The influence of the size and aspect ratio of anisotropic, porous CaCO3 particles on their uptake by cells

Abstract

Background: Recent reports highlighting the role of particle geometry have suggested that anisotropy can affect the rate and the pathway of particle uptake by cells. Therefore, we investigate the internalization by cells of porous calcium carbonate particles with different shapes and anisotropies.

Results: We report here on a new method of the synthesis of polyelectrolyte coated calcium carbonate particles whose geometry was controlled by varying the mixing speed and time, pH value of the reaction solution, and ratio of the interacting salts used for particle formation. Uptake of spherical, cuboidal, ellipsoidal (with two different sizes) polyelectrolyte coated calcium carbonate particles was studied in cervical carcinoma cells. Quantitative data were obtained from the analysis of confocal laser scanning microscopy images.

Conclusions: Our results indicate that the number of internalized calcium carbonate particles depends on the aspect ratio of the particle, whereby elongated particles (higher aspect ratio) are internalized with a higher frequency than more spherical particles (lower aspect ratio). The total volume of internalized particles scales with the volume of the individual particles, in case equal amount of particles were added per cell.

Fig. 1

The first row from the top: schematics of the geometry of the investigated particles. a “Small” spherical CaCO₃ particles, b “small” ellipsoidal CaCO₃ particles, c cuboidal CaCO₃ particles, d “big” ellipsoidal CaCO₃ particles, and e control “big” spherical SiO₂ particles. All particles have symmetry in the x–y plane. d ₁ describes the extension of the particles along the axis of symmetry, and d ₂ the extension perpendicular to this axis. The second row from the top: fluorescence microscopy images of the different particles used in the uptake study as recorded in solution for (a) through (e). The scale bars in all images correspond to 20 µm. The third row from the top: SEM images of the different particles used in the uptake study for (a) through (e). The scale bars in all images correspond to 2 μm. The fourth row from the top: TEM images of the different particles used in the uptake study for (a) through (e). The scale bars in all images correspond to 1 μm.

Fig. 2

Tuning the sizes of particles. a The size d = d₁ = d₂ of spherical CaCO₃ particles was tuned by varying the stirring time t of the CaCl₂ and Na₂CO₃ solutions at pH values of 5, 7, 9 in pure water solution with a sodium carbonate to calcium chloride ratio S = 1. b The size of the long axis d₁ of ellipsoidal CaCO₃ particles was tuned by varying the stirring time t_s at certain salt concentration ratios S = 2.5 (black curve); 5 (red curve); and 10 (blue curve) in water/ethylene glycol = 1/5 solutions of pH = 9.5. c The aspect ratio R (d₁/d₂) of the CaCO₃ particles as a function of S; R was tuned by varying the stirring time t_s in different water and water/EG (ethylene glycol) mixtures at pH = 9.5. The vertical bars represent the standard deviation values.

Fig. 3

Orthogonal view from different planes (x/y, x/z or y/z) of the confocal microscope images used to analyze the particle uptake. Examples correspond to: a “small” ellipsoidal CaCO₃ particles and b “big” spherical SiO₂ particles (used as control). Co-localization of fluorescently labeled CaCO₃ particles (with TRITC, in red) with the lysosomal marker Anti-LAMP1 labeled with DyLight 649 (artificially colored in yellow). The HeLa cell´s nucleus was stained with DAPI (in blue) and the cytoskeleton with Oregon Green^® 488 phalloidin (in green). Scale bars correspond to 20 µm.

Fig. 4

Confocal images of HeLa cells which internalized differently shaped particles (red fluorescence) after incubation for 24 h. a “Small” spherical CaCO₃ particles, b “small” ellipsoidal CaCO₃ particles, c cuboidal CaCO₃ particles, d “big” ellipsoidal CaCO₃ particles, and e “big” spherical SiO₂ particles (used as control). Particles are labeled with TRITC (red). Nuclei, lysosomes, and cytoskeletons are fluorescence labeled with DAPI (blue), Anti-LAMP1 labeled with DyLight 649 (artificially colored in yellow), and Oregon Green^® 488 phalloidin (green), respectively. 1, 2, 3, 4 and 5 mean the red, green, blue and yellow channels and the merged image, respectively. The scale bars in all images correspond to 20 μm.

Fig. 5

Influence of size on the uptake of ellipsoidal particles by HeLa cells. a Histogram of the frequency f(N) of cells which have internalized N particles per HeLa cell after 24 h of particle incubation with a concentration of 10 added particles per cell. b Corresponding cumulative probability plot p(N) showing a higher internalization rate for smaller ellipsoidal CaCO₃ particles. The vertical bars represent the standard deviation values. c Cumulative probability p(N) for N internalized particles per cell for particles with different shape (and size) after 24 h of particle incubation with a cocentration of 10 added particles per cell. Cuboidal CaCO₃ (black dots), “small” spherical CaCO₃ (green dots), “big” spherical SiO₂ (pink dots), “big” ellipsoidal CaCO₃ (red dots), and “small” ellipsoidal (black dots). All particles had the same surface modification (positively charged layer of PAH). The vertical bars represent the standard deviation values.