A staining agent for endoscopic surgery and its preparation method and application

Abstract

The application relates to the technical field of medical materials, in particular to a dyeing agent for endoscopic surgery and a preparation method and application thereof. The dyeing agent is an acetyl-GPLGIRG-ninhydrin enzyme-activated precursor, the acetyl-GPLGIRG-ninhydrin enzyme-activated precursor is obtained by the reaction of acetylated GPLGIRG polypeptide and ninhydrin, and the ninhydrin is connected to the carbon end of the acetylated GPLGIRG polypeptide. The specific cross-linking reaction of the ninhydrin component with the amino group of the protein, the biochemical difference of the diseased tissue and the normal tissue in the microstructure, the rapid, clear and persistent targeted blue marking of the diseased area are realized. Not only the problem that the traditional dyeing agent cannot specifically mark the disease is solved, but also the cross-linking characteristic is used, the "cushion" formed by the lifting liquid under the mucosa is more stable and persistent, and double advantages are provided for the endoscopic surgery.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a staining agent for endoscopic surgery, its preparation method, and its application. Background Technology

[0002] Endoscopic submucosal dissection (ESD) and endoscopic mucosal resection (EMR) are advanced minimally invasive techniques for treating early-stage gastrointestinal tumors and precancerous lesions. The key to the success of these techniques lies in clearly visualizing the lesion boundaries (staining) and performing effective submucosal elevation (lifting) to facilitate the complete and safe removal of the lesion tissue.

[0003] In current clinical practice, staining and enhancement are two separate steps:

[0004] 1. Staining: Commonly used dyes include indigo carmine, methylene blue, and brilliant blue. However, these synthetic dyes have some problems. For example, methylene blue has potential genotoxicity, and its long-term safety is controversial; while indigo carmine is relatively safe, its contrast is sometimes unsatisfactory.

[0005] 2. Lifting: Commonly used submucosal injection agents include physiological saline, hydroxypropyl methylcellulose (HPMC), and sodium hyaluronate. These lifting agents are colorless, making it difficult for the operator to visually and in real-time judge the diffusion range and boundaries of the injection solution under the mucosa. This may result in insufficient injection volume (poor elevation, increasing the risk of perforation) or excessive injection (causing unclear vision or tissue damage). Although small amounts of dyes (such as indigo carmine, methylene blue, brilliant blue, etc.) are sometimes mixed into the lifting agent, this is a "physical mixture" and has problems such as uneven dispersion, poor stability, and weak color contrast.

[0006] Genipin is a natural iridoid compound extracted from gardenia fruit. It reacts with primary amine compounds (amino acids, peptides, and proteins, etc.) to produce a blue product, commonly known as "gardenia blue." On July 16, 2025, the US FDA officially added gardenia blue to its "certification-exempt colorant" list. Gardenia blue is a safe and non-toxic natural food coloring and biocrosslinking agent. Currently, there are no reports of gardenia blue being used as a staining agent specifically for endoscopic surgery, particularly in combination with submucosal lifting agents to form functional compositions.

[0007] Existing technology CN 111110926 A discloses "an injectable colored gel pad for gastrointestinal mucosal stratification and its application." This gel pad requires the injection of a silk fibroin solution, chitosan solution, or gelatin solution into the submucosa of the digestive tract, followed by the injection of a genipin solution containing disodium glycerol phosphate. After a cross-linking reaction, a gel pad is formed in the submucosa of the digestive tract to separate the diseased mucosal layer from the muscle layer, facilitating the peeling of the diseased mucosal layer. However, this method has the following drawbacks: First, it requires two injection operations, which is cumbersome in practice; second, the reaction of genipin with the silk fibroin solution, chitosan solution, or gelatin solution is slow in natural reactions, requiring adjustment of the amount of genipin and disodium glycerol phosphate in the genipin solution to control the gel pad formation time; third, it is also necessary to identify the lesion area and accurately inject the colored gel pad into the lesion area; finally, residual genipin poses a potential risk of hepatotoxicity.

