An assembly-enhanced poly-polypeptide-based biohybrid material and its preparation method and application

Abstract

The application discloses an assembled synergistic poly-polypeptide-based biological hybrid material and a preparation method and application thereof, and the preparation steps comprise the following steps: (1) preparation of hybrid bacteria; (2) preparation of side-chain tobramycinized poly-polypeptide; and (3) preparation of poly-polypeptide-based biological hybrid bacterial clusters. The application realizes synergistic antibacterial ability, and promotes the ability of effectively controlling inflammation and wound healing. The unique poly-polypeptide-based biological hybrid structure utilizes a multiplexing and aggregation synergistic design idea, realizes the dual purposes of resisting bacterial infection and regulating host life functions, and significantly improves the comprehensive effect of antibacterial and anti-inflammatory treatment.

Description

Technical Field

[0001] The present invention relates to the field of drug-resistant bacterial infection treatment, and in particular to an assembly-synergistic poly-polypeptide-based biohybrid material, a preparation method and application thereof. Background Art

[0002] Over the past decade, the health and economic burden of infections caused by drug-resistant bacteria and their biofilms has increased dramatically. This is primarily due to the increasing prevalence of antibiotic resistance, which has led to a reduction in treatment options and increased costs. There is an urgent need to develop novel antimicrobial therapies with alternative therapeutic effects. In recent years, antimicrobial peptides (AMPs) have emerged as promising antimicrobial agents that primarily disrupt bacterial cell membranes through physical means rather than acting on intracellular factors to cause cell death. AMPs exhibit broad antimicrobial activity, primarily due to the presence of cationic and hydrophobic residues in their peptide chains, which enable electrostatic interactions with cell membranes, thereby disrupting bacterial structure. Furthermore, this mechanism of action makes it difficult for bacteria to develop drug resistance. However, despite these advantages, the clinical application of AMPs remains limited, primarily due to the high cost of peptide synthesis, susceptibility to protease degradation, and low selectivity in cytotoxicity against mammalian cells.

[0003] Polypeptides prepared by chemical polymerization methods have attracted considerable attention as AMP mimics in antimicrobial strategies. Due to their similar chemical and secondary structures (α-helices and β-sheets) to natural peptides, polypeptides may possess potential biomimetic activities (cell penetration, cell adhesion, and antimicrobial activity), good biodegradability, and biocompatibility, leading to their widespread application in the biomedical field. Cheng et al. reported a class of cationic antimicrobial helical polypeptides with a radial amphiphilic structure, which enables the polypeptides to effectively bind to negatively charged bacterial surfaces and protect the peptide backbone from premature degradation by proteases, resulting in high antimicrobial activity. (Xiong M, Lee MW, Mansbach RA, et al. Helical antimicrobial polypeptides with radial amphiphilicity [J]. Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, 2015, 112 (43): 13155-13160.) Yang et al. studied the synergistic antimicrobial activity of silver and six-arm polylysine nanohybrid materials. Due to the complexation and synergistic effect of silver and polylysine, the antibacterial nanoparticles showed lower cytotoxicity and faster sterilization speed than pure and pure, which was attributed to the potential enhanced ability of these particles to destroy bacterial membranes. However, these high density of positive charges will limit their application in vivo. (Zhen JB, Kang PW, Zhao MH, et al. Silver Nanoparticle Conjugated Star PCL-b-AMPs Copolymer as Nanocomposite Exhibits Efficient AntibacterialProperties[J]. Bioconjugate Chemistry, American Chemical Society, 2020, 31(1): 51-63.) To solve this problem, Cheng et al. synthesized pathogen-activatable radial amphiphilic polypeptides, which restored the helical structure and positive charge of the polypeptide through bacterial phosphatase activation, showing a higher selective membrane-destructive effect on bacteria while reducing its cytotoxicity to normal mammalian cells.(Xiong M, Han Z, Song Z, et al. Bacteria-Assisted Activation of Antimicrobial Polypeptides by a Random-Coil to Helix Transition[J]. Angewandte Chemie International Edition, 2017, 56(36): 10826-10829.) However, its control of bacterial biofilms, complications and regulation of host immunity remains a challenge.

