A low eutectic bioadhesive based on a blend of peptides from periplaneta americana and ophiophagus hannah

Abstract

The application provides a low-eutectic bio-adhesive based on mixed polypeptides of Periplaneta americana and snake slough and a preparation method. Based on the application status of Periplaneta americana and snake slough in surgical diseases and external use drugs, the bio-adhesive fuses the optimal proportion of two animal polypeptide extracts and a low-eutectic solvent, and the three form a uniform composite system through a low-eutectic effect and a self-assembly process, is solidified at a diseased part through a preheating-cooling mode and plays a role. The low-eutectic solvent plays multiple roles as a cosolvent, a plasticizing excipient, a penetration enhancer, a bacteriostatic agent and an auxiliary functional substance. The preparation process is fast, the conditions are mild, special equipment is not needed and the process is easy to scale. The adhesive formula is simplified to the maximum extent, the raw materials are green, the system is stable, no other components are specially added, the operation is simple during use and the bio-adhesive is friendly and safe as an auxiliary treatment means. The whole system fully utilizes the unique activity and nursing efficacy of various components and can play a synergistic treatment role.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, and in particular relates to a bioadhesive containing two types of active polypeptides derived from animal raw materials and a eutectic solvent, and a method for preparing the same. Background Technology

[0002] American cockroach is a traditional insect-based Chinese medicine. Its extract is rich in small molecule peptides, amino acids, and various active substances, exhibiting significant tissue repair activity. Basic research and clinical applications have confirmed that American cockroach extracts, represented by Kangfuxin Liquid, have good therapeutic effects on burns, trauma, ulcers, and postoperative wound repair (Gao Yangyang, et al.; Chinese Journal of Experimental Traditional Medical Formulae, 2021, 27 (4): 220-228). With the continuous growth of related drug production capacity, the amount of waste residue from American cockroach alcohol extraction has also increased year by year. The waste residue of American cockroach still contains a large amount of active substances such as proteins and peptides, which have considerable redevelopment and utilization value. However, these residues are usually treated as waste, which not only wastes resources but also brings certain environmental pressures. In terms of pharmacological activity, the peptides, polysaccharides, and other substances remaining in the residue of American cockroach have been proven to still have anti-inflammatory, antibacterial, antioxidant, cell and granulation tissue generation, and wound growth and healing activities. These properties lay the foundation for the clinical development of novel drug materials that promote tissue repair and wound healing.

[0003] Snake slough, the dried epidermal membrane naturally shed by colubridae, is mainly composed of keratin, collagen, amino acids, fatty acids, and sterols. Clinical studies have found that it has a good therapeutic effect on herpes zoster, promoting the scab formation and resolution of blisters and relieving pain. In addition, snake slough is often used as a component of compound topical powders for the treatment of external injuries, possessing analgesic, hemostatic, and wound-healing properties. Modern pharmacological studies have confirmed that snake slough extract contains antibacterial, anti-inflammatory, and wound-healing active ingredients. Specifically, snake slough keratin, after appropriate hydrolysis, yields polypeptide fragments with specific structures and functions, which can promote fibroblast proliferation and epidermal regeneration, accelerating wound healing and demonstrating excellent medicinal value. In recent years, researchers have begun to focus on the hydrolysates of snake slough keratin, which possess unique advantages in topical preparations such as wound dressings and tissue adhesives. In addition, these macromolecules can also form β-sheet secondary structures through self-assembly (Tan, et al.; Mini-Reviews InMedicinal Chemistry, 2023, 23(4): 399-411), giving the material good film-forming properties and mechanical stability.

[0004] Eutectic solvents are eutectic mixtures formed by hydrogen bond interactions between hydrogen bond acceptors and hydrogen bond donors in a certain stoichiometric ratio, with melting points significantly lower than those of their individual components. As a novel type of green solvent, eutectic solvents offer advantages such as simple preparation, low cost, good biocompatibility, and biodegradability, showing broad application prospects in areas such as drug solubilization, transdermal absorption enhancement, and biomaterial preparation (Zainal-Abidin, et al.; Journal of Controlled Release, 2019, 316: 168-195). In the field of medical materials, the application research of eutectic solvents is increasingly in-depth. As pharmaceutical excipients, eutectic solvents can be used to improve drug solubility and transdermal absorption performance. In biomaterial preparation, eutectic solvents can serve as green preparation media for pharmaceutical excipients such as microcrystalline cellulose, resulting in materials exhibiting good release characteristics. Furthermore, some eutectic solvents themselves possess certain antibacterial activities and can be used as multifunctional excipients in biomedical materials, playing multiple roles such as plasticizing, permeation enhancement, and antibacterial action.

