibroin in the drug-loaded silk fibroin emulsion was 5%, the volume fraction of squalene was 60%, and the concentration of omeprazole was 50 mg / mL.

[0058] Comparative Example 1 Drug-loaded silk fibroin nano-microparticles

[0059] S1, the soluble silk fibroin was dissolved with deionized water to obtain a silk fibroin solution with a concentration of 10 wt%;

[0060] Omeprazole (OMP) was dissolved in 80wt% polyethylene glycol 400 to obtain a drug-loaded polyethylene glycol 400 solution with a concentration of 5mg / mL;

[0061] S2, the silk fibroin solution was mixed with the drug-loaded polyethylene glycol 400 solution, and then mixed with 50wt% polyethylene glycol 10000 (volume ratio 1:1) and inverted to mix uniformly, incubated at room temperature, centrifuged, and the collected omeprazole-silk fibroin nanoparticles were freeze-dried and reconstituted in water to obtain drug-loaded silk fibroin nanometer particles with a particle size of less than 1μm.

[0062] Comparative Example 2 drug-loaded albumin-dextran emulsion

[0063] S1, the albumin-dextran covalent complex was dissolved in deionized water to obtain a complex solution with a concentration of 10wt%;

[0064] Omeprazole (OMP) was dissolved in squalene, stirred at 37℃ for more than 2h in the dark until omeprazole was completely dissolved in squalene, to obtain a drug-loaded squalene solution with a concentration of 36.36mg / mL;

[0065] S2, the complex solution was mixed with the drug-loaded squalene solution, and then pure water was added to make up to 100mL (the mass concentration of albumin-dextran was 0.6%, the volume fraction of squalene was 11%, and the concentration of omeprazole was 4mg / mL), and the weighed solution was stirred in a high-speed homogenizer at 3000rmp for 5min to obtain a drug-loaded albumin-dextran primary emulsion;

[0066] S3, the drug-loaded albumin-dextran primary emulsion was poured into a high-pressure homogenizer, and treated at 400bar overpressure once and 700bar overpressure four times to obtain a drug-loaded albumin-dextran emulsion with a particle size of less than 500nm.

[0067] Test Example 1

[0068] (1) Based on Example 1, the particle size of the emulsion at different stages under different conditions was tested by a nanoparticle size potential analyzer, and the results are shown in Figure 1 . Figure 1 The left graph is the particle size of the drug-loaded silk fibroin primary emulsion when the homogenization time is 1min, 3min, 5min, 7min, and 9min. When the homogenization time is 1min, the particle size of the primary emulsion is less than 1200nm, and after 2min, it is less than 800nm. In this application, the stirring speed is 200rmp-20000rmp, which can meet the requirement of primary emulsion particle size less than 5μm. The optimal homogenization time is 5min. Figure 1The right graph is the particle size of the drug-loaded silk fibroin emulsion when the homogenization time is 1 min, 3 min, 5 min, 7 min and 9 min, respectively. Further optimization of the homogenization times, after the first homogenization at 400 bar, the particle size of the drug-loaded silk fibroin emulsion when the homogenization times at 700 bar are 0, 1, 2, 3, 4 and 8 times, respectively. When the homogenization time is 1 time, the particle size can be close to 500 nm, and after 2 times, the particle size of the emulsion is stable at about 350 nm. In the present application, more than 2 times of 500 bar-1000 bar overpressure can meet the requirement of particle size less than 500 nm.

[0069] (2) Based on Example 5, the appropriate oil-silk ratio was determined by analyzing the emulsion particle size value, the concentration of silk fibroin solution was 0.3%, 0.6%, 1.1%, 1.5% and 3%, and the volume fraction of oil phase was 1%, 5%, 10%, 20%, 40% and 60%. The particle size of the nanoemulsion was detected, and the particle size of the emulsion under different oil-silk ratios was analyzed. The smaller the particle size of the nanoemulsion, the more it can overcome the effect of gravity through Brownian motion, and it will not precipitate during storage, and it also prevents the generation of flocculated material, so that the system is uniform. However, nanoemulsion is a thermodynamically unstable system, the smaller the droplet size, the higher the interfacial energy, the more conducive to the occurrence of austenitic ripening, which will make the fluid in the small droplets transfer to the large droplets, eventually leading to the coarsening of the emulsion. Therefore, it is necessary to select an emulsion with moderate particle size and the most stable emulsion state for drug loading. The particle size of the emulsion under different oil-silk ratios is shown in Table 1:

