Preparation and application of a microbial alkaline protein and adhesive proteoglycan hydrogel

Abstract

The application discloses a kind of preparation and application of microbial basic protein and adhesive proteoglycan hydrogel, belong to the field of biomaterials.Basic protein is extracted from microbial cells in the present application, using supramolecular assembly method, basic protein and acidic polysaccharide electrostatic assembly protein glycan gel material with strong tissue adhesion, the method is simple, widely available, and the cost is almost negligible.The tissue adhesive provided by the present application has immediate tissue adhesion performance, excellent biocompatibility and excellent antibacterial ability and hemostatic performance, which is beneficial to cope with different types of bleeding scene and irregular wound healing.Through cell compatibility and blood compatibility test, the results show that the material has excellent biocompatibility, and the basic protein has super strong bacteriostatic ability, avoids the invasion of foreign bacteria in the wound repair process, and has strong healing ability, greatly shortens the healing period of wound.

Description

Technical Field

[0001] This invention relates to the preparation and application of a basic protein derived from microbial cells and a proteoglycan hydrogel with strong tissue adhesion, belonging to the field of biomaterials. Background Technology

[0002] Inspired by the excellent underwater adhesion abilities of natural marine organisms such as mussels, barnacles, and sandcastle worms, which utilize a large number of highly diverse proteins to achieve strong underwater adhesion, sandcastle worms, in particular, rely on complex aggregates induced by polyelectrolytes with opposite charges for adhesion. However, the yield of such adhesive proteins extracted from marine organisms is limited. Furthermore, histones, histone-like proteins, and protamines are among the most positively charged proteins in nature. Therefore, we extracted basic proteins from microbial cells and then interacted them with anionic substances such as acidic polysaccharides (hyaluronic acid, chondroitin sulfate, heparin, and sea cucumber polysaccharides) via electrostatic interactions to form hydrogel adhesives. These proteoglycan components, mimicking the extracellular matrix, form aggregates through supramolecular electrostatic interactions that exhibit strong adhesion, excellent biocompatibility, and antibacterial properties, as well as immediate hemostasis and wound tissue regeneration capabilities. Summary of the Invention

[0003] To address the shortcomings of the existing technologies, this invention provides a method for preparing and applying a proteoglycan colloidal material with strong tissue adhesion. The purpose is to solve the technical problem of the lack of adhesive materials in the existing technologies that can generate immediate adhesion to wet tissues, have antibacterial properties to prevent wound infection, and are biocompatible.

[0004] The first technical solution provided by this invention is a method for preparing tissue adhesives, which utilizes proteoglycan materials formed by the electrostatic self-assembly of basic proteins extracted by microorganisms and anionic acidic polysaccharides.

[0005] In one embodiment of the present invention, the basic protein electrostatically assembled with the acidic polysaccharide is a basic protein extracted from different bacterial cells; the acidic polysaccharide includes one or more of hyaluronic acid, sea cucumber polysaccharide, and sulfonated glycosaminoglycans.

[0006] In one embodiment of the present invention, in the electrostatic self-assembly method, the volume ratio of basic protein to hyaluronic acid is 1:(0.5~1.5), or the volume ratio of basic protein to sulfonated glycosaminoglycan is 1:(0.5~1.5).

[0007] In one embodiment of the present invention, the sulfonated glycosaminoglycan includes chondroitin sulfate A, chondroitin sulfate C, and heparin.

[0008] In one embodiment of the present invention, the basic protein is derived from the waste microbial cells discarded after centrifugation at the end of laboratory fermentation, including *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Corynebacterium glutamicum*. The cost is almost negligible, while significantly mitigating environmental pollution problems.

[0009] In one embodiment of the present invention, the hyaluronic acid used has three molecular weights: high molecular weight hyaluronic acid (1000-1300 kDa), medium molecular weight hyaluronic acid (600-900 kDa), and low molecular weight hyaluronic acid (0.8-10 kDa).

[0010] In one embodiment of the present invention, the specific steps are as follows: basic protein is mixed with anionic glycosaminoglycan, the supernatant is removed by centrifugation to obtain proteoglycan with strong tissue adhesion, which is then freeze-dried to obtain powder and dried and stored at room temperature.

[0011] In one embodiment of the present invention, the concentration of hyaluronic acid is 20 mg / mL, the concentration of basic protein is 50 mg / mL, and the concentrations of chondroitin sulfate A, chondroitin sulfate C, and heparin are all 50 mg / mL.

[0012] In one embodiment of the present invention, the centrifugation speed is 10,000 rpm, the temperature is 4°C, and the time is 10-20 min.

[0013] In one embodiment of the present invention, the freeze-drying time is 5-10 min, the sample temperature is -20℃, the cold trap temperature is -60℃, and the vacuum degree is less than 10 Pa.

[0014] The second technical solution provided by the present invention is a tissue adhesive prepared using the method described in the first technical solution.

[0015] The third technical solution provided by the present invention is the application of the tissue adhesive described in the second technical solution in the preparation of antibacterial materials, wound hemostasis, cell culture, tissue adhesion and / or wound healing products.

[0016] In one embodiment of the present invention, the antibacterial type includes Gram-positive bacteria and / or Gram-negative bacteria.

[0017] In one embodiment of the present invention, the antibacterial types include Escherichia coli and Staphylococcus aureus.

[0018] In one embodiment of the present invention, the tissue adhesion includes one or more of skin tissue, visceral tissue, muscle tissue, connective tissue, and mucous membrane tissue.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention extracts basic proteins from microbial cells, which are rich in basic amino acids, especially lysine, giving them a large positive charge. These proteins are then combined with negatively charged acidic polysaccharides using a supramolecular assembly method to prepare proteoglycan materials with antibacterial capabilities. The amino acids in the basic proteins significantly enhance various molecular interactions within the proteoglycans. Simultaneously, the basic amino acids endow the tissue adhesive with excellent antibacterial properties. The tissue adhesive provided by this invention exhibits immediate tissue adhesion, excellent biocompatibility, and outstanding antibacterial and hemostatic properties, making it beneficial for addressing different types of bleeding scenarios and healing irregular wounds.

[0020] This invention provides a proteoglycan gel material with strong tissue adhesion, which is electrostatically assembled from basic proteins and acidic polysaccharides using a supramolecular assembly method. The method is simple to prepare, the sources are widely available, and the cost is negligible. Cell compatibility and blood compatibility tests show that the material has excellent biocompatibility. Simultaneously, the basic proteins possess strong antibacterial capabilities, preventing the invasion of foreign bacteria during wound repair, and exhibit powerful healing abilities, significantly shortening the wound healing period. Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE image of histones extracted from Escherichia coli and Pichia pastoris.

[0022] Figure 2 A gel is formed by mixing hyaluronic acid solution and alkaline protein solution. Figure 3 The adhesiveness of the gel formed by hyaluronic acid and basic protein.

[0023] Figure 4 It is an image of an aggregate formed by sulfonated glycosaminoglycans and basic proteins.

[0024] Figure 5 It is a characterization of the adhesion of proteoglycans to the weights.

[0025] Figure 6 It is the adhesive effect of proteoglycans formed by covalent cross-linking.

[0026] Figure 7 It is the antibacterial effect of proteoglycans.

[0027] Figure 8 It refers to the cell compatibility of proteoglycans.

[0028] Figure 9 It is the hemostatic effect of proteoglycan on mouse liver.

[0029] Figure 10 It refers to the adhesion effect of proteoglycan on mouse viscera. Detailed Implementation

[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0031] Raw materials used in the examples: 1. Commercial strains of *Escherichia coli* BL21(DE3) and *Pichia pastoris* GS115 were collected in the laboratory. *Escherichia coli* ATCC25922 and *Staphylococcus aureus* ATCC6538 were purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0032] 2. All analytical grade reagents were purchased from Sinopharm Group, and hyaluronic acid was obtained from the fermentation of Corynebacterium glutamicum in our laboratory.

[0033] 3. C57BL6J mice were purchased from Vital River.

[0034] Example 1: Extraction of microbial basic proteins Extraction and purification of basic proteins: The bacterial cells were collected by centrifugation of *Escherichia coli* BL21(DE3) and *Pichia pastoris* GS115 cultures, and the OD values ​​of the bacterial cells were determined using Tris-HCl buffer. 600 The concentration was adjusted to 50, and then high-pressure homogenization was used for cell wall disruption. Basic protein extraction was performed using an acidified ethanol solution (60% ethanol, 2.65% concentrated hydrochloric acid, 37.35% water). The mixture was centrifuged at 10,000 r / min for 20 min to obtain the supernatant. The supernatant was then added to a protein extraction solution (acetone solution with a final concentration of 0.0049% concentrated hydrochloric acid), at a ratio of 1:3. The mixture was then incubated at -20°C for 2 h. The protein precipitate was then centrifuged at 10,000 r / min for 20 min to obtain a crude basic protein extract. This extract was washed with acetone and air-dried to obtain the protein. The protein was appropriately diluted to a concentration less than 1 mg / mL and subjected to SDS-PAGE. Results for *E. coli* and *Pichia pastoris* are shown in the figure. Figure 1 .

[0035] Amino acid content determination: Take 1 mL of diluted Pichia pastoris histone sample, add 1 mL of concentrated hydrochloric acid, and then add 6 mL of 6M HCl; purge with nitrogen to bring the solution to a gentle boil, and tighten the cap of the hydrolysis tube; place in an oven at 120 ℃ for hydrolysis for 22-24 h. Transfer all the sample from the hydrolysis tube to a volumetric flask, add 4.8 mL of 10M NaOH to neutralize, and dilute to 25 mL with distilled water; filter with double-layer filter paper, take 1 mL of the clear filtrate into a 1.5 mL centrifuge tube, and take 400 μL of the supernatant into a liquid phase sample bottle for amino acid content determination. The results are shown in Table 1, with lysine having the highest proportion, reaching 17.22%.

[0036] Table 1 shows the main amino acid content of basic proteins.

[0037] Example 2: Electrostatic assembly and covalent crosslinking of basic proteins and glycosaminoglycans Electrostatic interaction between basic proteins and hyaluronic acid: A 2% (w / v) hyaluronic acid solution and a 5% (w / v) Pichia pastoris histone solution were mixed at a 1:1 volume ratio and incubated for 30 min. The mixture solidified to form a gel. The results are shown in [Figure number missing]. Figure 2 .

[0038] In addition, when a crude histone solution from Pichia pastoris was added to a hyaluronic acid solution, the two substances aggregated. During gel formation, water precipitated from the solution, and the crude histone and hyaluronic acid in the solute formed aggregates with adhesive properties. The results are shown below. Figure 3 .

[0039] Electrostatic interaction between basic protein and sulfonated glycosaminoglycans: Different sulfonated glycosaminoglycans (5% (w / v) chondroitin A sulfate, 5% (w / v) chondroitin C sulfate, and 5% (w / v) heparin sodium solution) were prepared. 5% (w / v) basic protein was mixed with each of the different sulfonated glycosaminoglycans at a 1:1 volume ratio. Vigorous aggregation occurred in the mixed solutions. As a control, no aggregation reaction occurred when the sulfonated glycosaminoglycans were mixed with the negatively charged anionic surfactant SDBS. The results are shown in [Figure number missing]. Figure 4 .

[0040] The adhesion ability of the formed agglomerates was then tested. Taking the agglomerate formed by CSC and basic protein as an example, the agglomerate was centrifuged and then spread onto a weight. It immediately adhered to a 200g weight, but failed to adhere to a 200g weight. After curing overnight, it was able to adhere to a 500g weight. In contrast, individual CSA, CSC, heparin, and basic protein did not adhere to the weight. See the results below. Figure 5This demonstrates that this proteoglycan material, formed by supramolecular interactions between basic proteins and anionic glycosaminoglycans, possesses extremely strong adhesive properties.

[0041] Covalent cross-linking of basic proteins with hyaluronic acid: Basic proteins extracted from microbial cells are rich in lysine. Therefore, we use a covalent cross-linking method to form a hydrogel network with higher cohesion between hyaluronic acid and histones.

[0042] 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was added to a 0.1 L (w / v) HA solution, and the mixture was stirred at room temperature for 2 hours to activate the carboxyl groups on the HA. Simultaneously, a 0.1 L (w / v) histone solution was prepared, and 0.4 g of N-hydroxysuccinimide (NHS) was added. The mixture was stirred for 5 minutes, and the pH was adjusted to 8.0 using 1 M NaOH solution. Subsequently, the 10% HA solution and the 6% histone solution were mixed at a 1:1 volume ratio. The mixture was vigorously stirred to ensure homogeneity, then poured into a mold and allowed to stand at room temperature for crosslinking for 2 hours. The resulting hydrogel was transferred to a dialysis bag (7000 Da molecular weight cutoff) and dialyzed with distilled water for 3 hours, changing the dialysate hourly to remove unreacted reagents. The hydrogel formed by covalent crosslinking of basic protein and hyaluronic acid was inverted in a plate, and its adhesion was observed. The results are shown in [Figure number missing]. Figure 6 .

[0043] Example 3: Antibacterial properties and biocompatibility of proteoglycans 1. Antibacterial properties Filter paper method: *Escherichia coli* ATCC25922 and *Staphylococcus aureus* ATCC6538 were cultured overnight (approximately 10 h). The bacterial suspensions were diluted 100-fold and spread onto antibiotic-free LB agar plates. Sterile filter paper was then soaked in a proteoglycan gel formed from sterile water, basic protein, basic protein (Pichia pastoris crude histone obtained in Example 1), and hyaluronic acid, respectively, and affixed to the plate, labeled 1, 2, and 3. After overnight incubation, the size of the inhibition zone was observed.

[0044] Oxford cup method: *Escherichia coli* ATCC25922 and *Staphylococcus aureus* ATCC6538 were cultured overnight (approximately 10 h). 600 μL of each bacterial culture was added to 15 mL of LB broth, mixed thoroughly, and then poured onto a plate containing a layer of LB broth (three Oxford cups were placed). Sterile water, basic protein, and a proteoglycan gel formed from basic protein and hyaluronic acid were then added to the wells, labeled 1, 2, and 3 on the plate. After overnight incubation, the size of the inhibition zone was observed.

[0045] Antibacterial effect can be seen Figure 7 It can be seen that both the basic protein and the proteoglycan gel it forms have antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0046] 2. Blood compatibility Red blood cells were obtained by centrifuging anticoagulated rabbit blood at 1000 rpm for 10 min. The cells were then repeatedly washed with sterile PBS buffer until the supernatant became clear, and the supernatant was removed. The red blood cells were then diluted to a 5% (v / v) concentration with PBS buffer for use as a red blood cell suspension. Four different proteoglycans were added to 0.5 mL of whole blood diluent (5.0% PBS), followed by 0.4 mL of PBS buffer. After incubation at 37°C for 2 hours, the cells were centrifuged at 1000 rpm for 10 min. The supernatant was collected, and its absorbance at 540 nm was measured using a microplate reader. Deionized water and PBS buffer were used as positive and negative controls, respectively. The blood compatibility results of the proteoglycans are shown in [the table below]. Figure 7 The hemolysis rate was calculated using the following formula, where sample, control, and positive represent the absorbance of the sample, negative control, and positive control, respectively. The hemolysis rate results are shown in [link to results]. Figure 8 The hemolysis rates were all within the acceptable range for biological materials (<5%).