[0008] Developing a novel natural staining agent that is simple to operate, highly safe, produces excellent staining results, effectively identifies lesion areas, and can be perfectly combined with enhancement agents to achieve real-time intraoperative visualization has significant clinical implications and market value. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention cleverly utilizes the specific cross-linking reaction between genipin and protein amino groups, as well as the biochemical differences in microstructure between diseased and normal tissues, to achieve rapid, clear, and durable targeted blue labeling of lesion areas. This invention not only solves the problem of traditional staining agents' inability to specifically label lesions, but also, due to its cross-linking properties, the "cushion" formed by the elevating solution under the mucosa is more stable and durable, providing a dual advantage for endoscopic surgery (such as ESD). This invention also incorporates the amino acid sequence GPLGIRG, which can be digested by specific proteases at the tumor site, enabling specific identification of lesion sites. The specific details of this invention are as follows:

[0010] In a first aspect, the present invention provides a staining agent for endoscopic surgery, said staining agent being an acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor. The acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor is obtained by reacting an acetylated GPLGIRG polypeptide with genipin or a derivative thereof, wherein genipin or a derivative thereof is linked to the C-terminus of the acetylated GPLGIRG polypeptide.

[0011] Furthermore, the acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor is obtained by reacting acetylated GPLGIRG polypeptide with genipin in a molar ratio of 1:1 to 2.

[0012] Furthermore, the acetylated GPLGIRG polypeptide is linked to genipin via chemical bonds or non-chemical bonds.

[0013] Further, the genipin derivative is a genipin amino acid derivative. Preferably, the genipin derivative is prepared by reacting genipin with an amino acid, wherein the amino acid is selected from at least one of glycine, alanine, lysine, arginine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid.

[0014] It should be noted that the amino acids mentioned do not include proline.

[0015] It should be noted that in the staining agent described in this invention, genipin or its derivatives are mainly used to react with free amino acids in the lesion area to obtain gardenia blue for staining.

[0016] In a second aspect, the present invention provides a composition comprising the aforementioned staining agent and a submucosal lifting matrix.

[0017] Furthermore, the concentration of the staining agent is from 0.001% to 0.2% (w / v). Within this concentration range, it provides a bright, durable blue mark without compromising the rheological properties of the matrix or causing tissue toxicity due to excessive concentration.

[0018] Furthermore, the submucosal lifting matrix is ​​selected from at least one of physiological saline, glycerol, sodium hyaluronate, sodium carboxymethyl starch, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), sodium glycerophosphate, sodium alginate, fibrinogen solution, hydroxyethyl starch, phosphorylated amylopectin, poloxamer, trehalose, gellan gum, chitosan, carboxymethyl chitosan, acetylcysteine, lithium magnesium silicate, PLGA-PEG-PLGA block copolymer, and polypeptide.

[0019] In one specific embodiment of the present invention, the submucosal lifting matrix is ​​sodium hyaluronate.

[0020] Furthermore, the composition also includes one or more excipients acceptable in the medical or pharmaceutical fields, such as pH adjusters, osmotic pressure regulators, stabilizers, thickeners, preservatives, etc.

[0021] The pH adjuster, used for acidification, is selected from hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, and their buffer salts. The reagents used for alkalinity adjustment include sodium hydroxide, potassium hydroxide, sodium bicarbonate, disodium hydrogen phosphate, Tris, and their buffer salts. The pH value of the composition is preferably 6.5-7.8, more preferably 7.0-7.4.

[0022] The osmotic pressure regulator is a medically acceptable carrier that is isotonic with human blood plasma or has an osmotic pressure similar to that of a 0.85% injectable sodium chloride solution.

[0023] The thickener is selected from at least one of propylene glycol, butylene glycol, sorbitol, erythritol, xylitol, maltitol, mannitol, glycerin, polyglycerol ether-26, polyoxypropylene glycerol ether, polyoxypropylene ethylene oxide glycerol ether, and polyethylene glycol.

[0024] The preservatives are selected from parabens (such as methylparaben, ethylparaben and their mixtures), benzyl alcohol, benzalkonium chloride / benzalkonium bromide, phenols (such as phenol, cresol and their mixtures). The concentration of the preservatives must be strictly controlled within the safe range specified in the pharmacopoeia, usually 0.01% to 0.5% (w / v).

