A Novel Broad-Spectrum Nanoimmunotherapeutic Approach for Combating Multidrug Resistant Bacteria

PROJECT SUMMARY Multidrug-resistant (MDR) bacterial infections have emerged as an urgent global public health threat. The ESKAPE pathogens, comprising six virulent MDR bacteria, are major contributors to life-threatening nosocomial infections. Traditional antibiotic therapies are becoming increasingly ineffective due to the emergence of antibiotic resistance and the stagnation of new antibiotic development. Therefore, there is an urgent need for innovative non-antibiotic antimicrobial approaches to combat these MDR pathogens. To meet this urgent need, we developed an innovative nanoimmunotherapy mediated by a novel red blood cell membrane (RBCM)-coated hemoglobin (Hb)-encapsulating nanoparticle saturated with oxygen (termed RHNP). RHNP functions by sensitizing bacteria to host oxidant killing while simultaneously recruiting neutrophils and enhancing their oxidant killing capability. Our preliminary studies have shown that this approach was highly effective in treating antibiotic- resistant bacterial infections in several mouse models. The objective of this project is to further develop RHNP to achieve broad-spectrum antimicrobial capability against Gram-negative MDR ESKAPE bacterial pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Klebsiella pneumoniae (CRKP), and MDR Pseudomonas aeruginosa (MDRPA). We hypothesize that (1) RHNP can offer a broad- spectrum non-antibiotic immunotherapeutic approach for combating MDR bacteria, and (2) RHNP can exhibit synergistic effects with conventional antibiotics. In Aim 1, we will design, fabricate, characterize, and optimize the RHNP. We will optimize the RHNP formulation (i.e., Hb-cholesterol (Chol)@RBCM NP) by optimizing the ratios of Hb/Chol and RBCM/Hb-Chol. RHNPs will be fabricated via both a batch and a continuous process. RHNPs with desirable physiochemical characteristics and biocompatibility will be identified for studies described in Aims 2 and 3. In Aim 2, we will first examine the antimicrobial efficacy and mechanisms of RHNP in treating the MDR planktonic bacteria. We will then study the synergistic effects and synergistic mechanisms of RHNP/antibiotic combination therapy against MDR planktonic bacteria. Subsequently, the antimicrobial efficacy of RHNP with or without antibiotics against bacterial biofilms will be investigated. In Aim 3, we will study the therapeutic efficacy of RHNP alone or in combination with antibiotics in three rodent infection models representing different healthcare-associated infections. The effects of neutrophils and macrophages on the therapeutic outcome will also be assessed to further clarify the immunotherapeutic mechanisms. The safety of RHNP will be evaluated by hematological and biochemical parameters, inflammatory cytokines, and organ histopathology in mice. RHNP represents a transformative approach to treating MDR infections, offering a universal, safe, and effective immunotherapy option. Its potential to enhance the effectiveness of existing antibiotics could also transform current antibiotic treatment protocols. This project has the potential to save countless lives and will significantly alleviate the burden on healthcare systems worldwide.