Labeling and Tracking of Individual Human Mesenchymal Stromal Cells Using Photoconvertible Fluorescent Microcapsules.

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as "gold standard" to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 µm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling.

Single Mesenchymal Stromal Cell Migration Tracking into Glioblastoma Using Photoconvertible Vesicles.

Reliable cell labeling and tracking techniques are imperative for elucidating the intricate and ambiguous interactions between mesenchymal stromal cells (MSCs) and tumors. Here, we explore fluorescent photoconvertible nanoengineered vesicles to study mMSC migration in brain tumors. These 3 µm sized vesicles made of carbon nanoparticles, Rhodamine B (RhB), and polyelectrolytes are readily internalized by cells. The dye undergoes photoconversion under 561 nm laser exposure with a fluorescence blue shift upon demand. The optimal laser irradiation duration for photoconversion was 0.4 ms, which provided a maximal blue shift of the fluorescent signal label without excessive laser exposure on cells. Vesicles modified with an extra polymer layer demonstrated enhanced intracellular uptake without remarkable effects on cell viability, motility, or proliferation. The optimal ratio of 20 vesicles per mMSC was determined. Moreover, the migration of individual mMSCs within 2D and 3D glioblastoma cell (EPNT-5) colonies over 2 days and in vivo tumor settings over 7 days were traced. Our study provides a robust nanocomposite platform for investigating MSC-tumor dynamics and offers insights into envisaged therapeutic strategies. Photoconvertible vesicles also present an indispensable tool for studying complex fundamental processes of cell-cell interactions for a wide range of problems in biomedicine.

Multiple dyes applications for fluorescent convertible polymer capsules as macrophages tracking labels.

Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.

Enhanced cytotoxicity caused by AC magnetic field for polymer microcapsules containing packed magnetic nanoparticles.

Magnetic hyperthermia (MH) is a perspective tool to treat the tumor while the magnetic material is delivered. The key problems in MH development is to ensure an effective local heating within cancer cell without overheating other cells. In order to do that one has to reach substantial local accumulation of magnetic nanoparticles (MNPs) and/or magnetically sensitive objects with advanced heat properties. Absorbing heat energy for destroying tumor cells can be generated only if there is sufficient amount of locally placed MNPs. In this work, we propose polyelectrolyte microcapsules modified with iron oxide nanoparticles as an approach to tie magnetic materials in high concentration locally. These microcapsules (about 3 microns in diameter) can be readily internalized by various cells. The human fibroblasts uptake of the microcapsules and cytotoxic effect upon the influence of alternating magnetic field (AMF) while magnetic capsules are inside the cells is under study in this work. The cytotoxicity of the magnetic microcapsules was compared with the cytotoxicity of the MNPs while free in the solution to evaluate the effect of bounding MNPs. A cytotoxic effect on cells was found in the case of preliminary incubation of fibroblasts with capsules while the AMF is applied. In the case of MNPs in an equivalent dose per mass of magnetic material, there was no cytotoxic effect noticed after the treatment with the field. It is noteworthy that during the treatment of cells with the AMF, the increase in temperature of the incubation medium was not registered. The morphological changes on fibroblasts were consistent with the data of the viability assessment. Thus, the synthesized capsules are shown as a means for local enhancement of magnetic hyperthermia in the treatment of tumor diseases.