Micrococcal nuclease-triggered antibiotics release: a prophylactic implant coating against S. aureus infections

项目摘要/摘要 假体周围感染是骨科手术中最严重的并发症之一，发生在1-4%的 初次全关节置换和高达30%的翻修。金黄色葡萄球菌(S. 金黄色葡萄球菌)是骨科感染中最常见的微生物罪魁祸首，由于它们的 有在种植体上形成生物膜的倾向，以及臭名昭著的侵入周围小管网络的能力 骨头。现有的预防性抗生素注射涉及不安全但无效的大剂量药物和 可能会导致抗药性的发展。利用对金黄色葡萄球菌不稳定的寡核苷酸连接子 微球菌核酸酶(MN)裂解，我们最近开发了一种水凝胶，能够按需释放 共价束缚的万古霉素。当作为水凝胶涂层应用于Ti6Al4V髓内钉时， 将MN植入接种金黄色葡萄球菌的小鼠股动脉内，及时触发万古霉素释放 在植入物表面和IM空间内杀死细菌，然后才有机会定居或入侵 包围骨骼，从而在3周内防止生物膜形成和骨髓炎发展 检查过了。这种涂层中万古霉素的共价拴系剂量比 临床使用的典型预防性抗生素含量。拟议研究的目标是进一步设计这一点。 激发按需药物释放系统提高其血清稳定性并严格检测其疗效 使用两种临床相关的种植体感染提供对假体周围感染的持续保护 模特们。在目标1中，通过选择性2‘-O-甲基化对寡核苷酸连接物进行化学修饰，并 硫代修饰在保持哺乳动物血清核酸酶稳定性的同时提高其稳定性 对MN裂解的必要敏感性。在目标2中，体外优化的核苷酸连接子将在 MN敏感水凝胶涂层，用于按需输送万古霉素的Ti6Al4V IM针脚。这个 这种预防性涂层对金黄色葡萄球菌提供及时和持续保护的有效性和安全性 假体周围感染将使用大鼠股骨管感染模型在6个月内进行严格评估。在……里面 目的3：这种按需释放抗生素策略在减少高位假体周围感染方面的效果。 手术清除先前感染的大鼠股动脉管后的比率将使用大鼠IM进行检查 种植体翻修手术模式。感染程度与针状涂层和生物发光S。 通过生物发光成像和皮质的µCT定量对金黄色葡萄球菌接种进行纵向监测 在2周、1、2、3和6个月时骨增厚，并通过终点量化取材上的细菌 术后1个月、3个月、6个月行股骨穿针、股骨扭转试验和股骨组织学检查。涂层的长期安全性 由终末点的全身器官病理学检查。全身性一次性注射万古霉素 在一部分受感染的动物身上进行的试验比预防涂层中的试验高出数百倍 接受未涂覆的IM针脚，以允许直接比较这种预防性涂层与 标准护理。实现对假体周围感染或反复感染的更持久的保护 那么全身注射万古霉素将被认为是成功的，同时实现了对6 没有局部和系统性副作用的几个月将被视为例外。如果验证成功，则 通过MN触发的万古霉素释放使细菌能够及时和持续地根除 在应对骨科植入相关感染的艰巨挑战方面的安全性和有效性 绕过了臭名昭著的难以治疗的生物膜和骨髓炎。

PROJECT SUMMARY/ABSTRACT Periprosthetic infections is one of the most serious complications in orthopedic surgeries, occurring in 1-4% of primary total joint replacement and up to 30% of revisions. Infections caused by Staphylococcus aureus (S. aureus), the most prevalent microbial culprit in orthopedic infections, are particularly hard to treat due to their tendency to form biofilms on implant and notorious ability to invade the canalicular network of surrounding bone. Existing prophylactic antibiotic deliveries involve high drug doses that are unsafe yet ineffective and could lead to the development of drug resistance. Utilizing an oligonucleotide linker labile to S. aureus micrococcal nuclease (MN) cleavage, we recently developed a hydrogel capable of on-demand release of covalently tethered vancomycin. When applied as a hydrogel coating to Ti6Al4V intramedullary (IM) pin and inserted to mouse femoral canal inoculated with S. aureus, the MN-triggered release of vancomycin timely killed the bacterial on implant surface and within IM space before they had a chance to colonize or invade surrounding bone, thereby preventing biofilm formation and osteomyelitis development in the 3 weeks examined. The covalent tethering dose of vancomycin in this coating was orders of magnitude lower than the typical prophylactic antibiotic content used clinically. The goal of the proposed study is to further engineer this exciting on-demand drug release system to enhance its serum stability and rigorously examine its efficacy in providing sustained protection against periprosthetic infections using two clinically relevant implant infection models. In Aim 1, the oligonucleotide linker is chemically modified by selective 2'-O-methylation and phosphorothioate modifications to achieved enhanced mammalian serum nuclease stability while maintaining necessary sensitivity to MN cleavage. In Aim 2, the in vitro optimized nucleotide linker will be implemented in MN-sensitive hydrogel coating and applied to Ti6Al4V IM pins for on-demand delivery of vancomycin. The efficacy and safety of this prophylactic coating in providing timely and sustained protection against S. aureus periprosthetic infections will be rigorously evaluated over 6 months using a rat femoral canal infection model. In Aim 3, the efficacy of this on-demand antibiotic release strategy in reducing the high periprosthetic infection rates following surgical debridement of previously infected rat femoral canal will be examined using a rat IM implant revision surgery model. The degree of infections as a function of pin coating and bioluminescent S. aureus inoculation are longitudinally monitored by bioluminescent imaging and µCT quantification of cortical bone thickening at 2 weeks, 1, 2, 3 and 6 months, and by end-point quantification of bacteria on the retrieved pin, torsion test of explanted femur and femoral histology at 1, 3 and 6 months. Long-term safety of the coating is examined by systemic organ pathology at the endpoints. Systemic injections of vancomycin at a dose several hundred-fold higher than that in the prophylactic coating are carried out in a subset of infected animals receiving uncoated IM pins to allow direct comparison of the efficacy of this prophylactic coating vs. that of the standard care. Achieving more sustained protection against periprosthetic infections or recurrent infections than systemic vancomycin injections will be considered a success while achieving extended protection for 6 months without local and systemic side effects will be considered exceptional. If successfully validated, the timely and sustained eradication of bacteria enabled by MN-triggered vancomycin release could bring together safety and efficacy in addressing the daunting challenge of orthopedic implant-associated infections by bypassing the notoriously hard-to-treat biofilms and osteomyelitis.