[0005] Bioadhesives are materials that can adhere to biological tissues. They are convenient to use, quickly achieving tissue closure and hemostasis while reducing secondary injury to patients, and have received widespread attention in clinical applications as an alternative to traditional suturing techniques. Existing bioadhesives are mainly divided into two categories: natural bioadhesives and synthetic bioadhesives. Natural bioadhesives, such as fibrin glue and gelatin-based adhesives, have good biocompatibility and cause mild inflammatory and foreign body reactions (Su Xinru, et al.; Chinese Invention Patent, CN202511240126.0). However, they generally suffer from drawbacks such as low adhesive strength in wet environments, unstable quality, and poor thermal stability. Furthermore, fibrin glue is derived from blood products, raising concerns about biosafety, and its high cost limits its widespread application. Synthetic bioadhesives, such as cyanoacrylate, polyethylene glycol, and polyurethane adhesives, have advantages such as high adhesive strength, rapid curing, and stable performance (Mo Xiumei, et al.; Chinese Invention Patent, CN201810218280.1). Meanwhile, these materials often exhibit poor biocompatibility, potentially leading to severe inflammatory responses and immune rejection; their degradation products may be cytotoxic, and the exothermic polymerization process can cause thermal damage to surrounding tissues. Although synthetic polymeric bioadhesives offer certain advantages in mechanical properties and mechanical regulation, their shortcomings in wet adhesion, biocompatibility, and mechanical compatibility still fail to meet the needs of clinical applications. Achieving excellent biocompatibility, suitable mechanical strength, controllable degradation performance, and biological functions that promote tissue repair remains a significant challenge in the field of bioadhesive research and development. Therefore, developing a novel bioadhesive based on natural sources, possessing both repair-promoting activity and good mechanical properties, and capable of controlled curing through a simple method, has significant clinical implications and application value. Summary of the Invention

[0006] The purpose of this invention is to provide a low-eutectic bioadhesive based on a mixture of American cockroach and snake slough polypeptides and its preparation method. Based on the current applications of American cockroach and snake slough in surgical diseases and topical medications, this bioadhesive integrates an optimal ratio of two animal polypeptide extracts and a low-eutectic solvent. These three components form a homogeneous composite system through the low-eutectic effect and self-assembly process, which solidifies at the affected area and exerts its effect through a preheating-cooling process. The low-eutectic solvent acts as a co-solvent, plasticizer, penetration enhancer, antibacterial agent, and auxiliary functional agent, playing multiple roles. The preparation process is rapid, mild, requires no special equipment, and is easily scalable. The adhesive formulation is simplified to the maximum extent, using environmentally friendly raw materials, with a stable system and no other specially added components. It is simple to use and safe and user-friendly as an adjunctive treatment. The entire system fully utilizes the unique activity and therapeutic effects of multiple components, achieving a synergistic therapeutic effect.

[0007] Technical solution: In order to achieve the above objectives, a low-melting bioadhesive based on a mixture of American cockroach and snake molt peptides and its preparation method are proposed.

[0008] This invention discloses a low-eutectic bioadhesive based on a mixture of American cockroach and snake slough polypeptides. Its characteristic feature is that the active components, comprising extracts of active peptides from American cockroaches and snake slough polypeptides, are combined with a low-eutectic solvent to form a homogeneous composite system through the low-eutectic effect and self-assembly process. The adhesive is applied to the affected area via a preheating-cooling process. The specific preparation process is as follows:

[0009] (1) The main functional components, American cockroach and snake slough polypeptide, are mixed and compounded at a ratio of 3:7~5:5 g / g. This ratio is used to balance the bioactivity and mechanical adhesion strength of the bioadhesive.

