载庆大霉素壳聚糖磁纳米微粒治疗骨关节感染的基础研究

Bone and joint infection is a refractory disease that is associated with progression and recurrence. With increasing numbers of foreign bodies implanted in orthopedic surgery, such as prosthetic joints,implant-related infection poses a threat to patients. The bacterial biofilms is believed to be an important reason to cause the recurrence of chronic osteomyelitis. The phenomenon that bacteria can be internalized by the osteoblast was considered to be another mechanism for the persistence and recurrence of chronic osteomyelitis. Bacteria internalized by the osteoblast may be sequestered from the majority of antibiotics as well as the immune system. Since S. aureus is capable of remaining alive in the osteoblast for a long period of time, the bacteria released from the dead osteoblast may induce the recurrence of osteomyelitis. Most antibiotics can not penetrate into the eukaryotic cell membrane or bacterial biofilms. Intravenous antibiotics use is often ineffective to achieve high concentration in local infections era. In response to these problems, we prepare drug-loaded magnetic nanoparticles by chemical co-precipitation method and study their effect in bone and joint infection. Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. In our early vitro study, we concluded that use of a low concentration of magnetic iron oxide nanoparticles was safe to osteoblasts. Chitosan is used to coat on iron oxide nanoparticles and their cytocompatibility with osteoblasts increase. Due to its characteristics, gentamicin loaded chitosan magnetic nanoparticles would accumulate in local areas when an external magnetic field is applied and the antibiotics concentration in bacteria biofilm would increase. Gentamicin loaded chitosan magnetic nanoparticles would enter into eukaryotic cell by endocytosis and expected to kill intracellular bacteria. Therefore, gentamicin loaded chitosan magnetic nanoparticles provide a new method of drug delivery and infection treatment. Experiments will also be performed in vivo to study gentamicin loaded chitosan magnetic nanoparticles in treating bone and joint infection.

慢性骨关节感染易复发，抗生素是治疗感染的重要手段，但静脉应用抗生素往往不能在感染局部达到有效浓度，多数抗生素存在无法透过细胞膜和细菌生物膜的缺陷。针对上述问题，本课题首次提出使用磁纳米颗粒在外磁场作用下靶向治疗骨关节感染；通过化学共沉淀法设计一种安全、靶向、缓释的抗生素磁纳米药物。将Fe3O4的表面包裹壳聚糖后增加了磁纳米微粒与成骨细胞的细胞相容性；并利用磁纳米药物的优点和特有的磁场响应性使其在特定部位释放，有利于进入细菌生物膜内部以提高生物膜局部的药物浓度。利用细胞的胞吞作用介导抗生素进入感染的成骨细胞内，为杀灭胞内菌提供新的药物传递方式。载庆大霉素壳聚糖磁纳米微粒有望针对细菌生物膜和成骨细胞的胞内感染起到良好的治疗作用，这为临床上治疗慢性骨髓炎和假体感染、减少复发提供了一种药物改性修饰的新思路；同时，通过动物体内实验对载药磁纳米微粒的应用进行有益探索和尝试。

骨与关节的感染长期以来一直是困扰骨科医生的难题。细菌生物膜被认为是引起慢性骨髓炎复发和不愈的重要原因。近年来发现金葡菌能够入侵到成骨细胞内而长期存活引起细胞内感染。并可以随着成骨细胞的死亡而释放引起感染的复发。磁纳米药物系统可以靶向定植于特定的部位而释放，在靶向给药方面具有独特的优势因而在治疗感染方面具有应用前景。本课题针对目前临床上骨科慢性感染难治的特点。制备载抗生素磁纳米微粒，旨在对目前引起临床上骨科慢性感染的两大机制（生物膜和成骨细胞内细菌）应用磁纳米载药技术在体外实验水平上检验磁纳米药物治疗骨科感染的可能性。通过制备壳聚糖磁纳米粒子，并对其进行表征。通过不同浓度的磁纳米粒子与人成骨细胞体外地共培养；通过观察和检测成骨细胞的增殖和分化指标包括透射电镜、普鲁士蓝染色、总蛋白测定、AKP、LDH、细胞外钙离子沉积等来检验包埋壳聚糖后的磁纳米粒子对成骨细胞的细胞相容性。用金黄色葡萄球菌（ATCC 25923）作为病原菌制备静态生物膜，用扫描电镜、共聚焦显微镜、生物膜内活细菌计数、结晶紫染色定量等方法检测壳聚糖磁纳米微粒对金葡菌生物膜形成的影响。 在体外用金葡菌感染成骨细胞建立成骨细胞的细胞内感染模型；将载庆大霉素壳聚糖磁纳米粒子加入感染金葡菌的成骨细胞培养24小时后，用透射电镜观察细胞内金葡菌的形态学变化并裂解成骨细胞计数细胞内存活金葡菌的数量。结果显示壳聚糖包埋的四氧化三铁磁纳米粒子对成骨细胞具有良好的细胞相容性。根据成骨细胞的分化指标实验浓度的壳聚糖磁纳米粒子对成骨细胞有良好的生物相容性。壳聚糖磁纳米粒子可以抑制金葡菌生物膜的形成，增加生物膜中死细菌的比例，减少生物膜活细菌的数量，降低生物膜的厚度。载庆大霉素的壳聚糖磁纳米微粒对成骨细胞内的金葡菌有显著的杀灭作用。同时对兔胫骨骨髓炎具有一定的治疗作用。本课题为临床上治疗慢性骨髓炎和假体感染提供了一定的实验基础和新的治疗思路。