Self-assembled hydrogel loaded with antibacterial healing-promoting recombinant polypeptide as well as preparation method and application of self-assembled hydrogel

Abstract

The invention relates to the technical field of hydrogel preparation, in particular to self-assembled hydrogel loaded with antibacterial healing-promoting recombinant polypeptide as well as a preparation method and application of the self-assembled hydrogel. The hydrogel disclosed by the invention provides a novel treatment strategy and a novel material method for repairing radioactive skin injury wounds. Compared with pure antibacterial hydrogel, the hydrogel has the advantages that the healing promoting function is added; compared with growth factor dressings, the antibacterial dressing has antibacterial ability, and can solve two core problems of infection and repair at the same time; compared with inorganic antibacterial materials such as nano-silver, the antibacterial material has no heavy metal accumulation risk and is higher in biological safety.

Description

Technical Field

[0001] This invention relates to the field of hydrogel preparation technology, and in particular to a self-assembled hydrogel loaded with antibacterial and healing-promoting recombinant peptides, its preparation method, and its application. Background Technology

[0002] Skin is the largest organ in the human body and its most basic protective barrier. Damaged skin can allow microorganisms to invade and cause infection, and wound infection is a major cause of increased morbidity and mortality. Therefore, functional recovery after skin injury has always been a focus of tissue engineering research. Radiation-induced skin injury, as a typical chronic and difficult-to-heal wound, presents a significant challenge in clinical treatment due to its recurrent ulceration and difficulty in healing in the later stages. The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, posing an even greater challenge to antibacterial treatment of wounds. Promoting healing, anti-infection, and wound dressings are current research directions in the treatment of chronic healing wounds. However, materials and methods for "antibacterial and promoting healing" in the treatment of radiation-induced skin injury with ulceration and difficulty in healing are still lacking.

[0003] Hydrogels are polymeric materials with a three-dimensional cross-linked network structure. They are formed by soluble polymers through chemical cross-linking (such as covalent bonding) or physical cross-linking (such as hydrogen bonding and van der Waals forces) to create a stable network structure. This unique structure endows hydrogels with certain mechanical properties, allowing them to swell rapidly in water while retaining a large volume of water without dissolving. Simultaneously, their polymer chains contain numerous active groups, providing a basis for gel modification, drug delivery, and release. Furthermore, hydrogels exhibit good biocompatibility, do not affect human metabolism, and are biodegradable, making them ideal wound healing materials.

[0004] Radiation-induced skin injury is a relatively common occupational disease caused by radiation, primarily resulting from external exposure to X-rays / gamma rays or beta rays. It is widespread in the fields of nuclear fuel cycle and radiotherapy. Repeated ulceration and prolonged non-healing of radiation-induced skin injury wounds pose a significant challenge to clinical treatment. Bacterial infection is unavoidable during the wound healing process, and the proliferation of multidrug-resistant bacteria due to antibiotic overuse presents an even greater challenge to antibacterial treatment of these wounds.

[0005] Currently, wound dressing products are mainly traditional dressings, generally made of cotton, soft linen, and flax, such as adhesive bandages. These dressings are only used to keep the wound dry and have no significant effect on wound repair. Simple antibacterial materials (such as dressings containing nano-silver or antibiotics) can only kill pathogens and cannot regulate repair processes such as fibroblast proliferation and angiogenesis, and are prone to heavy metal accumulation or bacterial resistance. On the other hand, simple healing-promoting materials (such as collagen dressings containing growth factors) lack antibacterial ability and can easily become a nutrient substrate for bacteria, leading to secondary infections. The combined use of the two also has problems with poor compatibility and complicated operation. Summary of the Invention

[0006] To address the technical deficiencies in the existing technology, the present invention adopts the following technical solution: The first aspect of the present invention provides a self-assembled hydrogel loaded with an antibacterial and healing-promoting recombinant peptide, which is formed by self-assembly of RADA16 peptide, antibacterial and healing-promoting recombinant peptide, and buffer solution.

[0007] In some preferred embodiments, the final concentration of the antibacterial and healing-promoting recombinant polypeptide is 0.5-2 × 10⁻⁶. -7 The final concentration of the RADA16 peptide is 5-10 mg / mL.

[0008] The second aspect of this invention provides a method for preparing a self-assembled hydrogel loaded with an antibacterial and healing-promoting recombinant peptide, comprising the following steps: S1: dissolving the RADA16 peptide to prepare a RADA16 stock solution with a concentration of 10 mg / mL; S2: dissolving and diluting the antibacterial and healing-promoting recombinant peptide with a buffer solution to prepare a solution with a concentration of 2 × 10⁻⁶ mg / mL. -7 S3: Mix the RADA16 storage solution and the antibacterial and healing-promoting recombinant peptide working solution in equal volumes, sonicate, and allow it to stand at 20-37℃ to self-assemble into a gel.

[0009] In some preferred embodiments, in steps S1 and S2, the dissolution is achieved by ultrasonic treatment.

[0010] In some preferred embodiments, in step S3, the ultrasonic treatment time is 5-15 minutes, and the settling time is not less than 1 hour.

[0011] A third aspect of the present invention provides the use of a self-assembling hydrogel in the preparation of a medicament or medical device for promoting skin wound healing.

[0012] In some preferred embodiments, the skin wound is a radiation-induced skin injury.

[0013] In some preferred embodiments, the radiation-induced skin damage is caused by irradiation with high-energy electron beams.

[0014] A fourth aspect of the present invention provides a medical dressing for treating radiation-induced skin damage, comprising a therapeutically effective amount of self-assembled hydrogel and a pharmaceutically or medical device-acceptable carrier or matrix.

[0015] In some preferred embodiments, the effective amount of the self-assembled hydrogel is 1-99%wt.

[0016] The technical solution adopted in this invention can achieve the following beneficial effects: This invention provides a self-assembled hydrogel loaded with antibacterial and healing-promoting recombinant peptides and its preparation method, offering a new treatment strategy and material method for the repair of radiation-induced skin injuries. Compared to simple antibacterial hydrogels, it adds healing-promoting functions; compared to growth factor dressings, it also possesses antibacterial capabilities, simultaneously addressing the two core issues of infection and repair; compared to inorganic antibacterial materials such as nano-silver, it has no risk of heavy metal accumulation and offers higher biocompatibility.

[0017] In the treatment of chronic, refractory wounds, functional hydrogels loaded with antibacterial and healing-promoting recombinant peptides can eliminate common pathogens such as Staphylococcus aureus and Escherichia coli that colonize wounds, and are less likely to induce bacterial resistance. At the same time, they promote fibroblast proliferation, accelerate vascular endothelial cell migration to induce angiogenesis, and promote wound healing. In addition, the hydrogel matrix creates a stable local microenvironment for the healing of chronic, refractory wounds.

[0018] In the treatment of acute infected wounds (burns, postoperative incision infections, etc.), hydrogels can quickly cover the wound, forming a physical barrier to prevent external bacteria from invading. The loaded recombinant peptides are rapidly released and exert antibacterial effects, inhibiting bacterial reproduction and controlling the spread of acute infection. On the other hand, the good biocompatibility of hydrogels can avoid irritating newly formed tissues. In conjunction with the peptides, hydrogels can promote the migration and proliferation of epidermal cells, accelerate the epithelialization process of the wound, reduce scar tissue formation, and promote wound healing.

[0019] In the preparation of antibacterial and healing-promoting dressings, these dressings have both physical protection and bioactivity. The hydrogel matrix can achieve different properties by adjusting the raw material composition and preparation process. At the same time, dressing products of different forms can be designed according to the needs of different wounds, such as sheet dressings for large-area burns, filling dressings for deep wounds, and spray dressings for mucosal wounds. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, forming part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 This is a diagram showing the gelation effect of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0021] Figure 2 This is a transmission electron microscope (TEM) image of the internal nanofiber structure of a functional hydrogel loaded with antibacterial and healing recombinant peptides.

[0022] Figure 3 This is a surface structure diagram of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides under a scanning electron microscope (SEM).

[0023] Figure 4 This is a diagram showing the mechanical properties of a functional hydrogel loaded with antibacterial and healing recombinant peptides.

[0024] Figure 5 This is a cytotoxicity identification diagram of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0025] Figure 6 This is a diagram illustrating the cell migration-promoting properties of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0026] Figure 7 This is a biocompatibility identification diagram of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0027] Figure 8 This is a diagram illustrating the antibacterial activity of a functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0028] Figure 9 This is an observational diagram of wound healing after intervention with functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides.

[0029] Figure 10 This is a pathological observation of wound healing after intervention with functional hydrogel loaded with antibacterial and healing-promoting recombinant peptides. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0031] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0032] The preparation method of the antibacterial and healing-promoting recombinant polypeptide described in this invention is derived from Chinese Invention Patent 202311867731.1.

[0033] Example 1: Preparation of a functional self-assembled hydrogel loaded with antibacterial and healing-promoting recombinant peptides (1) Preparation of RADA16 storage solution: Dissolve RADA16 lyophilized powder in an appropriate volume of ultrapure water, sonicate for 10 min, and prepare storage solution (concentration of 10 mg / ml). (2) Preparation of antibacterial and healing-promoting recombinant peptide solution: Take the lyophilized powder of antibacterial and healing-promoting recombinant peptide, add an appropriate volume of PBS to dissolve it, sonicate for 10 min to prepare a solution with a concentration of 1 mmol / L, and then serially dilute to obtain a concentration of 2 × 10⁻⁶. -7 A solution of mmol / L; (3) Induction of gelation: RADA16 stock solution (concentration of 10 mg / ml) and 2×10 -7 Equal volumes of antibacterial and healing-promoting recombinant peptides at mmol / L were mixed, sonicated for 10 min, and incubated at 37°C or room temperature for 1 h to obtain the antibacterial and healing-promoting recombinant peptide loaded with 1×10 mmol / L. -7 A functional self-assembled hydrogel (RADA16 concentration of 10 mg / ml) was observed by inverting the sample to check its gelation effect. Figure 1 As shown, the control group RADA16 (left) and the hydrogel loaded with antibacterial and healing-promoting recombinant peptides (right) have very little fluidity when inverted, and appear as a jelly.

[0034] Example 2: Physicochemical property testing of functional hydrogels loaded with antibacterial and healing-promoting recombinant peptides 1. Observation of nanofiber structure characteristics inside hydrogel using transmission electron microscopy (TEM) Take the prepared hydrogel sample and, using the pendant drop method, place a drop (100 μL) of sample suspension onto a copper grid. Let it stand for several minutes, then blot away excess liquid with filter paper. Add phosphotungstic acid negative staining solution and stain for 1–2 minutes. Blot away the negative staining solution with filter paper. Wash the copper grid 1–2 times with distilled water. After drying, observe under a transmission electron microscope. Figure 2 As shown, both the control group RADA16 hydrogel and the hydrogel loaded with antibacterial and healing-promoting recombinant peptides exhibit nanofiber structures, with the nanofibers of the hydrogel loaded with antibacterial and healing-promoting recombinant peptides being shorter than those of the control group RADA16 hydrogel.

[0035] 2. Scanning electron microscopy (SEM) observation of hydrogel surface structure Take 100 μL of the prepared hydrogel sample, rapidly freeze-dry it, and then observe its microstructure under a scanning electron microscope. Figure 3 As shown, the microstructure of the hydrogel samples all exhibited typical three-dimensional mesh characteristics, forming an interconnected porous network system. The control group RADA16 hydrogel showed a densely packed network structure under the microscope, while the network structure of the hydrogel loaded with antibacterial and healing-promoting recombinant peptides had relatively larger pore sizes.

[0036] 3. Rheometer analysis of the mechanical properties of hydrogels The prepared hydrogel samples were dropped onto the rheometer cone plate, and the storage modulus (G') and loss modulus (G'') of the samples were measured under the limiting conditions of a frequency range of 10⁻⁰.⁻¹ rad / s and a constant stress of 1 Pa. Figure 4 As shown, the storage modulus (G') of the hydrogel loaded with antibacterial and healing-promoting recombinant peptides and the control group RADA16 hydrogel are both greater than the loss modulus (G''). Furthermore, G' and G'' increase continuously with increasing angular frequency, indicating that both have the ability to self-assemble into gels and have good mechanical properties.

[0037] Example 3: Application of functional hydrogels loaded with antibacterial and healing-promoting recombinant peptides in radiation-induced skin injuries 1. Cytotoxicity analysis Skin fibroblasts were digested in cell culture flasks to prepare a cell suspension with a final concentration of 5 × 10⁵ cells / ml. 300 μL of this suspension was added to each well of a 24-well plate and incubated overnight. The next day, the culture medium was aspirated from the wells. Using complete culture medium as a blank control, 300 μL of medium containing RADA16 and a recombinant antibacterial peptide was added to the corresponding wells for incubation. After 1 and 3 days of co-culture, the 24-well plates were removed, washed three times with PBS, and then 200 μL of 1.2 mmol / L calcein-AM (for staining viable cytoplasm) and 200 μL of 1 μg / ml PI staining solution (for binding to dead cell nuclei) were added. The plates were incubated in the dark for 25 min, washed three times with PBS, and then DAPI staining solution (for nucleus localization) was added. After incubation in the dark for 10 min, the plates were washed three times with PBS and observed under a fluorescence microscope. Figure 5 As shown, almost all cells in the RADA16 and antibacterial and healing-promoting recombinant peptide hydrogel groups survived (green represents live cells, red represents dead cells), and there was no significant difference compared with the blank control group, indicating that the antibacterial and healing-promoting recombinant peptide hydrogel did not exhibit significant biotoxicity.

[0038] 2. Analysis of the cell migration-promoting activity of hydrogels loaded with antibacterial and healing-promoting recombinant peptides BJ cell suspension at a concentration of 1×10⁶ cells / mL was seeded into 6-well plates and cultured overnight. Once cell confluence reached 80% or higher, the medium was replaced with serum-free medium and cultured for 2 h. A streak was then performed along the bottom of the plate using a pipette tip. Control and experimental group samples were added separately, and cell growth at the streaks was observed under an inverted microscope at 0 h, 6 h, 12 h, 24 h, and 48 h of culture. Results are as follows: Figure 6 As shown, the healing speed of the hydrogel group loaded with antibacterial and healing-promoting recombinant peptides was significantly better than that of the control group.

[0039] 3. Biocompatibility analysis of hydrogels loaded with antibacterial and healing-promoting recombinant peptides Two ml of healthy rabbit red blood cells were used to prepare a 2% red blood cell suspension using 98 ml of 0.9% sodium chloride injection. In a centrifuge tube containing 2.5 ml of the 2% red blood cell suspension, 2.5 ml of water for injection (positive control), 0.9% sodium chloride injection (negative control), RADA16, and a hydrogel loaded with antibacterial and healing-promoting recombinant peptides were added. The mixture was then immediately incubated at 37°C, with observations every 1 hour for 3 hours. Results are as follows: Figure 7 As shown, the supernatant of the RADA16 and antibacterial healing-promoting recombinant peptide hydrogel groups and the negative control group was clear and transparent, and no hemolysis occurred, indicating that the antibacterial healing-promoting recombinant peptide hydrogel has good blood compatibility.

[0040] 4. Identification of the antibacterial activity of hydrogel loaded with antibacterial and healing-promoting recombinant peptides (1) Take logarithmic growth phase Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) respectively and inoculate them into fresh LB liquid medium, with a final concentration of 1×106 CFU / ml; (2) Take the prepared antibacterial and healing-promoting recombinant polypeptide hydrogel, and prepare an soaking solution with liquid culture medium at a ratio of 1:10. Use the soaking solution to conduct antibacterial experiments. (3) The soaking solution was diluted with culture medium to 1.88 μg / ml, 18.8 μg / ml, 56.4 μg / ml, and 94 μg / ml, respectively. 100 μl of hydrogel soaking solution (experimental group) and blank culture medium (control group) were added to each well of a 96-well plate (containing 1 μl of spare bacterial solution), and the plates were incubated at 37℃. OD600 nm was measured at different time points. Figure 8 As shown, the experimental groups showed antibacterial effects after co-culturing with E. coli and S. aureus for 4 hours.

[0041] 5. Application of antibacterial and healing-promoting recombinant peptide hydrogels in radiation-induced skin injuries (1) Establishment of an electron beam-induced radiation-induced skin injury model in rats One day prior to the experiment, rats were weighed, marked, and numbered. During the experiment, rats were anesthetized via intraperitoneal injection of 2.5% sodium pentobarbital (0.2 ml per 100 g). After anesthesia took effect, the skin on the rump and back of the rats was shaved, and they were placed in irradiation cages designed in our laboratory. Non-irradiated areas were shielded with aluminum plates. Irradiation was performed using an Elekta Synergy medical linear electron accelerator with a high-energy electron beam of 4 Mev, an absorbed dose rate of 300 cGy / min, a skin-source distance of 100 cm, a single absorbed dose of 45 Gy, and a circular irradiation field with a diameter of 32 mm. The rats' rump and back were irradiated to establish an animal model of radiation-induced skin injury. After irradiation, the rats were kept warm and, once awake, transferred to rearing cages at the Rodent Laboratory of the China Institute of Radiation Protection. Experimental procedures were strictly performed according to the experimental animal handling guidelines of the GLP Center of the China Institute of Radiation Protection.

[0042] (2) Application of antibacterial and healing-promoting recombinant peptide hydrogels in radiation-induced skin injuries ① A randomized controlled trial was conducted, with rats divided into 5 groups: Control group, negative control group (PBS buffer), positive control group (Biafen), RADA16 hydrogel group, and hydrogel group loaded with antibacterial and healing-promoting recombinant peptides. The drugs were applied daily after irradiation, and the rats' eating habits, mental state, and skin damage were observed. Figure 9 As shown, about one week after irradiation, the skin of rats in each group showed obvious erythema and dryness in the irradiated area; two weeks after irradiation, the skin of rats in the irradiated area showed obvious ulceration, some with exudate, some with obvious tenderness, and wet desquamation; four weeks after irradiation, the irradiated wounds stopped expanding, and the ulceration of the wounds in the experimental group began to gradually improve; eight weeks after irradiation, the group loaded with antibacterial and healing-promoting recombinant polypeptide hydrogels was basically healed.

[0043] ② Eight weeks after irradiation, tissue samples were taken from each group of rats. Anesthesia was administered via intraperitoneal injection of 2.5% sodium pentobarbital (0.2 ml of 2.5% sodium pentobarbital per 100g). Full-thickness skin tissue from the irradiated field and surrounding area was collected, rinsed with 0.9% sodium chloride injection to remove residual blood and loose hair, laid flat on filter paper to absorb moisture, and fixed in 10% neutral formalin solution for paraffin section preparation.

[0044] ③ Pathological observation of wound healing: In each group, approximately 5 mm × 5 mm full-thickness skin tissue samples were excised from the wound surface, the peri-wound junction, and the peri-wound area and placed in a dehydration box for thorough dehydration in a fully automated tissue dehydrator. Afterwards, the skin tissue was longitudinally embedded in paraffin and prepared as slides. After drying, the sections underwent dewaxing, hydration, hematoxylin-eosin staining, dehydration, clearing, and mounting. The histopathological changes of the hydrogel in response to electron beam-induced radiation-induced skin damage were observed under an optical microscope. Results are as follows: Figure 10 As shown, in the negative control group, the epidermis in the damaged area was significantly thickened, and the hyperkeratosis was not significantly improved. A few skin appendages were occasionally seen, and the number of inflammatory cells in the dermis was reduced. In the positive control group, the degree of epidermal thickening and keratosis was reduced in the Biafen and RADA16 hydrogel groups, and the number of inflammatory cells infiltrating the dermis was reduced. Some newly formed hair follicles, blood vessels, and sweat glands and other skin appendages were visible. The degree of epidermal keratosis and thickening in the hydrogel group loaded with antibacterial and healing-promoting recombinant peptides was even milder, the inflammatory response in the dermis was less, and the number of skin appendages was significantly increased.

[0045] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A self-assembled hydrogel loaded with antibacterial and healing-promoting recombinant peptides, characterized in that, It is formed by self-assembly of RADA16 peptide, antibacterial and healing-promoting recombinant peptide, and buffer.

2. The self-assembling hydrogel according to claim 1, characterized in that, The final concentration of the antibacterial and healing-promoting recombinant polypeptide is 0.5-2×10⁻⁶. -7 The final concentration of the RADA16 peptide is 5-10 mg / mL.

3. A method for preparing a self-assembled hydrogel loaded with antibacterial and healing-promoting recombinant peptides as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Dissolve the RADA16 peptide to prepare a RADA16 stock solution with a concentration of 10 mg / mL; S2: Dissolve and dilute the antibacterial and healing-promoting recombinant peptides using buffer to prepare a concentration of 2×10⁻⁶. -7 S3: Mix the RADA16 storage solution and the antibacterial and healing-promoting recombinant peptide working solution in equal volumes, sonicate, and allow it to stand at 20-37℃ to self-assemble into a gel.

4. The preparation method according to claim 3, characterized in that, In steps S1 and S2, the dissolution is achieved by ultrasonic treatment.

5. The preparation method according to claim 3, characterized in that, In step S3, the ultrasonic treatment time is 5-15 minutes, and the settling time is no less than 1 hour.

6. The use of the self-assembling hydrogel of claim 1 or 2 in the preparation of a medicament or medical device for promoting skin wound healing.

7. The application according to claim 6, characterized in that, The skin wound is a radiation-induced skin injury.

8. The application according to claim 7, characterized in that, The radiation-induced skin damage was caused by exposure to high-energy electron beams.

9. A medical dressing for treating radiation-induced skin damage, characterized in that, The self-assembled hydrogel of claim 1 or 2 contains a therapeutically effective amount, and is a carrier or matrix in the pharmaceutical or medical device field.

10. The medical dressing according to claim 9, characterized in that, The effective amount of the self-assembled hydrogel is 1-99%wt.

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