[0010] (2) Mix the compound of American cockroach and snake slough polypeptide with a low eutectic solvent at a mass-volume ratio of 1:3~1:5 g / mL, and disperse it fully at 55~60℃ with 300~500W ultrasonic for 10~20min; after forming a uniform low eutectic bio-adhesive composite system, sterilize and seal for later use.

[0011] The present invention discloses a low-melting bioadhesive based on a mixture of American cockroach and snake slough polypeptides, characterized in that the American cockroach active polypeptide extract is derived from the enzymatic hydrolysis product of pharmaceutical waste residue, with a molecular weight range of 500~1000 Da; this component is rich in small molecule active peptides, which mainly play the role of promoting tissue repair, anti-inflammation and healing.

[0012] The present invention discloses a low-melting bioadhesive based on a mixture of American cockroach and snake slough polypeptides, characterized in that the snake slough polypeptide extract is derived from the enzymatic hydrolysis product of snake slough keratin, with a molecular weight range of 2000~3000 Da, and the polypeptide has the ability to self-assemble into a β-sheet structure.

[0013] The present invention discloses a low-eutectic bioadhesive based on a mixed polypeptide of American cockroach and snake slough, characterized in that the low-eutectic solvent is a mixture of betaine and phytic acid or betaine and lactic acid, with a molar ratio of the two components of 3:1 to 1:3. This low-eutectic solvent plays multiple roles in the present invention as a co-solvent, plasticizer, penetration enhancer, antibacterial agent, and auxiliary functional compound.

[0014] The present invention discloses a low-melting bio-adhesive based on a mixture of American cockroach and snake slough polypeptides, characterized in that its application method is as follows: before use, the bio-adhesive is preheated to 60°C in a water bath and shaken; then slowly cooled to near room temperature and applied to the affected area; finally, an ice pack is used to quickly cool the affected area, and self-assembly and curing are completed after 5-10 minutes. Attached Figure Description

[0015] To more completely illustrate the technical solutions of specific embodiments of the present invention, the accompanying drawings required in the embodiments will be briefly described below. The described drawings are merely a part of the embodiments of the present invention; those skilled in the art should be able to obtain other drawings of the embodiments based on these drawings. Wherein:

[0016] Figure 1 The American cockroach polypeptide extract and snake slough polypeptide extract described in this invention;

[0017] Figure 2 This refers to the curing process of the eutectic bio-adhesive described in this invention;

[0018] Figure 3 This shows the trend of average particle size change of the polypeptide before and after self-assembly in this invention;

[0019] Figure 4 The adhesive strength of the eutectic bioadhesives with different compositions described in this invention;

[0020] Figure 5 This invention describes the in vitro simulated release behavior of peptides in the eutectic bioadhesive described in this invention.

[0021] Figure 6 This is the stability evaluation result of the eutectic bio-adhesive described in this invention. Detailed Implementation

[0022] The following is a detailed description of the present invention. While specific embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0023] Example 1

[0024] 10 g of defatted 120-mesh dry powder of American cockroach was soaked in 150 mL of 75% ethanol and extracted by ultrasonication at 300W for 30 min. The extraction was filtered and repeated once, and the two extracts were combined. The orange-yellow transparent extract was concentrated by rotary evaporation until no alcohol odor was detected. Three times the volume of 95% ethanol was added and the mixture was placed at 4℃ for 12 h. The mixture was centrifuged at 4000 rpm for 5 min, the precipitate was collected and dissolved by stirring with Tris-HCl buffer solution, and then dialyzed through a semipermeable membrane with a molecular weight cutoff of 1000 Da. The extracellular fluid was collected, concentrated under reduced pressure, and then freeze-dried to obtain the American cockroach polypeptide extract.