[0070] Table 1

[0071] Group Silk fibroin concentration (%) Volume fraction of oil phase (%) Particle size value (nm) 1 0.3 1 318.1±19.0 2 0.3 5 378.2±11.2 3 0.3 10 328.3±7.1 4 0.6 5 476.3±8.0 5 0.6 10 589.4±9.6 6 0.6 20 406.3±4.1 7 1.1 10 283.8±6.2 8 1.1 20 297.5±17.8 9 1.1 40 208.3±3.1 10 1.5 20 323.8±13.0 11 1.5 40 353.2±9.2 12 1.5 60 198.3±5.1 13 3 5 234.5±6.8 14 3 20 268.0±6.2 15 3 60 331.0±11.1

[0072] As can be seen from Table 1, the increase of the concentration of silk fibroin solution reduces the emulsion droplet size, and the higher the concentration of silk fibroin solution, the more conducive to the adsorption of silk fibroin on the oil-water interface, forming a stronger viscoelastic interface, which enhances the stability of the emulsion; when the concentration of silk fibroin solution is constant, the increase of the volume fraction of oil phase increases the final droplet surface area after homogenization, thereby reducing the adsorption amount of silk fibroin at the oil-water interface, leading to the coalescence of droplets and the increase of the average droplet size. At the same time, the collision of droplets will also be more frequent with the increase of droplets, thereby accelerating the coalescence rate and further increasing the droplet size. It is found that the mass concentration of silk fibroin in the drug-loaded silk fibroin emulsion is 0.1%-5%, the volume fraction of oil phase material is 0.1%-60%, and the concentration of oil-soluble drug molecules is 0.2 mg / mL-50 mg / mL, which meets the requirements.

[0073] Test Example 2 Drug loading rate and loading rate

[0074] (1) Determination of loading efficiency (EE): Using UV-Vis spectrometer, according to the pre-prepared omeprazole standard curve. The calculation formula is: EE (%) = (1 - Wfree / Wtotal) x 100%; wherein, Wfree is the amount of omeprazole in the supernatant of washing, Wtotal is the total amount of omeprazole added.

[0075] (2) Determination of drug loading efficiency (DL): First, the dry powder of nanoemulsion is obtained by vacuum drying. A certain amount of dry powder is accurately weighed, dissolved with a known volume of appropriate solvent, and the omeprazole concentration in the dissolved solution is determined by UV-Vis spectrometer. The calculation formula is: DL (%) = (Wdrug / Wdry) x 100%; wherein, Wdrug is the amount of omeprazole in the dry powder, Wdry is the total amount of dry powder.

[0076] The drug loading efficiency and loading efficiency of the drug-loaded materials of Example 1 (drug concentration of 2 mg / mL, 4 mg / mL, 8 mg / mL, 10 mg / mL, 20 mg / mL of drug-loaded silk fibroin emulsion) and Comparative Examples 1-2 are shown in Table 2:

[0077] Table 2

[0078] Group Drug loading rate (%) Loading rate (%) Example 1-2 13.0 100 Example 1-4 28.3 99.4 Example 1-8 50.5 98.6 Example 1-10 61.5 97.4 Example 1-20 65.3 96.8 Comparative Example 1 4.78 60.43 Comparative Example 2 26.4 89.4

[0079] From the data in Table 2, it can be seen that the drug loading efficiency (encapsulation efficiency) and loading efficiency (encapsulation efficiency) of the emulsion form of the example are much higher than that of the nanoparticle form. At the same time, there is no significant difference with the drug-loaded albumin-dextran emulsion prepared by other natural polymer materials such as albumin-dextran.

[0080] Test Example 3 Protective Effect of Carrier on Drug

[0081] The drug-loaded materials of Examples 1-7 and Comparative Examples 1-2 are mixed with an equal volume of simulated gastric juice, and the mixture is placed in a temperature-controlled oscillator with a temperature setting of 37°C to simulate human conditions. At different time points (such as 6h, 12h, 24h, 36h), sample, neutralize the gastric juice with sodium hydroxide. The logarithmic phase growth of gastric mucosal epithelial cells (GES-1) is digested with 0.25% trypsin and blown into a single cell suspension for counting, with 1.5 x 10 4The cells were inoculated in 24-well plates and cultured for 24 h. Fresh culture medium containing 5% ethanol was added to induce gastric mucosal epithelial cells (GES-1) for 6 h, and 60 μg / mL (omeprazole equivalent of nanomaterial (1 mL of culture medium containing 66.66 μL of drug-loaded nanomaterial) of drug-loaded materials of Example 1 and Comparative Examples 1 and 2 were added for incubation for 24 h. An omeprazole solution having a concentration of 60 μg / mL was used as a control group, and the levels of TNF-α and IL-6 were detected by ELSA to evaluate the drug activity in terms of the expression inhibitory effect of the drugs on inflammatory factors. The drug activities after treatment with gastric juice for 12 h and 24 h are shown in Table 3:

[0082] Table 3

[0083]

[0084]

[0085] Silk fibroin nanoparticles and nanoemulsions as drug delivery systems exhibit significant potential in protecting the activity of active drugs. The spherical structure of silk fibroin material provides a closed environment, which helps to isolate the drug from harmful factors such as extreme changes in pH, enzyme attack, and the action of oxidizing agents, thereby protecting the structure and activity of the drug. As can be seen from Table 3, the protective effect of nanoemulsions is comparable to that of nanoparticles with a dense structure and is much higher than that of other polymers.

[0086] Drug release evaluation of Test Example 4

[0087] Based on Example 2, a standard curve of absorbance-concentration of curcumin in the release solution was prepared. Specifically, curcumin was dissolved in benzhydrol to a concentration of 1 mg / mL as a mother solution, and then diluted with the release solution (3 mL of methanol, 5 mL of 5% Tween 80, and 92 mL of PBS at pH 7.4). Five equal ratio concentration gradients (common ratio of 1 / 2) were set, and the absorbance values were detected at 425 nm by an enzyme marker. The measured absorbance values of curcumin at different concentrations were plotted as a trend line by the scatter method, and the linear regression equation was calculated. A certain amount of curcumin-silk microspheres was weighed and suspended in the release solution, and then transferred and dispensed into 2 mL centrifuge tubes (concentration of 10 mg / mL). After the centrifuge tubes were sealed, they were placed in a shaking bed in a 37°C oven. The centrifuge tubes were removed at specified times (1, 6, 12, 24, 48, 72, 96, 120, 180, 240, 300 h), and the release solution was replaced by centrifugation. The centrifuge tubes were then placed in the shaking bed in the oven. The release solution was removed and its light absorption value was measured at 425 nm by an enzyme marker. The standard curve of absorbance-concentration of curcumin (y = 0.13911 ± 0.00559x + 0.0136 ± 0.01154, R 2= 0.99518) to calculate the content of curcumin in the release liquid. According to the release amount of curcumin in different time, the cumulative release rate of curcumin was calculated.

[0088] Figure 2 is the drug concentration, release amount and release rate of curcumin at the sampling point, and Figure 2 It can be seen that whether it is low concentration drug loading (1.1 mg / mL) or high concentration drug loading (11 mg / mL), the silk fibroin nanoemulsion has good sustained release effect on the hydrophobic drug curcumin, and the release shows a first-order release, which is important for subsequent application. In the first-order release mode, the release rate of the drug does not change with time, which helps to maintain the blood drug concentration in a relatively stable range, thereby reducing the dose frequency, improving the patient's compliance, and possibly reducing the side effects caused by the sharp fluctuation of blood drug concentration. By maintaining the concentration of the drug within the therapeutic window, first-order release can enhance the efficacy of the drug. This is particularly important for chronic disease treatment that requires long-term maintenance of stable blood drug concentration. First-order release mode can also help reduce drug waste. This not only optimizes drug use, but also may reduce treatment costs.

[0089] Test Example 5 Evaluation of Ulcer Targeting

[0090] In order to explore the gastric retention of drug-loaded silk fibroin emulsion, a gastric ulcer model was established in C57 mice, and the main method of model establishment was ethanol-induced ulceration. Then, the samples of rhodamine-labeled non-drug-loaded silk fibroin emulsion (SO), rhodamine-labeled drug-loaded silk fibroin emulsion (OMP) and free rhodamine (RhB, concentration consistent with cross-linked emulsion) were administered by gavage to observe the gastric retention effect of the drug in the gastric ulcer mice. All sample groups were set up with 5 parallel groups at each time period to verify the effectiveness of the experimental results. At 4 / 24 / 48 / 72 h after gavage treatment, the mice were dissected to remove the stomach tissue, and the stomach tissue was cut at the greater curvature and gently washed to remove food residues. The small animal live imaging instrument was used to take pictures and observe the fluorescence intensity of rhodamine at an excitation wavelength of 535 nm. The fluorescence scale was adjusted to the same scale, and the fluorescence intensity of the stomach section of each sample group was compared (scale Min = 6000, Max = 40000).

