Lipid-polymer hybrid nanoparticles as an anti-infective delivery system

Antimicrobial resistance poses a significant global health challenge, highlighting the urgent need for innovative drug delivery systems that enhance the efficacy of existing antibiotics and new antimicrobial agents. This thesis investigates the development, optimization, and evaluation of lipid-polymer hybrid nanoparticles (LPHNPs) as a dual-delivery platform for cefotaxime (CTX), a β-lactam antibiotic, and RN7IN6, a synthetic antimicrobial peptide (AMP). The overarching goal was to create an advanced co-delivery system to improve treatment outcomes against Gram-positive and Gram-negative pathogens. Initial investigations focused on chitosan nanoparticles (CS NPs) and POPE:POPG:CL liposomes as separate systems, with the aim of combining them to form the core and lipid shell of LPHNPs, respectively. Methodologically, microfluidic mixing was employed for the precise and scalable synthesis of CS NPs, POPE:POPG:CL liposomes, and LPHNPs. Various formulation parameters were systematically optimized to ensure desirable physicochemical properties such as particle size, polydispersity index (PDI), and Z-potential. High-performance liquid chromatography (HPLC) was used for drug quantification, while antimicrobial efficacy was assessed using minimum inhibitory concentration (MIC) and time-kill assays against clinically relevant bacterial strains. Two types of CS, being chitosan hydrochloride (CHCL) and chitosan low molecular weight (CHT), were explored for CTX encapsulation within NPs. Despite the similarity in physicochemical and antibacterial properties of both CTX encapsulating CS NPs, CHCL-based NPs were chosen due to the CHCL aqueous solubility, enabling more efficient production and streamlined formulation for LPHNP development. The optimized CHCL NPs achieved a high CTX loaded concentration while maintaining small and uniform particle characteristics. Meanwhile, bacteriomimetic POPE:POPG:CL liposomes, designed to mimic bacterial membranes, were evaluated for the incorporation of a novel AMP being RN7IN6. Results revealed that while free RN7IN6 demonstrated potent antimicrobial activity against various bacterial strains, its adsorption onto liposome surface presented challenges such as aggregation, instability, and reproducibility issues. xiv Consequently, RN7IN6 was incorporated within the lipid bilayer to address these limitations. Although RN7IN6-loaded formulations demonstrated limited peptide release and no significant antimicrobial activity against Staphylococcus aureus and Escherichia coli, the POPE:POPG:CL system might be promising as the lipid shell of LPHNPs to enhance interaction with bacteria. Encapsulation within LPHNPs was driven by the synergistic potential observed between CTX and RN7IN6, forming the foundation for developing a co-loaded delivery system. The resulting CTX and RN7IN6 co-loaded LPHNPs demonstrated superior antibacterial activity compared to free and encapsulated CTX, as evidenced by the inhibition of E. coli bacterial growth. Similarly, for S. aureus, the encapsulation of RN7IN6 within LPHNPs alongside CTX demonstrated increased antimicrobial activity compared to CTX-only loaded LPHNPs. The development of CTX and RN7IN6 co-loaded LPHNPs showcases the advantages of using hybrid nanocarriers to overcome the limitations of traditional antimicrobial therapies. These findings emphasize the potential of LPHNPs not only to deliver dual-drug formulations effectively but also to enhance synergistic antibacterial effects. However, further refinement is necessary to understand the contribution of RN7IN6 to the overall antimicrobial activity of the co-loaded LPHNPs. Moreover, future studies should focus on investigating the mechanisms underlying the interaction between LPHNPs and bacterial cell membranes, optimizing release kinetics by tuning the lipid composition of LPHNPs lipid shell, and assessing the potential for these nanocarriers to combat a broader spectrum of multidrug-resistant pathogens.