[0004] In addition to antimicrobial peptides and biomimetic polypeptides, the new generation of antimicrobial materials also includes nanomaterials such as metal ions and their metal oxides, light-induced antimicrobial nanomaterials, organic-inorganic nanohybrid materials, and novel biomaterials. The antimicrobial mechanisms of metal ions and metal nanoparticles include inducing bacterial membrane disruption, damaging bacterial proteins and DNA, and generating reactive oxygen species. Despite the widespread use of metals and their metal oxides for antimicrobial resistance, there is little literature describing the potential for drug resistance, high antimicrobial efficacy, and risks. Furthermore, when metal-based nanomaterials are used without any supporting materials, they often have limitations, such as aggregation due to their high surface reactivity, which reduces antimicrobial efficacy, high biotoxicity, and low targeting. Consequently, much research in recent years has focused on integrating them into biomaterials for effective improvement and combination therapy. Zhang et al. reported a materials-assisted microbial strategy to prepare a biohybrid material modified with zinc oxide nanorods to eradicate Gram-negative bacterial biofilms. The collision with Bdellovibrio bacteria triggered the production of reactive oxygen species by zinc oxide nanorods, enhancing the removal of plaque biofilms (Tang Y, Huang QX, Zheng DW, et al. Engineered Bdellovibrio bacteriovorus: a countermeasure for biofilm-induced periodontitis [J]. Materials Today, 2022, 53: 71-83). However, this approach still carries the potential risk of probiotic infection and lacks the ability to regulate the host's own defenses. Summary of the Invention

[0005] Technical problems to be solved: Infections and excessive inflammation caused by drug-resistant bacteria pose a major challenge to clinical antibiotic treatment, as well as the current problems of the need to optimize the efficacy of polypeptides and biohybrid materials and the cumbersome preparation. The present invention aims to provide an assembly-synergistic polypeptide-based biohybrid material and its preparation method and application. This method utilizes the gradual biological self-assembly and self-growth of metal ions after co-mixing with probiotics, the further reduction of surface self-assembled metal particles by tannic acid polyphenols, and the chemical surface-specific coupling modification of borated polypeptides to construct a multifunctional polypeptide biohybrid bacterial cluster system. This system combines the optical and electrical properties of inorganic nanomaterials, the pharmaceutical physicochemical properties of tobramycin-modified antibacterial polypeptides, and the multiple functions of probiotic bioactivity regulators. At the same time, it achieves the assembly synergy of the increased local concentration of organic components in the hybrid material and the nano-confined enhancement mechanism of inorganic components through a simple site-specific coupling method, providing a new way to treat drug-resistant bacterial infections with a new type of biohybrid material.

[0006] Technical solution: A preparation method of an assembly-enhanced poly-polypeptide-based biohybrid material, the preparation steps comprising: (1) preparation of hybrid bacteria: suspending probiotics in PBS to a final concentration of OD not less than 0.3, sequentially adding at least two metal salt solutions and incubating them for 4-24 hours, wherein the final concentration of the metal salt is 100-250 μmol / L, wherein each incubation is followed by centrifugation and PBS washing; subsequently, adding tannic acid polyphenols and incubating them, wherein the final concentration of the tannic acid polyphenols is 100-250 μmol / L, to obtain hybrid bacteria with polyphenolized metal particles on the surface; (2) preparation of poly-polypeptides with tobramycin on the side chain: dissolving poly-L-aspartic acid benzyl ester and tobramycin in dimethyl sulfoxide solvent according to proportion to carry out aminoester exchange reaction, wherein The molar ratio of poly (L-aspartic acid benzyl ester) to tobramycin is 1:(1-5), and tobramycin boronated poly (PT) is obtained after purification; (3) Preparation of poly (polypeptide-based biohybrid bacterial clusters: tobramycin boronated poly (polypeptide) and 5-aldehyde-2-thiophene boronic acid are mixed in PBS, and the molar number of 5-aldehyde-2-thiophene boronic acid is 1-5 times that of the poly (polypeptide-based) repeating unit, respectively. The amine-aldehyde condensation reaction is carried out for 1-3 hours (PTS), and then hybrid bacteria with surface metal particles polyphenolized are added to the above solution to make the final concentration OD not less than 0.3, and continue to incubate for 1-3 hours (UGT); finally, centrifugation and washing are carried out to obtain an assembly-enhanced poly (polypeptide-based biohybrid material): poly (polypeptide-based biohybrid bacterial cluster (PTS-UGT).

