WO2026001787A1 - 一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用 - Google Patents

一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用

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Publication number
WO2026001787A1
WO2026001787A1 PCT/CN2025/101784 CN2025101784W WO2026001787A1 WO 2026001787 A1 WO2026001787 A1 WO 2026001787A1 CN 2025101784 W CN2025101784 W CN 2025101784W WO 2026001787 A1 WO2026001787 A1 WO 2026001787A1
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WIPO (PCT)
Prior art keywords
polypeptide
self
caries
hydrogel
assembled
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Pending
Application number
PCT/CN2025/101784
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English (en)
French (fr)
Inventor
阎文锦
张洁
邓亚博
魏佳琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innova Pepplus Biotech LLP
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Innova Pepplus Biotech LLP
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Publication of WO2026001787A1 publication Critical patent/WO2026001787A1/zh
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This application belongs to the field of biochemical technology, and specifically relates to a polypeptide with anti-caries properties, a polypeptide self-assembled hydrogel, and its applications.
  • Antimicrobial peptides are a large class of bioactive polypeptides that possess resistance to harmful organisms (or pathogens) such as bacteria, fungi, parasites, and viruses. Because they generally carry a sufficient amount of positive charge and are often hydrophobic, they can bind to negatively charged biomembranes under electrostatic interactions, penetrating and disrupting the membrane structure, leading to cell death. Unlike the single-target bactericidal mechanism of traditional antibiotics, antimicrobial peptides can disrupt pathogens at multiple sites, significantly reducing the emergence of drug-resistant bacteria. They also possess broad-spectrum antimicrobial activity, making them one of the best alternatives to antibiotics in the future.
  • This application also provides a peptide self-assembled hydrogel with anti-caries properties and its application.
  • This application provides a polypeptide with anti-caries properties, the amino acid sequence of which is shown in SEQ ID NO.1.
  • this application provides a polypeptide self-assembly hydrogel with anti-caries properties, wherein the polypeptide self-assembly hydrogel contains the aforementioned polypeptide with anti-caries properties.
  • polypeptide self-assembly hydrogel is formed by the self-assembly of the polypeptide in a solvent.
  • the solvent is PBS buffer.
  • this application provides the application of a polypeptide with anti-caries properties in the preparation of anti-caries drugs.
  • this application provides the application of a polypeptide self-assembled hydrogel with anti-caries properties in the preparation of anti-caries drugs.
  • this application provides an anti-caries drug, wherein the effective component of the anti-caries drug includes the aforementioned polypeptide with anti-caries properties and/or a polypeptide self-assembled hydrogel with anti-caries properties.
  • this application provides the application of a polypeptide with anti-caries properties in inhibiting or killing Streptococcus mutans.
  • this application provides the application of a polypeptide self-assembled hydrogel with anti-caries properties in inhibiting or killing Streptococcus mutans.
  • this application provides a Streptococcus mutans inhibitor, wherein the active ingredient of the inhibitor includes the aforementioned polypeptide with anti-caries properties and/or a polypeptide self-assembled hydrogel with anti-caries properties.
  • reaction solution was reacted at 23–37°C for a period of time to obtain a polypeptide self-assembled hydrogel with anti-caries properties.
  • the reaction time of the reaction solution is 5 to 30 minutes.
  • polypeptide A3 exhibits excellent antibacterial activity against a variety of pathogens, especially Streptococcus mutans.
  • the minimum inhibitory concentration (MIC) of polypeptide A3 against Streptococcus mutans is significantly lower than that against Escherichia coli and Staphylococcus aureus.
  • Polypeptide A3 can also be self-assembled to prepare polypeptide hydrogels, achieving adhesion and sustained release at the site of tooth decay. It can serve as an effective component of anti-caries drugs, providing a new option for the prevention and treatment of early caries in clinical practice.
  • Figure 2 shows the effect of compound A3 on the proliferation of cell line L929.
  • Figure 3 shows the preparation process of the antimicrobial peptide self-assembled hydrogel (inverted vial experiment).
  • Figure 4 shows the antibacterial activity of the self-assembled hydrogel of the antimicrobial peptide.
  • Figure 6 shows the experimental results of the self-assembled hydrogel CCK-8 of antimicrobial peptides.
  • Figure 8 shows the staining results of live/dead cells in the self-assembled hydrogel of antimicrobial peptides.
  • Hydrogels are highly hydrophilic three-dimensional network gels that can rapidly expand in water but cannot dissolve. They can absorb large amounts of water while maintaining their original structure after expansion. As a soft material, hydrogels are widely used in the biomedical field. Compared with traditional synthetic hydrogels, peptide self-assembled hydrogels have many advantages. Similar to other molecular self-assembly systems, peptide self-assembly relies on non-covalent interactions. First, the structure and function of the hydrogel can be adjusted by regulating the structure of the peptide. Second, peptide hydrogels possess stable mechanical properties, a porous structure, and good biocompatibility, which are beneficial for the migration and growth of the loaded cells. Finally, the large specific surface area of peptides can promote the adsorption of drugs and biomolecules.
