Antibacterial peptide-berberine co-assembled hydrogel and application thereof in preparation of anti-infective drugs

Abstract

The invention provides antibacterial peptide-berberine co-assembled hydrogel and application thereof in preparation of anti-infection drugs, lauric acid is connected to the N terminal and the C terminal of antibacterial peptide KKWWW for amidation to obtain antibacterial peptide 2KW, then BBR is added, and the antibacterial peptide 2KW and the antibacterial peptide 2KW are co-assembled through hydrophobic interaction and hydrogen-bond interaction to form the hydrogel, namely 2KW-BBR. The 2KW-BBR hydrogel can perfectly fit and completely cover the damaged surface of the peritoneum by virtue of the unique physical characteristics of the 2KW-BBR hydrogel, and the effective isolation of the 2KW-BBR hydrogel from surrounding tissues is realized by virtue of a compact net structure in the 2KW-BBR hydrogel. Compared with traditional antibiotics such as polymyxin B, severe pulmonary toxicity risks are often accompanied in the treatment process, and 2KW-BBR shows extremely low toxicity characteristics, so that the traditional antibiotics have higher safety and wider application range. In addition, due to the excellent antibacterial ability, the polypeptide can be used for preparing drugs for treating wound infection caused by MRSA or resisting postoperative intestinal adhesion, and has wide application prospects in clinical research and development of anti-infection drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and relates to an antibacterial peptide - berberine co - assembled hydrogel. The present invention also relates to the application of the hydrogel in the preparation of anti - infective drugs, especially in the preparation of drugs for treating wound infections caused by MRSA and preventing postoperative intestinal adhesions. Background Art

[0002] The abuse of antibiotics has led to the emergence of bacterial drug resistance and superbugs. The development of new antibiotics lags far behind the frequency of the emergence of new drug - resistant bacteria. Drug - resistant bacterial infections seriously threaten human health (Science. 2022, 375: 889 - 894., Science. 2011, 333: 1764 - 1767.). More importantly, long - term antibiotic injection may induce the generation of multi - drug - resistant (MDR) bacteria and cause systemic damage to other organs. For example, methicillin - resistant Staphylococcus aureus (MRSA) is a highly virulent Gram - positive bacterium with multiple transmission routes that can infect multiple tissues and organs, causing skin wound infections (J Nanobiotechnology. 2023, 21: 162.). In addition to bacterial infections, increased inflammatory stress and capillary damage are two other important factors leading to the difficult healing of diabetic foot wounds. Bacterial contamination of wounds can lead to an increase in the secretion of pro - inflammatory cytokines, which will prolong the inflammatory period and produce chronic wounds even if the bacteria are not cleared. Therefore, it is extremely important to eliminate or inhibit bacteria in wound infections (Int J Biol Macromol. 2023, 235: 123847.). Although traditional dressings such as gauze, polymer bandages, and cotton wool are widely used for skin wound treatment, they cannot prevent bacterial infections, and their frequent replacement will damage new skin tissues.

[0003] Supramolecular hydrogels based on amphiphilic peptides and their derivatives have received extensive attention as biofunctional materials (Chem. 2018, 4: 2084 - 2107). Compared with polymer hydrogels that cause adverse reactions and even toxicity, self-assembling peptide hydrogels exhibit better biocompatibility and biodegradability and are more easily processed physiologically. Various peptide sequences and structures have been used to construct self-assembling hydrogels; due to their good biocompatibility and minimal immunity, they are widely used in drug delivery, cell culture, and biosensing (ACS Nano 2020, 14: 12133 - 12147, Polymers 2023, 15: 1160). Berberine (BBR) exhibits significant antibacterial activity against bacteria, fungi, protozoa, and viruses. In addition, berberine has been identified as an efflux pump substrate. When used in combination with antibiotics, as the intracellular concentration of berberine increases, its own antibacterial efficacy enhances; at the same time, due to the competitive inhibition of efflux pumps (such as NorA and RamR) on the bacterial cell membrane by berberine, the antibacterial effect of antibiotics is also improved. Studies have shown that berberine can inhibit the growth of multidrug-resistant Staphylococcus aureus by binding to these efflux pumps. More importantly, as a natural antibacterial agent, berberine can not only inhibit microbial adhesion and biofilm formation, but also does not induce bacteria to develop drug resistance, and can also eliminate drug-resistant plasmids and restore the sensitivity of bacteria to other antibacterial drugs. Summary of the Invention

[0004] One object of the present invention is to provide an antibacterial peptide-berberine co-assembled hydrogel.

