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.

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.

Macrophage In Vitro and In Vivo Tracking via Anchored Microcapsules.

A new promising trend in personalized medicine is the use of autologous cells (macrophages or stem cells) for cell-based therapy and also as a "Trojan horse" for targeted delivery of a drug carrier. The natural ability of macrophages for chemotaxis allows them to deliver cargo to the damaged area, significantly reducing side effects on healthy organ tissues. Therefore, it is important to develop tools to track their behavior in the organism. While labeled containers can serve as anchored tags for imaging macrophages in vivo, they can affect the properties and functions of macrophages. This work demonstrates that 3 µm sized capsules based on biocompatible polyelectrolytes and fluorescently labeled with both Cy7 and RITC dyes do not affect cell functionalization in vitro, such as viability, proliferation, and movement of transformed monocyte/macrophage-like cells (RAW 264.7) and primary bone marrow derived macrophages (BMDM) at maximal loading of five capsules per cell. In addition, capsules allowed fluorescent detection of ex vivo loaded cells 24 h after the tail vein injection in vivo and visualization of microcapsule-laden macrophages ex vivo using confocal microscopy. We have delivered about 62.5% of injected BMDM containing 12.5 million capsules with 3.75 µg of high-molecular-weight cargo (0.3 pg/capsule) to the liver. Our results demonstrate that 3 µm polyelectrolyte fluorescently labeled microcapsules can be used for safe macrophage loading, allowing cell tracking and drug delivery, which will facilitate development of macrophage-based cell therapy protocols.

Doxorubicin-Loaded Core-Shell UiO-66@SiO2 Metal-Organic Frameworks for Targeted Cellular Uptake and Cancer Treatment.

Beneficial features of biocompatible high-capacity UiO-66 nanoparticles, mesoporous SiO2, and folate-conjugated pluronic F127 were combined to prepare the core-shell UiO-66@SiO2/F127-FA drug delivery carrier for targeted cellular uptake in cancer treatment. UiO-66 and UiO-66-NH2 nanoparticles with a narrow size and shape distribution were used to form a series of core-shell MOF@SiO2 structures. The duration of silanization was varied to change the thickness of the SiO2 shell, revealing a nonlinear dependence that was attributed to silicon penetration into the porous MOF structure. Doxorubicin encapsulation showed a similar final loading of 5.6 wt % for both uncoated and silica-coated particles, demonstrating the potential of the nanocomposite's application in small molecule delivery. Silica coating improved the colloidal stability of the composites in a number of model physiological media, enabled grafting of target molecules to the surface, and prevented an uncontrolled release of their cargo, with the drawback of decreased overall porosity. Further modification of the particles with the conjugate of pluronic and folic acid was performed to improve the biocompatibility, prolong the blood circulation time, and target the encapsulated drug to the folate-expressing cancer cells. The final DOX-loaded UiO-66@SiO2/F127-FA nanoparticles were subjected to properties characterization and in vitro evaluation, including studies of internalization into cells and antitumor activity. Two cell lines were used: MCF-7 breast cancer cells, which have overexpressed folate receptors on the cell membranes, and RAW 264.7 macrophages without folate overexpression. These findings will provide a potential delivery system for DOX and increase the practical value of MOFs.