In Vitro Evaluation of Novel Nano-Sized Colloidal Assemblies Incorporating Hydrophobic Tobramycin Ion Pairs: Enhanced Cellular Uptake with Preserved Antimicrobial Activity Toward Oral Drug Delivery

Tobramycin is a highly hydrophilic aminoglycoside antibiotic with limited cellular permeability and negligible oral bioavailability, necessitating parenteral administration. This study aimed to develop drug delivery systems based on nano-sized colloidal assemblies (NCAs) incorporating tobramycin ion pairs to enhance its lipophilicity, potential for transition to the oral route, and antimicrobial activities. Tobramycin was ionically paired with oleic acid, lauric acid, and fluorescein and formulated into NCA preconcentrates (F1–F5) using combinations of Tween 80, DMSO, and propylene glycol. The resulting formulations formed stable nanodroplets upon dilution (9.50–16.30 nm) with narrow size distributions (polydispersity index; PDI < 0.3) and moderate negative zeta potentials (−4.99 to −11.13 mV). In vitro release studies indicated sustained drug release for ion-paired systems compared to the rapid release of free tobramycin. Cytotoxicity evaluation in Caco-2 cells demonstrated high biocompatibility at 1:10,000 and 2:10,000 dilutions, while concentration-dependent toxicity at higher doses suggested enhanced intracellular delivery. Cellular uptake studies revealed significantly higher tobramycin internalization (p < 0.001) from formulations F1–F3, with uptake values in the range of 81.76–96.14% compared to free drug, which showed zero or negligible uptake. Fluorescein-labeled formulations (F4 and F5) further confirmed enhanced uptake, demonstrating strong intracellular fluorescence. This was supported by visual observation, UV–Vis absorbance (70.5–84.8% relative to positive control), and confocal microscopy imaging. Antimicrobial activities against P. aeruginosa and S. aureus were comparable between formulations F1–F5 and free tobramycin (inhibition zones of 16–18 mm), utilizing the same tobramycin concentration in the diluting medium. These findings validate the effectiveness of the formulated NCAs in facilitating intracellular delivery of tobramycin while preserving biocompatibility and similar antimicrobial activities. Moreover, the uptake of fluorescein provides indirect evidence supporting the enhanced internalization of tobramycin in analogous ion-paired formulations. This strategy holds promise for overcoming intestinal barriers and improving oral bioavailability, potentially enabling the transition of tobramycin from parenteral to oral administration.