  • this application provides a polypeptide with anti-caries properties.
  • This application uses computer-generated antimicrobial peptides to capture their antimicrobial characteristics, and artificial intelligence learns these characteristics to establish a predictive model. From a series of generated non-natural antimicrobial peptides, a polypeptide with high antimicrobial activity is selected, its amino acid sequence being RRRWRWRIGIRIY, and labeled as compound A3.
  • compound A3 exhibits excellent antibacterial activity against a variety of pathogens, particularly *Streptococcus mutans*.
  • the minimum inhibitory concentration (MIC) of peptide A3 against *Streptococcus mutans* was significantly lower than that against *Escherichia coli* and *Staphylococcus aureus*.
  • compound A3 can self-assemble into a peptide hydrogel in PBS solution. This material retains the original antibacterial properties of the antimicrobial peptide while possessing excellent biocompatibility, and also achieves adhesion and sustained release at dental caries sites.
  • Studies on the stability of the peptide hydrogel in different pH environments showed that its in vitro degradation rate slows down as the pH decreases. This is beneficial for the slow release of the drug in acidic caries environments, providing a new approach for the clinical inhibition of early caries.
  • This embodiment describes the synthesis and purification of a polypeptide with anti-caries properties.
  • polypeptide i.e., compound A3
  • compound A3 was synthesized using the classic solid-phase polypeptide synthesis method, and the specific steps are as follows:
  • Swelling resin Weigh a certain amount of resin and add it to the peptide synthesizer. Then add an appropriate amount of redistilled DCM and stir for 30 minutes to make the resin completely swell. After drying, wash the resin 4 times with redistilled DMF for 3 minutes each time, and then dry it.
  • Peptide purification The peptide was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). The crude peptide powder was dissolved, filtered, and loaded onto the elution system, which was an aqueous solution of 15%-95% acetonitrile at a flow rate of 10 mL/min for 80 min. The peak shape changes at 220 nm and 254 nm were observed. The eluent from the main peak was collected and labeled. After freeze-drying, the pure peptide was obtained. Purity analysis was performed using RP-HPLC.
  • This embodiment determines the minimum inhibitory concentration of compound A3.
  • the standard strains used for the assay of the antibacterial activity of the compounds were obtained from the U.S. Culture Collection.
  • the minimum inhibitory concentration (MIC) of the compound against bacteria was slightly modified according to the Clinical and Laboratory Standards Institute (CLSI) microdilution method. In short, a single bacterial colony was picked and incubated in 4 ml of the appropriate culture medium at 37°C and 180 rpm for 18 h on a shaker. The bacterial culture was then diluted to 1 ⁇ 105 CFU/ml with the appropriate culture medium. The MIC of the compound against the selected strains was determined using the microbroth dilution method: serially diluted solutions of the compound were added to sterile 96-well plates, with a final bacterial concentration of 1 ⁇ 104 CFU/ml in each well.
  • CCSI Clinical and Laboratory Standards Institute
  • Table 2 shows the minimum inhibitory concentration of compound A3 against standard bacterial strains.
  • This embodiment conducts an experiment to determine the minimum bactericidal concentration of compound A3:
  • This embodiment demonstrates the cytotoxicity experiment of compound A3.
  • the cytotoxicity of compound A3 was determined by measuring its effect on the proliferation of mouse epithelial-like fibroblasts L929 using the CCK-8 assay.
  • the cell lines used in this example were provided by the Experimental Center of the School of Basic Medical Sciences, Lanzhou University. L929 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C in a cell culture incubator with 5 % CO2.
  • Cell suspension (8 ⁇ 103 cells per well) was seeded in 96-well plates. 100 ⁇ l of high-glucose DMEM medium containing 10% fetal bovine serum was added to each well. The plates were incubated until cell attachment. The old medium was discarded, and the compound was serially diluted with complete medium. 100 ⁇ l of the diluted compound solution was added to each well, with three replicates per group. A control group without compound was used. The plates were incubated for 24 h, 48 h, and 72 h, respectively. The original medium was then discarded, and 10 ⁇ l of CCK-8 solution and 100 ⁇ l of complete medium were added. The plates were gently shaken and incubated for another 1–2 h.
  • This embodiment describes the preparation of a peptide self-assembled hydrogel with anti-caries properties.
