WO2019140796A1 - 一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用 - Google Patents

一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用 Download PDF

Info

Publication number
WO2019140796A1
WO2019140796A1 PCT/CN2018/083402 CN2018083402W WO2019140796A1 WO 2019140796 A1 WO2019140796 A1 WO 2019140796A1 CN 2018083402 W CN2018083402 W CN 2018083402W WO 2019140796 A1 WO2019140796 A1 WO 2019140796A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdc
endotracheal tube
antimicrobial peptide
coating
peptide
Prior art date
Application number
PCT/CN2018/083402
Other languages
English (en)
French (fr)
Inventor
卢雪梅
桂水清
张伦
金小宝
朱家勇
Original Assignee
广东药科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东药科大学 filed Critical 广东药科大学
Publication of WO2019140796A1 publication Critical patent/WO2019140796A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M16/00—Devices for influencing the respiratory system of patients by gas treatment, e.g. mouth-to-mouth respiration; Tracheal tubes
    • A61M16/04—Tracheal tubes
    • A61M16/0402—Special features for tracheal tubes not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08—Materials for coatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08—Materials for coatings
    • A61L29/085—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M16/00—Devices for influencing the respiratory system of patients by gas treatment, e.g. mouth-to-mouth respiration; Tracheal tubes
    • A61M16/04—Tracheal tubes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/606—Coatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0205—Materials having antiseptic or antimicrobial properties, e.g. silver compounds, rubber with sterilising agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0238—General characteristics of the apparatus characterised by a particular materials the material being a coating or protective layer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2207/00—Methods of manufacture, assembly or production

