Intracellular uptake of nanocrystals: Probing with aggregation-induced emission of fluorescence and kinetic modeling

Abstract

Nanocrystal formulations have been explored to deliver poorly water-soluble drug molecules. Despite various studies of nanocrystal formulation and delivery, much more understanding needs to be gained into absorption mechanisms and kinetics of drug nanocrystals at various levels, ranging from cells to tissues and to the whole body. In this study, nanocrystals of tetrakis (4-hydroxyphenyl) ethylene (THPE) with an aggregation-induced emission (AIE) property was used as a model to explore intracellular absorption mechanism and dissolution kinetics of nanocrystals. Cellular uptake studies were conducted with KB cells and characterized by confocal microscopy, flow cytometry, and quantitative analyses. The results suggested that THPE nanocrystals could be taken up by KB cells directly, as well as in the form of dissolved molecules. The cellular uptake was found to be concentration- and time-dependent. In addition, the intracellular THPE also could be exocytosed from cells in forms of dissolved molecules and nanocrystals. Kinetic modeling was conducted to further understand the cellular mechanism of THPE nanocrystals based on first-order ordinary differential equations (ODEs). By fitting the kinetic model against experimental measurements, it was found that the initial nanocrystal concentration had a great influence on the dynamic process of dissolution, cellular uptake, and exocytosis of THPE nanocrystals. As the nanocrystal concentration increased in the culture media, dissolution of endocytosed nanocrystals became enhanced, subsequently driving the efflux of THPE molecules from cells.

Keywords: Absorption mechanism; Aggregation-induced emission; Dissolution kinetics; Fate; Intracellular uptake; Nanocrystal; Pharmacokinetics; Tetrakis(4-hydroxyphenyl) ethylene.

Graphical abstract

Figure 1

Structures of tetraphenylethylene (TPE, A) and tetrakis (4-hydroxyphenyl) ethylene (THPE, B).

Figure 2

Schematic depicting transportation of THPE across cell membrane, including both molecules and nanocrystals. C denotes concentration and k rate constant, with subscripts nc and mol representing nanocrystal and molecule, and en, ex, in, and d representing endocytosis, exocytosis, intracellular and dissolution.

Figure 3

SEM image of tetrakis (4-hydroxyphenyl) ethylene nanocrystals THPE-NCs.

Figure 4

The AIE feature of THPE-NCs (A) Vials of THPE-NCs in water/ethanol mixtures of various v/v ratios under UV illumination (B) Fluorescence measured by fluorospectrometer and dissolved THPE measured by HPLC in the mixtures (C) The relationship between fluorescence and concentration of THPE-NCs.

Figure 5

Confocal images of KB cells cultured with THPE-NCs at (A) 2.7 and (B) 24 μg/mL for 1, 3, 7 h, and additional 1, 2, and 5 h incubation with only fresh medium after 3 h of treatment. THPE was shown in blue and cell membrane in red. Scale bar = 100 μm.

Figure 6

Intracellular fluorescence intensity (IFI) measured by flow cytometry after 0.5, 1, 2 and 3 h of incubation with THPE-NCs, respectively, as well as an additional 1 and 2 h incubation after removing NCs from the culture media at 3 h. Statistically significant difference between a data point and its previous time point of the same concentration is marked by ∗P < 0.05 or ∗∗P<0.01).

Figure 7

Relative intracellular concentration of THPE-NCs derived from IFI and PK modeling at 0.5, 1, 2 and 3 h of incubation with 2.67 (A), 8 (B), and 24 (C) μg/mL of THPE-NCs, respectively, as well as for an additional 1 and 2 h incubation after removing NCs from the culture media at 3 h. The value at 0.5 h is used as the reference.

Figure 8

Cellular uptake measurement of KB cells by HPLC after 0.5, 1, 2 and 3 h of incubation with 2.67 (A), 8 (B), and 24 (C) μg/mL of THPE-NCs, respectively, as well as an additional 1 and 2 h incubation after removing NCs from the culture media at 3 h.

Figure 9

The fitted (line) and predicted curves (dash line) of cellular uptake in KB cells after 0.5, 1, 2 and 3 h incubation with 2.67 (A), 8 (B), and 24 (C) μg/mL of THPE-NCs, respectively, as well as an additional 1 and 2 h incubation after removing the drug from the culture media at 3 h. Experimentally measured data are shown as dot.