  • a certain amount of antimicrobial peptide A3 was dissolved in sterile water to prepare a 25 mM stock solution. Then, the A3 stock solution was added to PBS of different concentrations (0 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM) at pH 7.4 to prepare reaction solutions with a final concentration of 5 mM or 10 mM. The reaction was carried out at 23°C for 5 min, and the fluidity of the solution was observed.
  • the antimicrobial peptide A3 stock solution was added to 8 mM PBS at pH 7.4 at different final concentrations (0 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM) and reacted at 23°C for 5 min. The fluidity of the solution was observed. The results are shown in Figure 3.
  • both 5 mM and 10 mM A3 concentrations can form peptide self-assembled hydrogels.
  • the hydrogel morphology is unstable due to insufficient PBS ion concentration.
  • both 5 mM and 10 mM A3 concentrations can form stable peptide self-assembled hydrogels.
  • the PBS concentration is controlled at 8 mM, the fluidity and transparency of the entire system gradually decrease with increasing A3 concentration.
  • a stable peptide self-assembled hydrogel can be formed at an A3 concentration of 5 mM.
  • This embodiment tests the antibacterial activity of the self-assembled hydrogel of the antimicrobial peptide.
  • A3 self-assembled hydrogels (A3 concentration 5 mM, PBS concentration 8 mM) was tested using *Streptococcus mutans*.
  • the bacterial suspension was gently shaken to mix. 100 ⁇ l of the bacterial suspension was then added sequentially to a sterile 96-well plate, with three parallel control wells. The absorbance (OD600) at 600 nm was measured using a multi-mode microplate reader. Simultaneously, 100 ⁇ l of the bacterial suspension was diluted 106 times in BHI medium and added to BHI agar plates. The plates were spread evenly with a spreader and incubated at 37°C. Colony growth was recorded after 18 h. The results are shown in Figures 4 and 5.
  • the antibacterial activity of the A3 self-assembled hydrogel was judged by the change in the OD600 value of the bacterial solution.
  • the OD600 value of the control group gradually increased, indicating that the bacteria were active.
  • the OD600 value of the experimental group did not change with the extension of the incubation time, indicating that the A3 self-assembled hydrogel could effectively inhibit the proliferation of Streptococcus mutans.
  • This embodiment tests the cytotoxicity of antimicrobial peptide self-assembled hydrogels.
  • the cytotoxicity of the self-assembled hydrogel of antimicrobial peptides to L929 cells was determined using the CCK-8 assay: L929 cells were seeded in 96-well plates at a density of 4 ⁇ 104 /ml and incubated for 24 h. The supernatant was then discarded, and 100 ⁇ l of DMEM medium (equilibrated with the hydrogel and control group PBS for 24 h) and the corresponding proportion of fetal bovine serum were added to each well for further incubation for 24 h, 48 h, and 72 h. After incubation, the supernatant was discarded, and 100 ⁇ l of CKK-8 application solution was prepared at a ratio of CCK-8 solution to complete medium of 1:10. After 1.5 h of incubation, the OD at 450 nm was measured to evaluate the cytotoxicity of the self-assembled hydrogel of antimicrobial peptides. The results are shown in Figure 6.
  • the cytotoxicity of the hydrogel was determined by an LDH (lactate dehydrogenase) release assay: L929 cells were seeded in 96-well plates at a density of 4 ⁇ 104 /ml and incubated for 24 h. The supernatant was then discarded, and 100 ⁇ l of DMEM medium (equilibrated with PBS for 24 h prior to the hydrogel and negative control group) and the corresponding proportion of fetal bovine serum were added. Incubation continued for 24 h, 48 h, and 72 h. The LDH release reagent (C0016-1, Beyotime) from the LDH release kit was used as the maximum enzyme activity control (positive control).
  • LDH release reagent C0016-1, Beyotime
  • LDH release (OD490(hydrogel) - OD490(blank)) / (OD490(MAX) - OD490(blank)), to determine the effect of antimicrobial peptide gelation on its cytotoxicity. The results are shown in Figure 7.
  • the amount of cell death and cytotoxicity were determined by the amount of LDH released. There were no significant differences between the experimental group and the PBS control group at 24h, 48h, and 72h, indicating that the A3 self-assembled antimicrobial peptide has good cell compatibility.
  • Live/dead cells were stained green/red using a live/dead cell staining method, and then the cell density and state were observed using a fluorescence microscope to determine the cytotoxicity of the antimicrobial peptide self-assembled hydrogel.
  • L929 cells were seeded in 96-well plates at a density of 4 ⁇ 104 cells /ml and incubated for 24 h. Then, the supernatant was discarded, and DMEM pre-equilibrated with hydrogel and control PBS for 24 h was added for further incubation for 24 h, 48 h, and 72 h. After incubation, the supernatant was discarded, and the cells were stained with the corresponding fluorescent dyes. Finally, cell morphology was observed under a fluorescence microscope and photographed. Live cells were stained with calcein AM, and dead cells were stained with BOBO-3 iodide for 15 min. The results are shown in Figure 8.

