Encapsulin Based Self-Assembling Iron-Containing Protein Nanoparticles for Stem Cells MRI Visualization

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR-T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long-term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles’ synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron-containing nanoparticles in mesenchymal stem cells—without affecting cell viability—and increased contrast properties of MSCs in MRI

New Approach to Non-Invasive Tumor Model Monitoring via Self-Assemble Iron Containing Protein Nanocompartments

According to the World Health Organization, breast cancer is the most common oncological disease worldwide. There are multiple animal models for different types of breast carcinoma, allowing the research of tumor growth, metastasis, and angiogenesis. When studying these processes, it is crucial to visualize cancer cells for a prolonged time via a non-invasive method, for example, magnetic resonance imaging (MRI). In this study, we establish a new genetically encoded material based on Quasibacillus thermotolerans (Q.thermotolerans, Qt) encapsulin, stably expressed in mouse 4T1 breast carcinoma cells. The label consists of a protein shell containing an enzyme called ferroxidase. When adding Fe2+, a ferroxidase oxidizes Fe2+ to Fe3+, followed by iron oxide nanoparticles formation. Additionally, genes encoding mZip14 metal transporter, enhancing the iron transport, were inserted into the cells via lentiviral transduction. The expression of transgenic sequences does not affect cell viability, and the presence of magnetic nanoparticles formed inside encapsulins results in an increase in T2 relaxivity

Study of Cytotoxicity and Internalization of Redox-Responsive Iron Oxide Nanoparticles on PC-3 and 4T1 Cancer Cell Lines

Redox-responsive and magnetic nanomaterials are widely used in tumor treatment separately, and while the application of their combined functionalities is perspective, exactly how such synergistic effects can be implemented is still unclear. This report investigates the internalization dynamics of magnetic redox-responsive nanoparticles (MNP-SS) and their cytotoxicity toward PC-3 and 4T1 cell lines. It is shown that MNP-SS synthesized by covalent grafting of polyethylene glycol (PEG) on the magnetic nanoparticle (MNP) surface via SS-bonds lose their colloidal stability and aggregate fully in a solution containing DTT, and partially in conditioned media, whereas the PEGylated MNP (MNP-PEG) without S-S linker control remains stable under the same conditions. Internalized MNP-SS lose the PEG shell more quickly, causing enhanced magnetic core dissolution and thus increased toxicity. This was confirmed by fluorescence microscopy using MNP-SS dual-labeled by Cy3 via labile disulfide, and Cy5 via a rigid linker. The dyes demonstrated a significant difference in fluorescence dynamics and intensity. Additionally, MNP-SS demonstrate quicker cellular uptake compared to MNP-PEG, as confirmed by TEM analysis. The combination of disulfide bonds, leading to faster dissolution of the iron oxide core, and the high-oxidative potential Fe3+ ions can synergically enhance oxidative stress in comparison with more stable coating without SS-bonds in the case of MNP-PEG. It decreases the cancer cell viability, especially for the 4T1, which is known for being sensitive to ferroptosis-triggering factors. In this work, we have shown the effect of redox-responsive grafting of the MNP surface as a key factor affecting MNP-internalization rate and dissolution with the release of iron ions inside cancer cells. This kind of synergistic effect is described for the first time and can be used not only in combination with drug delivery, but also in treatment of tumors responsive to ferroptosis