[0091] The rhodamine fluorescence intensity results of the stomach section are shown in Figure 3 As shown in Figure 3It can be seen that in the gastric ulcer model group (GU), the larger the yellow area on the gastric cross-section of the unloaded silk fibroin emulsion group (GU-SO) and the drug-loaded silk fibroin emulsion group (GU-OMP) after 4 / 24 / 48 / 72 hours of gastric retention, compared to the free rhodamine group (GU-RhB), indicates stronger fluorescence intensity. This suggests that the unloaded silk fibroin emulsion has better adhesion than the free rhodamine group, regardless of whether it is drug-loaded. Furthermore, the fluorescence intensity of the gastric cross-section in each group decreases with time. After 72 hours of gastric retention, the fluorescence intensity of both the unloaded and drug-loaded silk fibroin emulsions remains high compared to the free rhodamine group, indicating that this form of unloaded silk fibroin emulsion can prolong the gastric retention time, up to 72 hours. Therefore, it has significant potential as a carrier for drug delivery. Comparison of gastric cross-sectional fluorescence images between mice in the gastric ulcer group (GU) and healthy mice (H) revealed that, regardless of whether it was the free rhodamine group (RhB), the unloaded silk fibroin emulsion group (SO), or the drug-loaded silk fibroin emulsion group (GU-OMP), the fluorescence intensity of the gastric cross-section in the ulcer group was stronger after 72 hours of gastric retention. This indicates that inflammatory adhesion is more effective than non-inflammatory adhesion, and the fluorescence intensity of the unloaded and drug-loaded silk fibroin emulsions was stronger than that of the free rhodamine group.

[0092] The actual cross-sectional image of the stomach is as follows: Figure 4 As shown. From Figure 4 As can be seen, the unloaded silk fibroin emulsion largely adhered to the ulcer area, consistent with the fluorescence image results. Compared to the loaded silk fibroin emulsion group, the unloaded silk fibroin emulsion group exhibited a greater adhesion area for free rhodamine. This further demonstrates that the silk fibroin emulsion of the present invention possesses a passive targeted adhesion effect, forming a protective layer on the inflamed wound surface and preventing secondary irritation of the ulcer surface by gastric acid secretion.

[0093] Test Example 6: Cell Phagocytosis

[0094] Rhodamine-labeled silk fibroin was prepared using the EDC-NHS method. A 5 mg / mL solution of rhodamine was prepared and diluted to 1 mg / mL. 72.72 mg of EDC and 199.98 mg of NHS were added to 9.09 mL of a 2% silk fibroin solution, mixed thoroughly, and stirred for 15 min. Then, 0.91 mL of rhodamine (concentration: 1 mg / mL) was added to the solution and cross-linked via EDC / NHS. The mixture was stirred in the dark for 4 h. The cross-linked solution was dialyzed for 48 h, with six changes of pure water (low temperature, dark protection).

[0095] Based on Example 1, a rhodamine-labeled drug-loaded silk fibroin emulsion was prepared (rhodamine-labeled silk fibroin: unlabeled silk fibroin = 1:49), and the emulsion was diluted (86.25 μL emulsion + 913.75 μL culture medium). Cell crawling slides were placed in 24-well plates, and GES-1 was added at 1 × 10⁻⁶. 4 Cells were seeded at a density of 1 mL / well into 24-well plates containing cell spreaders and cultured for 24 h in a cell culture incubator at 37°C with 5% CO2. Sample diluent was added to each well at a rate of 1 mL / well, with 6 replicates. After 24 h of incubation, the cell plates were removed, the sample diluent was aspirated, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 10 min, followed by three washes with PBS for 8 min each. 200 μL of Triton-X 100 was added to penetrate the cells for 15 min, followed by three washes with PBS for 4 min each. Then, 200 μL of PBS staining solution containing 1% 488-labeled phalloidin was added to each well for 45 min of staining. After staining, the cells were washed three times with PBS for 8 min each. Finally, 200 μL of DAPI staining agent was added to each well for 4 min of staining. The stained cell spreaders were then removed and placed center-side up in a laser confocal microscopy dish for cell morphology observation using an FV100 laser confocal microscope.