[0025] Example 2

[0026] 10g of 100-mesh dried snake slough powder was soaked in 200mL of 60% ethanol and extracted at 50℃ for 2 h. After extraction, the mixture was filtered while hot, and the residue was rinsed three times with water. The residue was dried by air blowing, and ultrapure water and 2500 U / g immobilized alkaline protease were added at a solid-liquid ratio of 1:3g / mL. The mixture was enzymatically hydrolyzed at 42℃ for 4 h. Then, 10 times the volume of ultrapure water was added to the hydrolysate, and the mixture was extracted at 50℃ for 2 h. After extraction, the mixture was filtered while hot, and finally dialyzed through a semipermeable membrane with a molecular weight cutoff of 2000 Da. The membrane fluid was collected, concentrated under reduced pressure, and then freeze-dried to obtain the snake slough polypeptide extract.

[0027] The products obtained in Examples 1 and 2 are as follows Figure 1 As shown, the American cockroach polypeptide extract is a light yellow amorphous powder, and the snake slough polypeptide extract is a beige amorphous granules. Both have a certain degree of hygroscopicity and should be dried and sealed for later use.

[0028] Example 3

[0029] American cockroaches and snake slough peptides were compounded at a ratio of 3:7 g / g and mixed with a betaine-phytic acid eutectic solvent at a molar ratio of 3:1 at a mass-volume ratio of 1:3 g / mL. The mixture was then dispersed at 55°C with ultrasonic waves at 300W for 10 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0030] Example 4

[0031] American cockroaches and snake slough peptides were compounded at a ratio of 3:7 g / g and mixed with a betaine-lactic acid eutectic solvent at a molar ratio of 1:1 at a mass-volume ratio of 1:3 g / mL. The mixture was then dispersed at 55°C with ultrasonic waves at 300W for 10 min to form a uniform eutectic bio-adhesive composite system. The mixture was then sterilized and sealed for later use.

[0032] Example 5

[0033] American cockroaches and snake slough peptides were compounded at a ratio of 3:7 g / g and mixed with a betaine-phytic acid eutectic solvent at a molar ratio of 3:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0034] Example 6

[0035] American cockroaches and snake slough peptides were compounded at a ratio of 3:7 g / g and mixed with a betaine-lactic acid eutectic solvent at a molar ratio of 1:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0036] Example 7

[0037] American cockroaches and snake slough peptides were compounded at a ratio of 4:6 g / g and mixed with a betaine-phytic acid eutectic solvent at a molar ratio of 3:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0038] Example 8

[0039] American cockroaches and snake slough peptides were compounded at a ratio of 4:6 g / g and mixed with a betaine-lactic acid eutectic solvent at a molar ratio of 1:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0040] Example 7

[0041] American cockroaches and snake slough peptides were compounded at a ratio of 5:5 g / g and mixed with a betaine-phytic acid eutectic solvent at a molar ratio of 3:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0042] Example 8

[0043] American cockroaches and snake slough peptides were compounded at a ratio of 5:5 g / g and mixed with a betaine-lactic acid eutectic solvent at a molar ratio of 1:1 at a mass-volume ratio of 1:5 g / mL. The mixture was then dispersed at 60℃ using ultrasonication at 300W for 20 min to form a uniform eutectic bio-adhesive composite system, which was then sterilized and sealed for later use.

[0044] Figure 2 The image shows a comparison of the state of the eutectic bio-adhesive prepared in Example 3 before and after it was preheated to 60°C in a water bath, then slowly cooled to near room temperature, and then rapidly cooled and cured.

[0045] Figure 3 The average particle size change trend of the peptides in the eutectic bioadhesive prepared in Example 3 before and after temperature-induced self-assembly is shown. Specifically, the average particle size was measured by dynamic light scattering method in three stages: preheating, slow cooling, and fast cooling. It can be seen that the size of the peptides increased significantly due to self-assembly at different temperatures.

[0046] Figure 4The adhesion test results of the bio-adhesives prepared in Examples 3, 4, 5, and 6 are shown. The specific procedure was as follows: Two pieces of thermoplastic elastomer (TPETPR) synthetic rubber (1cm*3cm, 5g) were taken to simulate human tissue. One piece was attached to the lower end of a pan on one side of a balance, while the other was fixed to the balance base. The preheated bio-adhesive, cooled to near room temperature, was then applied to the contact surfaces of the two rubber pieces. The mixture was then rapidly cooled and pressed down with a 50g weight for 10 minutes. The weight was then removed, and additional weights were continuously added to the other pan of the balance to apply gravity. Separation of the two rubber pieces within 5 seconds was used as the standard, and the added weight reflected the adhesion force of the test object. The results show that the bio-adhesive prepared in Example 3 exhibited the strongest adhesion force. Based on the chemical structure of the relevant components, the tissue adhesion mechanism of this type of bio-adhesive may mainly involve hydrogen bonding, electrostatic interactions, and van der Waals forces.