Definitions

  • the invention belongs to the field of biomedical materials, and in particular relates to an endotracheal tube carrying an antimicrobial peptide MDC coating and a preparation method and application thereof.
  • Tracheal intubation is a simple and effective method for establishing a clear airway in the rescue of critically ill patients.
  • the artificial airway established by tracheal intubation has become the most important "lifeline" in patients.
  • the most common and serious complication of mechanical ventilation, ventilator-associated pneumonia has a high morbidity and mortality. Once it is not effectively controlled, patients will have to face removal of the tracheal tube, re-intubation or tracheotomy. Opening, greatly increasing the patient's treatment risk and physical and mental torture, the medical economic burden will also increase greatly.
  • the sol-gel method is used to prepare a silicone-loaded silver antibacterial sol, and then the endotracheal tube is used as a substrate to perform surface antibacterial coating.
  • the silicone-loaded silver antibacterial coating tracheal intubation catheter is prepared, and the tracheal tube not only has high antibacterial property, but also has almost no silver ions deposited on the coating, the surface is smooth and does not adhere to the sputum, and the side reaction is extremely low, etc.
  • CN 103933617 A "Preparation of an Endotracheal Tube with Antibacterial Adhesion" by preparing an iodine-containing photocatalytic antibacterial sol, surface hydroxylation modification of a PVC endotracheal tube, on the surface of a PVC endotracheal tube The steps of modifying the photocatalytic antibacterial film are carried out, so that the surface modified tracheal tube has good antibacterial adhesion performance and visible light-induced sterilization performance, long-lasting antibacterial effect and high chemical stability.
  • Antimicrobial peptides are small molecular peptides produced by the biological immune system against external pathogen infections. They are widely found in insects, plants, animals and humans. The insect antibacterial peptide cecropin is one of the earliest antibacterial peptides discovered by humans. Antimicrobial peptides have a range of compelling biological activities, including antibacterial, anti-inflammatory, anti-viral, anti-parasitic, tumor-inhibiting and immunomodulatory activities.
  • the antibacterial peptide Musca domestica cecropin is an insect antibacterial peptide cloned from the cDNA library of Musca domestica larvae fat body.
  • the ORF region of this gene is 192 bp in length and encodes a 63 amino acid precursor protein.
  • the ⁇ 23 amino acid is a signal peptide ending in a conserved 4 peptide, and its mature peptide contains 40 amino acids.
  • Previous studies have found that the antimicrobial peptide MDC shows strong in vitro antibacterial activity against many standard strains and clinical resistant strains, and can destroy bacterial cell membranes. Or through the cell membrane to act on the intracellular target site, the mechanism of action is unique, cytotoxic to normal human cells, and side effects are small.
  • an object of the present invention to provide an endotracheal tube carrying an antimicrobial peptide MDC coating and a method of preparing the same.
  • Another object of the present invention is to provide the use of the endotracheal tube carrying the antimicrobial peptide MDC coating in artificial tracheal intubation and ventilator.
  • the antibacterial peptide MDC provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1. Specifically, the amino acid sequence is: GWLKKI GKKIE RVGQH TRDAT IQTIG VAQQA ANVAA TLKG.
  • the antibacterial peptide MDC provided by the invention is prepared by solid phase chemical synthesis, in particular, the polypeptide is synthesized by a polypeptide synthesizer; then the polypeptide is synthesized by solid phase synthesis; and the synthesized polypeptide is purified by reverse phase high performance liquid chromatography.
  • the synthesized polypeptide is identified by electrospray mass spectrometry to complete the preparation of the polypeptide.
  • the invention also provides a preparation method of the endotracheal tube carrying the antimicrobial peptide MDC coating, which comprises the following steps:
  • the antimicrobial peptide chitosan composite membrane prepared in the step (1) is dip-coated or cast in a tracheal tube substrate, and naturally dried or cured at 50 ° C to obtain an endotracheal tube.
  • the chitosan gel is used as a fixed and sustained-release matrix
  • the antimicrobial peptide MDC is used as an antibacterial active ingredient to form a coating film liquid
  • the chitosan is a polymer compound purified from crab shell.
  • Chitin a kind of polyglucagon prepared by deep N-acetylation, is a kind of medical polymer polysaccharide with good biocompatibility, biodegradability and biological activity.
  • the further prepared medical chitosan gel is non-toxic, non-irritating, pyrogen-free, non-immune antigenic, non-hemolyzed and has thermal stability, no adverse reactions such as mutagenicity, lethality, and slight bacteriostatic action.
  • an antimicrobial peptide MDC immobilized and sustained-release matrix it exerts a long-term antibacterial effect.
  • the invention selects the antibacterial peptide MDC as the biological coating, obtains the antibacterial property of the surface of the tracheal intubation catheter, effectively prevents bacterial colonization and growth, reduces or delays the occurrence of tracheal intubation ventilator-associated pneumonia, and prolongs the catheter indwelling time, The patient rescues valuable time and improves the success rate of treatment. It can also reduce the medical economic burden and alleviate the patient's physical and mental pain.
  • the tracheal tube carrying the antimicrobial peptide MDC coating designed and developed by the invention has no biological adverse reaction and meets the requirements of clinical tracheal intubation, and has broad application prospects.
  • the present invention has the following beneficial effects:
  • the tracheal tube carrying the antimicrobial peptide MDC coating provided by the invention has remarkable antibacterial property, and the sterilization rate is over 98%.
  • the antibacterial peptide MDC has a unique mechanism of action, is not easy to produce drug resistance, and breaks through the traditional antibacterial drug. Limitations on the role of bacterial biofilms provide clinically available endotracheal tubes with safe and effective antimicrobial properties.
  • the preparation method of the tracheal tube carrying the antimicrobial peptide MDC coating provided by the invention is simple, safe, has no adverse reaction, and has broad application prospects.
  • Figure 1 is a high performance liquid chromatogram of the antimicrobial peptide MDC.