[0096] The cell morphology of GES-1 cells after culturing with rhodamine-labeled silk fibroin emulsion for 24 hours is as follows: Figure 5 As shown. From Figure 5 As can be seen, GES-1 cells exhibit good morphology, resembling epithelial cells, and are relatively large in size. Furthermore, a large amount of rhodamine-labeled silk fibroin emulsion has entered the interior of GES-1 cells. Firstly, during the preparation of rhodamine-labeled silk fibroin using the EDC-NHS method, free rhodamine molecules have already been removed by dialysis. Secondly, rhodamine carries a positive surface charge, while silk fibroin carries a negative surface charge. Through electrostatic and cross-linking interactions, the silk fibroin binds very tightly to rhodamine, preventing any free rhodamine from entering the cell interior.

[0097] Test Case 7 Inflammation Suppression

[0098] Unloaded squalene emulsion and drug-loaded squalene emulsion (OMPE emulsion, OMP concentration 1 / 2 / 4 / 8 / 12 mg / mL) were diluted 100-fold with 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The OMP aqueous solution (OMP) was diluted to a concentration of 10 / 20 / 40 μg / mL. Macrophage resuscitation, culture, and digestion procedures were the same as above. After cell culture, the cells were sputtered at 1×10⁻⁶... 5Cells were seeded into 24-well plates at a specific density. After 24 hours, cell status was observed. If cells were adherent and in good condition, the waste culture was removed, and macrophage polarization was performed. 50 μL of 1 ng / μL lipopolysaccharide (LPS) solution and 250 μL of 1640 medium were added to each well. Six replicates were established per group. In addition to the sample group, two groups were set up as negative and positive controls: pure culture medium (control) and LPS-containing culture medium (LPS). After 24 hours of polarization, the differentiation of antennae at both ends of the cells indicated completion of polarization. The LPS culture waste was removed, and 1 mL of sample diluent was added to each well. After incubation for 24 hours, the cell supernatant was collected by centrifugation at 10000 rpm for 5 minutes. The expression of inflammatory factors IL-6 and TNF-α was detected using an ELISA kit.

[0099] The expression results of inflammatory cytokines after incubation in different samples are as follows: Figure 6 As shown. From Figure 6 It can be seen that the secretion of pro-inflammatory factors TNF-α and IL-6 was significantly increased in the control group and LPS group (p≤0.001). However, the secretion of pro-inflammatory factors IL-6 and TNF-α was significantly decreased in the squalene aqueous solution group, the unloaded silk fibroin emulsion group, the OMP aqueous solution group, the drug-loaded silk fibroin emulsion group, and the dexamethasone aqueous solution group after polarization and incubation compared to the LPS group, indicating that squalene and omeprazole can inhibit the expression of pro-inflammatory factors. Among the OMP aqueous solution group and the drug-loaded silk fibroin emulsion group with drug concentrations of 10 / 20 / 40 μg / mL, the OMP aqueous solution group with a drug concentration of 4 mg / mL and the drug-loaded silk fibroin emulsion group had lower secretion of inflammatory factors compared to the dexamethasone aqueous solution group, and the drug-loaded silk fibroin emulsion group with a drug concentration of 40 μg / mL had the lowest secretion of inflammatory factors, demonstrating that the encapsulation effect of the unloaded silk fibroin emulsion can synergistically regulate the inflammatory response and improve the inflammatory regulation capacity. Drug-loaded silk fibroin emulsions enhance inflammation regulation, helping to reduce inflammatory responses at the ulcer surface and protect the wound from secondary irritation. This promotes the transition of the healing process from the inflammatory phase to the proliferative phase, accelerating wound healing.

[0100] After testing, the cells in each group were stained, and cell morphology was observed. The results are as follows: Figure 7 As shown. From Figure 7 It can be seen that the unpolarized macrophages (control group) were basically round in morphology, while the LPS group, after polarization with the addition of lipopolysaccharide, had predominantly elongated spindle-shaped cells with pseudopodia. The other groups were sample groups incubated with different samples after polarization. It was found that the cell morphology in each sample group changed from the morphology of the differentiated tentacles at both ends after polarization back to a round shape, indicating that the samples could regulate the differentiation of induced inflammatory M1 macrophages.

