兰州大学机构库 >口腔医学院
自适应纳米诊疗系统用于预防与治疗植入物感染的研究
Alternative TitleApplication of self-adaptive theranostics nanosystem in the prevention and treatment of implant-related infections
易俊丹
Subtype硕士
Thesis Advisor唐荣冰
2023-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline口腔医学
Keyword植入物感染 Implant-related infections 自适应纳米诊疗系统 Self-adaptive theranostics nanosystem 相变材料 Phase change materials 细菌生物膜 Bacterial biofilm 免疫记忆 Immune memory
Abstract

随着医疗技术和生物材料的不断进步,越来越多的医疗植入物被应用于外科手术。然而,罹患手术并发症的人数逐年上升,其中植入物感染是生物材料植入术后最常见和最严重的并发症之一,它可能导致植入物失效,甚至导致残疾和死亡。金黄色葡萄球菌(Staphylococcus aureus, S. aureus)是植入物感染中最常见的致病菌,作为一种广泛存在于人类皮肤和医院环境中的细菌,S. aureus可以在围手术期进入手术部位定植导致植入物周围的早期感染,也可以从其他部位通过血源性扩散至植入物区域引发迟发性感染。此外,S. aureus生物膜产生的乳酸可以抑制巨噬细胞中的组蛋白去乙酰化酶11(HDAC11)活性,从而提高白介素-10(IL-10)等抗炎细胞因子的转录水平。这些细胞因子促使巨噬细胞的极化状态从M1促炎表型转变为M2抗炎表型,有利于致病菌的生存并容易导致感染的复发。因此,研发彻底清除生物膜并逆转免疫抑制的新型策略是预防和治疗植入物感染亟待解决的重要临床问题。

目的:本实验旨在设计一种基于相变材料的自适应纳米诊疗系统CaAlg/LOD/POD@MNO/FAs(CLPM),探究其表征、温控药物释放性能、酶促反应性能、细胞相容性、体外抗菌性能、对巨噬细胞极化的影响及机制,以及体内用于治疗和预防植入物感染的效果。

方法:

使用机械搅拌和离子交联的方法合成自适应纳米诊疗系统CLPM,通过激光粒度仪检测、透射电镜(TEM)观察、傅里叶变换红外光谱仪(FTIR)检测、荧光标记及激光扫描共聚焦显微镜(CLSM)观察对CLPM的结构、形貌等进行表征。

通过观察多次相变循环后的TEM图像、检测在含血清培养基中的7天内粒径变化和测定zeta电位,验证CLPM的稳定性。通过体外温控药物释放实验验证CLPM的温度响应性,通过酶促反应实验验证CLPM对乳酸的降解能力,通过细胞形态观察和CCK8实验证明CLPM的细胞相容性。

通过细菌活/死染、平板涂布法、微观结构观察、流变性能检测等方法,使用流式细胞仪、扫描电子显微镜、透射电子显微镜、激光扫描共聚焦显微镜、流变仪等仪器,探讨了CLPM分别在37°C和39°C下对S. aureus的浮游状态和生物膜状态的抗菌作用。

通过使用不同处理后的S. aureus生物膜上清液与巨噬细胞共培养,利用免疫荧光染色方法在CLSM下观察不同表型的巨噬细胞的标记物(iNOS: M1; Arg-1: M2),同时使用酶联免疫吸附(ELISA)方法测定巨噬细胞上清液中不同细胞因子(IL-6、TNF-α、IL-10和TGF-β)的变化判断CLPM在体外对巨噬细胞极化的影响。此外,建立小鼠皮下植入物感染模型,取经过不同处理后的小鼠植入物周围组织进行免疫荧光染色,判断CLPM在体内对巨噬细胞极化的作用。

建立小鼠皮下植入物感染模型和大鼠胫骨内植入物感染模型,研究CLPM在体内对植入物感染的治疗作用。

建立小鼠皮下植入物感染复发模型,研究CLPM在体内对感染复发的预防作用以及对记忆B细胞、效应B细胞比例、血清IgG和IL-1β水平的影响。对CLPM处理后的巨噬细胞进行转录组学分析,研究CLPM促进免疫记忆的机制。对小鼠皮下植入物感染模型中的CLPM治疗组的钛片周围组织进行总蛋白提取,使用蛋白质免疫印迹法研究关键信号通路相关蛋白的表达情况。

结果:本实验完成了自适应纳米诊疗系统CLPM的合成,并验证了其具有良好的核壳结构,具有较好的稳定性、分散性、温度响应性、乳酸降解能力、细胞相容性等。CLPM具有优秀的体外抗浮游细菌和抗生物膜性能,在体内与体外均可逆转巨噬细胞的极化方向使其向M1表型转换。动物实验证明,CLPM可以有效治疗植入物感染,并可以预防植入物感染的复发。转录组学研究证明,CLPM对免疫记忆的促进作用可能源自共刺激分子与促炎细胞因子表达上调,参与抗原呈递与处理的几种信号通路如NOD样受体信号通路、TNF信号通路和MAPK信号通路被激活。对CLPM治疗组小鼠的钛片周围组织进行蛋白质免疫印迹分析,结果显示JNK和p38磷酸化水平和促炎细胞因子IL-6、IL-1β和TNF-α表达水平提高,进一步验证了CLPM在体内对相关信号通路的促进作用。