[0025] In a third aspect, the present invention provides a method for preparing the staining agent, the method comprising the following steps:

[0026] The acetylated GPLGIRG peptide Ac-GPLGIRG was dissolved in anhydrous DMF, and the C-terminus of Ac-GPLGIRG was activated by adding an activator. Genipin or its derivative was added directly or dissolved and then added dropwise to the activation reaction solution, with the molar ratio of the acetylated GPLGIRG peptide to genipin or its derivative being 1:1~2. DIEA was added, the mixture was brought to room temperature, and the reaction was continued with stirring in the dark for 12-24 hours to obtain the acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor.

[0027] Furthermore, the activator is a carbodiimide, preferably at least one of EDC / NHS or DCC.

[0028] Furthermore, the method also includes the step of separating and purifying the obtained acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor.

[0029] In one embodiment of the present invention, the separation and purification step involves slightly acidifying and quenching the reaction solution with dilute acid (such as 1% TFA aqueous solution), concentrating it by rotary evaporation, precipitating it with cold diethyl ether / n-hexane, and collecting the solid by centrifugation.

[0030] The crude product was dissolved in DMSO or methanol / water, purified by preparative reversed-phase HPLC (C18 column, acetonitrile / water gradient elution), the target peak was collected, and the product was lyophilized to obtain a colorless or very light-colored Ac-GPLGIRG-Gen enzyme activator precursor.

[0031] In a fourth aspect, the present invention provides the use of the said staining agent or the said composition in the preparation of a staining agent for endoscopic submucosal staining.

[0032] Furthermore, the endoscopic procedures include endoscopic submucosal dissection (ESD) and / or endoscopic mucosal resection (EMR).

[0033] The beneficial effects of the present invention include, but are not limited to:

[0034] This invention utilizes the specific cross-linking reaction between genipin and protein amino groups, as well as the biochemical differences in the microstructure between diseased and normal tissues, to achieve rapid, clear, and long-lasting targeted blue labeling of diseased areas.

[0035] This invention not only solves the problem that traditional staining agents cannot specifically mark lesions, but also provides a dual advantage for endoscopic surgery (such as ESD) due to the cross-linking properties of the lifting fluid forming a more stable and durable "cushion" under the mucosa.

[0036] The present invention also incorporates an amino acid sequence GPLGIRG that can be digested by specific proteases at the tumor site, enabling it to specifically identify the lesion site.

[0037] The composition of the present invention has both "lifting" and "staining" functions, which simplifies the surgical procedure, shortens the surgical time, and improves the surgical efficiency. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0039] Figure 1 These are the mass spectrum and chromatogram of the acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor in Example 1 of this invention, wherein Figure A is its chromatogram and Figure B is its mass spectrum;

[0040] Figure 2 This is a schematic diagram of the in vitro simulated mucosal staining experiment results of the submucosal lifting agent composition containing the Ac-GPLGIRG-Gen enzyme activation precursor in Example 3 of the present invention. In the figure, A and D are the staining results of the experimental group, negative control group 1, negative control group 2, and negative control group 3, respectively.

[0041] Figure 3This is a schematic diagram of the in vitro simulated submucosal injection experiment results of the submucosal lifting agent composition containing the Ac-GPLGIRG-Gen enzyme activation precursor in Example 4 of the present invention. In this figure, Figure A shows the bulging effect after injection in the experimental group (left) and the control group (right), and Figure B shows the effect after peeling in the experimental group (left) and the control group (right).

[0042] Figure 4 Figure A is a schematic diagram of the experimental results of the animal gastric cancer model in Embodiment 5 of the present invention, wherein Figure A is a schematic diagram of the injection operation of ESD surgery, and Figure B is a schematic diagram of the result of the lesion area bulging.

