单硝酸异山梨酯靶向纳米颗粒杀灭慢性鼻-鼻窦炎金黄色葡萄球菌生物膜机制的研究

Chrionic rhinosinusitis (CRS) is a common disease in rhinology. Biofilms are considered a common and important cause of persistent infections. Although received techinically successful endoscopic sinus surgery (ESS) and standard comprehansive medical treatment, some patients still suffer from persistent symptoms. In the field of rhinology，it has proven that Staphylococcus aureus biofilms are associated with the unfavorable evolution after ESS, more severe disease parameters, and intracellular infection, suggesting that S. aureus biofilms may contribute greatly to the recalcitrant nature of CRS. Nitric Oxide (NO), a gas naturally produced in high concentrations in healthy sinuses, is found in significantly lower concentrations in CRS patients. NO has demonstrated antibacterial properties, plays a role in innate immunity and mucociliary clearance and is thought to provide a significant contribution to maintaining the balance of normal flora in non-diseased sinuses. .Preliminary data showed that NO can effectively eliminate S. aureus biofilms in vitro. Active targeting presents a promising approach to the purpose of the high-efficiency “on-site” drug delivery, which can be realized by conjugating molecular probes or antibodies that specifically target the biofilm onto the surface of the nanoparticles encapsulating the NO donor, isosorbide mononitrate (ISMN)..Repeated and prolonged antibiotic therapy becomes a global concern with emergence of drug-resistant strains.In this project, experimental methods including fluorescence labelling,immunohistochemistry, fluorescence in situ hybridization (FISH) and observation with confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) will be applied to study the mechanism, efficacy and safety of nanoparticle both in vitro and in vivo. To date, there are no published papers regarding the effects of ISMN loaded nanoparticles conjugated with anti-Staphylococcus alpha-toxin on S. aureus biofilms in the in vitro model or in vivo rhinosinusitis sheep model. This work would be a prior study in the treatment of recalcitrant CRS.

慢性鼻-鼻窦炎（CRS）是鼻科常见病，部分CRS患者虽经过鼻内镜鼻窦手术及综合规范化治疗，其临床疗效并未明显改善。已证实金黄色葡萄球菌生物膜是CRS重症化和难治化的重要原因之一。我们的前期试验证实一氧化氮能够有效杀伤金葡菌生物膜。本研究针对目前综合治疗对金葡菌生物膜阳性CRS患者疗效欠佳的现状，提出生物膜特异性主动靶向纳米颗粒能够安全有效杀灭金葡菌生物膜的假说，为了验证这一假说，我们拟构建金葡菌α毒素抗体修饰的包裹一氧化氮供体药物单硝酸异山梨酯（ISMN）的纳米颗粒，并用不同方法进行修饰，产出不同体积和电性的纳米颗粒；通过荧光标记，免疫组化，荧光原位杂交，激光共聚扫描显微镜及扫描电镜等方法，在细胞、组织（体外）及慢性鼻-鼻窦炎绵羊动物模型体内等进行实验，探讨新型主动靶向纳米颗粒杀灭金葡菌生物膜的作用机制及治疗金葡菌生物膜相关CRS的可行性，为难治性CRS治疗提供新的策略。

慢性鼻窦炎是鼻科常见疾病，严重影响患者的生活质量。金黄色葡萄球菌生物膜加重疾病的炎症负荷，对鼻窦手术疗效产生不良影响，增加术后复发几率，是造成慢性鼻窦炎难治性的重要原因。本项目构建特异性针对金黄色葡萄球菌生物膜的靶向纳米颗粒，为慢性鼻窦炎的治疗提供了新的思路。我们构建pET28a/Hla重组质粒，将HLA蛋白进行原核表达，以纯化的HLA重组蛋白为抗原免疫BALB/c小鼠，融合小鼠脾脏细胞与骨髓瘤细胞SP2/0，杂交瘤细胞株以BALB/c小鼠为载体制备了金黄色葡萄球菌α毒素单克隆抗体（AA），以其为特异性分子探针识别金黄色葡萄球菌。我们发现AA有较强的特异性，可以做为靶向基团连接到纳米颗粒上。进而我们首先采用单因素优化制备单硝酸异山梨酯（ISMN）脂质体，正交试验设计筛选出最优的处方，通过戊二醛交联的方法将特异性抗体与纳米颗粒进行连接，制备了ISMN脂质体靶向纳米颗粒。在此基础上我们制备了ISMN-聚乙交酯丙交酯纳米颗粒，通过聚乙烯乙二醇将特异性抗体与纳米颗粒进行连接，制备ISMN-聚乙交酯丙交酯-聚乙烯乙二醇-金黄色葡萄球菌α毒素抗体（ISMN-PLGA-PEG-AA）纳米颗粒。然后检测两种特异性纳米颗粒的理化特征，发现它们的粒径大小均一(ISMN脂质体:136.8±7.414nm,ISMN-PLGA-PEG-AA:176.9±48.46nm)，表面电位分布均匀(ISMN脂质体:-1.145±0.095,ISMN-PLGA-PEG-AA:-26.4±8.77mV)，药物包封率较高(ISMN脂质体:19.03±1.595％,ISMN-PLGA-PEG-AA:21.60±2.162%)，长期保存稳定性良好。在体外抗生物膜实验中，纳米颗粒可以将生物膜的结构彻底破坏(96.67±2.08％)。为了验证两种纳米颗粒体内杀灭生物膜效果，我们分别构建了家兔和绵羊金黄色葡萄球菌生物膜相关慢性鼻窦炎模型，并用纳米颗粒进行干预，证实靶向纳米颗粒可以清除生物膜降低体内炎症反应（45mg/ml ISMN免疫脂质体和ISMN-PLGA-PEG-AA冲洗一周后IL-4、IL-8、IL-17和IFN-γ的水平和mRNA表达水平明显降低，且降低程度均优于单纯药物），在慢性鼻窦炎及金黄色葡萄球菌生物膜所造成的感染性疾病的治疗中具有广阔的前景。