[0101] Test Example 8: Gastric Ulcer Damage Repair Experiment

[0102] To investigate whether drug-loaded silk fibroin emulsion has a therapeutic effect on gastric ulcers, this experiment established a gastric ulcer model by gavage of SD rats with ethanol. The modeled rats were then treated with gavage, and the ulcer repair was observed at different time points. The specific steps are as follows: First, 2 / 4 mg / mL drug-loaded silk fibroin emulsion (OMP-E) and omeprazole solution (OMP, with physiological saline as the solvent) were prepared. Second, SD rats that had been fasted for 48 hours but allowed free access to water were gavaged with ethanol at a dose of 5 mL / kg to establish a gastric ulcer model. One hour after model establishment, samples were gavaged, with three SD rats in each group gavaged at each time point. A total of three time points were observed: 1, 3, and 5 days. Four experimental groups were treated with 2 / 4 mg / mL drug-loaded silk fibroin emulsion and omeprazole solution, and a physiological saline control group was included. A healthy rat control group was established to compare the effectiveness of the gastric ulcer model establishment and treatment. One day after gavage, SD rats at that time point were dissected. Drug concentrations of 2 mg / mL were denoted as L, and 4 mg / mL as H. The rats were numbered GU-NaCl-1-1 / 2 / 3, GU-OMP-L-1-1 / 2 / 3, GU-OMP-H-1-1 / 2 / 3, GU-OMP-EL-1-1 / 2 / 3, and GU-OMP-EH-1-1 / 2 / 3. After dissection, the stomach tissue was removed and cut along the greater curvature. Food residue was gently rinsed away from the stomach tissue section, and the tissue was observed and photographed. The stomach tissue was then divided in half along the midline. One half was immersed in 10% neutral formalin solution for subsequent HE staining and immunohistochemical detection, while the other half was flash-frozen in liquid nitrogen at -80℃ for ELISA detection. The remaining SD rats on days 3 and 5 were treated with gavage daily until the designated time points for dissection and sampling, following the same sampling procedure as on day 1. Tissue samples taken on day 3 were named GU-NaCl-3-1 / 2 / 3, GU-OMP-L-3-1 / 2 / 3, GU-OMP-H-3-1 / 2 / 3, GU-OMP-EL-3-1 / 2 / 3, and GU-OMP-EH-3-1 / 2 / 3. Tissue samples taken on day 5 were named GU-NaCl-5-1 / 2 / 3, GU-OMP-L-5-1 / 2 / 3, GU-OMP-H-5-1 / 2 / 3, GU-OMP-EL-5-1 / 2 / 3, and GU-OMP-EH-5-1 / 2 / 3. Healthy SD rats were dissected simultaneously on days 1, 3, and 5, and stomach tissue and internal organs were collected, designated H-1, H-3, and H-5. After all samples were collected, the gastric tissue photographs of each group were processed using ImageJ software to calculate the total area of ​​gastric ulcers (A) and the total area of ​​gastric tissue (B), and the percentage of gastric ulcer area was calculated as A / B × 100%. Then, using the saline group as the model group, the ulcer area of ​​the model group (A0) and the ulcer area of ​​the treatment group (A1) were calculated, and the ulcer inhibition rate was calculated as (A0-A1) / A0 × 100%.

[0103] Cross-sectional images of rat stomach tissues after gavage administration of different samples for 1 / 3 / 5 days are shown below. Figure 8 As shown. From Figure 8 It can be seen that, compared with the healthy control group, on the first day, all the model groups established by ethanol gavage had varying degrees of cord-like coagulation; compared with the drug-loaded silk fibroin emulsion and omeprazole solution groups, the saline group had the largest ulcer area, and the drug-loaded silk fibroin emulsion group had less surface coagulation coverage than the omeprazole solution group.

[0104] Results of gastric ulcer repair experiments with different samples administered by gavage for 1 / 3 / 5 days are as follows: Figure 9 As shown. From Figure 9 It can be seen that the saline group showed significant differences from the other four groups (***p≤0.001), while the ulcer area was significantly reduced in both the low-concentration and high-concentration drug-loaded silk fibroin emulsion groups compared to the omeprazole solution group (***p≤0.001). Furthermore, the ulcer inhibition rate of the low-concentration drug-loaded silk fibroin emulsion reached 43.10±2.10% on day 1, significantly different from the 15.76±4.02% in the low-concentration omeprazole solution group (**p≤0.01). Similarly, the ulcer inhibition rate of the high-concentration drug-loaded silk fibroin emulsion reached 62.91±0.90% on day 1, significantly different from the 33.85±0.65% in the high-concentration omeprazole solution group (**p≤0.01). This indicates that administration via silk fibroin emulsion encapsulation is more effective than immediate direct administration. On day 3, the saline control group still showed significant surface coagulation and telangiectasia compared to the other groups (**p≤0.01), while the ulcer areas in the omeprazole solution groups and the drug-loaded silk fibroin emulsion groups were all smaller. Ulcer inhibition rates showed that the ulcer inhibition rates in the low-concentration drug-loaded silk fibroin emulsion group, low-concentration omeprazole solution group, high-concentration drug-loaded silk fibroin emulsion group, and high-concentration omeprazole solution group were 50.31±3.20%, 81.40±8.74%, 83.06±0.92%, and 93.15±0.98%, respectively. This indicates that on day 3 of gavage treatment, both emulsion-loaded and direct administration achieved good therapeutic effects, but the drug-loaded silk fibroin emulsion group showed a more significant therapeutic effect compared to the omeprazole solution group. Based on the cross-sectional images of the gastric tissue and the statistical data on ulcer area and ulcer inhibition rate, on the 5th day of gavage treatment, except for the saline group which had a small area of ​​red blood streaks, the other four groups had no ulcer points, and the ulcer inhibition rate reached more than 95%, indicating that the gastric ulcers were basically healed.

