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

Abstract

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.

Keywords: Carbon nanoparticle; Cell imaging; Cell labeling; Deethylation; Encapsulation; Fluorescent label; Macrophage; Microcapsule; Photoconversion; Photoconvertible label.

Graphical abstract

Fig. 1

(a) Scheme of the dye-loaded thermally treated capsules preparation in PVA gel. (b) Graph of the capsule diameters before and after thermal treatment with the dyes RhB, Rh6G, PyB, Fl, AY, AO, and TR with corresponding CLSM images (scale bar is 2 μm).

Fig. 2

(a) Angularly averaged fluorescence intensities of the capsules with dyes RhB, Rh6G, and PyB (detection range 580–620/670 nm, red channel) and Fl, AY, AO, and TR (detection range 505–600 nm, green channel). λ-scans of capsules with (b) RhB, (c) Rh6G, and PyB (488 nm excitation). λ-scans of capsules with (d) Fl, AY, AO, and TR (405 nm excitation). Red and green rectangles correspond to the detection ranges 580–620/670 nm and 505–600 nm, respectively.

Fig. 3

(a) Normalized changes in the fluorescence intensity of the dyes before and after the bleaching test. CLSM images of thermally treated microcapsules in PVA gel with RhB (b), Rh6G (c), and PyB (d) before and after photoconversion (scale bar is 5 μm). Changes in the angularly averaged fluorescence intensity of the capsules before and after laser irradiation in the red and green channels of RhB (e,f), Rh6G (h,i), and PyB (k,l). Capsule emission spectra under 488 nm excitation before and after laser conversion for RhB (g), Rh6G (j), and PyB (m) (green and red rectangles correspond to the 505–540 and 580–620/670 nm detection ranges of the confocal microscope, respectively).

Fig. 4

Mass spectra of RhB and Rh6G solutions (a, b) and RhB/Rh6G solutions with DS after thermal treatment and UV irradiation (c, d).

Fig. 5

Assessment of the metabolic activity of RAW 264.7 (a) and BMDM (b) cells by adding empty, heat-treated empty, and heat-treated capsules with RhB or Rh6G dyes at 1, 5, and 10 capsules per cell. (c,d) Fluorescence microscopy images combined with brightfield images of RAW 264.7 and BMDM cells with 5 capsules/cell (RhB and Rh6G). Images were taken after the addition of capsules, 0.5, 2, and 24 h later (scale bar is 20 μm).

Fig. 6

Dependence of the number of cells with internalized capsules containing RhB on the number of added capsules.

Fig. 7

Bright-field microscopy images of the wound closure for RAW 264.7 (a) and BMDM (b) with and without the capsules. Graph of wound closure over time for RAW 264.7 (c) and BMDM (d).

Fig. 8

CLSM images of RAW (a,b) 264.7 and BMDM (c,d) cells before and after photoconversion of capsules inside the cells (scale bar is 10 μm).