A pre-gel solution, a preparation method thereof, a gelation verification method and application thereof

Abstract

The present application relates to the technical field of medical materials, and specifically discloses a pre-gel solution, a preparation method thereof, a gel formation verification method and application. The pre-gel solution contains bovine serum albumin or silk fibroin, and also contains ferrous ions. The response factor for forming a colloid is active oxygen. The pre-gel solution is used for in-situ gel formation and delivery of probiotics and / or drugs while eliminating active oxygen, so as to solve the problems of ulcerative colitis and intestinal flora imbalance.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and more specifically, to a pregel solution, its preparation method, gelation verification method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that mainly affects the colon and rectum, causing inflammation and ulceration of the intestinal mucosa. It is often accompanied by symptoms such as abdominal pain, diarrhea, and stools mixed with mucus and blood, which seriously affect the quality of life of patients and bring huge economic and social burdens.

[0003] Previous studies have found that intestinal mucosal barrier dysfunction and gut microbiota dysbiosis significantly promote the occurrence and progression of ulcerative colitis (UC). Excessive intestinal oxidative stress induced by reactive oxygen species (ROS) is considered a major factor in the pathogenesis and progression of UC. Excessive ROS can induce lipid peroxidation and DNA mutations, impair protein function, alter epithelial permeability, and disrupt the intestinal epithelial barrier, ultimately leading to the initiation or exacerbation of UC, resulting in intestinal endothelial cell damage. Furthermore, excessive ROS can also cause dysregulation of pro-inflammatory species-sensitive pathways in immune cells and induce gut microbiota dysbiosis. A disordered microbiota can induce a chronic inflammatory state, increase toxicity production, and disrupt host metabolism; therefore, gut microbiota dysbiosis in the colonic microenvironment is also closely related to UC.

[0004] Currently, transanal treatment of colitis (UC) is the primary treatment for localized lesions of the rectum and colon, mainly delivering nonsteroidal anti-inflammatory drugs (NSAIDs), hormones, or immunosuppressants to control inflammation and improve symptoms. Enemas, as a basic form of targeted drug delivery to the inflamed colon, are commonly used for mild to moderate colitis. However, the main component of existing enemas is saline, which is difficult to retain in the intestines, leading to easy excretion and thus low drug utilization and poor efficacy. Existing hydrogel precursors are difficult to gel rapidly after injection into the intestines, resulting in easy excretion and a lack of ROS elimination function.

[0005] In addition to transrectal drug delivery, studies have found that transrectal probiotic delivery can actively regulate the gut microbiota and immune system, showing significant advantages in the treatment of UC. Probiotics can colonize colon tissue and help restore a normal gut microbiome to treat gastrointestinal-related diseases.

[0006] Environmentally sensitive hydrogels are a rapidly developing new drug delivery formulation in recent years. They can form a semi-solid with good adhesion at the application site based on different physiological environments, resulting in a long local retention time that facilitates sustained drug release. Their preparation process is simple and easily industrialized, and they can be administered via a wide range of routes, showing great promise for clinical applications. Existing environmentally sensitive hydrogels can be specifically classified according to different response factors, including temperature, pH, ion, light, and multi-sensitivity hydrogels, with temperature-sensitive hydrogels being the most common. Commonly used environmentally sensitive polymers include chitosan, poly(N-isoacrylamide), and poloxamer. However, no studies have yet developed environmentally sensitive hydrogels based on reactive oxygen species (ROS) as the response factor. Summary of the Invention

[0007] The present invention aims to overcome the deficiencies of at least one of the above-mentioned prior art, and provides a pregel solution and its preparation method, gelation verification method and application, for in-situ gelation and delivery of probiotics and / or drugs while eliminating reactive oxygen species, so as to solve the problems of ulcerative colitis and intestinal flora imbalance.

[0008] The technical solution adopted in this invention is to provide a pregel solution containing bovine serum albumin or silk fibroin, and also containing ferrous ions, wherein the response factor for the formation of colloids in the pregel solution is reactive oxygen species.

[0009] Furthermore, the mass percentage concentration of bovine serum albumin or silk fibroin in the pregel solution is 1% to 5%.

[0010] Preferably, the mass percentage concentration of bovine serum albumin or silk fibroin in the pregel solution is 5%.

[0011] Furthermore, the mass percentage concentration of ferrous ions is 0.2%-1%, and the sources of ferrous ions include, but are not limited to, ferrous chloride and ferrous sulfate.

[0012] Furthermore, the coagulation time of the pregelation solution does not exceed 1 second.

[0013] Furthermore, the storage modulus of the colloid formed by the pregel solution does not exceed 0.02 MPa.

