The characterization of nanoparticles (NPs) has become increasingly important due to their wide-ranging applications in fields such as biomedicine and drug delivery. NPs have emerged as promising candidates for drug delivery systems due to their unique physicochemical properties, which enable them to interact with biological systems at the molecular level. Among these, soft nanocarriers, such as niosomes, and hard nanocarriers, such as Iron Oxide Nanoparticles (IONPs), offer distinct advantages for targeted therapy and diagnostics. This study provides a comprehensive, multi-disciplinary evaluation of two distinct types of nanoparticles: soft nanocarriers (niosomes, NVs) and hard nanocarriers (IONPs), by examining their physicochemical properties, cellular uptake, and cytotoxicity profiles. This comparative analysis seeks to highlight the different behaviour of soft and hard nanoparticles in drug delivery applications, with a particular focus on the impact of surface modifications. The addition of chitosan to sample NVsB not only resulted in an increase in particle dimensions but also shifted the ζ-potential to positive values which could enhance the interactions with cell membranes, improving cellular uptake. As desired, the obtained ζ-potential value of NVsB-Chit was comparable to that of the commercial coated ferrofluid. In addition to the traditional characterization techniques, this study integrates advanced analytical methods, such as Atomic Force Microscopy (AFM), complementing traditional techniques such as Dynamic Light Scattering (DLS), to assess the nanoscale topography of both types of nanoparticles. Cytotoxicity studies on Calu-3 lung adenocarcinoma cells were conducted to evaluate the biocompatibility of the nanoparticles, demonstrating that NVs and FluidMAG exhibited minimal cytotoxic effects, particularly at lower concentrations. Cell internalization was confirmed for IONPs by magnetic cell separation whereas confocal microscopy analysis has been conducted for calcein-loaded NVs intracellular visualization. By integrating structural, chemical, and biological evaluations, we take an interdisciplinary approach which could also enable us to explore how variations in nanoparticle design (such as surface charge, size and coating) affect their performance in drug delivery and diagnostics. Moreover, combining physicochemical characterizations (e.g., hydrodynamic diameter, zeta potential and nanoparticles morphology) with biological evaluations (e.g., cellular uptake and safety profiles) allows for a holistic assessment of these nanocarriers to gain a comprehensive understanding of their behaviour and performance. This aspect is crucial for designing more efficient, safer, and targeted nanomedicines.