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Abstract

提供了一种具有抗龋性能的多肽和，其氨基酸序列如SEQ ID NO.1所示。还提供了一种具有抗龋性能的多肽自组装水凝胶及其应用，该肽自组装水凝胶含有SEQ ID NO.1所示多肽。

Description

一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用
相关申请的交叉引用
本申请要求于2024年6月24日提交中国专利局的申请号为2024108177997、名称为“一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请属于生物化学技术领域，特别涉及一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用。
背景技术
抗菌肽是一大类对细菌、真菌、寄生虫和病毒等有害生命体(或病原体)具有抗性功能的活性多肽，因其普遍带有足量的正电荷并常伴随疏水性，可在静电作用下与含有负电的生物膜结合，穿透并破坏膜结构致细胞死亡。与传统抗生素的单一靶点杀菌原理不同，抗菌肽可在病原体上进行多靶点破坏，能极大程度降低耐药菌的产生，且具有广谱抗菌性，是未来替代抗生素的最佳选择之一。近年来，关于抗菌肽在口腔中的应用研究已在世界各地广泛进行，在过去十年中，通过抗菌肽控制口腔病原体的研究有着重大飞跃。到目前为止，许多天然或合成的抗菌肽已经显示出对体外变异链球菌、浮游细菌和生物膜生长的抑制作用，它们通过抑制变异链球菌等微生物的生长、降低细菌耐酸性、抑制微生物产酸、抑制生物膜形成和促进再矿化等作用机制防治龋病。这些研究表明，抗菌肽有可能成为一种安全且有前途的龋齿解决方案。
发明内容
为了获得一种新的变异链球菌抗菌肽，本申请提供了一种具有抗龋性能的多肽，该多肽对变异链球菌具有优异的抗菌活性，为临床上早期龋病的防治提供了新的选择。
本申请还提供了一种具有抗龋性能的多肽自组装水凝胶及其应用。
本申请通过以下技术方案实现：
本申请提供一种具有抗龋性能的多肽，所述多肽的氨基酸序列如SEQ ID NO.1所示。
基于同一发明构思，本申请提供一种具有抗龋性能的多肽自组装水凝胶，所述多肽自组装水凝胶中含有上述一种具有抗龋性能的多肽。
进一步的，所述多肽自组装水凝胶通过所述多肽在溶剂中自组装形成。
可选的，所述溶剂为PBS缓冲液。
基于同一发明构思，本申请提供一种具有抗龋性能的多肽在制备抗龋药物中的应用。
基于同一发明构思，本申请提供一种具有抗龋性能的多肽自组装水凝胶在制备抗龋药物中的应用。
基于同一发明构思，本申请提供一种抗龋药物，所述抗龋药物的有效成分包括上述一种具有抗龋性能的多肽和/或一种具有抗龋性能的多肽自组装水凝胶。
基于同一发明构思，本申请提供一种具有抗龋性能的多肽在抑制或杀灭变异链球菌中的应用。
基于同一发明构思，本申请提供一种具有抗龋性能的多肽自组装水凝胶在抑制或杀灭变异链球菌中的应用。
基于同一发明构思，本申请提供一种变异链球菌抑制剂，所述抑制剂的有效成分包括上述一种具有抗龋性能的多肽和/或一种具有抗龋性能的多肽自组装水凝胶。
基于同一发明构思，本申请还提供一种具有抗龋性能的多肽自组装水凝胶的制备方法，所述制备方法包括：
将一种具有抗龋性能的多肽与PBS缓冲液混匀，获得反应液；
所述反应液于23～37℃下反应一段时间，获得具有抗龋性能的多肽自组装水凝胶。
优选的，在所述反应液中，所述多肽浓度为4～10mmol/L，磷酸盐浓度为6～10mmol/L；
所述反应液的反应时间为5～30min。
本申请实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
1.本申请一种具有抗龋性能的多肽，命名为A3，多肽A3对多种病原菌均具有优异的抗菌活性，尤其是变异链球菌，多肽A3对变异链球菌的最小生长抑制浓度显著低于对大肠杆菌和金黄色葡萄球菌的抑制浓度，多肽A3还能通过自组装制备多肽水凝胶，实现在牙齿龋坏部位的黏附和缓释，可作为抗龋药物的有效成分，为临床上早期龋病的防治提供新的选择。