[0014] The hydrogel developed in this invention has excellent biocompatibility and a simple preparation method. Specifically, this invention utilizes a protein rich in tyrosine and ferrous ions dissolved and mixed in pure water to obtain a pregel solution. In the presence of hydrogen peroxide or hydroxyl radicals, the tyrosine residues in the protein are rapidly cross-linked to quickly form a hydrogel.

[0015] In one embodiment of the present invention, a BSA-Fe pregel solution is obtained by dissolving and mixing bovine serum albumin (BSA) powder and ferrous sulfate in water. A BSA-Fe hydrogel is then formed in the presence of hydrogen peroxide or hydroxyl radicals. The pregel solution is a liquid, facilitating transrectal injection in animal experiments or clinical applications. Since reactive oxygen species (ROS) are closely related to intestinal endothelial cell damage, a certain concentration of ROS exists in the inflamed intestinal environment. Therefore, after the pregel solution is injected into the intestine, ferrous ions react with hydrogen peroxide in the ROS via a Fenton reaction to generate hydroxyl radicals, which crosslink tyrosine residues in BSA into dityrosine. Simultaneously, the hydroxyl radicals in the ROS also crosslink tyrosine residues into dityrosine, thus forming a hydrogel. This gelation process removes ROS from the inflamed colon tissue, effectively improving the prognosis of ulcerative colitis.

[0016] In another embodiment of the invention, replacing bovine serum albumin with tyrosine-rich silk fibroin (SF) also allows for rapid colloid formation.

[0017] In another embodiment of the invention, ferrous chloride is used instead of ferrous sulfate to form a colloid quickly.

[0018] The BSA-Fe hydrogel provided by this invention exhibits high responsiveness, rapid gelation speed, and suitable elasticity. An aqueous solution of bovine serum albumin and ferrous sulfate can form a BSA-Fe hydrogel in vitro at an extremely rapid rate (less than 1 second) under the action of 0.03% hydrogen peroxide solution. Furthermore, the BSA-Fe hydrogel can rapidly form even at a bovine serum albumin concentration of 1%. In addition, at 25°C, the BSA-Fe hydrogel formed with 5% bovine serum albumin has a storage modulus as low as 0.012 MPa, which can avoid damage to intestinal tissue, intestinal obstruction, and intestinal discomfort.

[0019] Furthermore, the pregel solution also includes probiotics, and / or prebiotics, and / or medications. Based on the above principles, adding probiotics, prebiotics, and medications to the BSA-Fe pregel solution helps regulate the intestinal microecology and improve the prognosis of intestinal diseases. Preferably, to further improve the prognosis of ulcerative colitis and regulate the intestinal microecology, the probiotics can be selected at a concentration of (1~3)×10⁻⁶. 9The prebiotic can be selected from fructooligosaccharides, inulin, etc., and the drug can be selected from 5-aminosalicylic acid, sodium salicylate, etc. In one embodiment of the present invention, the probiotic includes EcN. EcN is one of the few Gram-negative bacteria among probiotics, and is mainly used clinically for gastrointestinal dysfunction such as Crohn's disease and ulcerative colitis. Its mechanism is that EcN can colonize the human intestine and prevent pathogens from invading the intestinal mucosa, thus protecting and repairing the intestinal mucosal barrier. EcN also participates in the body's immune regulation, balances the secretion of immune factors, enhances the host's immunity, and thus alleviates and treats inflammation. Therefore, the delivered probiotic EcN can also improve the homeostasis of the intestinal microbiota. In addition to probiotics, the pregel solution provided by the present invention can also be used to stably carry prebiotics and drugs to the intestine, and has good application prospects.

[0020] Another object of the present invention is to provide a method for preparing the pregelation solution, comprising the following steps:

[0021] S1. Incubate the probiotics at a constant temperature overnight, then collect them by centrifugation to obtain the precipitate;

[0022] S2. Bovine serum albumin or silk fibroin, ferrous sulfate and pure water are mixed and completely dissolved in a mass ratio of (2~10):(0.4~2):(100~500) to obtain a mixture. The mixture is added to the precipitate obtained in step S1 for resuspension to obtain a pregel solution.

[0023] Another object of the present invention is to provide a method for verifying the gelation of the pregel solution, comprising the following steps:

[0024] S3. Pour the pregel solution into the mold, add hydrogen peroxide solution, and the resulting mixed solution quickly forms a hydrogel. Record the solidification time, and use a rotational rheometer to confirm the gelation of the hydrogel and obtain its storage modulus value.

[0025] S4. Fluorescent dye was added to the pregel solution to obtain an enema solution. A mouse colitis model was constructed. Mice with colitis were given an enema, and fluorescence images at different time points after the enema were collected using an in vivo imaging device, and the fluorescence intensity was recorded.

