Exosome-facilitated nanoplatform for enhanced antibiotic delivery to eradicate intracellular multidrug-resistant Escherichia coli in neonatal sepsis.

Neonatal sepsis is a pathogenic infection with an estimated global fatality rate of 18% and poses a greater threat in preterm infants, where early-onset cases often result from vertical transmission from the maternal genital tract. Conventional high-dose antibiotic therapy may lead to adverse effects and reduced therapeutic efficacy due to suboptimal intracellular delivery and interactions with extracellular components before effective cellular uptake. To overcome this limitation, exosome-based nanocarriers deliver antibiotics directly into cells, preventing systemic dissociation in the blood. In this study, human serum-derived exosomes were employed to deliver colistin sulfate (ExoCol) against intracellular multidrug-resistant (MDR) Escherichia coli associated with neonatal sepsis. Exosomes were isolated from a serum sample through the PEG precipitation method. Colistin sulfate (Col) was encapsulated within the exosomes and both the free exosomes (Exos) and colistin sulfate-loaded exosomes (ExoCol) were characterized using Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Fourier Transform Infrared Spectroscopy (FTIR), Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), western blotting, Scanning Electron Microscopy (SEM), and UV-visible spectrophotometry. Macrophage RAW 264.7 cells were treated with Exos and ExoCol, and cellular uptake was analyzed using confocal microscopy. Low to high concentrations of Exos (10-100 µg/mL), Col (0.5-6 µg/mL), and ExoCol (Exo: 100 µg/mL & Col: 0.5-6 µg/mL)-treated macrophage cells were assessed to check cell viability by MTT assay and intracellular drug release study by UV-visible spectrophotometry. The cells were then infected with MDR Escherichia coli, and the same treatments were given to evaluate antibacterial efficacy using CFU count, intracellular live-dead bacterial imaging by confocal microscopy, and single-cell sorting by flow cytometry. After 24 h of the treatment, ExoCol-treated macrophage cells maintained higher cell viability (∼85-95%) than the free antibiotic-treated cells (60-80%), and ExoCol demonstrated significantly greater intracellular drug release (∼85-95%) as compared to free colistin sulfate (∼45-55%). Moreover, ExoCol eradicated more intracellular bacteria than free colistin sulfate, as indicated by a higher reduction in bacterial count (8.8 × 102 CFU/mL vs. 2.25 CFU/mL, respectively). In conclusion, these findings imply that antibacterial therapy mediated by exosomes reduces cytotoxicity and increases bacterial eradication, with significant intracellular delivery. Therefore, ExoCol approaches as a promising nanotherapeutic for the treatment of neonatal sepsis. Future studies will involve in vivo models to further optimize doses and validate therapeutic efficacy and treatment strategies.