CN113694255B - 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 - Google Patents
一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 Download PDFInfo
- Publication number
- CN113694255B CN113694255B CN202111029281.XA CN202111029281A CN113694255B CN 113694255 B CN113694255 B CN 113694255B CN 202111029281 A CN202111029281 A CN 202111029281A CN 113694255 B CN113694255 B CN 113694255B
- Authority
- CN
- China
- Prior art keywords
- solution
- gl13k
- medical implant
- nano silver
- nano
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Images
Classifications
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/30—Inorganic materials
- A61L27/306—Other specific inorganic materials not covered by A61L27/303 - A61L27/32
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/06—Titanium or titanium alloys
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
-
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/02—Inorganic materials
- A61L31/022—Metals or alloys
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/082—Inorganic materials
- A61L31/088—Other specific inorganic materials not covered by A61L31/084 or A61L31/086
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- 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
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
- A61L2300/104—Silver, e.g. silver sulfadiazine
-
- 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
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- 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
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/02—Methods for coating medical devices
-
- 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
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/06—Coatings containing a mixture of two or more compounds
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Nanotechnology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Surgery (AREA)
- Vascular Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Crystallography & Structural Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Biophysics (AREA)
- Composite Materials (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Manufacturing & Machinery (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明涉及一种医用植入物表面有机‑无机抗菌复合涂层及其制备方法,步骤如下:制备纳米银粉;在硼砂‑氢氧化钠缓冲液中加入纳米银粉得到纳米银溶液,在水中加入抗菌肽GL13K得到GL13K溶液,接着将两者混合,在4℃下储存4天,GL13K自组装形成纳米纤维,得复合物溶液;将经过预处理的医用植入物放入48孔板中,每个孔板中加入250μL纳米银‑抗菌肽GL13K复合物溶液,用封口膜封闭孔板边缘,在4℃下培养12 h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即得复合涂层。本发明将纳米银和抗菌肽GL13K两者结合形成复合涂层,可达到优势互补的效果。
Description
技术领域
本发明涉及医用材料技术领域,尤其涉及一种医用植入物表面有机-无机抗菌复合涂层及其制备方法。
背景技术
感染是各类金属植入手术(钛板、钛钉、种植体、种植钉、人工关节等)最严重的并发症之一,常导致植入物植入失败,是亟需解决的问题。植入物感染发展过程中最关键的致病因素是细菌生物膜的形成,它在细菌粘附后立即开始,一旦细菌在植入物表面黏附聚集形成细菌生物膜,将具有极强的抗生素抵抗性。根据“竞争表面”理论,细菌和宿主细胞是以竞争的方式定植在植入物表面,如果植入物材料本身具有抗菌能力,就能在植入初期削弱细菌的竞争力,为宿主细胞争取更多的定植时间,极大提高植入成功率。可降低感染的钛表面抗菌修饰涂层是目前研究热点,研究人员尝试通过向涂层中添加抗生素(庆大霉素、米诺环素利福平等)、非抗生素有机抗菌剂(氯己定、氯二甲酚)、纳米粒子(银、铜、氧化锌)等不同的抗菌物质,赋予植入物抑制细菌黏附、杀灭细菌的能力来预防和控制植入物相关感染,涂层方法虽多样,但仍存在抗菌性不佳、耐药性、操作复杂等诸多问题,亟需高效可靠的解决方案。
抗菌肽(antimicrobial peptides,AMPs)是人体内产生的有免疫活性的天然有机多肽,由于其抗菌机理独特、对耐药菌株有广谱抗菌活性,且不易产生新耐药菌,被认为是天然新一代抗生素。抗菌肽GL13K是一种从人腮腺分泌蛋白中提取的正电荷两亲性小分子多肽,近些年,其在钛植入物表面的抗菌作用取得了突破性研究成果,将它涂层于钛植入物表面,显示出抑制生物膜生长和对植入物感染多种相关病原体的杀灭作用。最重要的是它能有效杀灭多种耐药菌,不会导致新的耐药菌产生。同时,该涂层与钛植入物结合牢固(具有抗水解和机械挑战的能力),细胞相容性好(对成骨细胞、人牙龈成纤维细胞、红细胞等多种细胞无细胞毒性),并且涂层方法简单易行。
但是,抗菌肽是一种被动有机抗菌涂层,其抗菌性仅当细菌接触其表面时发挥作用,难以杀灭周围感染组织的细菌,而且对于许多肽而言,其抗菌性在生理盐和血清条件下会降低甚至丧失。
纳米银(Silver nanoparticles,AgNPs)是无机抗菌剂中的典型代表,它由于小尺寸效应、量子效应和极大的比表面积,具有广谱抗菌、强效持久杀菌、渗透杀菌、促进伤口修复再生、低耐药性等特点,在钛植入物表面涂层中具有广泛应用。将纳米银涂层于钛植入物表面后,对变形链球菌、牙龈卟啉单胞菌和白色念珠菌等多种细菌表现出良好的抗菌效果。纳米银对细菌等原核细胞的毒性大,而对真核细胞的相容性较好,因此银在具备优异抗菌性能的同时对生物体的细胞毒性较低,在一定剂量下未观察到其细胞毒性。
但是,纳米银易团聚,团聚后杀菌性大大降低,且目前将纳米银涂层到钛植入物表面技术繁琐复杂,经涂层或表面处理后纳米银抗菌性也会降低。更重要的是由于它在临床上具有广泛应用,已分离出多株具有银抗性的细菌
因此,需要开发通过不同机制抗菌的联合药物疗法。
发明内容
解决的技术问题:针对现有技术存在的缺点,本发明提供一种医用植入物表面有机-无机抗菌复合涂层及其制备方法,根据两种抗菌机制不同的被动有机抗菌剂抗菌肽GL13K和主动无机抗菌剂纳米银各自的优缺点,将两者结合形成复合涂层,可达到避开劣势,优势互补的效果,形成具有双重抗菌、协同抗菌、耐药性低、操作简便的有机/无机复合抗菌涂层。
技术方案:一种医用植入物表面有机-无机抗菌复合涂层的制备方法,该方法的制备步骤如下:
(1)纳米银粉的制备:以柠檬酸盐作为还原剂、糖作为稳定剂,通过还原硝酸银制备得到纳米银粉;
(2)纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液的制备:在pH值为9.8的硼砂-氢氧化钠缓冲液中加入纳米银粉,配置成0.2mg/mL的纳米银溶液,在水中加入抗菌肽GL13K,震荡均匀配置成100mg/mL的GL13K溶液,接着将GL13K溶液加入到纳米银溶液中,其中GL13K溶液与纳米银溶液的体积比为1:99,得到的混合溶液中纳米银和GL13K的最终浓度分别为0.2mg/mL和1mg/mL,将混合溶液在4℃下储存4天,即发生GL13K的自组装,并形成纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液;