[0026] Furthermore, the mass percentage concentration of hydrogen peroxide in the mixed solution is 0.01% to 0.05%. The pregel solution provided by this invention is sensitive to ROS; therefore, the concentration of hydrogen peroxide solution in the gelation validation method needs to be low to simulate the low ROS concentration under intestinal inflammation conditions.

[0027] Another object of the present invention is to provide the application of the pregel solution in the colonic delivery of prebiotics and probiotics.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) The pregel solution provided by the present invention can gel in situ while eliminating reactive oxygen species. It has a sensitive response, fast gelation speed, suitable elasticity, avoids leakage out of the body, and is suitable for transrectal injection.

[0030] (2) The pregel solution contains probiotic EcN, which helps probiotics colonize the intestines after forming a hydrogel, regulates the intestinal microecology, enhances intestinal immunity, and thus relieves and treats inflammation. The hydrogel has a sustained-release effect, so it can also be used to carry drugs.

[0031] (3) The preparation method of the present invention is simple and low in cost, and has good application prospects in the treatment of intestinal inflammation. Attached Figure Description

[0032] Figure 1 These are actual images of the bovine serum albumin hydrogel before and after gelation as described in Example 1.

[0033] Figure 2 This is a scan graph of the oscillation frequency of the BSA-Fe hydrogel in Example 1.

[0034] Figure 3 This is a scanning electron microscope (EDS) image of the BSA-Fe hydrogel from Example 1.

[0035] Figure 4 Fluorescence images of mice in the control group (Water) and the experimental group (DSS) at different time points.

[0036] Figure 5 This is a comparison of fluorescence intensity at different time points between the control group (Water) and the experimental group (DSS) of C57BL / 6 mice, calculated using IVIS. Detailed Implementation

[0037] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the invention. To better illustrate the following embodiments, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions; it is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0038] Example 1

[0039] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0040] S1. Incubate EcN overnight in LB medium at 37 °C. Collect EcN by centrifugation at 4000 r for 5 min.

[0041] S2. Weigh 250 mg of bovine serum albumin and 25 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0042] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 μL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0043] Figure 1 Images of the BSA-Fe hydrogel described in Example 1 before and after gelation are shown. Gelation was confirmed using a rotational rheometer, and its storage modulus was obtained. Figure 2 The image shows a scanned image of the oscillation frequency of the bovine serum albumin hydrogel described in Example 1. SEM and EDS tests were performed on the BSA-Fe hydrogel prepared in Example 1, as shown below. Figure 3 As shown, the prepared BSA-Fe hydrogel has a porous structure. The energy dispersive spectroscopy (EDS) spectrum shows that the elements in the BSA-Fe hydrogel are uniformly distributed, with iron being uniformly distributed in small amounts in the hydrogel.

[0044] Example 2

[0045] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0046] S1. Incubate EcN overnight in LB medium at 37 °C. Collect EcN by centrifugation at 4000 r for 5 min.

[0047] S2. Weigh 200 mg of bovine serum albumin and 25 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0048] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 μL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0049] Example 3

[0050] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0051] S1. Incubate EcN overnight in LB medium at 37 °C. Collect EcN by centrifugation at 4000 r for 5 min.

[0052] S2. Weigh 150 mg of bovine serum albumin and 25 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0053] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 μL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0054] Example 4

[0055] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0056] S1. Incubate EcN overnight in LB medium at 37°C. Collect EcN by centrifugation at 4000 r for 5 min.

[0057] S2. Weigh 100 mg of bovine serum albumin and 10 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The resulting EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0058] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 uL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0059] Example 5

[0060] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0061] S1. Incubate EcN overnight in LB medium at 37 °C. Collect EcN by centrifugation at 4000 r for 5 min.

[0062] S2. Weigh 50 mg of bovine serum albumin and 25 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The resulting EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0063] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 uL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0064] Example 6

[0065] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0066] S1. Incubate EcN overnight in LB medium at 37 °C. Collect EcN by centrifugation at 4000 r for 5 min.

[0067] S2. Weigh 250 mg of bovine serum albumin and 50 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending. The resulting EcN concentration is 2 × 10⁻⁶. 9 CFU / mL BSA-Fe pregel solution;

[0068] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 uL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0069] Example 7

[0070] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0071] S1. EcN was incubated overnight at 37 °C in LB medium. The concentration of EcN was then measured to be 2 × 10⁻⁶. 9 CFU / mL, EcN was collected by centrifugation at 4000 r for 5 minutes.

[0072] S2. Weigh 250 mg of silk fibroin (SF) and 50 mg of ferrous sulfate, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending to obtain SF-Fe pregel solution.

[0073] S3. After pouring the SF-Fe pregel solution into the mold, add 100 uL of hydrogen peroxide solution to quickly form the final SF-Fe hydrogel.