[0005] Another object of the present invention is to provide the application of the above hydrogel in the preparation of drugs for treating wound infections caused by MRSA and preventing postoperative intestinal adhesions.

[0006] To achieve its objects, the present invention adopts the following technical solutions:

[0007] I. Preparation of an antibacterial peptide-berberine co-assembled hydrogel

[0008] The antibacterial peptide-berberine co-assembled hydrogel provided by the present invention is obtained by connecting lauric acid to the N-terminus of the antibacterial peptide "KKWWW" and amidating the C-terminus to maintain a high net positive charge, obtaining the antibacterial peptide, denoted as 2KW, and then adding BBR. Since BBR contains an aromatic ring, it can form a co-assembled antibacterial hydrogel with the antibacterial peptide 2KW through hydrophobic and hydrogen bond interactions, denoted as: 2KW-BBR. The structural formulas of 2KW and BBR are as follows.

[0009]

[0010] The above antibacterial peptide 2KW is prepared by the classical solid-phase synthesis method.

[0011] II. Application of Antimicrobial Peptide-Berberine Co-Assembled Hydrogel in the Preparation of Clinical Anti-Infective Drugs

[0012] 1. Scanning Electron Microscopy Experiment

[0013] In this experiment, the applicant used a scanning electron microscope (SEM) to visually observe the morphological changes after the co-assembly of antimicrobial peptide and berberine to form a hydrogel. The specific experimental method is as follows: 1 mM of BBR was added to 1 mL of PBS solution with 4 mM and 6 mM of 2KW respectively, and gently pipetted for 1 min to make it evenly distributed, and placed on a round glass slide. The samples were dried, sputter-coated with gold, and analyzed using an Apreo S scanning electron microscope (Thermo Fisher Scientific, MA, USA).

[0014] As Figure 1 shown, 2KW-BBR could form a hydrogel at a concentration of 4 mM, but part of the BBR was not encapsulated; while at a concentration of 6 mM, the hydrogel structure formed by 2KW-BBR was more compact. Therefore, the 1 mM BBR and 6 mM 2KW co-assembled gel system was selected for subsequent experiments to further study its properties.

[0015] 2. In Vitro Bacteriostatic Experiment

[0016] The minimum inhibitory concentration of antimicrobial peptides against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, MRSA, Acinetobacter baumannii) was determined by the classical microdilution method. Antibiotics Methicillin, Polymyxin B, Cephalothin, Gentamicin, Streptomycin and Rifampicin were used as positive controls, and the experiment was repeated in parallel 3 times. The results are shown in Table 1.

[0017] Table 1 Minimum Inhibitory Concentration of the Antimicrobial Peptide of the Present Invention against Common Standard Strains

[0018]

[0019] The results in Table 1 show that the antimicrobial peptide 2KW has strong inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, MRSA, Acinetobacter baumannii), showing broad-spectrum antibacterial activity.

[0020] 3. LPS Binding Experiment

[0021] Endotoxin is one of the main components of the outer membrane of Gram-negative bacteria. Therefore, the applicant designed a competitive inhibition experiment to verify whether the antimicrobial peptide of the present invention can act on endotoxin by incubating with endotoxin at different concentrations. The results are shown in Figure 2 .