  • Figure 2 is a mass spectrum of the antimicrobial peptide MDC.
  • Figure 3 shows the morphology of Acinetobacter baumannii biofilm on the surface of the blank control tracheal tube by scanning electron microscopy.
  • Figure 4 is a scanning electron microscope observation of the morphology of the biofilm of Acinetobacter baumannii on the surface of the tracheal tube in the control group of chitosan gel.
  • Fig. 5 is a scanning electron microscope observation of the morphology of the biofilm of Acinetobacter baumannii on the surface of the tracheal tube of the antimicrobial peptide MDC chitosan gel composite coating.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • the amino acid sequence of the antimicrobial peptide MDC is: Ac-GWLKKI GKKIE RVGQH TRDAT IQTIG VAQQA ANVAA TLKG-NH2, as shown in SEQ ID NO: 1, contains 40 amino acids, the theoretical isoelectric point is 10.56, and the theoretical molecular weight is 4299.04.
  • the preparation of the antimicrobial peptide MDC is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. The specific steps are as follows:
  • Fmoc-X (X is the first amino acid of the C-terminus of the antibacterial peptide MDC) is added to the Wang resin, and then the Fmoc group is removed to obtain an X-Wang resin; and then Fmoc-Y-Trt-OH ( 9-fluorenylmethoxy-trimethyl-Y, Y is the second amino acid of the antibacterial peptide MDC C-terminus; according to this procedure, it is synthesized from the C-terminus to the N-terminus, until the synthesis is completed, and the side from which the Fmoc group is removed is obtained. Chain protected resin;
  • eluent A is 0.1% TFA/water solution
  • eluent B is 0.1% TFA/acetonitrile solution
  • elution concentration is 25% B-40% B
  • elution time For 12min, the flow rate is 1mL / min, and then collect the main peak as above, freeze-dried, that is, the refined antimicrobial peptide MDC;
  • the purified antimicrobial peptide MDC was analyzed by reversed-phase high performance liquid chromatography and electrospray ionization mass spectrometry.
  • the reversed-phase high performance liquid chromatogram is shown in Fig. 1.
  • the mass spectrum is shown in Fig. 2, and the results show that the antimicrobial peptide MDC has a purity greater than 95% and a molecular weight of 4929.36, which is substantially consistent with the theoretical molecular weight.
  • NCTC colone929 cells (mouse fibroblasts) were cultured in 10% fetal calf serum MEM medium and prepared with an anti-tuberculosis MDC coating in the tracheal tube sample extract, and the blank PVC endotracheal tube catheter sample extract was used as the extract.
  • Negative control 5 g / L phenol solution as a positive control.
  • test results the cell morphology of the negative control was normal, the adherent growth was good, there were discrete particles in the cytoplasm, no cell lysis; the cells of the positive control grew poorly, more than 90% of the cells were round or lysed; the antimicrobial peptide MDC was contained.
  • the coated tracheal tube sample extract has normal cell morphology, good adherent growth, discrete particles in the cytoplasm, and no cell lysis.
  • the tracheal tube sample containing the antimicrobial peptide MDC coating and the blank PVC endotracheal tube catheter sample extract were evaluated as non-toxic, and the classification was judged to be grade 0 (the cytotoxic reaction was grade 0 or grade 1 was acceptable).
  • the cells were cultured for 48 hours to monitor the OD570 and the relative degree of increase.
  • the tracheal tube samples containing the antimicrobial peptide MDC coating and the blank PVC endotracheal tube catheter sample extracts were all graded to grade 0, and the endotracheal tube containing the antimicrobial peptide MDC coating. The results of the sample cytotoxicity test were acceptable.
  • the acute systemic toxicity of the tracheal tube sample containing the antimicrobial peptide MDC coating was observed.
  • the blank PVC endotracheal tube catheter sample and the 0.9% sodium chloride injection extract of the tracheal tube sample carrying the antimicrobial peptide MDC coating were administered in a single injection by tail vein injection with the corresponding extraction medium as a blank control.
  • the toxicity of the mice was observed at 4, 24, 48 and 72 hours after injection. The results showed that there was no abnormal reaction in the blank control solution group, the blank PVC endotracheal tube catheter sample and the tracheal tube sample extract solution group containing the antimicrobial peptide MDC coating.
  • Acinetobacter baumannii As a representative of Acinetobacter baumannii, the clinical ICU ventilator-associated Acinetobacter baumannii biofilm infection was simulated, and an in vitro model of Acinetobacter baumannii biofilm was established.
  • a blank tracheal tube sample without any treatment was used as a blank control group, and a chitosan gel control group was set up to simulate the fluid flow state in the ventilator catheter.
  • Each group of tracheal tubes was immersed in 1.0 ⁇ 10 5 Acinetobacter baumannii.
  • the results of the comparative experiment confirmed that the bacteria in the blank tracheal tube sample grew normally, and the bacterial growth of the tracheal tube sample in the chitosan gel control group was not significantly reduced, and the sterilization rate was about 5%, while the antimicrobial peptide MDC chitosan gel composite coating trachea The bacterial growth of the catheter samples was significantly reduced or even disappeared, and the sterilization rate was over 98%.
  • the morphological structure of the biofilm on the surface of the tracheal tube was observed by scanning electron microscopy.
  • the results showed that the blank control tracheal tube (Fig. 3) showed that a large number of bacteria adhered into a mass to form a massive biofilm;
  • the tracheal tube (Fig. 4) showed a decrease in biofilm thickness and area;
  • the antibacterial peptide MDC chitosan gel composite coating tracheal tube (Fig. 5) only showed scattered bacteria and bacterial cells deformed, broken, and detached from the outer membrane. , content leakage;
  • results of the agar plate colony counting method also showed that the number of viable cells on the surface of the tracheal tube of the antimicrobial peptide MDC chitosan gel composite coating was significantly reduced compared with the blank control group and the chitosan gel control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Pulmonology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Emergency Medicine (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Anesthesiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种载有抗菌肽MDC涂层的气管导管及其制备方法和应用。该气管导管以抗菌肽MDC为抗菌剂，以几丁糖凝胶为固定和缓释基质，对气管导管进行表面抗菌涂层，制成载有抗菌肽MDC涂层的气管导管。抗菌肽MDC序列如SEQ ID NO：1所示。该载有抗菌肽MDC涂层的气管导管，具有显著的抗菌性能，能有效预防细菌定植和生长。因此，将该载有抗菌肽MDC涂层的气管导管应用于人工气管插管及呼吸机中，将减少或延缓气管插管呼吸机相关性肺炎的发生，减少医疗经济负担、减轻患者的身心痛苦，具有广阔前景。

Description

一种载有抗菌肽MDC涂层的气管导管及其制备方法和应用 技术领域
本发明属于生物医用材料领域，具体涉及一种载有抗菌肽MDC涂层的气管导管及其制备方法和应用。
背景技术
气管插管是危重病患者抢救中建立通畅气道的简捷有效的方法，气管插管所建立的人工气道，成为病人身上最重要的一条“生命线”。临床中每天需要进行气管插管机械通气的患者不计其数，其并发症中的植入物相关感染成为临床医生亟待解决的问题。机械通气过程中最常见且最严重的并发症—呼吸机相关性肺炎，发病率、病死率高，一旦发生又得不到有效控制，患者将不得不面临拔除气管导管、重新插管或气管切开，大大增加了患者治疗风险和身心折磨，医疗经济负担也将极大增加。目前采取的抗感染治疗、气道湿化、有效护理等措施并没有很好的减少呼吸机相关性肺炎的发生。研究表明，气管插管导致的呼吸道感染及呼吸机相关性肺炎常见的病原菌是大肠埃希菌、鲍曼不动杆菌、金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌等，而且这些细菌在气管插管患者中可以相继或重叠出现，表现出多重耐药甚至泛耐药，尤其是细菌生物被膜一旦形成，由于生物膜的屏障作用及生物膜内细菌低代谢等特点，其耐药性可提高成百上千倍，使得疗效堪忧，患者常常由于无法有效控制呼吸道感染而死亡。
目前，国内外学者将目光聚焦在气管插管导管的表面抗菌改性上，不改变原有的导管外形使其获得抗菌性能，减少细菌生长，以此防治呼吸机相关性肺炎。公开号为CN 103948973 A的中国专利申请“一种具有安全高效抗菌性能的医用气管导管”以正硅酸乙酯为硅源，以γ-甲基丙烯酰氧丙基三甲氧基硅烷、甲基三乙氧基硅烷为有机相前驱体，以无机纳米银为抗菌剂，经溶胶凝胶方法，制成有机硅载银抗菌溶胶，再以气管插管导管为基材，进行表面抗菌涂层，制备而成有机硅载银抗菌涂层气管插管导管，该气管导管不仅具有高效的抗菌性，而且涂层银离子几乎不析出，表面光滑不粘附痰液，副反应极低等优势，符合临床气管插管的需要。公开号为CN 103933617 A的中国专利申请“一种具有抗细菌粘附的气管导管的制备方法”通过制备含碘光催化抗菌溶胶、对PVC气管导管进行表面羟基化改性、在PVC气管导管表面形成光催化抗菌膜改性等步骤，使表面改性后的气管导管具有良好的抗细菌粘附性能和可见光诱导灭菌性能，抗菌效果持久，化学稳定性高。
然而，上述已公开的气管导管表面抗菌改性的制备方法比较复杂，且均采用无机化学类 抗菌剂，存在一定的安全隐患，难以避免残余毒性的问题。
近年来抗菌肽(Antimicrobial peptides,AMP)的出现为气管导管表面抗菌涂层的研究带来了新的思路。抗菌肽是生物免疫系统产生的一类抵抗外界病原体感染的小分子多肽，广泛存在于昆虫、植物、动物及人体内，其中昆虫抗菌肽cecropin是人类发现最早的一类抗菌肽。抗菌肽具有一系列引人注目的生物学活性，包括抗菌、抗炎、抗病毒、抗寄生虫、抑制肿瘤细胞及免疫调节活性等。抗菌肽Musca domestica cecropin(MDC)是本课题组从家蝇幼虫脂肪体cDNA文库中克隆的一种昆虫抗菌肽，该基因的ORF区全长为192bp，可编码63个氨基酸的前体蛋白，1～23位氨基酸是以保守4肽结尾的信号肽，其成熟肽含有40个氨基酸，前期研究发现抗菌肽MDC对许多标准菌株及临床耐药菌株显示了极强的体外抗菌活性，能够破坏细菌细胞膜或穿过细胞膜作用于胞内靶位点，作用机制独特，对正常人体细胞毒、副作用小。
目前，没有关于以抗菌肽MDC作为生物涂层的抗菌成分用于制备气管插管导管的相关报道。
发明内容
为解决现有技术存在的问题，本发明的目的在于提供一种载有抗菌肽MDC涂层的气管导管及其制备方法。
本发明的另一个目的在于提供所述的载有抗菌肽MDC涂层的气管导管在人工气管插管及呼吸机中的应用。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
本发明提供的抗菌肽MDC，其氨基酸序列如SEQ ID NO:1所示，具体地，所述的氨基酸序列为：GWLKKI GKKIE RVGQH TRDAT IQTIG VAQQA ANVAA TLKG。
本发明提供的抗菌肽MDC是采用固相化学合成法制得，具体地，通过多肽合成仪合成多肽粗品；然后用固相合成法合成多肽；再将合成的多肽使用反相高效液相色谱进行纯化，并利用电喷射质谱法对合成的多肽进行鉴定，从而完成多肽的制备。
本发明还提供一种所述的载有抗菌肽MDC涂层的气管导管的制备方法，其包括以下步骤：
(1)将医用几丁糖溶于注射用水，制成质量体积浓度为3～5％的凝胶液，然后加入一定量的抗菌肽溶液，使抗菌肽终浓度为0.2mg/mL，在30℃下以300转/分钟的速度搅拌混匀，制备涂层膜液，在室温下，用超声波振荡20分钟，除去涂层膜液中的气泡，即得抗菌肽几丁糖复合膜液；
(2)将步骤(1)制得的抗菌肽几丁糖复合膜液浸涂或流延于气管导管基材中，自然烘干或50℃固化干燥，即得气管导管。
上述的制备方法中，以几丁糖凝胶作为固定和缓释基质，抗菌肽MDC作为抗菌有效成分，制成涂层膜液，所述的几丁糖是由蟹壳提纯的高分子化合物几丁质(chitin)，经脱N—乙酰基再深加工后制成的一种聚氨基葡萄糖，是一种具有良好生物相容性、生物可降解性及生物学活性的医用高分子多糖类物质。进一步制得的医用几丁糖凝胶无毒、无刺激性、无热原性、无免疫抗原性，不溶血且具有热稳定性，无致突变、致死等不良反应，还有轻微抑菌作用，在本发明中作为抗菌肽MDC固定和缓释基质，使其发挥长期抗菌效果。
本发明选取抗菌肽MDC作为生物涂层，使气管插管导管的表面获得抗菌性能，有效预防细菌定植和生长，将减少或延缓气管插管呼吸机相关性肺炎的发生，延长导管留置时间，为患者抢救争取宝贵时间，提高救治成功率，也可减少医疗经济负担、减轻患者的身心痛苦。此外，本发明研发设计的载有抗菌肽MDC涂层的气管导管，无生物不良反应，符合临床气管插管的需要，具有广泛的应用前景。
与现有技术相比，本发明具有以下有益效果：
(1)本发明提供的载有抗菌肽MDC涂层的气管导管具有显著的抗菌性，杀菌率达98％以上，所述的抗菌肽MDC作用机制独特，不易产生耐药性，突破传统抗菌药物对细菌生物膜作用的局限，为临床提供具有安全高效抗菌性能的气管导管。
(2)本发明提供的载有抗菌肽MDC涂层的气管导管的制备步骤简单，安全性高、无不良反应，应用前景广阔。
附图说明
图1为抗菌肽MDC的高效液相色谱图。
图2为抗菌肽MDC的质谱图。
图3为扫描电镜观察空白对照气管导管表面鲍曼不动杆菌生物膜形态结构。
图4为扫描电镜观察几丁糖凝胶对照组气管导管表面鲍曼不动杆菌生物膜形态结构。
图5为扫描电镜观察抗菌肽MDC几丁糖凝胶复合涂层气管导管表面鲍曼不动杆菌生物膜形态结构。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1固相化学合成法合成抗菌肽MDC
抗菌肽MDC的氨基酸序列为：Ac-GWLKKI GKKIE RVGQH TRDAT IQTIG VAQQA ANVAA TLKG-NH2，如SEQ ID NO:1所示，含有40个氨基酸，理论等电点为10.56，理论分子量为4299.04。抗菌肽MDC的制备从C端到N端逐一进行，通过多肽合成仪来完成，具体步骤为：
(1)首先将Fmoc-X(X是抗菌肽MDC C端的第一个氨基酸)接入到Wang树脂，然后脱去Fmoc基团后得到X-Wang树脂；再将Fmoc-Y-Trt-OH(9-芴甲氧羧基-三甲基-Y，Y为抗菌肽MDC C端第二个氨基酸)；按照这个程序依次从C端合成到N端，直至合成完毕，得到脱去Fmoc基团的侧链保护的树脂；
(2)在上述得到的肽树脂中，加入切割试剂，20℃避光下反应2h，过滤；沉淀用TFA(三氟乙酸)洗涤，将洗液与上述滤液混合，旋转蒸发仪浓缩，再加入10倍左右体积的预冷无水乙醚，-20℃沉淀3h，析出白色粉末物，以2500g离心10min，收集沉淀，再用无水乙醚洗涤沉淀，真空干燥，得到多肽，其中切割试剂由TFA、水和TIS(三异丙基氯硅烷)按照质量比95:2.5:2.5混合而成；
(3)使用0.2mol/L硫酸钠(磷酸调节至pH7.5)进行柱平衡30min，用90％乙腈水溶液溶解多肽，过滤，C18反相常压柱，采用梯度洗脱(洗脱剂为甲醇和硫酸钠水溶液按照体积比为30:70～70:30混合)，流速为1mL/min，检测波为220nm，收集主峰，冻干；
(4)再利用反相C18柱进一步纯化，洗脱液A为0.1％TFA/水溶液；洗脱液B为0.1％TFA/乙腈溶液，洗脱浓度为25％B～40％B，洗脱时间为12min，流速为1mL/min，再同上收集主峰，冻干，即得精制的抗菌肽MDC；
(5)将上述得到精制抗菌肽MDC经过反相高效液相色谱和电喷雾质谱法分析，反相高效液相色谱图如图1所示，质谱图如图2所示，结果显示，抗菌肽MDC的纯度大于95％，分子量为4299.36，与理论分子量基本一致。
实施例2载有抗菌肽MDC涂层的气管导管制备
(1)将医用几丁糖溶于注射用水，制成质量体积浓度为4％的凝胶液，然后加入一定量的抗菌肽溶液，使抗菌肽终浓度为0.2mg/mL，在30℃下以300转/分钟的速度搅拌混匀，制备涂层膜液，在室温下，用超声波振荡20分钟，除去涂层膜液中的气泡，即得抗菌肽几丁糖复合膜液；
(2)将步骤(1)制得的抗菌肽几丁糖复合膜液浸涂或流延于PVC气管导管基材中，自然烘干或50℃固化干燥，即得载有抗菌肽MDC涂层的气管导管。
试验例一、安全性评价
(1)细胞毒性试验
按照GB/T16886.5-2003规定的细胞毒性试验方法，对载有抗菌肽MDC涂层的气管导管样品进行细胞毒性试验。用10％的小牛血清MEM培养基配制载有抗菌肽MDC涂层的气管导管样品浸提液培养NCTC colone929细胞(小鼠成纤维细胞)，并以空白PVC气管插管导管样品浸提液作阴性对照，5g/L苯酚溶液作阳性对照。
试验结果：阴性对照的细胞形态正常，贴壁生长良好，胞浆内有离散颗粒，无细胞溶解；阳性对照的细胞生长不佳，90％以上细胞呈圆形或细胞溶解；载有抗菌肽MDC涂层的气管导管样品浸提液中细胞形态正常，贴壁生长良好，胞浆内有离散颗粒，无细胞溶解。载有抗菌肽MDC涂层的气管导管样品和空白PVC气管插管导管样品浸提液都评为无毒，判定分级为0级(细胞毒性反应为0级或1级为合格)。培养48小时监测细胞OD570和相对增值度，载有抗菌肽MDC涂层的气管导管样品和空白PVC气管插管导管样品浸提液都判定分级为0级，载有抗菌肽MDC涂层的气管导管样品细胞毒性试验结果为合格。
(2)急性全身毒性试验
按照GB/T16886.11-2011标准，观察载有抗菌肽MDC涂层的气管导管样品对小鼠急性全身毒性反应。空白PVC气管插管导管样品和载有抗菌肽MDC涂层的气管导管样品的0.9％氯化钠注射液浸提液以尾静脉注射方式单次给予，并以相应浸提介质作为空白对照。注射后4、24、48和72h，观察小鼠毒性反应情况。结果发现：空白对照液组、空白PVC气管插管导管样品和载有抗菌肽MDC涂层的气管导管样品浸提液组均未见异常反应。结果表明：载有抗菌肽MDC涂层的气管导管样品浸提液的急性全身毒性试验结果为符合无毒性要求，和空白PVC气管插管导管样品的急性全身毒性试验无差别。
试验例二、抗菌性评价
以鲍曼不动杆菌为代表，模拟临床ICU呼吸机相关性鲍曼不动杆菌生物膜感染，建立鲍曼不动杆菌生物膜体外模型。以不做任何处理的空白气管导管样品为空白对照组，同时设几丁糖凝胶对照组，模拟呼吸机导管中液体流动状态，将各组气管导管浸渍于1.0×10 5鲍曼不动杆菌菌液中，培养24h，取10ul浸渍液在LB培养基上涂布接种，35℃过夜培养后，计数菌落数，观察细菌的相对生长情况，以显示其抗菌性能。
对比实验的结果证实：空白气管导管样品细菌正常生长，几丁糖凝胶对照组气管导管样品细菌生长减少不明显，杀菌率在5％左右，而抗菌肽MDC几丁糖凝胶复合涂层气管导管样品细菌生长明显减少甚至消失，杀菌率达98％以上。
其中，扫描电镜观察气管导管表面生物膜形态结构如图3-图5所示，结果显示：空白对照气管导管(图3)可见大量细菌黏附成团，形成大块状生物膜；几丁糖凝胶对照组气管导管(图4)生物膜厚度及面积均有所减少；抗菌肽MDC几丁糖凝胶复合涂层气管导管(图5)仅见散在细菌且菌细胞发生变形、断裂、外膜脱离，内容物泄漏；
此外，琼脂平板菌落计数法结果亦显示：抗菌肽MDC几丁糖凝胶复合涂层气管导管表面活菌数较空白对照组和几丁糖凝胶对照组均显著减少。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (6)

  1. 一种气管导管，其特征在于，所述的气管导管表面载有抗菌肽MDC作为生物涂层。
  2. 根据权利要求1所述的气管导管，其特征在于，所述的抗菌肽MDC的序列如SEQ ID NO:1所示。
  3. 根据权利要求1或2所述的气管导管的制备方法，其特征在于，包括以下步骤：
    (1)将医用几丁糖溶于注射用水，制成质量体积浓度为3～5％的凝胶液，然后加入一定量的抗菌肽溶液，使抗菌肽终浓度为0.2mg/mL，在30℃下以300转/分钟的速度搅拌混匀，制备涂层膜液，在室温下，用超声波振荡20分钟，除去涂层膜液中的气泡，即得抗菌肽几丁糖复合膜液；
    (2)将步骤(1)制得的抗菌肽几丁糖复合膜液浸涂或流延于气管导管基材中，自然烘干或50℃固化干燥，即得气管导管。
  4. 根据权利要求3所述的气管导管的制备方法，其特征在于，所述的气管导管基材为聚氯乙烯。
  5. 根据权利要求1-4任一所述的气管导管在人工气管插管及呼吸机中的应用。
  6. 一种抗菌肽MDC，其序列如SEQ ID NO:1所示。
PCT/CN2018/083402 2018-01-19 2018-04-17 一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用 WO2019140796A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810054269.6 2018-01-19
CN201810054269.6A CN108452418A (zh) 2018-01-19 2018-01-19 一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2019140796A1 true WO2019140796A1 (zh) 2019-07-25

Family

ID=63221009

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/083402 WO2019140796A1 (zh) 2018-01-19 2018-04-17 一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN108452418A (zh)
WO (1) WO2019140796A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111973805B (zh) * 2019-08-16 2022-03-01 苏州吉美瑞生医学科技有限公司 抗菌肽hCAP18/LL-37在抗感染生物工程肺中的应用
IT202000006481A1 (it) * 2020-03-27 2021-09-27 Sanidrink S R L Condotti tubolari antimicrobici
CN112057718A (zh) * 2020-08-24 2020-12-11 浙江隆泰医疗科技股份有限公司 一种用于呼吸系统的阻隔材料

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101428157A (zh) * 2008-12-23 2009-05-13 福州大学 一种具有抗细菌粘附和光诱导杀菌性能的医用气管导管制备方法
CN103933617A (zh) * 2014-05-04 2014-07-23 福州大学 一种具有长效抗细菌粘附的气管导管及其制备方法
CN103948973A (zh) * 2014-04-21 2014-07-30 蒋旭宏 一种具有安全高效抗菌性能的医用气管导管
CN107459568A (zh) * 2017-08-22 2017-12-12 广东药科大学 一种Musca domestica cecropin 衍生肽M27‑39及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101628133A (zh) * 2009-08-11 2010-01-20 中国人民解放军第三军医大学第一附属医院 载缓释抗菌素涂层医用导管及其制备方法
CN102675428A (zh) * 2012-04-20 2012-09-19 广东药学院 肝靶向穿膜抗病毒融合多肽及其编码基因与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101428157A (zh) * 2008-12-23 2009-05-13 福州大学 一种具有抗细菌粘附和光诱导杀菌性能的医用气管导管制备方法
CN103948973A (zh) * 2014-04-21 2014-07-30 蒋旭宏 一种具有安全高效抗菌性能的医用气管导管
CN103933617A (zh) * 2014-05-04 2014-07-23 福州大学 一种具有长效抗细菌粘附的气管导管及其制备方法
CN107459568A (zh) * 2017-08-22 2017-12-12 广东药科大学 一种Musca domestica cecropin 衍生肽M27‑39及其应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 21 January 2007 (2007-01-21), JIN, X.B.: "Musca domestica cecropin precursor, mRNA, complete cds", XP055625117, Database accession no. EF175878 *
DATABASE PROTEIN 18 December 2017 (2017-12-18), ANONYMOUS: "Cecropin-2 [Ceratitis capitata]", XP055625098, retrieved from NCBI Database accession no. XP_004534330 *
DATABASE PROTEIN 18 December 2017 (2017-12-18), ANONYMOUS: "Cecropin-2 [Ceratitis capitata]", XP055625106, retrieved from NCBI Database accession no. XP_004534333 *
DATABASE PROTEIN 18 December 2017 (2017-12-18), ANONYMOUS: "Cecropin-2-like [Ceratitis capitata]", XP055625109, retrieved from NCBI Database accession no. XP_004534331 *
DATABASE PROTEIN 5 October 2014 (2014-10-05), LU , X.: "HTPP-MDC fusion peptide, partial [synthetic construct)", XP055625115, retrieved from NCBI Database accession no. AIR95616 *
LU , X. ET AL.: "Bactericidal activity of Musca domestica cecropin (Mdc) on mu- ltidrug-resistant clinical isolate of Escherichia coli", APPL. MICROBIOL. BIOTECHNOL., vol. 95, no. 4, 28 December 2011 (2011-12-28), pages 939 - 945, XP035090905 *
LU , X. M.: "Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli", APPL. MICROBIOL. BIOTECHNOL., 25 May 2010 (2010-05-25), pages 2169 - 2176, XP019841604 *
LU XUEMEI: "Cloning Expression and Antimicrobial Activities of Musca Domestica Cecropin Fused with Human Lysozyme", BASIC SCIENCES, CHINA MASTER'S THESES, no. 6, A006-102, 15 June 2011 (2011-06-15) *

Also Published As

Publication number Publication date
CN108452418A (zh) 2018-08-28

Similar Documents

Publication Publication Date Title
WO2019140796A1 (zh) 一种载有抗菌肽mdc涂层的气管导管及其制备方法和应用
Ceresa et al. Medical-grade silicone coated with rhamnolipid R89 is effective against Staphylococcus spp. biofilms
Nie et al. Bioinspired and biocompatible carbon nanotube-Ag nanohybrid coatings for robust antibacterial applications
CN103948973B (zh) 一种具有安全高效抗菌性能的医用气管导管
Rapsch et al. Identification of antimicrobial peptides and immobilization strategy suitable for a covalent surface coating with biocompatible properties
Zhang et al. Advances in antibacterial functionalized coatings on Mg and its alloys for medical use—A review
CN112998884B (zh) 一种长效抗菌材料、牙科膜片和长效隐形矫治器
WO2015022874A1 (ja) 水素含有抗微生物剤
CN104740690B (zh) 一种海洋生物载药纳米抗菌超滑涂层
CN101653635A (zh) 纳米抗菌材料涂层的人工气管切开套管
CN101554505A (zh) 纳米抗菌材料涂层的人工气管插管
CN114181293A (zh) 一种人源抗菌肽ll-37改造体及其应用
León-Buitimea et al. Nanomaterials-based combinatorial therapy as a strategy to combat antibiotic resistance
CN101628133A (zh) 载缓释抗菌素涂层医用导管及其制备方法
Bai et al. Metallic antibacterial surface treatments of dental and orthopedic materials
Laganà et al. Antibacterial activity of nanoparticles and nanomaterials: a possible weapon in the fight against healthcare-associated infections.
Bartmański et al. The chemical and biological properties of nanohydroxyapatite Coatings with antibacterial nanometals, obtained in the electrophoretic process on the Ti13Zr13Nb alloy
CN104740141B (zh) 一种抗菌喷剂及其制备方法
Lethongkam et al. Biogenic nanosilver-fabricated endotracheal tube to prevent microbial colonization in a veterinary hospital
Yin et al. Antiviral and antibacterial sulfated polysaccharide–chitosan nanocomposite particles as a drug carrier
CN103933617B (zh) 一种具有抗细菌粘附的气管导管的制备方法
CN111249444B (zh) 一种用于抑制白色念珠菌的制剂
Marcut et al. Improving the Hydrophobicity of Plasticized Polyvinyl Chloride for Use in an Endotracheal Tube
WO2022041634A1 (zh) 一种预防病毒传染的复方制剂及其配制/使用方法与应用
Mina-Aponzá et al. Study of titanium–silver monolayer and multilayer films for protective applications in biomedical devices

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18901412

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18901412

Country of ref document: EP

Kind code of ref document: A1