【作者】 杨静；

【导师】 吴富根；

【作者基本信息】 东南大学 ， 生物医学工程， 2023， 硕士

【摘要】 抗生素滥用导致治疗细菌感染的有效药物减少,由此造成的细菌感染治疗失败成为威胁人类生命安全的严峻挑战。有研究报道,细菌被杀伤后其表面形貌的保留是细菌产生耐药性的重要诱因之一,因此开发可对细菌外表面结构造成破坏并导致细菌死亡的新型抗菌药物将成为应对该挑战的有效方案。含季铵盐基团的分子/材料可以破坏细菌外膜/壁结构,同时二氧化硅纳米颗粒具有良好的生物安全性,因此制备含季铵盐基团的二氧化硅纳米颗粒用于开发新的抗菌材料具有重要意义。本论文选取二甲基十八烷基[3-（三甲氧基硅基）丙基]氯化铵（Si-QAC）作为主要原料通过一步加热搅拌的方法合成季铵化二氧化硅材料,并探究其抗菌效果和应用。具体工作如下:（1）利用Si-QAC和四乙氧基硅烷（TEOS）合成具有超高产率的二氧化硅抗菌纳米颗粒:本工作将TEOS和Si-QAC加入纯水中,并通过一步加热搅拌法成功获得带有长链季铵盐基团的二氧化硅纳米颗粒（nanoparticle,NP）。当TEOS和Si-QAC的投料摩尔比为4:1时获得的纳米颗粒（命名为TS4 NPs）粒径均一（～33.5 nm）且呈球形,并具有良好的水分散性、稳定性（室温下可保存超过520天）和生物安全性。实验结果表明,带长链烷烃季铵盐基团的TS4 NPs可以通过静电吸附和疏水相互作用致密地包裹在金黄色葡萄球菌表面,并诱导细菌聚集形成微米级聚集体;同时长链烷烃季铵盐基团可插入细胞磷脂双分子层破坏细菌外膜结构,进而诱导细胞内产生大量活性氧并裂解细菌DNA,导致细菌最终死亡。同时,TS4 NPs也可以有效地清除并抑制由金黄色葡萄球菌所形成的生物膜。（2）季铵盐基团与其他抗菌手段的联合抗菌:（a）与光敏剂联合:首先用3-氨丙基三乙氧基硅烷（APTES）与TEOS和Si-QAC成功合成了三组分二氧化硅纳米颗粒,并成功接枝上孟加拉玫瑰红（RB）,但涂板实验的结果表明RB与二氧化硅纳米颗粒之间并未形成明显的联合抗菌效果。（b）与抗生素联合:为避免抗生素的羧基中和来自Si-QAC的季铵盐基团的正电,因此选取3-（2,3-环氧丙氧）丙基三甲氧基硅烷（GPTMS）与TEOS、Si-QAC和万古霉素一起制备得到接枝有万古霉素的纳米颗粒并命名为TSG-Van NPs,而涂板结果显示,当TSG-Van NP杀死>99.99%的细菌时,所对应浓度的游离万古霉素对细菌并未产生明显抗菌效果,因此不能断言万古霉素是否在其中产生联合的抗菌效果。（c）与环氧基联合:使用TEOS、Si-QAC和GPTMS合成三组分二氧化硅,通过涂板实验以及最小抑菌/抗菌浓度的测定发现,当投料比GPTMS:Si-QAC:TEOS=4:2:4时制备所得的TSG₄₂₄NPs相比于通过其他投料比所制得的纳米颗粒而言具有最好的抗菌效果,且其抗菌效果明显优于TS4 NPs。此外,用同样的方法以TEOS和GPTMS为原料未形成良好的分散液,说明Si-QAC在制备具有良好水分散性的纳米颗粒中必不可少。综上所述,本论文基于Si-QAC成功设计合成了两种二氧化硅抗菌纳米颗粒,并对它们进行了表征。两种纳米颗粒均展示出优异的抗菌和抗生物膜效果,环氧基的加入也大大提升了季铵化纳米颗粒的抗菌效果。预期本论文能为纳米抗菌材料的开发提供新的启发。更多还原

