Development and Evaluation of Liposomal Nanobiotics for Combating Antibiotic Resistance Among Enterobacteriaceae

Barani Devi T,1 Dhiya Fathima K V,1 Sudarshan Kini,2 Vijaya Kumar Deekshit1,3 1Department of Infectious Diseases and Microbial Genomics, Nitte University Centre for Science Education and Research (NUCSER), Nitte (Deemed to be University), Paneer Campus, Mangalore, Karnataka, 575018, India; 2Department of Bio and Nanotechnology, Nitte University Centre for Science Education and Research (NUCSER), Nitte (Deemed to be University), Paneer Campus, Mangaluru, Karnataka, 575018, India; 3Department of Microbiology, KS Hegde Medical Academy (KSHEMA), Nitte (Deemed to be University), Mangaluru, Karnataka, 575018, IndiaCorrespondence: Vijaya Kumar Deekshit, Department of Infectious Diseases and Microbial Genomics, Nitte University Centre for Science Education and Research (NUCSER), Nitte (Deemed to be University), Paneer Campus, Mangaluru, 575018, India, Email [email protected]: The increasing prevalence of antimicrobial-resistant bacteria, such as Escherichia coli, Klebsiella pneumoniae, and non-typhoidal Salmonella, poses a significant healthcare problem, leading to increased mortality. Liposomal nanocarriers have already shown their potential in overcoming resistance and improving antibiotic delivery.Methods: In this study, liposome-based nanocarriers encapsulating conventional antibiotics were developed, characterised and evaluated for their antibacterial efficacy and interactions with bacteria. The liposomal nanobiotics were prepared using the thin-film hydration method and loaded with antibiotics.Results: The prepared nanobiotics of tetracycline, chloramphenicol and nalidixic acid showed average particle size of 120– 190 nm and entrapment ranging between 30% and 85% for the antibiotics used. Furthermore, the formulations exhibited minimal cytotoxic effects on HEK293 cells, indicating favourable biocompatibility. Compared to the free antibiotics the nanobiotics demonstrated significantly enhanced antibacterial activity against MDR isolates. The time kill curves showed significant reduction in viable bacterial count, while in vitro release profile showed sustained release of the encapsulated liposomes around 85% within 24 hours. The TEM analysis and PI/calceinAM assay demonstrated effective liposme-bacterial interaction and enhanced intracellular delivery of the encapsulated antibiotics. Further, the analysis of the efflux-pump associated genes showed impact of nanobiotic formulations on efflux pump gene expressions and suggests a role in mitigating the resistance mechanism. Also, antibiotics encapsulated in liposomes significantly reduced the bacterial load across all conditions in the food-spiking experiment with enhanced antibiotic delivery.Conclusion: The liposomal formulation of tetracycline, chloramphenicol and nalidixic acid showed better physicochemical properties, sustained release profile and increased antibacterial activity against MDR bacterial isolates compared to free drug. Overall findings suggest that encapsulation enhances the therapeutic potential of the conventional antibiotics and can be a promising strategy to overcome the multi-drug resistance in bacteria. The diagram illustrates antimicrobial resistance mechanisms: efflux pump, impermeable, enzymatic inactivation, target alteration, gene mutation and plasmid mediated. It presents nanobiotic delivery as an alternative approach, focusing on liposomal formulation and characterization. The process involves dissolving lipid and cholesterol in solvent, forming a dry lipid layer, hydration and creating liposomes using the thin film hydration method. Efficacy validation includes agar well diffusion, MIC determination, time kill curves, transmission electron microscopy, permeability assay, in vitro release study, cytotoxicity study and spiking assay on food matrices like sprouts, chicken and shrimps. It also covers bacterial enumeration and qPCR-DNA copy number variation.Diagram of antimicrobial resistance and nanobiotic delivery with liposomal formulation and efficacy validation.Keywords: Enterobacteriaceae, multi-drug resistance, nanobiotics, liposomes, antibacterial activity, nanocarrier delivery