(3)有机-无机抗菌复合涂层的制备:将经过预处理的医用植入物放入48孔板中,每个孔板中加入250μL纳米银-抗菌肽GL13K复合物溶液,用封口膜封闭孔板边缘,在4℃下培养12h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即在医用植入物表面形成有机-无机抗菌复合涂层。
上述所述的步骤(1)中纳米银粉的具体制备方法如下:将250mM的柠檬酸盐水溶液逐滴加入1mM硝酸银水溶液中,其中柠檬酸盐溶液与硝酸银溶液的体积比为1:50,在93-94℃的水浴中搅拌10min,在35280RCF的离心力下离心30min,重复离心三次收集沉淀的纳米银;接着在去离子水中重新溶解收集的纳米银,在2380RCF的离心力下离心10min,重复离心三次收集沉淀的纳米银;将最终收集的纳米银沉淀用去离子水稀释,用分光光度计测量光密度值(OD值),直到稀释至OD420=2.9,再每mL加入0.1g蔗糖,混合搅拌2h,在冻干机中干燥24h后得到纳米银粉,将纳米银粉密封后储存于-20℃备用。
上述所述的步骤(1)中柠檬酸盐为柠檬酸三钠。
上述所述的步骤(2)中的硼砂-氢氧化钠缓冲液均由0.025M硼砂水溶液和0.1MNaOH水溶液配制而成。
上述所述的步骤(3)中医用植入物的预处理方法如下:将医用植入物依次用丙酮浸泡10min、去离子水超声清洗15min、环己烷超声处理15min、氮气吹干备用;接着将医用植入物放入试管中,在试管中加入5mL浓度为5M的NaOH水溶液,加盖固定,置于60℃的温箱中碱蚀12h后,用去离子水、丙酮依次冲洗医用植入物,再用氮气干燥备用。
上述所述的医用植入物为Ti6Al4V微种植体。
由上述所述的的制备方法制备得到的医用植入物表面有机-无机抗菌复合涂层。
本发明采用的有机抗菌肽和无机纳米银的抗菌作用机制不同,可考虑将两者双重涂层,但抗菌肽与纳米银两者涂层工艺也完全不同,抗菌肽通常需要使用化学共价键枝节到钛基表面以形成稳定的涂层,纳米银常通过高温下蒸发、溅射或等离子喷涂等方法涂层,如果先涂层抗菌肽再涂层纳米银会造成抗菌肽高温下变性,而先涂层纳米银再涂层抗菌肽则难以共价枝节抗菌肽,所以如何将两者双重涂层成为难点。本发明首先通过物理化学方法使抗菌肽GL13K自组装成纳米纤维,再通过静电力使纳米银结合到抗菌肽纳米纤维极性端,纳米银负载镶嵌于抗菌肽中,形成GL13K-纳米银复合体,利用抗菌肽纳米纤维对钛基的强吸附作用,使两者可以同时在钛基表面形成稳定的涂层。本发明根据两种抗菌机制不同的被动有机抗菌剂抗菌肽GL13K和主动无机抗菌剂纳米银各自的优缺点,将两者结合形成复合涂层,可达到避开劣势,优势互补的效果,形成具有双重抗菌、协同抗菌、耐药性低、操作简便的有机/无机复合抗菌涂层。
有益效果:本发明提供的一种医用植入物表面有机-无机抗菌复合涂层及其制备方法,具有以下有益效果:本发明根据两种抗菌机制不同的被动有机抗菌剂抗菌肽GL13K和主动无机抗菌剂纳米银各自的优缺点,将两者结合形成复合涂层,可达到避开劣势,优势互补的效果,形成具有双重抗菌、协同抗菌、耐药性低、操作简便的有机/无机复合抗菌涂层。
附图说明
图1为采用能谱分析仪对eTi组、Ag组、GL组、Ag-GL组的涂层表面元素含量检测结果图。
图2为eTi组、Ag组、GL组、Ag-GL组涂层表面水接触角测量结果图。
图3为eTi组、Ag组、GL组、Ag-GL组的组织切片表面菌落计数图。
图4为eTi组、Ag组、GL组、Ag-GL组的组织切片表面菌落图。
图5为eTi组、Ag组、GL组、Ag-GL组的组织切片HE染色结果图。
具体实施方式
以下实施例和对比例所述的医用植入物均为Ti6Al4V微种植体,购自韩国JeilMedical Corp(韩国);硝酸银购自上海申博化工有限公司(中国);柠檬酸三钠购自西陇科学股份有限公司(中国);抗菌肽GL13K购自合肥国肽生物科技有限公司(中国)。其余物质均为市售常用物质。
下述硼砂-氢氧化钠缓冲液均由0.025M硼砂水溶液和0.1M NaOH水溶液配制而成。
实施例1
该实施例提供一种医用植入物表面有机-无机抗菌复合涂层的制备方法,该方法的制备步骤如下:
(1)纳米银粉的制备:将10mL 250mM的柠檬酸三钠水溶液逐滴加入500mL 1mM硝酸银水溶液中,在93-94℃的水浴中搅拌10min,在35280RCF的离心力下离心30min,重复离心三次收集沉淀的纳米银;接着在去离子水中重新溶解收集的纳米银,在2380RCF的离心力下离心10min,重复离心三次收集沉淀的纳米银;将最终收集的纳米银沉淀用去离子水稀释,用分光光度计测量光密度值(OD值),直到稀释至OD420=2.9,再每mL加入0.1g蔗糖,混合搅拌2h,在冻干机中干燥24h后得到纳米银粉,将纳米银粉密封后储存于-20℃备用;
(2)纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液的制备:在pH值为9.8的硼砂-氢氧化钠缓冲液中加入纳米银粉,配置成0.2mg/mL的纳米银溶液,在水中加入抗菌肽GL13K,震荡均匀配置成100mg/mL的GL13K溶液,接着将10μL GL13K溶液加入到990μL纳米银溶液中,得到的混合溶液中纳米银和GL13K的最终浓度分别为0.2mg/mL和1mg/mL,将混合溶液在4℃下储存4天,即发生GL13K的自组装,并形成纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液;