2.本申请一种具有抗龋性能的多肽自组装水凝胶，以多肽A3为原料，多肽A3在PBS溶液中通过自组装形成多肽水凝胶，所得材料既保持了抗菌肽A3原有的抗菌特性，又具有出色的生物相容性，同时还能够实现在牙齿龋坏部位的黏附和缓释，研究该多肽自组装水凝胶在不同pH环境中的稳定性，发现其体外降解的速度随pH降低而减慢，这有利于药物在酸性龋病环境中的缓慢释放，对临床上早期龋病的抑制具有重要意义。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为化合物A3对变异链球菌标准细菌菌株的最小杀菌浓度测定结果。
图2为化合物A3对细胞株L929增殖的影响图。
图3为抗菌肽自组装水凝胶的制备过程(小瓶倒置实验)。
图4为抗菌肽自组装水凝胶抗菌活性。
图5为抗菌肽自组装水凝胶作用后菌落生长情况。
图6为抗菌肽自组装水凝胶CCK-8实验结果。
图7为抗菌肽自组装水凝胶LDH释放实验结果。
图8为抗菌肽自组装水凝胶活/死细胞染色结果。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本申请，本申请的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本申请，而非限制本申请。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本申请所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
除非另有特别说明，本申请中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。
本申请整体思路如下：
传统抗菌肽在控制变异链球菌生长的同时，对其它共生菌也会产生同样的抑制作用，这极大影响了口腔的菌群平衡，对口腔健康产生极为不利的影响。另外，龋齿主要发病于牙冠部位，因为血管分布特点，系统性用药很难到达，局部涂抹是相对有效的药物递送方式，但绝大多数药物需要在一定的载药介质辅助条件下实现在牙齿部位的黏附和缓释。
水凝胶是高度亲水的三维网络凝胶，可以在水中迅速膨胀，但不能溶解，水凝胶可以吸收大量的水，同时在膨胀后保持原有的结构。水凝胶作为一种软性物质，广泛应用于生物医学领域，与传统合成水凝胶相比，多肽自组装水凝胶有着众多优点。与其他分子自组装系统类似，多肽的自组装依赖于非共价键的相互作用，首先，可以通过调节多肽的结构调整水凝胶的结构和功能；其次，多肽水凝胶具有稳定的机械性、多孔结构和良好的生物相容性，有利于所载细胞的迁移和生长；最后，多肽的大比表面积可以促进药物和生物分子的吸附。
基于此，本申请提供一种具有抗龋性能的多肽。本申请通过计算机抓取抗菌肽的抗菌特征，人工智能学习这些特征，建立预测模型，从生成的一系列非天然抗菌肽中筛选出1条高抗菌活性的多肽，其氨基酸序列为RRRWRWRIGIRIY，标记为化合物A3。
经试验发现，化合物A3对多种病原菌均具有优异的抗菌活性，尤其是变异链球菌，多肽A3对变异链球菌的最小生长抑制浓度明显小于对大肠杆菌和金黄色葡萄球菌的抑制浓度。并且，化合物A3可以在PBS溶液中自组装成多肽水凝胶，这种材料既保持了抗菌肽原有的抗菌特性，又具有出色的生物相容性，同时还能够实现在牙齿龋坏部位的黏附和缓释。研究多肽水凝胶在不同pH环境中的稳定性，显示出其体外降解的速度随pH降低而减慢，这有利于药物在酸性龋病环境中的缓慢释放，为临床上早期龋病的抑制提供了新思路。
下面将结合实施例及实验数据对本申请一种具有抗龋性能的多肽、多肽自组装水凝胶进行详细说明。
实施例1
本实施例一种具有抗龋性能的多肽的合成与纯化。
多肽(即化合物A3)的合成采用经典的多肽固相合成方法，具体步骤如下：
1)溶胀树脂：称取一定量的树脂加入多肽合成仪中，再加入适量重蒸的DCM搅拌30min，使树脂完全溶胀，抽干后，用重蒸的DMF将树脂洗涤4次，每次3min，抽干。
2)茚检：取少量树脂置于试管中，加入茚检试剂(茚三酮：吡啶：苯酚＝1:2:1)，于沸水中加热3min，若树脂颜色无变化，可进行下一步操作。
3)脱保护：在合成仪中加入脱保护试剂(哌啶：DMF＝1：4)，搅拌，重复3次，每次5min，使Fmoc保护基完全脱除，用重蒸的DMF洗涤树脂4次，每次3min。再次茚检，若树脂为蓝紫色，则证明脱保护成功。
4)缩合反应：称取3倍过量的Fmoc保护的氨基酸和缩合剂HOBT、HBTU，用重蒸的DMF溶解，加入6倍过量的启动剂DIEA，搅拌混匀后加入合成仪中，在惰性气体保护下搅拌1h，抽干，用重蒸的DMF洗涤树脂4次，每次3min，抽干，茚检，若树脂颜色无变化，则证明缩合反应成功。重复脱除Fmoc保护基和缩合反应的步骤，直至整条肽链全部合成。
5)多肽的切割：最后一个氨基酸缩合完成后，脱去Fmoc保护基，用重蒸的DMF洗涤树脂4次，每次3min，再用DCM和无水甲醇交替洗涤树脂各3次，每次3min，将树脂放入真空干燥箱中。待树脂完全干燥后，加入切割试剂(TFA:Tis:H2O＝95:2.5:2.5),缓慢搅拌3h，收集切割液。
6)多肽的萃取：将切割液减压旋干，放入-20℃冰箱保存15min，取出后随即加入适量冰乙醚进行沉淀，用水萃取，将水相收集于烧杯中，冷冻干燥后得到粗肽粉末。
7)多肽的纯化：用反相高效液相色谱仪纯化多肽，将粗肽粉末溶解，过滤后上样，洗脱体系为15％-95％乙腈的水溶液，流速为10mL/min，洗脱80min，观察220nm和254nm处的图谱峰形变化，收集主峰处的洗脱液并标记，冷冻干燥后得到纯肽。用反相高效液相色谱进行纯度分析，取少量纯肽溶解，上样，洗脱体系为5％-95％的乙腈的水溶液，流速为1mL/min，洗脱30min，对图谱进行积分，计算纯度，用质谱分析检测多肽的实际分子量。
所得化合物A3的氨基酸序列如表1所示。
表1合成的化合物的氨基酸序列及理论分子量
实施例2
本实施例进行化合物A3最小抑菌浓度的测定。
化合物抗菌活性测定所用的标准菌株：变异链球菌(ATCC 25175)、金黄色葡萄球菌(ATCC 25923)、表皮葡萄球菌(ATCC 12228)、大肠杆菌(ATCC 25922)、肺炎链球菌(ATCC 49619)均来自于美国菌种保藏中心。
化合物对细菌的最小抑菌浓度(MIC)值根据临床和实验室标准协会(CLSI)微量稀释法进行了少量修改。简而言之，挑取单个细菌菌落于4ml相应的培养基中，37℃，180rpm摇床上孵育18h，用相应的培养基将菌液稀释至1×105CFU/ml备用。采用微量肉汤稀释法检测化合物对所选菌株的最小抑菌浓度：即将倍比稀释后不同浓度的化合物溶液分别加到无菌的96孔板中每个孔中菌液终浓度为1×104CFU/ml。首先在每孔加入100μl BHI培养基，将2mg/ml的药液稀释到512μM，在第一行A中第1-5孔每孔添加100μl药液使其浓度变为256μM，梯度稀释至B-H孔浓度分别为128μM，64μM，32μM，16μM，8μM，4μM，2μM，并各自加入BHI培养基100μl至每孔体系为200μl，各孔加入20μl浓度为1×105CFU/ml的菌液，以保证最后每个孔中菌液终浓度为1×104CFU/ml。在每个菌株所对应的培养条件下培养24h，以在OD 600nm下测定小孔内无细菌生长的最低浓度定为MIC。以上实验有效重复3次。实验结果见表2。
表2化合物A3对标准细菌菌株的最小抑菌浓度
从表2可知：化合物A3对所测试的细菌均表现出了很好的抗菌活性。
实施例3
本实施例进行化合物A3最小杀菌浓度检测实验:
在上述MIC测定实验中，从小孔内取出等分试样50ul的变异链球菌菌液均匀涂布到合适的琼脂固体培养基上后，在合适的培养条件下将平板倒置培养24h，以在平板上不能观察到菌落生长的最低浓度定为最小杀菌浓度(MBC)。以上实验有效重复3次。实验结果如图1所示。
从图1可知：在8MIC浓度下，平板上不能观察到菌落生长，即为MBC浓度。
实施例4
本实施例进行化合物A3的细胞毒性实验。
化合物A3的细胞毒性是通过CCK-8方法测定对小鼠上皮样成纤维细胞L929的增殖影响而测定的。本实施例所用的细胞株均由兰州大学基础医学院实验中心提供，L929细胞使用含有10％胎牛血清的高糖DMEM培养基培养在37℃，5％ CO2的细胞培养箱中。
检测化合物A3对L929细胞的体外毒性：
在96孔板中接种细胞悬液(每孔细胞数为8×103个)，每孔加入含10％胎牛血清的100μl高糖DMEM培养基，放入培养箱中培养，待细胞贴壁后，吸弃旧培养基，使用完全培养基梯度稀释化合物，每孔加入100μ1稀释后化合物溶液，每组设置3个复孔，并以无化合物添加作为对照组，将此培养板置于培养箱中分别培养24h、48h、72h后弃去原有培养基，加入10μ1cck-8溶液和100μl完全培养基，轻轻振摇培养板，继续在培养箱中培养1-2h，检测波长在450nm处的吸光度，最后计算细胞毒性*(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]×100％。实验结果如图2所示。
从图2可知：抗菌肽A3作用于L929细胞后，在最小抑菌浓度下(8μM)，细胞存活率接近80％，细胞相容性良好。
实施例5
本实施例一种具有抗龋性能的多肽自组装水凝胶的制备。
为了探讨抗菌肽A3浓度、PBS浓度对水凝胶形成的影响，采取控制变量法，并通过小瓶倒转观察，探讨以上因素对抗菌肽自组装水凝胶形成的影响。
将一定量的抗菌肽A3溶解于无菌水中配制成25mM的储存液，然后将A3储存液加入pH为7.4的不同浓度的PBS(0mM，2mM，4mM，6mM，8mM，10mM)中配制成终浓度为5mM或10mM的反应液23℃下反应5min，观察溶液的流动性。再将抗菌肽A3储存液以不同的终浓度(0mM，0.5mM，1mM，2mM，3mM，4mM，5mM，10mM)加入到pH 7.4的8mM的PBS中23℃下反应5min,观察溶液的流动性。结果如图3所示。
从图3可知：当PBS浓度为6mM时，A3浓度为5mM或是10mM时均可形成多肽自组装水凝胶，但是A3为10mM浓度下由于PBS离子浓度不够，水凝胶形态不稳定，当PBS浓度为8mM时，A3浓度为5mM或是10mM时均可形成稳定的多肽自组装水凝胶。控制PBS的浓度为8mM时，随着抗菌肽A3的浓度的逐渐增加，整个体系的流动性和透明度逐渐降低，在A3为5mM时，可形成稳定的多肽自组装水凝胶，最终我们选定了A3浓度为5mM，PBS浓度为8mM所形成的多肽自组装水凝胶用作后续的实验。
实施例6
本实施例测试抗菌肽自组装水凝胶的抗菌活性。
使用变异链球菌(S.mutans)来检测A3自组装水凝胶(A3浓度为5mM，PBS浓度为8mM)的抗菌活性：
从BHI琼脂平板上挑取单个菌落，接种在4ml的BHI液体培养基中，在37℃温度180rpm恒温摇床下培养8-12h至对数期。吸取700μl的抗菌肽自组装水凝胶加入1.5ml无菌EP管中，以同体积PBS作为阴性对照组，将对数生长期的菌液稀释至1×105CFU/ml，在水凝胶或PBS中缓慢加入700μl菌液，在37℃恒温培养箱中孵育0h、3h、6h、9h、12h、24h后，将菌液轻轻摇晃均匀，取出100μl菌液依次加入无菌96孔板，设置三组平行对照孔，用多功能酶标仪检测600nm处的光吸收值(OD600)，同时取出100μl菌液在BHI培养基中稀释106倍后加入BHI琼脂平板中，用涂棒涂布均匀后放入37℃恒温培养箱培养，18h后记录平板上菌落的生长情况。结果如图4、5所示。
从图4、5可知：通过判断菌液OD600值的变化判断A3自组装水凝胶的抗菌活性，对照组菌液的OD600值逐渐增加，说明细菌的活力正常，而实验组的OD600值没有随孵育时间的延长而变化，说明A3自组装水凝胶能够有效抑制变异链球菌的增殖。
根据平板上菌落生长的情况，对照组平板上的菌落数逐渐增长，说明细菌的活力正常，3h时水凝胶组平板上的细菌没有生长，且随着时间的延长，平板上也没有出现任何菌落，说明A3自组装水凝胶可以快速杀死与其共同孵育的变异链球菌。
实施例7
本实施例测试抗菌肽自组装水凝胶的细胞毒性。
1.采用CCK-8法测定抗菌肽自组装水凝胶对L929细胞的细胞毒性：将L929细胞按照4×104/ml的密度种于96孔板，并在细胞培养箱孵育24h；随后弃掉上清，加入事先与水凝胶及对照组PBS平衡24h的DMEM培养基(每孔100μl)及相应比例的胎牛血清继续孵育24h,48h及72h，孵育结束后，弃掉上清，按照CCK-8溶液：完全培养基＝1:10的比例配置CKK-8应用液，每孔加入100μl，培养1.5h后测OD450nm，进而评价抗菌肽自组装水凝胶的细胞毒性。结果如图6所示。
从图6可知：L929细胞与A3自组装水凝胶共同孵育24h，48h，72h后，与对照组相比，均未表现出显著的差异，说明A3自组装抗菌肽具有良好的细胞相容性。。
2.通过LDH(乳酸脱氢酶)释放实验来判断水凝胶的细胞毒性：将L929细胞按照4×104/ml的密度种于96孔板，并在细胞培养箱孵育24h；随后弃掉上清，加入事先与水凝胶及阴性对照组PBS平衡24h的DMEM培养基(每孔100μl)及相应比例的胎牛血清继续孵育24h,48h及72h，使用LDH释放试剂盒中的LDH释放试剂(C0016-1，碧云天)作为样品最大酶活性对照(阳性对照)，孵育结束后，使用碧云天公司的LDH释放试剂盒测定所有细胞的LDH释放量。A3水凝胶的LDH释放用以下公式计算：LDH释放＝(OD490(水凝胶)-OD490(空白))/(OD490(MAX)-OD490(空白))，判断抗菌肽凝胶化对其细胞毒性的影响。结果如图7所示。
