Surface functionalized vitamin B₁₂ (VB₁₂) biocompatible nanoparticles exploit the well characterized uptake absorption pathway of VB₁₂ shielding it from enzymatic degradation and inadequate absorption. In this perspective, subsequent to escalated mucus interaction and diffusion analysis, the nanoparticles were investigated by immunostaining with anti CD320 antibody and internalization mechanisms were examined by selectively blocking specific uptake processes, we saw internalization to be energy dependent clathrin mediated, simultaneously highlighting the remarkable ability to bypass P-glycoprotein efflux. In particular, synthesized nanoparticles were evaluated for cytocompatibility by analyzing cellular proliferation, membrane viscoelasticity and fluidity analysis by FRAP and oxidative stress detection making them well suited for successful translation to clinical setups. Our previous in vitro antileishmanial results paramount for further in vivo and toxicity analysis paving for targeted therapeutic efficiency. The augmented surface hydrophilicity attributed by VB₁₂ and monomerization of AmB within the lipid core strengthen the oral bioavailability and stability as evidenced by FRET analysis.