[0043] Figure 5 These are schematic diagrams of the staining results of various organ sections in the animal model experiment of this invention. Figure A is a pathological section of the gastric antrum mucosa (HE, X100), Figure B is a pathological section of the gastric body mucosa (HE, X100), and Figure C is a pathological section of the esophageal mucosa (HE, X100). Detailed Implementation

[0044] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0045] Example 1. Preparation of Acetyl-GPLGIRG-Gen enzyme activation precursor

[0046] The peptide GPLGVRG (SEQ ID NO.1) is a specific recognition sequence for MMP-2 and is rapidly cleaved. The peptide GPQGIAGQ (SEQ ID NO.2) is a specific recognition sequence for matrix metalloproteinases MMP-2 / MMP-9, effectively distinguishing MMP-2 / 9 from other MMP family members, but its cleavage rate is significantly weaker. Through analysis of different sequences, the P1' Val of GPLGVRG was replaced with a larger Ile to better fill the deep S1' hydrophobic pocket of MMP-2 / 9, improving affinity and yielding the peptide GPLGIRG (SEQ ID NO.3). Current research indicates that various solid tumors (gastrointestinal, breast, lung, etc.) contain matrix metalloproteinases MMP-2 / MMP-9, providing feasibility for preparing enzyme activation precursors. A biotechnology company was directly commissioned to prepare the N-terminal acetylated peptide GPLGIRG (Ac-GPLGIRG) for subsequent experiments.

[0047] The purified Ac-GPLGIRG peptide was dissolved in anhydrous DMF to prepare a 25 mM solution. 2 mL of this Ac-GPLGIRG peptide solution was placed in a 10 mL round-bottom flask. The reaction flask was placed in an ice bath (0°C), connected to a nitrogen atmosphere, and wrapped with aluminum foil to protect it from light. Excess EDC·HCl and NHS were added with stirring, where the molar ratio of Ac-GPLGIRG peptide: EDC: NHS = 1:2:2. The reaction was carried out under ice bath, nitrogen protection, and light protection conditions with stirring for 2–4 h to activate the carboxyl groups of the peptide into active NHS esters. Genipin was dissolved in a small amount of anhydrous DMF and then slowly added dropwise to the activated reaction solution using a syringe or pipette, with a molar ratio of activated peptide: genipin = 1:1.5. Add an appropriate amount of DIEA to neutralize the hydrochloric acid produced in the reaction and maintain the pH of the reaction system at 7.8. The amount of DIEA added is twice the molar amount of EDC. Raise the temperature to room temperature and continue to stir the reaction under nitrogen protection in the dark for 12-24 hours.

[0048] The reaction solution was slightly acidified and quenched with dilute acid (e.g., 1% TFA aqueous solution) until the pH paper showed a weakly acidic solution (pH=4.5). The reaction solution was then transferred to a round-bottom flask and concentrated under reduced pressure using a rotary evaporator in a warm water bath (<30°C). Pre-cooled diethyl ether / n-hexane mixed solvent (volume ratio of diethyl ether:n-hexane = 1:2) was added to the concentrated viscous residue, where the volume ratio of the concentrated viscous residue to the mixed solvent was 1:1.5. The mixture was then centrifuged at high speed (12000 rpm, 10 minutes) at low temperature (4°C). The supernatant was carefully discarded, and the solid precipitate at the bottom of the tube was collected; this was the crude product Ac-GPLGIRG-Gen.

[0049] The crude product was dissolved in DMSO and filtered through a 0.22 μm filter membrane. It was then purified by preparative reversed-phase HPLC-MS, and the chromatographic peaks corresponding to the target product were collected and identified. The collected fraction was freeze-dried to obtain a colorless or very light-colored solid powder, which was the purified Ac-GPLGIRG-Gen enzyme activator precursor.

[0050] The chromatographic conditions are as follows:

[0051] Chromatographic column: C18 column;

[0052] Mobile phase: Phase A: Water (containing 0.1% TFA); Phase B: Acetonitrile (containing 0.1% TFA);

[0053] The elution procedure is shown in Table 1;

[0054] Table 1 Gradient elution program

[0055]

[0056] Flow rate: 5 mL / min;

[0057] Detection wavelength: 240 nm;

[0058] Column temperature setting: 35°C.

[0059] Test results as follows Figure 1 As shown.