结论:CLPM可以在体内外均可有效清除生物膜、逆转免疫抑制并促进免疫记忆,在植入物感染的预防与治疗中展现了巨大的应用潜力。

Other Abstract

As medical technology and biomaterials continue to advance, more and more medical implants are being used in surgical procedures. However, the number of surgical complications is increasing every year, and one of the most common and serious complications after biomaterial implantation is implant-related infections (IRIs), which can lead to implant failure and even disability and death. Staphylococcus aureus (S. aureus) is the most common causative agent of IRIs. As a bacterium widely present in human skin and hospital environments, S. aureus can enter the surgical site during the perioperative period to colonize the surgical site and cause early infection around the implant, or it can cause delayed infection from other sites through hematogenous spread to the implant area. In addition, lactate produced by S. aureus biofilms can inhibit histone deacetylase 11 (HDAC11) activity in macrophages, thereby increasing the transcriptional levels of anti-inflammatory cytokines such as interleukin-10 (IL-10). These cytokines contribute to a shift in the polarization state of macrophages from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype, which favors the survival of pathogenic bacteria and predisposes to the recurrence of infection. Therefore, the development of novel strategies for complete biofilm clearance and reversal of immunosuppression is an urgent and important clinical problem for the prevention and treatment of IRIs.

Objective: This experiment aims to design a phase change material-based self-adaptive theranostics nanosystem CaAlg/LOD/POD@MNO/FAs (CLPM) and investigate its characterization, temperature-controlled drug release performance, enzymatic response performance, cytocompatibility, in vitro antibacterial performance, effect on macrophage polarization and mechanism, and in vivo use for treatment and prevention of implant-related infections.

Methods:

(1) The self-adaptive theranostics nanosystem CLPM was synthesized by mechanical stirring and ionic cross-linking, and the structure and morphology of CLPM were characterized by laser particle sizer analysis, transmission electron microscopy (TEM) observation, Fourier transform infrared spectroscopy (FTIR) detection, fluorescence labeling and confocal laser scanning microscopy (CLSM) observation.

(2) The stability of CLPM was verified by observing TEM images after multiple phase change cycles, detecting particle size changes in serum-containing medium for 7 days, and measuring zeta potential. The temperature responsiveness of CLPM was verified by in vitro temperature-controlled drug release assay. The degradation ability of CLPM to lactic acid was verified by enzymatic reaction assay, and the cytocompatibility of CLPM was demonstrated by cell morphology observation and CCK8 assay.

(3) The antibacterial effects of CLPM on on the planktonic and biofilm states of S. aureus at 37°C and 39°C were investigated by bacterial live/dead staining, spread plate method, microstructure observation, and rheological performance assay using flow cytometry, scanning electron microscope (SEM), TEM, CLSM, and rheometer, respectively.

(4) By using different treated S. aureus biofilm supernatants co-cultured with macrophages, markers of different phenotypes of macrophages (iNOS: M1; Arg-1: M2) were observed under CLSM using immunofluorescence staining method, while different cytokines (IL-6, TNF -α, IL-10 and TGF-β) in macrophage supernatants were measured by enzyme-linked immunosorbent assay (ELISA) to determine the effect of CLPM on macrophage polarization in vitro. In addition, a mouse subcutaneous implant-related infection model was established, and immuno- fluorescence staining was performed on the peri-implant tissues of mice after different treatments to determine the effect of CLPM on macrophage polarization in vivo.

(5) A mouse subcutaneous implant-related infection model and a rat intra-tibial implant-related infection model were established to study the therapeutic effect of CLPM on IRIs in vivo.

(6) A mouse subcutaneous implant-related infection recurrence model was established to study the preventive effect of CLPM on infection recurrence in vivo and the effects on the ratio of memory B cells, effector B cells, serum IgG and IL-1β levels. Transcriptomic analysis of CLPM-treated macrophages was performed to investigate the mechanism of CLPM in promoting immune memory. Total protein extraction was performed on peri-implant tissues of the CLPM-treated groups in a mouse subcutaneous implant infection model to study the expression of proteins related to key signaling pathways by Western blot.

Results: In this experiment, we have completed the synthesis of CLPM, a  self-adaptive theranostics nanosystem, and verified that it has an excellent core-shell structure with good stability, dispersion, temperature responsiveness, lactate degradation ability, and cytocompatibility. CLPM has excellent in vitro anti-planktonic bacterial and anti-biofilm properties, and can reverse the polarization direction of macrophages in vivo and in vitro to convert them to the M1 phenotype. Animal studies have demonstrated that CLPM can effectively treat implant infections and prevent the recurrence of implant infections. Transcriptomic studies demonstrated that the promotion of immune memory by CLPM may originate from the upregulation of co-stimulatory molecules and pro-inflammatory cytokine expression, and several signaling pathways involved in antigen presentation and processing, such as NOD-like receptor signaling pathway, TNF signaling pathway and MAPK signaling pathway, were activated. Protein immunoblot analysis of the titanium periplasmic tissues from CLPM-treated mice showed increased levels of JNK and p38 phosphorylation and expression of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, further verifying the promoting effect of CLPM on related signaling pathways in vivo.

Conclusion: CLPM can effectively clear biofilms, reverse immunosuppression and promote immune memory both in vitro and in vivo, showing great potential for application in the prevention and treatment of implant-related infections.

MOST Discipline Catalogue医学 - 口腔医学
URL查看原文
Language中文
Other Code262010_220200927160
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/535724
Collection口腔医学院
Affiliation
兰州大学口腔医学院
Recommended Citation
GB/T 7714
易俊丹. 自适应纳米诊疗系统用于预防与治疗植入物感染的研究[D]. 兰州. 兰州大学,2023.
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