[0105] Test Example 9: H&E Staining

[0106] Based on the above experiments, the fixed samples were first dehydrated using a gradient of ethanol (75 / 85% vs. 90 / 95% vs. 100%). After dehydration, they were soaked in xylene solution until the tissue blocks became transparent. Paraffin embedding machines were then used to prepare paraffin blocks. After preparation, the paraffin blocks were sliced ​​into thin sections approximately 4-8 μm thick using a microtome. These sections were then laid flat and dried, soaked in xylene for 20 minutes, the solution was changed, and the process was repeated for another 20 minutes until the paraffin was completely dissolved. After removing residual xylene by soaking in anhydrous ethanol for 5 minutes, the samples were then sequentially dissolved in ethanol of different concentrations. The sections were treated with 95% / 85% / 75% solutions for 5 min. After treatment, they were washed three times with deionized water and then stained. First, 100 μL of hematoxylin staining solution was added to the sections, and after standing for 5 min, they were washed with deionized water. Differentiation solution was added to remove residual staining solution. After differentiation, the sections were rinsed with deionized water until they turned blue. Next, a small amount of eosin staining solution was added to the sections, and after staining for 5 min, they were dehydrated with a gradient of ethanol (85% / 90% / 95% / 100%), soaking for 5 min at each concentration. Then, they were treated with xylene three times, 5 min each time. After treatment, the sections were air-dried. After the sections were air-dried, they were mounted with neutral resin. Finally, the sections were observed under an upright optical microscope, and images were acquired and analyzed using a scanner (scale bar: 500 μm, 100 μm).

[0107] H&E staining images of rat gastric tissue sections after gavage administration of different samples for 1 / 3 / 5 days are shown below. Figure 10 As shown. From Figure 10 It can be seen that on day 1 of gavage, compared with the gastric tissue sections of healthy rats, the saline group, due to ethanol gavage, showed severe hemorrhagic erosion of the gastric mucosa and submucosal edema, with a large number of inflammatory cell infiltrations and loss of mucosal epithelial cells. Observation of inflammatory cell morphology revealed that most inflammatory cells were neutrophils. Compared with the model group, all sample groups showed a certain therapeutic effect on day 1. Among them, GU-OMP EL and GU-OMP EH showed fewer hemorrhagic lesions and inflammatory cell infiltrations, indicating that the drug-loaded silk fibroin emulsion group had a more significant therapeutic effect than the omeprazole solution group, and had a good timely therapeutic effect. On day 3 of gavage, except for the saline group, the inflammatory cell infiltration and bleeding points in all sample groups decreased significantly compared with day 1, especially in the drug-loaded silk fibroin emulsion group, with only some superficial lesions remaining. On the 5th day after gavage, the model group still had bleeding lesions and inflammatory cell aggregation, while no edema, hemorrhagic damage, epithelial cell loss and leukocyte infiltration were observed in any of the sample groups, indicating that the damaged sites had basically healed.

[0108] Test Case 10 Intestinal Targeting

[0109] To investigate the colonic targeting of drug-loaded silk fibroin emulsions in Example 3, a colitis model was induced using the chemical inducer dithiothreitol (DSS). Six- to eight-week-old C57BL / 6 mice were randomly divided into gavage and rectal administration groups. 4% DSS was dissolved in the mice's drinking water and provided continuously for 7 days. Subsequently, the drugs were administered via both gavage and rectal administration. The results of rhodamine fluorescence intensity in the colon are shown below. Figure 11 As shown. From Figure 11 It can be seen that, regardless of whether it is administered rectally or by gavage, strong fluorescence was observed 24 hours later, indicating that the drug-loaded nanoemulsion has good adhesion to the intestine.

