Background/Objectives: Magnetite nanoparticles represent promising candidates for a broad spectrum of biomedical applications, ranging from in vitro diagnostic assays to in vivo imaging, hyperthermia, and targeted drug and gene delivery, with some nanoagents already approved for clinical use. A critical determinant of their functionality is the nanoparticle coating, which facilitates beneficial interactions within biological systems. In the context of tumor-targeted therapeutic delivery, key design parameters—particularly surface coatings—can be optimized to enhance treatment efficacy by modulating blood circulation kinetics, biodistribution, and other critical properties. However, current preclinical screening methods primarily rely on cell culture models to identify potential nanocarriers, yet these systems often poorly correlate with actual in vivo performance. This discrepancy highlights the necessity of incorporating more biologically relevant testing platforms, such as high-throughput in vivo assays. Methods: In this work, we employed an original magnetic particle quantification (MPQ) technology to systematically evaluate the blood circulation kinetics and biodistribution patterns for magnetite nanoparticles with 17 different coatings across multiple organs and tissues, including the liver, spleen, lungs, kidneys, heart, tumor, brain, peripheral blood, muscle, and bone. This methodology offers high sensitivity, user-friendly operation, and provides quantitative measurements across a broad dynamic range of nanoparticle concentrations. These advantages enabled high-throughput acquisition of precise blood circulation and biodistribution data. In addition, histological analysis was conducted to evaluate nanoparticle penetration depth within tumor tissue. Results: Here we conducted a comprehensive study of the effect of 17 different polymer-, lectin-, and small molecule-based coatings on the behavior of magnetite nanoparticles in vivo. For each type of obtained nanoparticles, we implemented passive targeting as well as magnetic targeting, the latter using an external magnetic field localized in the tumor area. Conclusions: The collected dataset provides critical insights into how surface modifications influence nanoparticle performance in complex biological systems, offering valuable guidance for optimizing therapeutic nanocarrier design.

Bioluminescent reporters and biosensors are widely used to quantify gene expression, evaluate delivery systems and assess biodistribution because they provide low-background readouts over a broad dynamic range. The NanoLuc–furimazine system is particularly attractive owing to its high brightness and stability. Ex vivo analysis of organ homogenates offers a controlled approach for measuring tissue-associated reporter activity, yet matrix-dependent effects are often overlooked in quantitative interpretation. Here, we systematically evaluated how mouse organ homogenates affect NanoLuc–furimazine measurements by spiking defined amounts of purified NanoLuc into different organ matrices. Across matrices, optical profiles, blank signals, calibration slopes, and estimated limits of detection and quantification varied, resulting in substantial differences in the measured NanoLuc signal. Further analysis showed that NanoLuc-independent furimazine luminescence arises from lysis-buffer components and protein-containing environments. Matrix-matched blank subtraction reduced background-related bias, whereas matrix-specific calibration demonstrated differences in analytical response, particularly for weak signals and optically dense samples. These findings highlight the extent of matrix-dependent variability in ex vivo NanoLuc measurements and support more accurate interpretation of reporter signals in delivery, biodistribution, biosensing, and gene-expression studies.