[0047] Figure 5 This study demonstrates the in vitro simulated release behavior of peptides in the eutectic bioadhesive prepared in Example 3. Specifically, the Franz diffusion cell method from the latest 2025 edition of the Chinese Pharmacopoeia was used. A PDMS medical membrane, 0.5 mm thick and 1.5 cm x 1.5 cm in area, was selected to simulate human skin. The eutectic bioadhesive was in its cured state. 1 mL samples were accurately taken from the sampling port at different time points, and 1 mL of physiological saline was injected back into the receiving cell after sampling. Each experiment was repeated three times. The cumulative release per unit area (mg / cm²) was calculated based on the peptide concentration (mg / mL) in the sample. 2 The peptides were quantified using the Lowry method, as per the 2025 edition of the Chinese Pharmacopoeia. In vitro release results indicate that this eutectic bioadhesive has the ability to slowly release the main functional peptide, which is beneficial for maintaining its therapeutic effect at the affected area.

[0048] Considering the potential instability of peptide components, the stability of the eutectic bioadhesive stored in a sealed container at room temperature (20–35 °C) was evaluated based on the trend of peptide content changes. Due to the stabilizing effect and antibacterial and preservative properties of the eutectic solvent, without the addition of other components, Figure 6 The results showed that the peptide content did not decrease significantly over 90 days, and the overall properties, odor, taste, and color did not change significantly, which fully demonstrates that the system has good stability.

Claims

1. A low-melting-point bioadhesive based on a mixture of American cockroach and snake slough polypeptides, characterized in that, The active components, including active peptide extracts from American cockroaches and snake slough peptide extracts, are combined with a eutectic solvent to form a homogeneous composite system through the eutectic effect and self-assembly process. The system then exerts its effect on the affected area through a preheating-cooling process. The specific preparation process is as follows: (1) The main functional components, American cockroach and snake slough polypeptide, are mixed and compounded at a ratio of 3:7~5:5 g / g. This ratio is used to balance the bioactivity and mechanical adhesion strength of the bioadhesive. (2) Mix the compound of American cockroach and snake slough polypeptide with a low eutectic solvent at a mass-volume ratio of 1:3~1:5 g / mL, and disperse it fully at 55~60℃ with 300~500W ultrasonic for 10~20min; after forming a uniform low eutectic bio-adhesive composite system, sterilize and seal for later use.

2. The low-melting bioadhesive based on a mixture of American cockroach and snake slough peptides as described in claim 1, characterized in that, The active polypeptide extract from American cockroaches is derived from the enzymatic hydrolysis product of pharmaceutical waste residue, with a molecular weight in the range of 500~1000 Da.

3. The low-melting bioadhesive based on a mixture of American cockroach and snake slough peptides as described in claim 1, characterized in that, The snake slough structure polypeptide extract is derived from the enzymatic hydrolysis product of snake slough keratin, with a molecular weight in the range of 2000~3000 Da, and has the ability to self-assemble into β-sheet structures.

4. The low-melting bioadhesive based on a mixture of American cockroach and snake slough peptides as described in claim 1, characterized in that, The eutectic solvent is a mixture of betaine and phytic acid or betaine and lactic acid, with a molar ratio of 3:1 to 1:

3. It plays multiple roles as a cosolvent, plasticizer, penetration enhancer, antibacterial agent, and auxiliary functional agent.

5. The low-melting bioadhesive based on a mixture of American cockroach and snake slough peptides as described in claim 1, characterized in that, Before use, the eutectic bio-adhesive is preheated to 60°C in a water bath and shaken, then slowly cooled to near room temperature before being applied to the affected area. Ice packs are then used to rapidly cool the affected area, and self-assembly and curing are completed in 5-10 minutes.

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