[0110] Test Example 11: Regulation of Inflammatory Response

[0111] Based on the above experiments, this study investigated whether the drug-loaded silk fibroin emulsion of Example 4 could effectively reduce colonic inflammation and detected TNF-α and IL-6 in the tissue. Colonic tissue blocks were prepared into tissue homogenates using the following method: An appropriate amount of tissue block was taken and washed in pre-cooled PBS (0.02 mol / L, pH 7.0-7.4), the tissue block was cut into small pieces, and the weight was determined. The tissue block was then transferred to a 10 mL test tube, and pre-cooled PBS was added (tissue to PBS mass-to-volume ratio of 1:9, i.e., 1 g of tissue fragments added to 9 mL of PBS). The homogenate was thoroughly ground using a tissue homogenizer until no obvious tissue fragments remained. Cells were then disrupted by sonication (10%, 2 min). The homogenate was then centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The BCA protein concentration of all tissue samples was detected. The tissue supernatant was then diluted to an appropriate concentration for ELISA detection.

[0112] The results of ELISA detection of mouse tissue homogenates administered by gavage for 1 / 3 / 5 days are as follows: Figure 12 As shown. From Figure 12It can be seen that on day 1 of gavage, compared with the healthy control group, the secretion levels of pro-inflammatory factors TNF-α and IL-6 in both the model group and the sample group were increased, and the secretion level of pro-inflammatory factors in the model group was significantly higher than that in the sample group. The secretion levels of pro-inflammatory factors in the different concentrations of drug-loaded silk fibroin emulsion groups (UC-FM-EL / UC-FM-EH) were significantly lower than those in the different concentrations of famotidine solution groups (UC-FM-L / UC-FM-H). On day 3 of gavage, the secretion level of pro-inflammatory factors in the model group was still higher than that in the sample group, showing a significant difference. The secretion level of pro-inflammatory factor TNF-α in the different concentrations of drug-loaded silk fibroin emulsion groups was lower than that in the different concentrations of famotidine solution groups, and this difference was significant. However, the secretion of pro-inflammatory factor IL-6 was lower in both groups, with no statistically significant difference. On day 5 of gavage, the secretion levels of pro-inflammatory factors TNF-α and IL-6 in the model group were still higher than those in the healthy control group, while the secretion levels of pro-inflammatory factors in the sample group tended to be similar to those in the healthy control group, with no statistically significant difference, indicating that the damaged tissue had basically healed.

[0113] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A drug-loaded silk fibroin emulsion, characterized in that, It includes a silk fibroin emulsion and an oil phase material encapsulated within the silk fibroin emulsion; the oil phase material contains oil-soluble drug molecules dispersed within it. The drug-loaded silk fibroin emulsion has a silk fibroin mass concentration of 0.1%-5%, an oil phase material volume fraction of 0.1%-60%, and an oil-soluble drug molecule concentration of 0.2 mg / mL-50 mg / mL. The oil phase material is selected from one or more of soybean oil, corn oil, and squalane; The oil-soluble drug molecule is selected from one or more of famotidine, roxatidine, lafutidine, nizatidine, esomeprazole, omeprazole, lansoprazole, pantoprazole, rabeprazole, iprazolamazole, esomeprazole, dexamethasone, and curcumin.

2. The drug-loaded silk fibroin emulsion according to claim 1, characterized in that, The size of the drug-loaded silk fibroin emulsion is less than 500 nm.

3. A method for preparing the drug-loaded silk fibroin emulsion according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Dissolve oil-soluble drug molecules in an oil phase material to obtain a drug-loaded oil phase solution; S2. Mix the silk fibroin solution and the drug-loaded oil phase solution described in S1, and stir using a high-speed homogenizer to obtain a drug-loaded silk fibroin primary emulsion. S3. The drug-loaded silk fibroin primary emulsion described in S2 is processed in stages using a high-pressure homogenizer to obtain the drug-loaded silk fibroin emulsion.

4. The method for preparing the drug-loaded silk fibroin emulsion according to claim 3, characterized in that, In S2, the stirring speed is 200 rpm to 20000 rpm, and the time is more than 3 minutes.

5. The method for preparing the drug-loaded silk fibroin emulsion according to claim 3, characterized in that, In S3, the phased processing is specifically divided into two stages: the first stage is at least one overpressure of 100bar-450bar, and the second stage is at least two overpressures of 500bar-1000bar.

6. The use of the drug-loaded silk fibroin emulsion according to any one of claims 1-2 in the preparation of a medicament for treating gastrointestinal diseases, characterized in that, The gastrointestinal diseases include one or more of gastric ulcers and colitis.

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