(3)有机-无机抗菌复合涂层的制备:将微种植体依次用丙酮浸泡10min、去离子水超声清洗15min、环己烷超声处理15min、氮气吹干备用;接着将微种植体放入试管中,在试管中加入5mL浓度为5M的NaOH水溶液,加盖固定,置于60℃的温箱中碱蚀12h后,用去离子水、丙酮依次冲洗医用植入物,再用氮气干燥备用,接着将经过预处理的微种植体放入48孔板中,每个孔板中加入250μL纳米银-抗菌肽GL13K复合物溶液,用封口膜封闭孔板边缘,在4℃下培养12h后,从溶液中取出微种植体,去离子水小心冲洗,氮气干燥后,即在微种植体表面形成有机-无机抗菌复合涂层。
对比例1
对比例1为在微种植体表面未涂覆涂层。
对比例2
对比例2为在微种植体表面仅涂覆了纳米银涂层。
纳米银涂层的制备方法为:将经过预处理的微种植体放入48孔板中,每个孔板中加入250μL 0.2mg/mL纳米银溶液,用封口膜封闭孔板边缘,在4℃下培养12h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即在微种植体表面形成纳米银涂层。
此处纳米银溶液与实施例1中相同。
对比例3
对比例3为在微种植体表面仅涂覆了抗菌肽GL13K自组装纳米纤维涂层。
抗菌肽GL13K自组装纳米纤维涂层的制备方法为:将经过预处理的微种植体放入48孔板中,每个孔板中加入1mg/mL抗菌肽GL13K溶液,用封口膜封闭孔板边缘,在4℃下培养12h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即在微种植体表面形成抗菌肽GL13K自组装纳米纤维涂层。
此处抗菌肽GL13K溶液的制备步骤如下:将GL13K在去离子水中溶解,制备成100mg/mL的GL13K水溶液,再将GL13K水溶液加入到pH值为9.8的硼砂-氢氧化钠缓冲液中,配制成GL13K浓度为1mg/mL的抗菌肽GL13K溶液,在4℃下储存4天,即得抗菌肽GL13K溶液。
下边对实施例1制备得到的微种植体表面纳米银-抗菌肽GL13K复合涂层以及对比例1-3的各项性能进行检测。其中实施例1为Ag-GL组,对比例1为eTi组,对比例2为Ag组,对比例3为GL组。
1.涂层的表征
(1)元素组成分析
采用能谱分析仪(Energy dispersive spectrometer,EDS)分析微种植体涂层表面元素含量其结果入图1所示,并检测主要元素成分含量如下表1。由图1和表1可知,Ag组表面含有3.2%的Ag,Ag-GL组检测到2.8%的Ag,证明纳米银成功加载到微种植体上。GL组和Ag-GL组分别检测到10.3%和9.8%的N,证明GL13K加载到了微种植体表面。
表1
(2)亲疏水性分析
每组微种植体各3个,在其螺帽处滴加相同体积的去离子水,拍摄10s后的水接触角图像,测量接触角(Water contact angle,WCA)。结果如图2所示,Ag组的水接触角与碱蚀钛种植体eTi组结果接近,水滴滴落到螺帽表面后扩散铺开,几乎观察不到水滴停留的形态,表现出亲水性。而GL13K、Ag-GL组涂层的水接触角要明显大于eTi组,水滴可在表面长时间停留不发生改变,GL13K的加入使得微种植体表面表现出疏水性。
2.体内抗菌性
SD大鼠麻醉、备皮、消毒后,在双侧膝关节处内侧,沿着股骨长轴做2cm的切口,切开表皮,肌肉直达骨膜,钝性分离暴露骨面。大量生理盐水冲洗下使用低速打磨机在股骨远心端关节面中心预备直径1.2mm的植入窝,转速控制在800r/min。植入窝内加10μL浓度为109CFU/mL的耐甲氧西林金黄色葡萄球菌(MRSA),将eTi组、AgNPs涂层组、GL13K涂层组、AgNPs-GL13K复合涂层组各5枚微种植体,随机植入大鼠左右两侧股骨种植窝内,植入后检查微种植体是否松动,分层缝合,关闭创口。术后5天处死大鼠,取出微种植体,进行菌落计数实验;牙科慢机截取股骨标本,小心剔除多余的组织后放入到10%中性福尔马林溶液中固定48h,进行脱钙硬组织切片,HE染色观察。
菌落计数实验结果显示如图3、图4所示,eTi组微种植体上收集到的细菌最多;Ag组和GL组微种植体上的细菌量均少于Ti组上收集到的细菌(p<0.05);Ag-GL组微种植体上的细菌最少,与Ag组和GL组相比有显著性差异(p<0.05),Ag-GL组微种植体对MRSA的抗菌性能明显优于其他三组。
组织切片HE染色结果显示如图5所示,接种细菌5天后,eTi组微种植体周围骨组织明显破坏,骨髓腔内大量炎症细胞浸润,主要为淋巴细胞和中性粒细胞浸润,提示为重度炎症反应;Ag组炎症轻,骨髓腔内可见微种植体的螺纹形态,部分区域密质骨不规则吸收,螺纹连续性中断,炎症较eTi组明显减轻;GL组可见少量的炎症细胞浸润,微种植体螺纹形态扭曲,部分区域可见少量破骨细胞以及成骨细胞;Ag-GL组炎症最轻,未见明显骨质破坏,密质骨完整,骨髓腔内螺纹形态清晰,下方骨髓腔可见较多的蓝染的骨髓细胞。