[0007] Preferably, the metal salt is chloroauric acid or silver nitrate.

[0008] Preferably, the molar number of the above 5-formyl-2-thiopheneboronic acid is twice that of the polypolypeptide repeating unit.

[0009] The above-mentioned probiotics are intestinal, oral or skin probiotics.

[0010] The above-mentioned metal salts also include copper, iron, zinc, bismuth, platinum, manganese or selenium salts.

[0011] The above preparation method produces an assembly-synergistic poly-polypeptide-based biohybrid material.

[0012] The application of the above-mentioned assembled synergistic poly-polypeptide-based biohybrid material in the preparation of drugs for treating drug-resistant bacterial infections.

[0013] Beneficial effects: 1. The present invention is composed of a metal ion stepwise biological self-assembly combined with a natural ingredient boric acid-polyphenol coordination site-specific coupling aggregation assembly, which has higher biosafety and provides a simple and efficient method for preparing a new multifunctional synergistic biohybrid material.

[0014] 2. The present invention integrates the optical and electrical properties of inorganic nanomaterials of metal-inorganic nanosystems, the pharmaceutical physicochemical properties of tobramycin-modified antibacterial polypeptides, the synergistic physicochemical properties of organic-inorganic assemblies, and the function of probiotics in regulating body components through biological activity.

[0015] 3. This invention achieves synergistic antibacterial capabilities, promoting effective inflammation control and wound healing. This unique poly(peptide)-based biohybrid structure, utilizing multiplexing and aggregation-enhanced design principles, achieves the dual purpose of combating bacterial infection while modulating host biological functions, significantly enhancing the overall efficacy of antibacterial and anti-inflammatory treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Transmission electron microscopy images of poly(peptide-based) biohybrid bacterial clusters.

[0017] Figure 2 .Elemental mapping of poly(peptide) hybrid bacterial clusters.

[0018] Figure 3 .Surface-enhanced Raman spectroscopy and photothermal properties of poly(peptide-based) biohybrid bacterial clusters.

[0019] Figure 4 .Anti-inflammatory regulatory properties of poly(peptide-based) biohybrid bacterial clusters (macrophage inflammatory factor levels and phenotypic protein analysis).

[0020] Figure 5 .Antibacterial properties of poly(peptide)-based biohybrid bacterial clusters.

[0021] Figure 6 .H&E staining of wound-infected mice treated with poly(peptide-based) biohybrid bacterial clusters. DETAILED DESCRIPTION

[0022] In order to further understand the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention.

[0023] Unless otherwise specified, the reagents and instruments involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0024] Example 1

[0025] 1. Preparation of hybrid bacteria: A cultured Staphylococcus epidermidis (SE) solution was resuspended in PBS (final concentration OD = 0.3), and chloroauric acid and silver nitrate solutions were added sequentially at final concentrations of 100-250 μmol / L for co-incubation for 4-24 hours. Each co-incubated component was centrifuged and washed with PBS to remove the previous component (UG). Subsequently, a corresponding volume of tannic acid polyphenols was added for co-incubation (final concentration of 100-250 μmol / L) to further reduce the metal particles in the probiotic bacteria and the surface metal particles to provide polyphenol sites (UGT).

[0026] 2. Preparation of side chain tobramycin-modified polypeptides: First, prepare poly (L-aspartic acid benzyl ester) (J. Xiao, Z. Guo, G. Lv, Z. Yan, T. Liu, Y. Wang, H. Liu, J. Martínez, L. Yin, X. Liu, H. Jiang, Y. Weizmann, X. Wang, Adv. Healthc. Mater. 2024, 2401993.), then dissolve it in dimethyl sulfoxide solvent for aminoester exchange reaction (the molar ratio of poly (L-aspartic acid benzyl ester) tobramycin is 1:5), dialyze, purify and lyophilize to prepare tobramycin borated polypeptide (PT).