[0074] Example 8

[0075] A rapidly gelling, ROS-free hydrogel, specifically a hydrogel loaded with beneficial bacteria EcN, is prepared as follows:

[0076] S1. EcN was incubated overnight at 37 °C in LB medium. The concentration of EcN was then measured to be 2 × 10⁻⁶. 9 CFU / mL, EcN was collected by centrifugation at 4000 r for 5 minutes.

[0077] S2. Weigh 250 mg of bovine serum albumin and 50 mg of ferrous chloride, add 5 mL of pure water, and after they are completely dissolved, add this solution to the EcN obtained in step S1 for resuspending to obtain BSA-Fe pregel solution.

[0078] S3. Pour the BSA-Fe pregel solution into the mold and then add 100 uL of hydrogen peroxide solution to quickly form the final BSA-Fe hydrogel.

[0079] Example 9

[0080] To verify the prognostic effect of BSA-Fe pregel solution applied to living tissue, a mouse colitis model was constructed for a control experiment in this embodiment. The BSA-Fe pregel solution was prepared according to the methods described in steps S1-S2 of Example 1. 0.1 mg of the fluorescent dye Cy5.5 was added to every 2 mL of the BSA-Fe pregel solution to obtain an enema solution for later use. Six female C57BL / 6 mice weighing approximately 20-25 g were taken and divided into an experimental group and a control group. The three female C57BL / 6 mice in the experimental group were treated with 3% w / v sodium dextran sulfate to induce colitis for 5 days, while the three female C57BL / 6 mice in the control group received normal food and water. Both the experimental and control mice were given enemas, with each mouse receiving 50 μL of enema solution. Fluorescence images were acquired using a live imaging system at 8, 24, and 48 hours after enema to observe the fluorescence intensity of the experimental group (DSS) and the control group (Water). The experimental group showed higher fluorescence intensity and better retention, resulting in significant relief of colitis, while the control group showed no significant change in colitis symptoms.

[0081] The results are shown in the figure. Figure 4 Fluorescence images of mice in the control group (Water) and the experimental group (DSS) at different time points. Figure 5 This is a comparison of fluorescence intensity at various time points between the control group (Water) and the experimental group (DSS) of C57BL / 6 mice, calculated using IVIS. It can be seen that, in a horizontal comparison at the same time point, the amount of fluorescent dye in the intestines of the control group (Water) mice was less than that in the experimental group (DSS). This indicates that the intestinal surface of healthy mice does not contain ROS, and the BSA-Fe pregel solution cannot coagulate into a colloid and be excreted by the mice; while the intestinal surface of colitis mice contains ROS, and the BSA-Fe pregel solution reacts with it, coagulates into a colloid, and remains in the intestine.

[0082] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A pregel solution, characterized by, The pre-gel solution contains bovine serum albumin or silk fibroin and ferrous ions, and the response factor for the formation of the gel is active oxygen; The mass percentage concentration of bovine serum albumin or silk fibroin is 1-5%; The mass percentage concentration of ferrous ions is 0.2-1%, and the source of ferrous ions includes ferrous chloride and ferrous sulfate; The coagulation time of the pre-gel solution is not more than 1 second; The storage modulus of the gel formed by the pre-gel solution is not more than 20 kPa and not less than 12 kPa.

2. A pre-gel solution according to claim 1, wherein, It also includes probiotics, and / or prebiotics, and / or drugs.

3. A pre-gel solution according to claim 2, wherein, The probiotic includes EcN at a concentration of (1-3) x 10 9 CFU / mL.

4. The method for preparing a pre-gel solution according to claim 2 or 3, characterized in that, The method comprises the following steps: S1, incubate the probiotics overnight at a constant temperature, centrifuge and collect the precipitate; S2, mix bovine serum albumin or silk fibroin, ferrous sulfate and pure water in a mass ratio of (2-10):(0.4-2):(100-500) and completely dissolve to obtain a mixed solution, add the mixed solution to the precipitate obtained in step S1 for resuspension to obtain a pre-gel solution.

5. The method for verifying gelation of the pre-gel solution according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S3, pour the pre-gel solution into a mold, add a hydrogen peroxide solution with a mass percentage concentration of 0.01-0.05%, quickly form a hydrogel from the mixed solution, record the coagulation time, and confirm the gel formation of the hydrogel by a rotational rheometer and obtain the storage modulus value; S4, add a fluorescent dye to the pre-gel solution to obtain an enema solution, construct a mouse colitis model, enema the colitis mouse, collect the fluorescence images at different time points after enema by a live imaging instrument, and record the fluorescence intensity.

6. Use of the pre-gel solution of any one of claims 1-3 in the preparation of a carrier for the delivery of drugs, prebiotics and / or probiotics in the colorectum.

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