以上对本发明实施例进行了详细介绍,对于本领域的一般技术人员,依据本发明实施例的思想,在具体实施方式及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本发明的限制。
Claims (7)
1.一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于该方法的制备步骤如下:
(1)纳米银粉的制备:以柠檬酸盐作为还原剂、糖作为稳定剂,通过还原硝酸银制备得到纳米银粉;
(2)纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液的制备:在pH值为 9.8的硼砂-氢氧化钠缓冲液中加入纳米银粉,配置成0.2 mg/mL的纳米银溶液,在水中加入抗菌肽GL13K,震荡均匀配置成100 mg/mL的GL13K溶液,接着将GL13K溶液加入到纳米银溶液中,其中GL13K溶液与纳米银溶液的体积比为1:99,得到的混合溶液中纳米银和GL13K的最终浓度分别为0.2 mg/mL和1 mg/mL,将混合溶液在4 ℃下储存4天,即发生GL13K的自组装,并形成纳米银-抗菌肽GL13K自组装纳米纤维复合物溶液;
(3)有机-无机抗菌复合涂层的制备:将经过预处理的医用植入物放入48孔板中,每个孔板中加入250 μL纳米银-抗菌肽GL13K复合物溶液,用封口膜封闭孔板边缘,在4℃下培养12 h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即在医用植入物表面形成有机-无机抗菌复合涂层。
2.根据权利要求1所述的一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于所述步骤(1)中纳米银粉的具体制备方法如下:将250 mM的柠檬酸盐水溶液逐滴加入1 mM硝酸银水溶液中,其中柠檬酸盐溶液与硝酸银溶液的体积比为1:50,在93-94 ℃的水浴中搅拌10 min,在35280 RCF的离心力下离心30min,重复离心三次收集沉淀的纳米银;接着在去离子水中重新溶解收集的纳米银,在2380 RCF的离心力下离心10min,重复离心三次收集沉淀的纳米银;将最终收集的纳米银沉淀用去离子水稀释,逐滴加入去离子水,用分光光度计测量光密度值(OD值),直到稀释至OD420=2.9,再每mL加入0.1 g蔗糖,混合搅拌2 h,在冻干机中干燥24 h后得到纳米银粉,将纳米银粉密封后储存于-20 ℃备用。
3.根据权利要求2所述的一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于:所述步骤(1)中柠檬酸盐为柠檬酸三钠。
4.根据权利要求1所述的一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于:所述步骤(2)中的硼砂-氢氧化钠缓冲液均由0.025 M硼砂水溶液和0.1 M NaOH水溶液配制而成。
5.根据权利要求1所述的一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于所述步骤(3)中医用植入物的预处理方法如下:将医用植入物依次用丙酮浸泡10min、去离子水超声清洗15 min、环己烷超声处理15 min、氮气吹干备用;接着将医用植入物放入试管中,在试管中加入5 mL浓度为5 M的 NaOH水溶液,加盖固定,置于60℃的温箱中碱蚀12 h后,用去离子水、丙酮依次冲洗医用植入物,再用氮气干燥备用。
6.根据权利要求5所述的一种医用植入物表面有机-无机抗菌复合涂层的制备方法,其特征在于:所述医用植入物为Ti6Al4V微种植体。
7.一种由权利要求1-6任一项所述的制备方法制备得到的医用植入物表面有机-无机抗菌复合涂层。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111029281.XA CN113694255B (zh) | 2021-09-02 | 2021-09-02 | 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111029281.XA CN113694255B (zh) | 2021-09-02 | 2021-09-02 | 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113694255A CN113694255A (zh) | 2021-11-26 |
CN113694255B true CN113694255B (zh) | 2022-07-22 |
Family
ID=78657800