[0060] The results showed that the chromatogram (Figure A) had three peaks, representing free Ac-GPLGIRG, free genipin, and the Ac-GPLGIRG-Gen enzyme activation precursor, respectively. The fraction collected from the third peak was identified as the Ac-GPLGIRG-Gen enzyme activation precursor. The mass spectrum (Figure B) showed a mass charge-to-mass ratio of 907.5, confirming that it is indeed the Ac-GPLGIRG-Gen enzyme activation precursor.

[0061] Example 2. Preparation of a submucosal lifting agent composition containing Ac-GPLGIRG-Gen enzyme activation precursor (hereinafter referred to as the lifting agent composition).

[0062] Each 1000mL contains:

[0063] Sodium hyaluronate 1.0g (submucosal lifting matrix);

[0064] Ac-GPLGIRG-Gen enzyme activation precursor 0.2g (staining agent);

[0065] Sodium chloride 8.4g (isotonic conditioner);

[0066] Phosphate buffer (potassium dihydrogen phosphate 0.6g, dipotassium hydrogen phosphate 0.076g) (pH buffer);

[0067] Add water to a final volume of 1000 mL.

[0068] Preparation method: Weigh 1.0 g of sodium hyaluronate and slowly add it to a solution prepared from sodium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Stir until completely swollen to form a transparent gel matrix. Weigh 0.2 g of the Ac-GPLGIRG-Gen enzyme activator precursor prepared in Example 1 and add it to the above gel matrix. Stir continuously for 4 hours until completely dissolved and a homogeneous, transparent colloidal solution is formed. Adjust the volume to 1000 mL with purified water to achieve a final concentration of 0.1% (w / v) for sodium hyaluronate and 0.01% (w / v) for the Ac-GPLGIRG-Gen enzyme activator precursor. Filter aseptically (0.22 μm), dispense into vials, and store at room temperature protected from light.

[0069] Example 3. In vitro simulated mucosal staining experiment

[0070] Experimental group: simulated lesion area (containing MMP-9) + the enhancement agent composition of Example 2.

[0071] Negative control group 1: normal mucosal area + the lifting agent composition of Example 2.

[0072] Negative control group 2: Simulated lesion area (without MMP-9) + the booster composition of Example 2.

[0073] Positive control group: normal mucosal area + conventional dye (indigo carmine) for direct color development.

[0074] Obtain healthy pig stomachs (with full lining) from a reputable slaughterhouse. Rinse thoroughly with pre-cooled PBS or saline solution within 30 minutes and transport on ice. Cut along the greater curvature of the stomach, flatten it, and fix it to a cork board or silicone pad with tissue staples or glue, mucosal side up. Apply 50%-75% ethanol precisely to the target area for 30-60 seconds using a cotton swab. Immediately and gently rinse the affected area with copious amounts of saline solution to remove chemical reagents or tissue debris, and blot dry with filter paper. Add or spray a specific concentration of recombinant MMP-9 enzyme solution onto the affected area, incubate for 10 minutes, and then perform staining experiments according to the reagents used for each group.

[0075] The results are as follows Figure 2 As shown in Figure A, the lesion areas in the experimental group were immediately stained a clear blue. Figures B and C show that the negative control groups 1 and 2 showed no color development, demonstrating that the submucosal lifting agent composition containing the Ac-GPLGIRG-Gen enzyme activation precursor has good enzyme response activity. Figure D shows that the normal mucosa in the positive control group was directly stained blue by conventional dyes, without any distinguishing effect.

[0076] Example 4. In vitro simulated submucosal injection experiment

[0077] The method for preparing the porcine gastric mucosa simulating the lesion area is the same as in Example 3. Injections were then performed using an endoscopic injection needle:

[0078] Experimental group: 3 mL of the submucosal lifting agent composition containing Ac-GPLGIRG-Gen prepared in Example 2 + MMP-9.

[0079] Control group: 3 mL of 0.1% sodium hyaluronate solution without Ac-GPLGIRG-Gen (colorless control group, without Ac-GPLGIRG-Gen, other components and preparation method are the same as in Example 2) + MMP-9.