[0022] Figure 2 The results showed that endotoxin had a dose-dependent inhibitory effect on the antibacterial activity of antimicrobial peptide 2KW. Moreover, the applicant could also clearly see that due to the competitive inhibition of efflux pumps on the bacterial cell membrane by berberine, the binding ability of 2KW to LPS was enhanced, resulting in a decrease in bacterial survival rate. This indicates that 2KW-BBR can enhance the antibacterial effect against drug-resistant bacteria.

[0023] 4. Experiment on the treatment of MRSA-induced wounds in mice with 2KW-BBR hydrogel

[0024] Male BALB / C mice, weighing 18 - 22 g, were raised according to the experimental animal ethics management method of Lanzhou University. The mice were randomly divided into 6 groups, with 8 mice in each group, namely: normal control group, model group, positive drug group (vancomycin: 10 mg / kg), 2KW-BBR treatment group (10 mg / kg, where 2KW:BBR = 6 mM:1 mM). All mice were anesthetized with pentobarbital, and the back hair was removed using depilatory cream. Two circular wounds with a diameter of 8 mm were created on the back using a skin punch. Subsequently, a 50-μL saline solution containing 1×10 9 CFU / mL MRSA was injected to establish a back MRSA infection model. The drug administration group was first administered (10 μL) 6 hours after modeling, and the second and third administrations were carried out at 12 hours and 24 hours respectively. After 32 hours, the samples from the left wound were scraped to record the bacterial count (see Figure 3 ). The mice were sacrificed after continuous observation for 10 days, and the area of the right wound was measured and statistically analyzed.

[0025] According to Figure 3 data analysis, 2KW-BBR showed a significant therapeutic effect on MRSA-induced wound infection, and its efficacy was comparable to that of vancomycin, both of which could effectively control MRSA infection. However, further analysis of Figure 4 and Figure 5 showed that 2KW-BBR hydrogel showed a better effect than vancomycin in promoting wound healing.

[0026] 5. Anti-intestinal adhesion experiment

[0027] The in vivo anti-adhesion effect of 2KW-BBR hydrogel was evaluated in a rat lateral wall defect-cecal abrasion model. During the experiment, SD rats were first anesthetized with sodium pentobarbital at a dose of 50 mg / kg body weight. The abdominal hair was then shaved and disinfected three times with alternating povidone-iodine and 75% ethanol. Then, a 4-5 cm long incision was made along the midline of the abdominal wall, the cecum was separated, and its surface was gently rubbed with sterile surgical gauze until punctate bleeding occurred. At the same time, a 1 cm × 2 cm peritoneal defect was made on the corresponding lateral edge of the abdominal wall. For the 2KW-BBR hydrogel treatment group, 1 mL of hydrogel was directly injected into the damaged abdominal wall and cecum surface without hemostatic treatment; the negative control group was sprayed with 1 mL of sterile saline on the wound surface; the positive control group used commercially available sodium hyaluronate hydrogel. Each group contained 5 SD rats. The rats were killed on the 7th day after surgery and the peritoneum was opened to examine the adhesion.

[0028] like Figure 6 As shown, the 2KW-BBR hydrogel of the present invention exhibits a significant anti-postoperative intestinal adhesion effect, and its performance is better than the commercially available sodium hyaluronate hydrogel. Inevitable peritoneal injury, bleeding, inflammatory response and potential infection risk during surgery are the main factors leading to peritoneal adhesion. With its dense internal reticular structure, the 2KW-BBR hydrogel can effectively physically isolate the damaged tissue from the surrounding environment, thereby significantly reducing the occurrence of adhesion.

[0029] 6. In vivo acute toxicity test

[0030] The peptides were dissolved in saline, formulated at a concentration of 10 mg / mL, and administered to mice by a single intraperitoneal injection to assess acute toxicity. The LD was also calculated using the modified Karber method. 50 Value. The applicant randomly divided male BABL / C mice into groups (n=10). The polymyxin B group was divided into 3 dose groups of 22.00, 27.75, and 35.00 mg / kg, the 2KW group was divided into 3 dose groups of 110.00, 132.00, and 160 mg / kg, and the 2KW-BBR hydrogel group (2KW:BBR=6mM:1mM) was divided into 3 dose groups of 100.00, 118.00, and 140 mg / kg. The survival rate of mice in each group was monitored for 7 consecutive days.