【Abstract】 The number of effective drugs that can treat bacterial infections is decreasing because of the abuse of antibiotics,which leads to the failures of bacterial infection therapy and is thus becoming a serious threat to human life.Preservation of the intact cell morphology of bacteria is recognized as one of the most important causes of bacterial drug resistance,and therefore developing new antibacterial agents capable of fighting against bacteria via disrupting their cell envelope is highly desirable.Considering that the molecules/materials containing quaternary ammonium groups can disrupt the bacterial outer membrane/wall structure and silica nanoparticles have good biocompatibility,it is important to prepare silica nanoparticles containing quaternary ammonium groups for the development of new antibacterial materials.In this thesis,we chose dimethyloctadecyl[3-（trimethoxysilyl）propyl]ammonium chloride（Si-QAC）as a main raw material to fabricate silica nanomaterials via a one-step reaction under heating and stirring,and investigated their antibacterial activities and applications.This thesis includes the following two chapters:（1）Antimicrobial silica nanoparticles with an ultra-high yield synthesized by Si-QAC and tetraethoxysilane（TEOS）:In this work,TEOS and Si-QAC were dissolved in deionized water and silica nanoparticles（termed TS4 NPs）with long-chain quaternary ammonium groups were obtained by a one-step heating and stirring method.The nanoparticles were prepared at a TEOS/Si-QAC ratio of 4:1,and had an average particle size of～33.5 nm,good water dispersity,aqueous stability（at least 520 days at room temperature）,and biocompatibility.The experimental results showed that the TS4 NPs with long alkyl chain（C18）-bearing quaternary ammonium groups could densely coat the surface of S.aureus through electrostatic adsorption and hydrophobic interaction,induce the aggregation of bacteria to form micrometer-sized aggregates to exert their cell wall/membrane damaging effect,and induce the production of intracellular reactive oxygen species and bacterial DNA destruction,finally leading to the death of the bacteria.Meanwhile,the TS4 NPs could effectively eradicate mature S.aureus biofilms and inhibit the growth of S.aureus biofilms.（2）Combined use of quaternary ammonium group and other antibacterial methods:（a）In combination with photosensitizers:the silica nanoparticles were synthesized with3-aminopropyltriethoxysilane（APTES）,TEOS,and Si-QAC,and then successfully grafted with rose bengal（RB）.However,the results of agar plate count assay suggested that,the silica nanoparticles combined with RB did not exhibit higher antibacterial activity.（b）In combination with antibiotics:To avoid the neutralization between the positively charged quaternary ammonium group of Si-QAC and the negatively charged carboxyl group of the antibiotic,3-（2,3-epoxypropoxy）propyltrimethoxysilane（GPTMS）was selected to obtain TSG-Van NPs together with TEOS,Si-QAC,and vancomycin.The results of agar plate count assay indicated that when the TSG-Van NPs killed>99.99%of bacteria,the corresponding concentration of free vancomycin could not kill any bacteria.Therefore,we cannot determine whether vancomycin exerted antibacterial activity or not.（c）In combination with epoxy groups:The silica nanoparticles were synthesized using TEOS,Si-QAC,and GPTMS.The results of agar plate count assay and the determination of minimum inhibitory/bactericidal concentration values showed that the TSG₄₂₄NPs（obtained at a feeding ratio of GPTMS:Si-QAC:TEOS=4:2:4）had a better antimicrobial effect compared with TS4 NPs.Moreover,TEOS and GPTMS could not form a product with good water dispersity,indicating that Si-QAC is essential in the preparation of nanoparticles.In this thesis,we successfully designed and obtained two kinds of antibacterial silica nanoparticles with Si-QAC,and then characterized the properties of the nanoparticles.The two kinds of nanoparticles possessed great antibacterial and anti-biofilm activities,and the addition of epoxy groups dramatically enhanced the antibacterial activity of the quaternized nanoparticles.It is expected that this thesis may provide new insight into the development of antibacterial nanomaterials.更多还原