从图7可知：通过LDH的释放量来判断细胞的死亡量进而判断细胞毒性，实验组与PBS对照组相比，在24h，48h，72h时均未有显著的差异，说明A3自组装抗菌肽具有良好的细胞相容性。
3.通过活细胞/死细胞染色法，将活细胞/死细胞染成绿色/红色，然后使用荧光显微镜观察细胞的密度及状态，判断抗菌肽自组装水凝胶的细胞毒性。
首先将L929细胞按照4×104/ml的密度种于96孔板，并在细胞培养箱孵育24h；随后弃掉上清，加入事先与水凝胶及对照组PBS平衡24h的DMEM继续孵育24h,48h及72h，孵育结束后，弃掉上清，加入对应的荧光染料对细胞进行染色，最后在荧光显微镜下观察细胞形态，并拍照记录。其中，活细胞使用钙黄绿素AM进行染色；死细胞用BOBO-3碘化物染色,作用时间均为15min。结果如图8所示。
从图8可知：与A3自组装水凝胶共同孵育后，L929细胞的形态与增殖都没有受到影响。实验组所有细胞均表现出完整、健康的贴壁状态，且随着孵育时间的延长，实验组的细胞密度逐渐增加，表现出与对照组相当的增长趋势，进一步说明A3自组装抗菌肽具有良好的细胞相容性。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本申请的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本申请范围的所有变更和修改。
显然，本领域的技术人员可以对本申请进行各种改动和变型而不脱离本申请的精神和范围。这样，倘若本申请的这些修改和变型属于本申请权利要求及其等同技术的范围之内，则本申请也意图包含这些改动和变型在内。

Claims (12)

  1. 一种具有抗龋性能的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO.1所示。
  2. 一种具有抗龋性能的多肽自组装水凝胶，其特征在于，所述多肽自组装水凝胶中含有权利要求1所述的一种具有抗龋性能的多肽。
  3. 根据权利要求2所述的一种具有抗龋性能的多肽自组装水凝胶，其特征在于，所述多肽自组装水凝胶通过所述多肽在溶剂中自组装形成。
  4. 根据权利要求3所述的一种具有抗龋性能的多肽自组装水凝胶，其特征在于，所述溶剂为PBS缓冲液。
  5. 如权利要求1所述的一种具有抗龋性能的多肽在制备抗龋药物中的应用。
  6. 如权利要求2～4所述的一种具有抗龋性能的多肽自组装水凝胶在制备抗龋药物中的应用。
  7. 一种抗龋药物，其特征在于，所述抗龋药物的有效成分包括权利要求1所述的一种具有抗龋性能的多肽和/或权利要求2～4所述的一种具有抗龋性能的多肽自组装水凝胶。
  8. 如权利要求1所述的一种具有抗龋性能的多肽在抑制或杀灭变异链球菌中的应用。
  9. 如权利要求2～4所述的一种具有抗龋性能的多肽自组装水凝胶在抑制或杀灭变异链球菌中的应用。
  10. 一种变异链球菌抑制剂，其特征在于，所述抑制剂的有效成分包括权利要求1所述的一种具有抗龋性能的多肽和/或权利要求2～4所述的一种具有抗龋性能的多肽自组装水凝胶。
  11. 如权利要求2～4所述的一种具有抗龋性能的多肽自组装水凝胶的制备方法，其特征在于，所述制备方法包括：
    将一种具有抗龋性能的多肽与PBS缓冲液混匀，获得反应液；
    所述反应液于23～37℃下反应一段时间，获得具有抗龋性能的多肽自组装水凝胶。
  12. 根据权利要求11所述的一种具有抗龋性能的多肽自组装水凝胶的制备方法，其特征在于，在所述反应液中，所述多肽浓度为4～10mmol/L，磷酸盐浓度为6～10mmol/L；
    所述反应液的反应时间为5～30min。
PCT/CN2025/101784 2024-06-24 2025-06-18 一种具有抗龋性能的多肽、多肽自组装水凝胶及其应用 Pending WO2026001787A1 (zh)

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