DESIGNING BIOMIMETIC NANOPARTICLES TO TACKLE INTRACELLULAR PATHOGENS

Despite advancements in modern medicine, certain bacterial pathogens such as Mycobacterium abscessus (Mab) continue to evade therapeutic clearance by creating intracellular reservoirs within host macrophages. Conventional drug delivery systems often fall short due to their lack of precision, limited intracellular access, and rapid clearance by the immune system. To address these limitations, we developed a biomimetic nanocarrier platform termed Nanoghosts (NGs), derived from the plasma membranes of human monocytes (THP-1 cells and primary CD14+ monocytes). These NGs were designed to retain key membrane features while excluding nuclear and cytoplasmic content, with the goal of targeting intracellular Mab more effectively. We adopted a stepwise experimental strategy using orthogonal validation techniques to ensure a thorough understanding of NG properties. The fabrication process was optimized through multiple approaches, and membrane retention was verified by gas chromatography and phospholipid quantification at each stage. Physicochemical characterization revealed uniform spherical NGs with an average size of 108.1 ± 1.2 nm and a membrane bilayer thickness of approximately 6.3 ± 1.2 nm. Minimal protein corona formation was detected using fluorescence correlation spectroscopy (FCS), suggesting desired biomimetic characteristics. Lectin staining confirmed plasma membrane origin and preservation of surface glycocalyx profiles. Partial retention of surface markers like CD14 (CD14+ monocyte-derived NGs) and CD11a (THP-1 cells derived NGs) was observed. Taken together, the lectin staining results and the detection of surface markers using antibodies targeting their exoplasmic domains suggested that NGs retained a right-side-out membrane orientation. In addition to this, in preliminary attempts, Hybrid NGs, as a proof of concept, were also engineered to compensate for missing markers, expanding the platform’s modular potential. Biological assays confirmed that NGs neither harbored nor induced reactive oxygen species (ROS) and remained non-cytotoxic even at high concentrations, as validated by MTT and luciferase viability assays. Moreover, NGs did not induce strong polarization of macrophages. Cellular Uptake studies in macrophages, including Mab-infected models, confirmed effective internalization. Notably, PBMC-derived macrophages showed enhanced uptake under infected conditions. Despite these advances, loading therapeutic cargo into NGs proved to be a major challenge. Initial attempts were unsuccessful, likely due to drug incompatibility or analytical detection limitations. Future efforts will focus on optimizing cargo selection and exploring alternative loading strategies such as hybrid systems that combine NGs with other nanoparticle cores.