Chitosan Coated Liposomes for Non-Invasive Brain Delivery in Zebra Finch: Formulation Optimization and Evidence of Blood Brain Barrier Traversal

Abstract

Zebra finches are a well-established neuroscience model for studying learning and vocalization, yet minimally invasive strategies for delivering pharmacological agents to their brain remain limited. Nanoparticle-based drug delivery systems, particularly liposomes, offer a biocompatible and membrane-mimetic platform that has demonstrated blood–brain barrier (BBB) penetration in mammalian models; however, their ability to access brain tissue in zebra finches has not been previously established. In this study, fluorescently labelled liposomes were formulated using ethanol injection and characterized for size, stability, and morphology using dynamic light scattering, zeta potential measurements, and scanning electron microscopy to confirm the formation of stable vesicular structures. Optimization of the formulation reduced liposome diameter to 67 nm ± 1.53 SEM, significantly smaller than earlier preparations with a size of 85 nm ± 3.76 SEM (p < 0.01), with no significant size increase observed over 30 days, demonstrating stability. Following intravenous administration, brain tissues were analyzed by confocal fluorescence microscopy with z-stack reconstruction to localize liposome-derived lipid signals relative to blood vessels and parenchyma. Injected birds exhibited significantly higher fluorescence intensity in both brain vasculature and parenchyma compared with controls, and threedimensional reconstructions revealed dye colocalization outside blood vessels, indicating extravasation and BBB crossing. Moreover, the visualization of intact vesicular structures using scanning electron microscope further supported the claim that the colocalized entities are in fact liposomes. These findings show that systemically administered liposomes can successfully cross the zebra finch BBB and reach brain tissue while maintaining structural integrity. This delivery approach provides a reliable and minimally invasive platform for future pharmacological and neurobiological interventions in this model.