[0031] The modified Karber method was used to determine the LD50 of the antimicrobial peptide 2KW and 2KW-BBR hydrogel in male BALB B / c mice. 50 and compared with polymyxin B. Figure 7As shown in A, the survival curve indicates that most mice died within 3 hours after treatment with polymyxin B, while the mice in the groups treated with C12-2KW and 2KW-BBR hydrogels showed a gradual death, indicating that the 2KW and 2KW-BBR hydrogels have lower toxicity than polymyxin B. Based on Figure 7 the results shown in B, the LD 50 values (129.45 mg / kg, 114.02 mg / kg) of the antimicrobial peptide 2KW and 2KW-BBR hydrogel are 5.0 times and 1.4 times that of polymyxin B (25.88 mg / kg), respectively.

[0032] To further reveal the potential toxicity mechanism, the applicant conducted a comprehensive and in-depth analysis of the pathological sections of the heart, liver, spleen, lungs and kidneys of mice through histopathological examination. As Figure 8 shown, all organs of the normal control group showed the characteristics of complete cell structure, clear tissue morphology and distinct layers. In contrast, the treatment with polymyxin B significantly induced pulmonary toxicity, specifically manifested as obvious congestion, disordered alveolar structure and widened interstitium caused by infiltration of inflammatory cells; in addition, the drug also caused mild damage to the kidneys, manifested as cavitation of renal tubular epithelial cells and local tissue abnormalities. However, the antimicrobial peptides 2KW and 2KW-BBR hydrogels showed lower toxicity characteristics, suggesting that they may have higher safety.

[0033] An antimicrobial peptide-berberine co-assembled hydrogel provided by the present invention is prepared by first connecting lauric acid to the N-terminus of the antimicrobial peptide "KKWWW" to endow the molecule with stronger lipophilicity, and performing C-terminal amidation to maintain a high net positive charge, obtaining an antimicrobial peptide 2KW with significant biological activity and stability, and then adding BBR. Since BBR contains aromatic rings, it can form a co-assembled antimicrobial hydrogel 2KW-BBR with the antimicrobial peptide 2KW through hydrophobic interaction and hydrogen bond interaction. With its unique physical properties, the 2KW-BBR hydrogel can perfectly fit and completely cover the surface of peritoneal injury, just like a protective barrier customized for the damaged tissue, and its internal dense network structure realizes effective isolation from the surrounding tissue. In contrast, traditional antibiotics such as polymyxin B often carry a serious risk of pulmonary toxicity during treatment, while 2KW-BBR shows extremely low toxicity characteristics, suggesting its higher safety and wider scope of application. In addition, due to its extraordinary antibacterial ability, the hydrogel can effectively prevent the occurrence of potential infections during surgery and has broad prospects in the research and development of clinical anti-infective drugs. Description of the Drawings

[0034] Figure 1 are electron micrographs of the 2KW-BBR hydrogel and antibiotics of the present invention;

[0035] Figure 2 This is the graph of the LPS-binding ability of the antimicrobial peptide 2KW and 2KW-BBR hydrogel of the present invention;

[0036] Figure 3 This is the plate count graph of the 2KW-BBR hydrogel of the present invention and antibiotic treatment of MRSA infection on the back of mice;

[0037] Figure 4 This is the wound area graph of the 2KW-BBR hydrogel of the present invention and antibiotic treatment of MRSA infection on the back of mice;

[0038] Figure 5 This is the statistical graph of the wound area of the 2KW-BBR hydrogel of the present invention and antibiotic treatment of MRSA infection on the back of mice;

[0039] Figure 6 This is the effect graph of the 2KW-BBR hydrogel of the present invention in anti-intestinal adhesion;

[0040] Figure 7 This is the LD 50 graph of the antimicrobial peptide 2KW, 2KW-BBR hydrogel and polymyxin B of the present invention;

[0041] Figure 8 This is the hematoxylin-eosin staining graph of each organ of the in vivo toxicity of the antimicrobial peptide 2KW, 2KW-BBR hydrogel and polymyxin B of the present invention;

[0042] Figure 9 This is the mass spectrum graph of the antimicrobial peptide 2KW of the present invention. Detailed implementation manners

[0043] The following further illustrates the synthesis of the broad-spectrum antibacterial activity and low-toxicity antimicrobial peptide of the present invention through specific examples.

