Background Multidrug-resistant (MDR) gram-negative bacteria-related infectious diseases have caused an increase in the public health burden and mortality. Moreover, the formation of biofilms makes these bacteria difficult to control. Therefore, developing novel interventions to combat MDR gram-negative bacteria and their biofilms-related infections are urgently needed. The purpose of this study was to develop a multifunctional nanoassembly (IRNB) based on IR-780 and N, N′-di-sec-butyl-N, N′- dinitroso-1,4-phenylenediamine (BNN6) for synergistic effect on the infected wounds and subcutaneous abscesses caused by gram-negative bacteria. Methods The characterization and bacteria-targeting ability of IRNB were investigated. The bactericidal efficacy of IRNB against gram-negative bacteria and their biofilms was demonstrated by crystal violet staining assay, plate counting method and live/dead staining in vitro. The antibacterial efficiency of IRNB was examined on a subcutaneous abscess and cutaneous infected wound model in vivo. A cell counting kit-8 assay, Calcein/PI cytotoxicity assay, hemolysis assay and intravenous injection assay were performed to detect the biocompatibility of IRNB in vitro and in vivo. Results Herein, we successfully developed a multifunctional nanoassembly IRNB based on IR-780 and BNN6 for synergistic photothermal therapy (PTT), photodynamic therapy (PDT) and nitric oxide (NO) effect triggered by an 808 nm laser. This nanoassembly could accumulate specifically at the infected sites of MDR gram-negative bacteria and their biofilms via the covalent coupling effect. Upon irradiation with an 808 nm laser, IRNB was activated and produced both reactive oxygen species (ROS) and hyperthermia. The local hyperthermia could induce NO generation, which further reacted with ROS to generate ONOO−, leading to the enhancement of bactericidal efficacy. Furthermore, NO and ONOO− could disrupt the cell membrane, which converts bacteria to an extremely susceptible state and further enhances the photothermal effect. In this study, IRNB showed a superior photothermal-photodynamic-chemo (NO) synergistic therapeutic effect on the infected wounds and subcutaneous abscesses caused by gram-negative bacteria. This resulted in effective control of associated infections, relief of inflammation, promotion of re-epithelization and collagen deposition, and regulation of angiogenesis during wound healing. Moreover, IRNB exhibited excellent biocompatibility, both in vitro and in vivo. Conclusions The present research suggests that IRNB can be considered a promising alternative for treating infections caused by MDR gram-negative bacteria and their biofilms.

中文翻译：

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用于对抗多重耐药革兰氏阴性细菌及其生物膜的光活化和苯基硼酸功能化纳米组件

背景多重耐药（MDR）革兰氏阴性菌相关传染病已导致公共卫生负担和死亡率增加。此外，生物膜的形成使这些细菌难以控制。因此，迫切需要开发新的干预措施来对抗耐多药革兰氏阴性菌及其生物膜相关感染。本研究的目的是开发基于 IR-780 和 N,N'-二仲丁基-N,N'-二亚硝基-1,4-苯二胺 (BNN6) 的多功能纳米组件 (IRNB)，以实现协同效应由革兰氏阴性菌引起的感染伤口和皮下脓肿。方法研究IRNB的表征及其细菌靶向能力。通过结晶紫染色法、平板计数法和体外活/死染色法证明了IRNB对革兰氏阴性菌及其生物膜的杀菌功效。在体内皮下脓肿和皮肤感染伤口模型上检查了 IRNB 的抗菌效率。采用细胞计数试剂盒8测定、Calcein/PI细胞毒性测定、溶血测定和静脉注射测定来检测IRNB的体外和体内生物相容性。结果在此，我们成功开发了基于IR-780和BNN6的多功能纳米组装IRNB，用于协同光热疗法（PTT）、光动力疗法（PDT）和808 nm激光触发的一氧化氮（NO）效应。这种纳米组装体可以通过共价偶联效应在耐多药革兰氏阴性菌及其生物膜的感染部位特异性积累。在 808 nm 激光照射下，IRNB 被激活并产生活性氧 (ROS) 和高温。局部热疗可诱导NO生成，NO进一步与ROS反应生成ONOO−，从而增强杀菌效果。此外，NO和ONOO−可以破坏细胞膜，使细菌转变为极其敏感的状态，并进一步增强光热效应。在这项研究中，IRNB对革兰氏阴性菌引起的感染伤口和皮下脓肿表现出优越的光热-光动力-化疗（NO）协同治疗作用。这导致有效控制相关感染、缓解炎症、促进上皮再形成和胶原蛋白沉积以及伤口愈合过程中血管生成的调节。此外，IRNB 在体外和体内均表现出优异的生物相容性。结论 目前的研究表明，IRNB 可以被认为是治疗由 MDR 革兰氏阴性菌及其生物膜引起的感染的有前途的替代方案。