[0047] .

[0048] Example 4: Hemostatic effect and organ adhesion of proteoglycans 1. Liver hemostasis Three healthy C57BL6J mice (n=3) weighing 20-25g were randomly selected and anesthetized with isoflurane (2.5%). After 10 minutes of complete anesthesia, the abdominal and thoracic cavities were disinfected with 75% ethanol. The thoracic cavity was then opened to expose the liver surface. Weighed filter paper was placed under the liver. The mouse liver was punctured with a 0.5 mm diameter needle, and a proteoglycan hydrogel patch (10 mm in diameter and 2 mm in thickness) was applied to the puncture site. After the experiment, the filter paper was weighed, and the blood loss was calculated. Commercial cyanoacrylate tissue glue was used as a control. The hemostasis process was recorded with a camera. All experiments, including the blank control group, the commercial cyanoacrylate group, and the hydrogel group, were conducted in accordance with animal ethics guidelines. The hemostatic effect on the liver is shown in the figure. Figure 9 The blood loss in the proteoglycan hydrogel patch group (≈6.3 mg) was significantly lower than that in the untreated group (≈66.2 mg) and the medical cyanoacrylate group (≈64.1 mg).

[0049] 2. Organ adhesion Remove the mouse viscera (heart, liver, spleen, lung, and kidney). Place the viscera on a flat plate. Apply a proteoglycan gel formed from basic protein and hyaluronic acid close to the viscera, and lift the viscera using adhesive forces. Observe the adhesion effect. Results are shown below. Figure 10 Even without external pressure, it can successfully adhere to internal organs and tissues with gentle contact.

[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a tissue adhesive, characterized in that, The proteoglycan material is formed by the electrostatic self-assembly of basic proteins and anionic acidic polysaccharides extracted by microorganisms. The acidic polysaccharides are selected from one or more of hyaluronic acid, sea cucumber polysaccharides, and sulfonated glycosaminoglycans. The microorganisms include Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, Corynebacterium glutamicum, etc. The sulfonated glycosaminoglycans include chondroitin sulfate A, chondroitin sulfate C, and heparin.

2. The method according to claim 1, characterized in that, The volume ratio of the basic protein to the acidic polysaccharide is 1:(0.5~1.5).

3. The method according to claim 1, characterized in that, The concentration of hyaluronic acid is 20 mg / mL, the concentration of the basic protein is 50 mg / mL, and the concentration of the sulfonated glycosaminoglycan is 50 mg / mL.

4. The method according to claim 1, characterized in that, The specific steps are as follows: basic protein is mixed with anionic glycosaminoglycan, the supernatant is removed by centrifugation to obtain proteoglycan with strong tissue adhesion, which is then freeze-dried to obtain powder and stored at room temperature.

5. The method according to claim 4, characterized in that, The centrifugation was performed at a speed of 10,000 rpm, a temperature of 4°C, and a time of 10-20 min.

6. The method according to claim 4, characterized in that, The freeze-drying time is 5-10 min, the sample temperature is -20℃, the cold trap temperature is -60℃, and the vacuum degree is less than 10 Pa.

7. A tissue adhesive prepared using the method according to any one of claims 1 to 6.

8. The use of the tissue adhesive of claim 7 in the preparation of products for antibacterial materials, wound hemostasis, cell culture, tissue adhesion and / or wound healing.

9. The application according to claim 8, characterized in that, The types of bacteria to be treated include Gram-positive and / or Gram-negative bacteria.

10. The application according to claim 8, characterized in that, The tissue adhesion includes one or more of the following: skin tissue, visceral tissue, muscle tissue, connective tissue, and mucous membrane tissue.

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