[0044] Example 1: Synthesis of antimicrobial peptide 2KW

[0045] (1) Activation and pretreatment of resin

[0046] Weigh 0.465 g of MBHA resin (0.43 mM / g), add it to the polypeptide solid-phase synthesizer, swell it with DCM for 30 min, wash it with DMF, and identify the resin by ninhydrin color reaction. If it is colorless, it indicates that the resin is normal.

[0047] (2) Synthesis of Fmoc-2KW-MBHA

[0048] The swollen resin was washed with a DMF solution containing 20% piperidine to remove the Fmoc protecting group until the indene test resin showed a blue-violet color. Three-fold excess of Trp, three-fold excess of HOBt, HBTU, and six-fold excess of DIEA were dissolved in redistilled DMF and added to the synthesizer and stirred for 1 h. After the reaction time, the indene test resin being colorless and transparent indicated successful condensation, and Fmoc-Trp-MBHA was obtained.

[0049] Trp, Trp, Lys, Lys, and lauric acid were successively condensed according to the above method to obtain: lauric acid-Lys-Lys-Trp-Trp-Trp-MBHA.

[0050] (3) Peptide cleavage

[0051] The obtained lauric acid-Lys-Lys-Trp-Trp-Trp-MBHA was washed twice with DCM and methanol successively and the resin was thoroughly dried by suction. 10 mL of cleavage reagent (TFA:Tris:water = 9.5:0.25:0.25 (v:v:v)) was added and reacted for 3 h, followed by freeze-drying after acetonitrile and water (1:1).

[0052] (4) Peptide purification

[0053] The RP-HPLC purification conditions were mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, with linear gradient elution. The effluent of the target peak was collected, freeze-dried to obtain the antibacterial peptide 2KW, and its mass spectrum was as Figure 9 shown.

[0054] (5) Preparation of 2KW-BBR co-assembled hydrogel

[0055] 1 mM of BBR was respectively added to 4 mM and 6 mM of 2KW in 1 mL of PBS solution, gently pipetted for 1 min to make it evenly distributed, and left standing for 3 min.

Claims

1. An antibacterial peptide - berberine co - assembled hydrogel, characterized in that, The hydrogel is prepared by first connecting lauric acid to the N-terminus of the antimicrobial peptide "KKWWW" and amidating the C-terminus to obtain the antimicrobial peptide, denoted as 2KW, and then adding BBR, which co-assembles with the antimicrobial peptide 2KW through hydrophobic and hydrogen bond interactions, denoted as: 2KW-BBR.

2. The antibacterial peptide - berberine co - assembled hydrogel according to claim 1, wherein, The hydrogel is obtained by adding 1 mM of BBR and 6 mM of 2KW to 1 mL of PBS solution, gently pipetting for 1 min, and allowing to stand for 3 min.

3. Use of an antimicrobial peptide-berberine co-assembled hydrogel according to claim 1 or 2 in the preparation of an anti-infective drug.

4. Use of an antibacterial peptide - berberine co - assembled hydrogel as described in claim 3 in the preparation of an anti - infective drug, characterized in that, The hydrogel can be used to prepare a drug for treating wound infections caused by MRSA.

5. Use of an antibacterial peptide-berberine co-assembled hydrogel as described in claim 3 in the preparation of an anti-infective drug, characterized in that, The hydrogel can be used to prepare a drug for preventing postoperative intestinal adhesions.

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