CN110974961A - 一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 - Google Patents
一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 Download PDFInfo
- Publication number
- CN110974961A CN110974961A CN201911318058.XA CN201911318058A CN110974961A CN 110974961 A CN110974961 A CN 110974961A CN 201911318058 A CN201911318058 A CN 201911318058A CN 110974961 A CN110974961 A CN 110974961A
- Authority
- CN
- China
- Prior art keywords
- amylase
- mno
- aqueous solution
- peg
- 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.)
- Granted
Links
Images
Classifications
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6923—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Biophysics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Composite Materials (AREA)
- Molecular Biology (AREA)
- Medical Informatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Ceramic Engineering (AREA)
- Inorganic Chemistry (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Manufacturing & Machinery (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用,该纳米复合材料是由淀粉酶(amylase)、聚乙二醇(PEG)、吲哚菁绿(ICG)修饰的MnO2纳米片(即MnO2‑amylase‑PEG‑ICG NSs,简称MAPI)。该材料可以通过amylase降解生物膜胞外聚合物(EPS),破坏生物膜结构,进而增强ICG的光热杀菌效果。本发明所提供的MAPI具有良好的稳定性和优异的生物膜内细菌杀灭作用,而且细胞毒性小,满足生物医药应用的基本条件。
Description
技术领域
本发明属于纳米抗生物膜技术领域,特别涉及一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用。
背景技术
细菌感染对人类健康构成严重威胁,每年在美国造成200多万例疾病和超过 23,000例死亡。其中,约80%的人类细菌感染病与活体组织上形成的生物膜有关。在生物膜中,细菌通过自我合成的EPS结合在一起,EPS通常由多糖、蛋白质和胞外DNA(eDNA)组成。在这种粘性和牢固框架的保护下,生物膜内的细菌对抗生素、宿主免疫防御和环境压力的抗性明显比游离细菌更强。细菌生物膜如果无法完全被根除,往往会导致持续感染,甚至死亡。因此,如何有效清除细菌生物膜是医疗界亟待解决的难题。
另外,近年来抗生素的滥用导致细菌产生耐药性,从而使抗生素疗效大大降低。因此,效果优异的新型抗菌剂越来越受到人们的关注。随着纳米生物医学的不断发展,细菌感染疾病的新疗法应运而生:将生物方法与纳米技术相结合,设计新型纳米抗菌材料,更好的治疗细菌生物膜感染病。当前纳米材料用于治疗细菌生物膜的主要策略包括(IEEE Trans.Nanobiosci., 2016, 15, 294.):(1)纳米材料作为载体,将药物递送到生物膜内部;(2)纳米材料自身作为抗菌剂,通过释放金属离子来破坏细菌的蛋白质或DNA;(3)通过基于纳米材料磁热或光热性质的热疗来杀死生物膜内的细菌;(4)在纳米材料表面修饰带负电的分子增强其对生物膜的渗透能力等。但是,目前的纳米技术中存在以下几个问题:缺乏对生物膜靶向能力、副作用较大、EPS的阻碍导致疗效较差。
发明内容
为了解决现有技术中的问题,本发明提供一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用,该方法选取具有在低pH条件下降解性能的MnO2纳米片作为载体,负载具有降解EPS功能的amylase、提高材料生物相容性的PEG及具有优异光热性能的ICG,制备MAPI纳米片。MAPI纳米片中的MnO2在生物膜的酸性环境下降解,释放amylase和ICG,降解EPS中的多糖以破坏细菌生物膜EPS中的多糖结构,同时增强近红外光热杀菌效果,从而实现细菌生物膜的有效清除。
为实现上述目的,本发明采用的技术方案为:
一种基于酶降解增强光热清除细菌生物膜的纳米复合材料,纳米复合材料为淀粉酶amylase、聚乙二醇PEG、吲哚菁绿ICG修饰的MnO2纳米片MAPI。
一种基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,包括以下步骤:
步骤1:制备MnO2纳米材料的水溶液;
步骤2:将MnO2纳米材料的水溶液加入淀粉酶amylase的水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase纳米片;
步骤3:将MnO2-amylase纳米片的水溶液加入聚乙二醇PEG水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase-PEG纳米片;
步骤4:将MnO2-amylase-PEG纳米片的水溶液加入吲哚菁绿ICG水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase-PEG-ICG纳米片。
优选地,所述步骤1中,MnO2纳米材料为MnO2纳米片, MnO2纳米片的粒径为20~200nm。
优选地,所述淀粉酶amylase为α-淀粉酶、β-淀粉酶、γ-淀粉酶、异淀粉酶的一种或多种;所述聚乙二醇PEG为mPEG-SH、mPEG-COOH、mPEG-NH2HCl、mPEG-OH的一种或多种。
优选地,所述步骤2中,MnO2纳米材料的水溶液的浓度为1~100 μg/mL,所述淀粉酶amylase的水溶液的浓度为10~1000 μg/mL;所述MnO2纳米材料的水溶液和amylase的水溶液的体积比为1:0.5~5。
优选地,所述步骤3中,MnO2-amylase纳米片的水溶液的浓度为1~100 μg/mL,所述聚乙二醇PEG的水溶液的浓度为10~1000 μg/mL,MnO2-amylase纳米片的水溶液和聚乙二醇PEG的水溶液的体积比为1:0.5~5。
优选地,所述步骤4中,MnO2-amylase-PEG纳米片的水溶液的浓度为1~100 μg/mL,所述吲哚菁绿ICG的水溶液的浓度为2~200 μg/mL,MnO2-amylase-PEG纳米片的水溶液和吲哚菁绿ICG的水溶液的体积比为1:0.5~5。
优选地,所述步骤2-4中,搅拌均在室温下采用磁力搅拌器进行,搅拌时间为0.5-12h;离心的条件为6000~18000 rpm,离心的时间为45 min;离心纯化的条件为6000~18000rpm,离心纯化的时间为45 min,离心纯化的次数为3次。
一种基于酶降解增强光热清除细菌生物膜的纳米复合材料的应用,所述纳米复合材料的抗菌表现为杀死生物膜内细菌,并清除细菌生物膜。
进一步的,当细菌为耐甲氧西林金黄色葡萄球菌MRSA时,MnO2-amylase-PEG-ICG纳米片的作用浓度为5~100 μg/mL。
与现有技术相比,本发明具有以下有益效果:
(1)、本发明不含抗生素,通过ICG的光热效果清除细菌,不易产生细菌耐药问题。
(2)、细菌生物膜微环境响应性能。MnO2纳米片可在细菌生物膜弱酸性条件下降解,实现amylase和ICG的可控释放,对于细菌生物膜具有特异性识别能力,有助于减小细菌清除过程中对正常细胞的毒副作用。
(3)、细菌生物膜EPS降解性能。amylase可以降解EPS中的多糖组分,能够破坏生物膜的整体结构,减少其对光热治疗试剂的阻碍作用,有利于将细菌暴露在周围环境中,从而增强ICG的光热杀菌效果。
(4)、低的毒副作用。本发明采用MnO2纳米片作为载体,降解后的Mn离子容易从身体排除,而天然的amylase也可以被人体降解,ICG是获得美国食品药品监督管理局(FDA)批准的试剂,潜在毒性较小。
附图说明
图1是本发明实施例1 验证MAPI纳米片的生物相容性的结果示意图;
图2是本发明实施例2验证MAPI纳米片的抗生物膜性能的结果示意图,其中:(a)和(c)分别是实施例2验证MAPI纳米片的抗生物膜性能的结晶紫染色照片及对应的生物膜生物量,(b)和(d)分别是实施例2验证MAPI纳米片的抗生物膜性能的涂板照片及涂板数据统计;
图3是本发明实施例3验证MAPI纳米片的抗生物膜性能的扫描电子显微镜(SEM)图。
具体实施方式
下面结合实施例对本发明作更进一步的说明。
实施例1
MnO2-amylase-PEG-ICG(MAPI)纳米片的制备
1.MnO2纳米片的制备
取0.6 g MnCl2·4H2O(3 mM)晶体于50 mL反应瓶中,加入10 mL H2O。取12 mL TMA·OH(四甲基氢氧化铵,12 mM)溶液,加入2 mL H2O2(30%wt),并加水稀释至20 mL。将以上两溶液混合,溶液由无色透明变棕色,在磁力搅拌器上搅拌反应12 h。反应结束后,将溶液取出,在3000 rpm下离心10 min,取下层沉淀。并用乙醇和水在上述离心条件下各清洗三次,最后加入水溶液定容至30 mL。取10 mL清洗好的MnO2,加入20 mL H2O,利用探头超声仪超声10 h(功率:40%,时间间隔:5 s),将得到的溶液用在12000 rpm下离心45 min,弃去下层沉淀,将上清液在18000 rpm下离心1.5 h取下层沉淀,重复离心三次,最终得到MnO2 纳米片。
2.MnO2-amylase(MA)纳米片的制备
将5 mL MnO2纳米片水溶液(100 μg/mL)加入5 mL amylase水溶液(1 mg/mL),在室温条件下置于磁力搅拌器上搅拌12 h。将得到的溶液在18000 rpm条件下离心45 min,纯化3次得到MA纳米片。
3.MnO2-amylase-PEG(MAP)纳米片的制备
将5 mL MA纳米片水溶液(100 μg/mL)加入5 mL PEG水溶液(1 mg/mL),在室温条件下置于磁力搅拌器上搅拌12 h。将得到的溶液在18000 rpm条件下离心45 min,纯化3次得到MAP纳米片。
4.MnO2-amylase-PEG-ICG(MAPI)纳米片的制备
将5 mL MAP纳米片水溶液(100 μg/mL)加入5 mL ICG水溶液(200 μg/mL),在室温条件下置于磁力搅拌器上搅拌4 h。将得到的溶液在18000 rpm条件下离心45 min,纯化3次得到MAPI纳米片。
实施例中,所述amylase选择为α-amylase,所述PEG选择mPEG-NH2HCl。
实施例2
MAPI纳米片的生物相容性
将HeLa细胞在含有FBS(10%)、青霉素(80 U/mL)和链霉素(0.08 mg/mL)的DMEM培养基中培养。将收集的对数期细胞加入96孔板(每孔100 μL,细胞密度调整至103-104/孔),置于充有5% CO2的37℃恒温箱中孵育。待细胞贴壁后,去除培养液,每孔分别加入100 μL含有不同浓度的MAP (MnO2: 0, 5, 10, 20, 40, 80, 160 μg/mL; amylase: 0, 18, 36, 72,144, 288, 576 μg/mL)与MAPI(MnO2: 0, 5, 10, 20, 40, 80, 160 μg/mL; amylase: 0,18, 36, 72, 144, 288, 576 μg/mL; ICG: 0, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 μg/mL)的DMEM培养基(每个浓度设5个复孔),然后置于充有5% CO2的37℃恒温箱中避光孵育24h。之后,每孔加入20 μL MTT溶液(5 mg/mL,即0.5%MTT),继续在恒温箱中避光孵育4 h。终止孵育,吸去上清液,每孔加入150 μL二甲基亚砜,使结晶物完全溶解,用酶标仪测定每孔在490 nm处的吸光值。此方法的HeLa细胞活力计算公式:S=C/C0×100%,其中C表示测试样品的OD490,C0表示空白对照的OD490。
从图1中显示的结果可以看出,通过MTT测定HeLa细胞与MAP、MAPI孵育24小时后细胞的相对细胞活力,可发现细胞活性均保持在88%以上。该实验结果表明MAP NSs、MAPI NSs具有优良的生物相容性。
实施例3
MAPI纳米片清除细菌生物膜的效果
划取单菌落耐甲氧西林金黄色葡萄球菌(MRSA,ATCC43300)于5 mL 液体LB 培养基中,在恒温摇床下孵育生长10-12 h(转速:220 rpm,温度:37℃),得到对数生长期的MRSA悬液。将菌液用生理盐水在下清洗三遍(离心条件:10000 rpm,3 min),利用酶标仪定量后(OD600= 0.1时,菌液浓度为1×107 CFU/mL)。将洗过的菌液用含1%葡萄糖的LB培养基稀释至1×108 CFU/mL,分别加入96孔板(每孔200 μL)、含有硅片的6孔板(每孔3 mL),置于37℃恒温箱中孵育2 d,得到MRSA生物膜。
(A)结晶紫染色法
参照图2a和2c,将在96孔板中长好的生物膜去除上清液,每孔分别加入100 μL生理盐水、MnO2、amylase、ICG、MAP、MAPI、MBP、MBPI,置于37℃恒温箱孵育3 h。对于光照组,使用785 nm激光器在0.8 W/cm2下光照10 min。去除上清液,每孔加入100 μL福尔马林。固定10min后,去除上清液,每孔加入100 μL 0.2%结晶紫染液。染色30 min后,每孔用生理盐水洗涤三次,然后在倒置显微镜下进行成像,成像结果如图2a所示。之后,每孔加入200 μL乙醇以溶解结晶紫,3 h后通过酶标仪测每孔在590 nm处的吸光度,细菌生物量如图2c所示。
从图2a和2c显示的结果可以看出,可以观察到MnO2、amylase、ICG、MAP、MAPI、MBP(MnO2-BSA-PEG,BSA对生物膜无降解作用)、MBPI(MnO2-BSA-PEG-ICG)在未光照或光照条件(785 nm激光器,0.8 W/cm2,光照10 min)降解MRSA生物膜的效果(生物膜可被结晶紫染成紫色,而被降解的生物膜被染成无色)。其中,在未光照条件下,amylase、MAP、MAPI均可对生物膜造成一定的降解作用,说明amylase在修饰到MnO2 纳米材料上后未对其酶活性造成损害。同时,可以观察到MAPI经过光照处理后相比未经过光照处理对MRSA生物膜的降解作用更加明显,说明MAPI的光热作用可以增强其对生物膜的降解作用。而对于光热效果相同的MAPI、MBPI来说,经过光照处理后,前者对生物膜的降解作用明显更好,说明amylase可以增强光热对生物膜的降解作用。
(B)稀释平板法
参照图2b和2d,将在96孔板中长好的生物膜去除上清液,每孔分别加入100 μL生理盐水、MnO2、amylase、ICG、MAP、MAPI、MBP(MnO2-BSA-ICG)、MBPI(MnO2-BSA-ICG-ICG)(材料均悬浮于生理盐水中),置于37℃恒温箱孵育3 h。对于光照组,使用785 nm激光器在0.8 W/cm2下光照10 min。去除上清液,每孔加200 μL生理盐水,混匀后加到1.5 mL离心管中,分别进行梯度稀释10、102、103、104、105、106倍,然后将100 μL原液及稀释104、105、106倍的菌液加到含有固体LB培养基的玻璃培养皿上进行涂板定量。
从图2d显示的结果可以看出,在未光照条件下,MnO2、amylase、ICG、MAP、MAPI、MBP、MBPI对MRSA生物膜几乎无杀菌作用;而在光照条件下,MAPI对MRSA生物膜的杀菌效果可达到接近2个数量级,MBPI对MRSA生物膜的杀菌效果可达到1个数量级左右,可以看出光热效果相同的MAPI比MBPI的杀菌效果高一个数量及左右,说明amylase降解生物膜可以增强MAPI的光热杀菌效果。
(C)扫描电子显微镜(SEM)观察法
参照图3,将长好的生物膜去除上清液,每孔分别加入1 mL生理盐水、MnO2、MAP、MAPI、MBP、MBPI(材料均悬浮于生理盐水中,MnO2:78 μg/mL; amylase:280 μg/mL; ICG:25 μg/mL),并使用785 nm激光器在0.8 W/cm2下光照10 min,置于37℃恒温箱孵育3 h。去除上清液,每孔加入1 mL福尔马林固定10 min,再用不同浓度(30%,50%,70%,80%,90%,95%)乙醇对样品进行梯度脱水处理,每种浓度处理15 min,再用100%的乙醇处理两次,每次20 min。将处理好的样品通过SEM成像。
从图3显示的结果可以看出,在光照条件下,MAPI、MBPI对MRSA的细胞结构造成了明显的损伤,同时可以看出光热效果相同的MAPI比MBPI的杀菌效果更好,说明amylase降解生物膜可以增强MAPI的光热杀菌效果。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,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 (10)
1.一种基于酶降解增强光热清除细菌生物膜的纳米复合材料,其特征在于,纳米复合材料为淀粉酶amylase、聚乙二醇PEG、吲哚菁绿ICG修饰的MnO2纳米片MAPI。
2.一种基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,包括以下步骤:
步骤1:制备MnO2纳米材料的水溶液;
步骤2:将MnO2纳米材料的水溶液加入淀粉酶amylase的水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase纳米片;
步骤3:将MnO2-amylase纳米片的水溶液加入聚乙二醇PEG水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase-PEG纳米片;
步骤4:将MnO2-amylase-PEG纳米片的水溶液加入吲哚菁绿ICG水溶液,并搅拌,将得到的溶液离心,将离心得到的沉淀分散在水中,继续离心纯化,得到MnO2-amylase-PEG-ICG纳米片。
3.根据权利要求4所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述步骤1中,MnO2纳米材料为MnO2纳米片,MnO2纳米片的粒径为20~200nm。
4.根据权利要求2所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述淀粉酶amylase为α-淀粉酶、β-淀粉酶、γ-淀粉酶、异淀粉酶的一种或多种;所述聚乙二醇PEG为mPEG-SH、mPEG-COOH、mPEG-NH2HCl、mPEG-OH的一种或多种。
5.根据权利要求2所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述步骤2中,MnO2纳米材料的水溶液的浓度为1~100 μg/mL,所述淀粉酶amylase的水溶液的浓度为10~1000 μg/mL;所述MnO2纳米材料的水溶液和amylase的水溶液的体积比为1:0.5~5。
6.根据权利要求2所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述步骤3中,MnO2-amylase纳米片的水溶液的浓度为1~100 μg/mL,所述聚乙二醇PEG的水溶液的浓度为10~1000 μg/mL,MnO2-amylase纳米片的水溶液和聚乙二醇PEG的水溶液的体积比为1:0.5~5。
7.根据权利要求2所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述步骤4中,MnO2-amylase-PEG纳米片的水溶液的浓度为1~100 μg/mL,所述吲哚菁绿ICG的水溶液的浓度为2~200 μg/mL,MnO2-amylase-PEG纳米片的水溶液和吲哚菁绿ICG的水溶液的体积比为1:0.5~5。
8.根据权利要求2所述的基于酶降解增强光热清除细菌生物膜的纳米复合材料的制备方法,其特征在于,所述步骤2-4中,搅拌均在室温下采用磁力搅拌器进行,搅拌时间为0.5-12h;离心的条件为6000~18000 rpm,离心的时间为45 min;离心纯化的条件为6000~18000rpm,离心纯化的时间为45 min,离心纯化的次数为3次。
9.根据权利要求2-8任一所述的方法得到的纳米复合材料的应用,其特征在于,所述纳米复合材料的抗菌表现为杀死生物膜内细菌,并清除细菌生物膜。
10.根据权利要求9所述的纳米复合材料的应用,其特征在于,当细菌为耐甲氧西林金黄色葡萄球菌MRSA时,MnO2-amylase-PEG-ICG纳米片的作用浓度为5~100 μg/mL。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911318058.XA CN110974961B (zh) | 2019-12-19 | 2019-12-19 | 一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911318058.XA CN110974961B (zh) | 2019-12-19 | 2019-12-19 | 一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110974961A true CN110974961A (zh) | 2020-04-10 |
CN110974961B CN110974961B (zh) | 2022-02-08 |
Family
ID=70063038
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911318058.XA Active CN110974961B (zh) | 2019-12-19 | 2019-12-19 | 一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110974961B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111714636A (zh) * | 2020-07-23 | 2020-09-29 | 重庆师范大学 | 一种光动力和光热协同杀菌的片状四氧化三锰纳米材料及其制备方法 |
CN113287607A (zh) * | 2021-04-08 | 2021-08-24 | 南京林业大学 | 光热剂PACP-MnO2薄膜及其制备方法和应用 |
CN113713089A (zh) * | 2021-09-18 | 2021-11-30 | 西北大学 | 可消除伤口处生物膜的可溶解微针贴片及制备方法和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018053646A1 (en) * | 2016-09-23 | 2018-03-29 | Klox Technologies Inc. | Biophotonic compositions, methods, and kits for inhibiting and disrupting biofilms |
CN108186676A (zh) * | 2018-03-05 | 2018-06-22 | 南京邮电大学 | 一种治疗伤口感染及促愈合的纳米抗菌凝胶及其制备方法 |
-
2019
- 2019-12-19 CN CN201911318058.XA patent/CN110974961B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018053646A1 (en) * | 2016-09-23 | 2018-03-29 | Klox Technologies Inc. | Biophotonic compositions, methods, and kits for inhibiting and disrupting biofilms |
CN108186676A (zh) * | 2018-03-05 | 2018-06-22 | 南京邮电大学 | 一种治疗伤口感染及促愈合的纳米抗菌凝胶及其制备方法 |
Non-Patent Citations (2)
Title |
---|
WEI LI等: ""Near-Infrared Light-Enhanced Protease-Conjugated Gold Nanorods As A Photothermal Antimicrobial Agent For Elimination Of Exotoxin And Biofilms"", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
杭银辉等: ""负载吲哚菁绿二氧化锰纳米片光热特性及影响因素研究"", 《影像研究与医学应用》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111714636A (zh) * | 2020-07-23 | 2020-09-29 | 重庆师范大学 | 一种光动力和光热协同杀菌的片状四氧化三锰纳米材料及其制备方法 |
CN111714636B (zh) * | 2020-07-23 | 2023-08-18 | 重庆师范大学 | 一种光动力和光热协同杀菌的片状四氧化三锰纳米材料及其制备方法 |
CN113287607A (zh) * | 2021-04-08 | 2021-08-24 | 南京林业大学 | 光热剂PACP-MnO2薄膜及其制备方法和应用 |
CN113713089A (zh) * | 2021-09-18 | 2021-11-30 | 西北大学 | 可消除伤口处生物膜的可溶解微针贴片及制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110974961B (zh) | 2022-02-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11981571B2 (en) | Identification and optimization of carbon radicals on hydrated graphene oxide for ubiquitous antibacterial coatings | |
Ji et al. | Enhanced eradication of bacterial/fungi biofilms by glucose oxidase-modified magnetic nanoparticles as a potential treatment for persistent endodontic infections | |
Cao et al. | Nanostructured titanium surfaces exhibit recalcitrance towards Staphylococcus epidermidis biofilm formation | |
Su et al. | Strong antibacterial polydopamine coatings prepared by a shaking-assisted method | |
CN110974961B (zh) | 一种基于酶降解增强光热清除细菌生物膜的纳米复合材料及其制备方法与应用 | |
Bing et al. | Hydrogen-producing hyperthermophilic bacteria synthesized size-controllable fine gold nanoparticles with excellence for eradicating biofilm and antibacterial applications | |
CN110464873B (zh) | 具有消除表面生物膜功能的医用钛植入体的制备方法 | |
Guo et al. | Black phosphorus nanosheets for killing bacteria through nanoknife effect | |
Tao et al. | Zn‐incorporation with graphene oxide on Ti substrates surface to improve osteogenic activity and inhibit bacterial adhesion | |
Wu et al. | Interdisciplinary‐inspired smart antibacterial materials and their biomedical applications | |
Tong et al. | Synthesis of DNA-guided silver nanoparticles on a graphene oxide surface: Enhancing the antibacterial effect and the wound healing activity | |
Zhang et al. | In situ assembly of well-dispersed Ag nanoparticles on the surface of polylactic acid-Au@ polydopamine nanofibers for antimicrobial applications | |
Yang et al. | Fabrication of graphene oxide/copper synergistic antibacterial coating for medical titanium substrate | |
CN105596367A (zh) | 以壳聚糖-泊洛沙姆为凝胶基质的纳米银抗菌凝胶及其制备方法和应用 | |
Xu et al. | A poly (hydroxyethyl methacrylate)–Ag nanoparticle porous hydrogel for simultaneous in vivo prevention of the foreign-body reaction and bacterial infection | |
CN108310392B (zh) | 一种医用氧化石墨烯抗菌剂的制备方法 | |
Huang et al. | Functional modification of polydimethylsiloxane nanocomposite with silver nanoparticles-based montmorillonite for antibacterial applications | |
CN109108275B (zh) | 氨基糖修饰的抗菌金纳米颗粒、其制备方法及应用 | |
Dalavi et al. | Microspheres containing biosynthesized silver nanoparticles with alginate-nano hydroxyapatite for biomedical applications | |
Ali et al. | Graphdiyne–hemin-mediated catalytic system for wound disinfection and accelerated wound healing | |
Yuwen et al. | Amylase degradation enhanced NIR photothermal therapy and fluorescence imaging of bacterial biofilm infections | |
Huang et al. | Magnetic-controlled dandelion-like nanocatalytic swarm for targeted biofilm elimination | |
Li et al. | CuS nanoenzyme against bacterial infection by in situ hydroxyl radical generation on bacteria surface | |
Lin et al. | Silk fibroin-based coating with pH-dependent controlled release of Cu2+ for removal of implant bacterial infections | |
Samberg et al. | In vitro biocompatibility and antibacterial efficacy of a degradable poly (L-lactide-co-epsilon-caprolactone) copolymer incorporated with silver nanoparticles |
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 | ||
CB03 | Change of inventor or designer information | ||
CB03 | Change of inventor or designer information |
Inventor after: YuWen Lihui Inventor after: Gan Siyu Inventor after: Wang Lianhui Inventor after: Xiu Wei Jun Inventor after: Qiu Qiu Inventor before: YuWen Lihui Inventor before: Wang Lianhui Inventor before: Gan Siyu Inventor before: Xiu Wei Jun Inventor before: Qiu Qiu |
|
GR01 | Patent grant | ||
GR01 | Patent grant |