[0080] Post-injection results as follows Figure 3As shown in Figure A, the experimental group formed a well-defined, uniformly colored blue ridge after injection, and the ridge height was persistent. The control group formed a colorless, transparent ridge, the boundaries and extent of which were difficult to accurately determine under a microscope, especially in poor lighting or when there was slight bleeding. The results after peeling are shown in Figure B; the experimental group showed a clear blue ridge, while the control group was colorless.

[0081] Example 5 Animal Model Experiment

[0082] Animal: Miniature pigs are the "gold standard" model for endoscopic research of the digestive tract, as their anatomy, physiology, and mucosal structure are most similar to those of humans.

[0083] Tumor model construction: Human gastric cancer cell line MKN-45 (purchased from Shanghai Kanglang Biotechnology Co., Ltd., catalog number CKL-0292, cell purity >90%) was transplanted in situ into the submucosal layer of pig stomach to induce gastric tumors.

[0084] Experimental Groups:

[0085] Experimental group: Tumor-bearing animals were sprayed with the submucosal lifting agent composition containing Ac-GPLGIRG-Gen of Example 2 under gastroscopy.

[0086] Animal preparation and anesthesia: Fasting was performed before the experiment, followed by general anesthesia and endotracheal intubation, and vital signs were monitored.

[0087] Endoscopic examination and localization: Using a swine or pediatric endoscope to enter the gastric cavity, the location, size, and morphology of all suspected naturally occurring or induced lesions are recorded.

[0088] Composition injection and staining:

[0089] Through the endoscope's working channel, the corresponding reagents were injected into the target lesion and the surrounding normal mucosa according to the experimental group.

[0090] Observe and time immediately: Record the time when the lesion area begins to turn blue after spraying, the color intensity, the clarity of the boundary, and compare it with the surrounding unstained area.

[0091] Dynamic observation: Observe continuously for 5-10 minutes and record whether the color spreads or fades.

[0092] Following the safety testing experiment, the animals were closely monitored for recovery. At the end of the experiment (7 days later), the animals were euthanized and subjected to gross necropsy, with a focus on examining organs such as the stomach, liver, and kidneys. Tissue samples were taken for pathological examination to assess for any toxic damage.

[0093] The results are as follows Figure 4As shown in Figure B, the results of an ESD procedure using the Gardenia Blue Submucosal Lifting Agent Composition of the present invention are illustrated in Figure A. The specific operation is shown in Figure B, and the results are shown in Figure B. The results show that the blue lifting agent clearly marked the peeling boundary, and no diffusion or fading was observed within 13 minutes. Although other non-lesion areas were raised, they were not stained blue.

[0094] Pathological examination such as Figure 5 As shown in Figure A, the gastric antrum mucosa is microscopically examined. The image shows that the gastric antrum mucosa epithelium is intact, with abundant glands in the lamina propria. The regenerated mucosa completely covers the resection site, with no congestion, edema, or inflammatory reaction observed. Tissue healing is good, and no sample residue is observed. Figure B shows the gastric body mucosa microscopically examined. The gastric body mucosa epithelium is intact, with abundant glands in the lamina propria. The regenerated mucosa completely covers the resection site, with no congestion, edema, or inflammatory reaction observed. Tissue healing is good, and no sample residue is observed. Figure C shows the esophageal mucosa microscopically examined. The esophageal mucosa epithelium is intact, and the regenerated mucosa completely covers the resection site. No congestion, edema, or inflammatory reaction is observed. Tissue healing is good, and no sample residue is observed. This demonstrates that the submucosal lifting agent composition containing Ac-GPLGIRG-Gen of the present invention has essentially no toxic side effects.

[0095] Example 6. Genotoxicity test

[0096] Sample preparation:

[0097] Enzyme activation precursor group: Two samples are required.

[0098] Unactivated precursor: Prepare a series of concentration solutions directly with appropriate DMSO.

[0099] Enzyme digestion activation product: The precursor was incubated with a sufficient amount of recombinant MMP-9 enzyme at 37°C for a sufficient time. After the reaction was complete, the reaction solution was used as the test sample.

[0100] Commercially available filler groups: Dilute serially with solvents according to product instructions or clinical use concentrations.

[0101] Negative control: DMSO solvent.

[0102] Positive control: Depending on the strain, sodium azide (for TA100 / TA1535) can be used when metabolic activation is not required; 2-aminoanthracene (2-AA) should be used when metabolic activation is required.

[0103] Strains selected and identified: TA97, TA98, TA100, TA102, TA1535. Genotypic and biological identification of the strains is required before formal testing.

[0104] Metabolic activation system (S9 mixture): Each experiment must be set with two conditions: "with S9" (simulating in vivo metabolism) and "without S9".

[0105] Melt and maintain the top agar in a 45°C water bath. Add 2 mL of top agar to a sterile test tube, then add the following in sequence:

[0106] 0.1 mL of fresh bacterial culture of the specific strain. 0.1 mL of the test sample (or control) at the corresponding concentration.

[0107] Group with S9: Add 0.5 mL of S9 mixture; Group without S9: Add 0.5 mL of phosphate buffer.

[0108] After quickly mixing, pour the mixture onto the lowest-level glucose agar plate and spread it evenly. Prepare at least three parallel plates for each treatment (each sample / concentration / strain). Once the agar has solidified, invert all plates and incubate at 37°C in the dark for 48 hours. After incubation, count the number of revertant colonies on each plate using an automated colony counter or manually.

[0109] Information for each group is shown in Table 2.

[0110] Table 2 Information for Each Group

[0111]

[0112] The experimental results (Table 3) show that the Ac-GPLGIRG-Gen enzyme activation precursor and the enzyme activation precursor + MMP-9 (after enzyme digestion activation) provided by this invention, with or without the addition of S9 activation group, showed negative reactions to the tested strains TA97, TA98, TA100, TA1535, and TA102. The number of revertant colonies did not exceed twice that of the negative control, indicating no genotoxicity. In contrast, the commercially available filler (containing 0.001% methylene blue) showed a significant positive reaction to strain TA97, and strains TA98, TA100, TA1535, and TA102 showed varying degrees of thickening, indicating a certain degree of genotoxicity.

[0113] Table 3. Results of genotoxicity experiments for strains TA97, TA98, TA100, TA102, and TA1535

[0114]

[0115] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A staining agent for gastroscopy, characterized in that, The staining agent is an acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor, which is obtained by reacting an acetylated GPLGIRG polypeptide with genipin, wherein genipin is linked to the carbon terminus of the acetylated GPLGIRG polypeptide. The acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor is obtained by reacting an acetylated GPLGIRG polypeptide with genipin at a molar ratio of 1:1 to 2.

2. A composition for gastroscopy, characterized in that, The composition comprises the staining agent of claim 1 and the submucosal lifting matrix.

3. The composition according to claim 2, characterized in that, The concentration of the dye by mass volume percentage is 0.001% to 0.2%.

4. The composition according to claim 2, characterized in that, The submucosal lifting matrix is ​​selected from at least one of physiological saline, glycerin, sodium hyaluronate, sodium carboxymethyl starch, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), sodium alginate, hydroxyethyl starch, phosphorylated amylopectin, poloxamer, gellan gum, chitosan, carboxymethyl chitosan, and PLGA-PEG-PLGA block copolymer.

5. The composition according to claim 2, characterized in that, The mass-volume percentage concentration of the submucosal lifting matrix is ​​0.1% to 2%.

6. The composition according to claim 2, characterized in that, The composition also includes excipients acceptable in the medical or pharmaceutical fields.

7. A method for preparing the staining agent according to claim 1, characterized in that, The method includes the following steps: The acetylated GPLGIRG polypeptide Ac-GPLGIRG was dissolved in anhydrous DMF, and an activator was added to activate the C-terminus of Ac-GPLGIRG. Genipin or its derivative was added directly or dissolved and then added dropwise to the activation reaction solution. The molar ratio of the acetylated GPLGIRG polypeptide to genipin or its derivative was 1:1~2. DIEA was added, and the mixture was heated to room temperature and stirred in the dark for 12-24 hours to obtain the acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor.

8. The method according to claim 7, characterized in that, The method further includes the step of separating and purifying the obtained acetyl-GPLGIRG-genipin (Ac-GPLGIRG-Gen) enzyme activation precursor.

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