Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluorescent dyes absorbing and emitting in the visible and near-IR regions are promising for the development of fluorescent probes for labeling and bio-visualization of body cells. The ability to absorb and emit in the long-wavelength region increases the efficiency of recording the spectral signals of the probes due to the higher permeability of the skin layers. Compared to other fluorescent dyes, BODIPYs are attractive due to their excellent photophysical properties–narrow absorption and emission, intense fluorescence, simple signal modulation for the practical applications. As part of conjugates with biomolecules, BODIPY could act as a biomarker, but as therapeutic agent, which allows solving several problems at once-labeling or bioimaging and treatment based on the suppression of pathogenic microflora and cancer cells, which provides a huge potential for practical application of BODIPY conjugates in medicine. The review is devoted to the discussion of the recent, promising directions of BODIPY application in the field of conjugation with biomolecules. The first direction is associated with the development of BODIPY conjugates with drugs, including compounds of platinum, paclitaxel, chlorambucil, isoxazole, capsaicin, etc. The second direction is devoted to the labeling of vitamins, hormones, lipids, and other biomolecules to control the processes of their transport, localization in target cells, and metabolism. Within the framework of the third direction, the problem of obtaining functional optically active materials by conjugating BODIPY with other colored and fluorescent particles, in particular, phthalocyanines, is being solved. [ABSTRACT FROM AUTHOR]

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene. [ABSTRACT FROM AUTHOR]