# Recent Progress and Challenges Regarding Magnetic-Based Nanoparticles for Targeted Drug Delivery
## 5 Monitoring of Magnetic-Based Nanocarriers Interacting with Cells in Biological Systems
Although various biomolecules interact with the surface of NPs, proteins surround the surface immediately after NPs are introduced into the biological system, forming protein corona. The interactions on the protein-NP surface are dynamic, with continuous adsorption/desorption processes. The presence of proteins results in the surface modification of NPs and, consequently, their bioreactivity. Thus, this is an essential aspect determining the recognition of magnetic nanocarriers by cells and their further cellular internalization. Meanwhile, research is most often carried out confirming only the formation of protein corona (e.g., withhuman serum albumin) without further examination of this issue. In drug delivery systems, effective treatment relies significantly on the cellular uptake of drug-loaded nanocarriers. They primarily gain entry into the cells through endocytosis, as confirmed by transmission electron microscopy (TEM) [125]. The potential entry mechanisms vary based on the size of nanoparticles. Endocytosis can be categorized into two primary types: phagocytosis, typically associated with the uptake of particles larger than 0.5 um in diameter, and pinocytosis, encompassing macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and clathrin/caveolae-independent endocytosis. Particles exceeding a diameter of 200 nm are commonly internalized through the processes of phagocytosis or micropinocytosis mentioned above. Nanoparticles with a diameter below 10 nm and cationic nanoparticles, characterized by high charge density, gain entry into the cell through direct penetration and pore formation. Numerous approaches have been suggested and employed to investigate endocytosis processes. These methods encompass chemical and pharmacological inhibitors, specific biomarkers, genetic techniques, and diverse microscopy techniques such as fluorescence, electron, and atomic force microscopy [126; 127; 128]. The characteristics affecting the penetration of nanoparticles into cells or tissues are associated with their size, surface charge, and hydrophobicity [129]. Therefore, nanoparticles exceeding 150 nm in diameter typically accumulate in the spleen and liver; those within the 30-150 nm range in the bone marrow, stomach, heart, and kidneys; and those smaller than 20 nm primarily gather in the kidneys. Additionally, research indicates that positively charged and hydrophobic particles have shorter circulation times than their hydrophilic and negatively charged counterparts [130]. Other crucial factors include biocompatibility, bioaccumulation, colloidal stability, solubility, administration method, degradation rate, and the target cell/tissue [127]. The factor that can halt endocytosis is an initial incubation temperature of 4 \({}^{\circ}\)C. Subsequently, unbound nanoparticles are removed through washing, and the incubation temperature is reverted to 37 \({}^{\circ}\)C. It facilitates a more straightforward interpretation of internalization kinetics [126]. On the other hand, nanoparticles face challenges within the human body due to physiological barriers. Initially, when nanoparticles are injected into the bloodstream, they can accumulate, altering their magnetic properties and impeding further movement. The already mentioned protein corona can potentially lead to their aggregation before reaching the cell [126]. Additionally, their interaction with plasma proteins may trigger immune responses. Further limitations arise from the relationship between particle size and the anatomy of the target tissue. Within the brain, the rate of pinocytosis is restricted, and the robust intercellular connections forming the blood-brain barrier (BBB) hinder the passage of small particles possessing suitable physicochemical properties [129].