[0027]

[0028] 3. Preparation of Polypeptide-Based Biohybrid Bacteria: A polypeptide with tobramycin-modified side chains was first mixed with 5-formyl-2-thiopheneboronic acid in PBS (the molar ratio of 5-formyl-2-thiopheneboronic acid was 1, 2, or 5 times the molar ratio of the polypeptide repeating unit, respectively (preferably 2 times for better electroactivity)). The mixture was then subjected to an amine-aldehyde condensation reaction for 2 hours (PTS). Hybrid bacteria with surface metal particles (final concentration OD = 0.3) were then added to the above solution and incubated for a further 2 hours (UG) to allow boric acid-polyphenol coordination and multiple hydrogen bond-mediated assembly and aggregation. Finally, the mixture was centrifuged and washed to prepare polypeptide-based biohybrid bacteria (PTS-UG).

[0029] 4. Preparation of Polypeptide-Based Biohybrid Bacterial Clusters: Polypeptides with tobramycin-labeled side chains were first mixed with 5-formyl-2-thiopheneboronic acid in PBS (the molar number of 5-formyl-2-thiopheneboronic acid was 1, 2, or 5 times the molar number of the polypeptide repeating unit, respectively (preferably 2 times for better electroactivity)), and the amine-aldehyde condensation reaction was carried out for 2 hours (PTS). Subsequently, hybrid bacteria with surface metal particle polyphenolization (final concentration OD = 0.3) were added to the above solution and incubated for a further 2 hours (UGT), allowing boric acid-polyphenol coordination and multiple hydrogen bond-mediated assembly and aggregation. Finally, the mixture was centrifuged and washed to prepare polypeptide-based biohybrid bacterial clusters (PTS-UGT).

[0030] 5. Immunomodulatory properties of poly(peptide-based) biohybrid bacterial clusters: Each experimental group was incubated with macrophages RAW264.7 for 24 hours, and the supernatant was then collected. The IL-1β concentration was identified using an Elisa cytokine detection kit, and the adherent cells were collected and incubated with antibodies (anti-CD86-FITC, anti-CD206-PE, and anti-F4 / 80-APC, and the macrophage phenotypic protein content was identified by flow cytometry).

[0031] 6. Antibacterial properties of poly(peptide-based) biohybrid bacterial clusters: Each experimental group was incubated with drug-resistant Staphylococcus aureus at 37°C in a shaker overnight, and the antibacterial properties of each sample were then examined using the plate count method.

[0032] 7. In vivo therapeutic efficacy of poly(peptide-based) biohybrid bacterial clusters: A bacterial wound infection model was first established in mice. Each sample was then dripped onto the wounds for treatment (100 μL, 3 mice per group). Data were collected during the treatment period. After 10 days of treatment, H&E staining of infected mouse skin tissues was performed to assess healing and therapeutic efficacy.

[0033]

[0034] Preparation of Polypeptide-Based Biohybrid Bacterial Clusters and Principles of Their Assembly for Synergistic Therapy:

[0035] The secretions and active ingredients of probiotics have redox and biotherapeutic activities. First, gold and silver nanoparticles are formed on the surface of bacteria through the redox properties of metal salts such as chloroauric acid and silver nitrate.

[0036] The addition of tannic acid further reduces and provides polyphenol sites on the bacterial surface, forming surface metal particle polyphenolized hybrid bacteria with antimicrobial potential and photoelectric activity.

[0037] Tobramycin-modified antibacterial polypeptides with boronic acid groups on the side chains were prepared and co-incubated with polyphenol-modified hybrid bacteria on the surface of metal particles. The site-specific composite assembly of polypolypeptide-based biohybrid bacterial clusters was achieved through boronic acid-polyphenol coordination and multiple hydrogen bonding.

[0038] The polypeptide-based biohybrid bacterial cluster integrates the photoelectric nano-confinement enhancement of the metal-inorganic nano-antibacterial components, the synergistic antibacterial properties of the increased local concentration of polypeptides, and the host body regulation of the bioactive components on the surface of probiotics, thereby showing an assembly-synergistic therapeutic effect.

[0039] result:

[0040] 1. Transmission electron microscopy images of poly(peptide-based biohybrid bacteria) (PTS-UG) and poly(peptide-based biohybrid bacteria clusters) (PTS-UGT) Figure 1 ), demonstrated that metal particle probiotics (UG) can effectively improve the site-specific coupling efficiency and aggregation assembly effect with boronated antibacterial polypeptides after surface-specific polyphenolization (UGT) provides phenolic hydroxyl sites, thereby achieving bridging between hybrid bacteria and forming hybrid probiotic aggregates.

[0041] 2. Elemental mapping of poly(peptide-based biohybrid bacterial clusters) (PTS-UGT) Figure 2 ), further demonstrating the stepwise self-assembly growth and site-directed coupling aggregation of poly(peptide-based) biohybrid bacterial clusters to form hybrid probiotic aggregates.

[0042] 3. Electrochemical CV patterns, surface-enhanced Raman spectroscopy, and photothermal properties of poly(peptide-based) biohybrid bacterial clusters (PTS-UGTs) Figure 3 ), demonstrating that the poly(peptide-based) biohybrid bacterial cluster exhibited superior electrochemical activity, surface-enhanced Raman scattering (SERS) activity, and photothermal performance (wavelength = 808 nm, laser power = 1 W cm) towards crystal violet compared to individual hybrid bacteria (UGT or PTS-UG). -2 ).

[0043] 4. Anti-inflammatory regulatory properties of poly(peptide-based biohybrid bacterial clusters) (PTS-UGT) Figure 4 ), demonstrating that the poly(peptide-based) biohybrid bacterial cluster has a superior ability to reduce the inflammatory level of macrophages RAW264.7 compared to single hybrid bacteria.

[0044] 5. Antibacterial properties of poly(peptide-based) hybrid bacterial clusters (PTS-UGTs) Figure 5), demonstrated that the poly-polypeptide-based biohybrid bacterial cluster had a better in vitro synergistic antibacterial effect than the single hybrid bacteria (UGT or PTS-UG), and further studied the poly-polypeptide-based biohybrid bacterial cluster's better inhibition of bacterial biofilms than the single hybrid bacteria (UGT or PTS-UG), as well as the penetration and destruction effects on mature bacterial biofilms.

[0045] 6. Hematoxylin and eosin staining (HE staining) images of infected skin of mice after treatment with poly(peptide-based biohybrid bacterial clusters) (PTS-UGT) Figure 6 ), demonstrating that the poly(peptide-based) biohybrid bacterial cluster has superior in vivo synergistic antibacterial, anti-inflammatory and pro-healing effects compared to individual hybrid bacteria.

[0046] The above examples are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and substance of this invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A method for preparing an assembly-synergistic poly(peptide)-based biohybrid material, characterized in that: The preparation steps include: (1) preparation of hybrid bacteria: suspending Staphylococcus epidermidis in PBS to a final concentration of OD not less than 0.3, adding two metal salt solutions in sequence and incubating them for 4-24 hours, wherein the metal salts are chloroauric acid and silver nitrate, and the final concentration of the metal salts is 100-250 μmol / L, wherein centrifugation and PBS washing are performed after each incubation; then adding tannic acid polyphenols and incubating them, and the final concentration of tannic acid polyphenols is 100-250 μmol / L. μmol / L, to obtain hybrid bacteria with polyphenolized metal particles on the surface; (2) Preparation of side chain tobramycin-modified polypeptides: poly (L-aspartic acid benzyl ester) and tobramycin are dissolved in dimethyl sulfoxide solvent according to proportion to carry out aminoester exchange reaction, the molar ratio of poly (L-aspartic acid benzyl ester) to tobramycin is 1: (1-5), and tobramycin boronated polypeptides are obtained after purification; (3) Preparation of polypeptide-based biohybrid bacterial clusters: tobramycin boronated polypeptides and 5-aldehyde-2-thiophene boronic acid are mixed in PBS, the molar number of 5-aldehyde-2-thiophene boronic acid is 1-5 times that of the polypeptide repeating unit, and the amine-aldehyde condensation reaction is carried out for 1-3 hours, and then the hybrid bacteria with polyphenolized metal particles on the surface are added to the above solution to make the final concentration OD not less than 0.3, and continue to incubate for 1-3 hours; finally, centrifugation and washing are carried out to obtain assembly-enhanced polypeptide-based biohybrid materials: polypeptide-based biohybrid bacterial clusters.

2. The preparation method according to claim 1, characterized in that The molar number of the 5-formyl-2-thiopheneboronic acid is twice that of the polypolypeptide repeating unit.

3. The assembly-synergistic poly-polypeptide-based biohybrid material prepared by the preparation method according to claim 1 or 2.

4. Use of the assembled and synergistic poly-polypeptide-based biohybrid material according to claim 3 in the preparation of a drug for treating drug-resistant Staphylococcus aureus infection.

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