# Recent Progress and Challenges Regarding Magnetic-Based Nanoparticles for Targeted Drug Delivery
## 5 Monitoring of Magnetic-Based Nanocarriers Interacting with Cells in Biological Systems
Additional constraints arise from intracellular barriers, encompassing challenges such as lysosome and endosome degradation escape, as well as physiological factors like target tissue characteristics, blood flow in the area, vascular sources, body weight, method of nanoparticle injection, distance from the field source, and tumor volume. The accumulation of nanoparticles is also linked to a higher enhanced permeability and retention (EPR) rate in cancer cells and tissues, occurring more rapidly than in healthy counterparts. Studies have demonstrated that nanoparticles ranging in size from 10 to 100 nm tend to accumulate predominantly in tumors as opposed to healthy cells and tissues [130]. In the context of utilizing magnetic nanoparticles for diagnostics and therapy, the optimal approach for their studying in biological systems is to integrate imaging techniques that facilitate their visualization with those employed for the pathological examination of tissues. Various imaging techniques, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), have been extensively utilized to track the targeting and biodistribution of nanoparticles throughout the entire body [13; 112; 113]. On the other hand, magnetic particle imaging (MPI) enables the direct detection of the quantity and location of superparamagnetic nanoparticles within biological tissue, surpassing the sensitivity of MRI [129]. Other methods are employed for smaller areas. Fluorescence microscopy, both conventional and confocal, is one of such methods, chosen for its relatively straightforward experimental setup. Its utilization was identified in numerous original papers referenced in this review [46; 47; 48; 50; 51; 63; 71; 73; 80; 84; 88; 90; 95; 101; 102; 103; 104; 117]. It allows for studying extensive cell populations or relatively sizable tissue samples. However, its resolution (approximately 200 nm) does not allow for the observation of individual nanoparticles and their interactions with cells and specific tissue components. Moreover, the observed structures must undergo fluorescent labeling, introducing the risk of altering their bioreactivity. Another difficulty lies in the precise differentiation between nanoparticles bound to the plasma membrane and those internalized [125]. A similar issue pertains to flow cytometry (FC), a method used to quantify the association of nanoparticles with cells [89; 91; 103; 107; 108; 109]. However, it quantifies the total association and does not distinguish between internalized material and surface-bound particles. Flow cytometry holds the advantage of simultaneously observing numerous cells and fluorophores. Additionally, there have been recent advancements with improved resolution, including total internal reflection fluorescence microscopy (TIFR), illumination microscopy (SIM), expansion microscopy (ExM), and single-molecule localization microscopy (SMLM) [126]. Another method within electron microscopy is scanning electron microscopy (SEM). While it boasts high resolution (3-20 nm), it is limited to imaging only the sample's surface. In this context, SEM primarily characterizes the spatial relationship between nanoparticles and the cell surface. In turn, confocal laser scanning microscopy (CLSM) offers a spatial representation of cells and their constituents growing in vitro on 3D scaffolds [55; 65; 117]. On the other hand, transmission electron microscopy (TEM) is a robust technique for obtaining information on nanoparticle uptake, biodistribution, and interactions with cell and tissue components. While the achievable resolution is approximately 0.2 nm, the practical limitations imposed by preparing biological samples restrict the resolution to around 2 nm. Furthermore, observations can only be carried out on thin and small sample slices. An additional drawback is the limitation of obtaining only static information.