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111029281.XA Active CN113694255B (zh) | 2021-09-02 | 2021-09-02 | 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113694255B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114699325B (zh) * | 2022-02-21 | 2023-12-29 | 南昌大学附属口腔医院(江西省口腔医院) | 预防正畸牙釉质损伤的新型釉质粘结方法 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8309117B2 (en) * | 2002-12-19 | 2012-11-13 | Novartis, Ag | Method for making medical devices having antimicrobial coatings thereon |
DK2471827T3 (da) * | 2010-12-30 | 2013-11-11 | Universitaetsklinikum Freiburg | Kovalent bundne antimikrobielle polymerer |
CN102240416A (zh) * | 2011-04-25 | 2011-11-16 | 南京大学 | 一种肝素化方法及应用 |
CN105031741A (zh) * | 2015-03-03 | 2015-11-11 | 江苏海泽医疗科技发展有限公司 | 一种含抗感染药物涂层,具缓释药物功能的医用导管及其制备方法 |
CN106860911A (zh) * | 2017-03-16 | 2017-06-20 | 湖北大学 | 一种钛金属表面抗菌复合涂层及其制备方法 |
CN107648680A (zh) * | 2017-09-29 | 2018-02-02 | 无锡盛雅生物科技有限公司佛山分公司 | 提高生物相容性的抗菌涂层的制备方法 |
CN110064075B (zh) * | 2019-04-23 | 2020-10-13 | 北京科技大学 | 一种基于纳米银/d-半胱氨酸的自组装抗菌涂层及制备方法 |
CN112190594B (zh) * | 2020-10-15 | 2022-11-15 | 南昌大学附属口腔医院(江西省口腔医院) | 一种生物相容性良好的抗菌碳纳米管及其制备方法 |
-
2021
- 2021-09-02 CN CN202111029281.XA patent/CN113694255B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN113694255A (zh) | 2021-11-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | A surface-engineered polyetheretherketone biomaterial implant with direct and immunoregulatory antibacterial activity against methicillin-resistant Staphylococcus aureus | |
Li et al. | Dual-functional antimicrobial coating based on a quaternary ammonium salt from rosin acid with in vitro and in vivo antimicrobial and antifouling properties | |
Ding et al. | A dual-functional implant with an enzyme-responsive effect for bacterial infection therapy and tissue regeneration | |
Zhou et al. | Biomimetic AgNPs@ antimicrobial peptide/silk fibroin coating for infection-trigger antibacterial capability and enhanced osseointegration | |
Tian et al. | Amyloid-like protein aggregates combining antifouling with antibacterial activity | |
Li et al. | Polydopamine-induced nanocomposite Ag/CaP coatings on the surface of titania nanotubes for antibacterial and osteointegration functions | |
Sun et al. | A reduced polydopamine nanoparticle-coupled sprayable PEG hydrogel adhesive with anti-infection activity for rapid wound sealing | |
Cochis et al. | Silver-doped keratin nanofibers preserve a titanium surface from biofilm contamination and favor soft-tissue healing | |
Ye et al. | Effects of DNase I coating of titanium on bacteria adhesion and biofilm formation | |
Liu et al. | Inside-outside Ag nanoparticles-loaded polylactic acid electrospun fiber for long-term antibacterial and bone regeneration | |
Xu et al. | Single-step fabrication of catechol-ε-poly-L-lysine antimicrobial paint that prevents superbug infection and promotes osteoconductivity of titanium implants | |
CN107693843B (zh) | 生物医用活性钛及其合金植入材料的表面改性方法 | |
Gou et al. | Bio-inspired peptide decorated dendrimers for a robust antibacterial coating on hydroxyapatite | |
Zhang et al. | Antimicrobial peptide-loaded pectolite nanorods for enhancing wound-healing and biocidal activity of titanium | |
CN113181431B (zh) | 形成于基材表面的抗菌及骨整合涂层及在基材表面制备抗菌及骨整合涂层的方法 | |
Chen et al. | Dual-functional antimicrobial coating based on the combination of zwitterionic and quaternary ammonium cation from rosin acid | |
CN113694255B (zh) | 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 | |
Tao et al. | N-halamine-based multilayers on titanium substrates for antibacterial application | |
Peng et al. | Quaternised chitosan coating on titanium provides a self-protective surface that prevents bacterial colonisation and implant-associated infections | |
Wu et al. | Novel dam-like effect based on piezoelectric energy conversion for drug sustained release of drug-loaded TiO2@ BaTiO3 coaxial nanotube coating | |
Zhou et al. | Surface configuration of microarc oxidized Ti with regionally loaded chitosan hydrogel containing ciprofloxacin for improving biological performance | |
Durdu et al. | Characterization and investigation of properties of copper nanoparticle coated TiO2 nanotube surfaces on Ti6Al4V alloy | |
Mumtaz et al. | Biological applications of biogenic silk fibroin–chitosan blend zinc oxide nanoparticles | |
Mallik et al. | Coating of chitosan onto bone implants | |
Wei et al. | Controllable AgNPs encapsulation to construct biocompatible and antibacterial titanium implant |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |