Submicron-Sized In Situ Osmotic Pressure Sensors for In Vitro Applications in Biology

Abstract

Physical forces are important cues in determining the development and the normal function of biological tissues. While forces generated by molecular motors have been widely studied, forces resulting from osmotic gradients have been less considered in this context. A possible reason is the lack of direct in situ measurement methods that can be applied to cell and organ culture systems. Herein, novel kinds of resonance energy transfer (FRET)-based liposomal sensors are developed, so that their sensing range and sensitivity can be adjusted to satisfy physiological osmotic conditions. Several types of sensors are prepared, either based on polyethylene glycol- (PEG)ylated liposomes with steric stabilization and stealth property or on crosslinked liposomes capable of enduring relatively harsh environments for liposomes (e.g., in the presence of biosurfactants). The sensors are demonstrated to be effective in the measurement of osmotic pressures in pre-osteoblastic in vitro cell culture systems by means of FRET microscopy. This development paves the way toward the in situ sensing of osmotic pressures in biological culture systems.

Keywords: biosensing; imaging; liposomes; resonance energy transfer; semi-permeable membranes.

Figure 1

a) Chemical structures of the lipids used for the preparation of osmotic pressure sensors. b) Schematic illustration of the sensor working principle and the in situ osmotic pressure imaging in cell cultures.

Figure 2

FRET ratio R obtained with Lip‐DA liposomes loaded with H₂O, 0.05% NaCl, 0.1% NaCl, 0.2% NaCl, 0.45% NaCl and 0.9% NaCl and with a dye concentration of 50 µM (1:1 molar ratio) as a function of the external osmotic pressure П. The dashed vertical line indicates the physiological osmotic pressure around 0.7 MPa. The solid line shows exemplarily a linear fit to the data points for Lip‐DA‐0 within the linear range.

Figure 3

Characteristics and osmotic responses of Lip‐PEG10‐DA liposomes. Distributions of a) size and b) zeta potential of Lip‐PEG10‐DA‐0.05 liposomes in 0.05% NaCl as obtained by DLS and phase analysis light scattering (PALS), respectively. TEM images of c) Lip‐PEG10‐DA‐0 and d) Lip‐PEG10‐DA‐0.05 liposomes in dry state (inset, higher magnification) stained with 1% uranyl acetate. e) FRET ratio R obtained with Lip‐PEG10‐DA liposomes loaded with H₂O, 0.05%, 0.1% and 0.2% NaCl and a dye concentration of 50 µM (1:1 molar ratio) as a function of the external osmotic pressure generated by various concentrations of NaCl.

Figure 4

Characteristics and osmotic responses of crosslinked polymeric liposomes. a,d) TEM images of a) cLip‐DA‐0 and d) cLip‐PEG10‐DA‐0.05 liposomes in dry state (inset, higher magnification) stained with 1% uranyl acetate. b,e) Size distributions of b) cLip‐DA‐0 and e) cLip‐PEG10‐DA‐0.05 liposomes in water and 0.05% NaCl, respectively, in the presence of 0.3% Triton X‐100. c,f) FRET ratio R obtained with c) cLip‐DA‐0 and f) cLip‐PEG10‐DA‐0.05 liposomes loaded with a dye concentration of 25 µM (1:1 molar ratio) in H₂O and 0.05% NaCl, respectively, as a function of the external osmotic pressure generated by various external concentrations of NaCl or PEG20000. The solid line in (c) shows exemplarily a linear fit to the data points for cLip‐DA‐0 in NaCl in the range of 0–0.15 MPa.

Figure 5

Cytotoxicity and sensor functionality in cell culture media. a) Viability of MC3T3‐E1 cells after co‐incubation with 25, 50, 100, and 200 µg mL⁻¹ Lip‐PEG10‐DA‐0.05 liposomes for 1, 2 and 3 d, respectively. b) FRET ratio obtained with Lip‐PEG10‐DA‐0.05 sensors loaded with a dye concentration of 75 µM (1:1 molar ratio) after incubation in NaCl or MEM α cell culture media supplemented with 10% ASF with/without MC3T3‐E1 cells for 24, 48, and 72 h at 37 °C. Data in (a) are expressed as the mean ± standard deviation (SD), n = 5. NS indicates no significant difference at a level of p < 0.05.

Figure 6

Application of Lip‐PEG10‐DA‐0.05 sensors for osmotic pressure imaging in cell culture. Confocal laser scanning microscopy (CLSM) images of sensors (125 µg mL⁻¹)/cells in a,b) 0.05% NaCl and e,f) MEM α‐10% ASF with MC3T3‐E1 cells. The green fluorescence in (a) and yellow fluorescence in (b,f) represent the donor emission signal (Ex 458 nm, Em 468–538 nm) and the sensitized acceptor emission signal (Ex 458 nm, Em 571–700 nm), respectively. (e) shows the live MC3T3‐E1 cells with the nuclei stained with Hoechst 33342 (Ex 405 nm, Em 415–450 nm). (f) demonstrates the functioning of the sensors around the cells. c,g) FRET ratio and d,h) osmotic pressure mapping with the sensors in 0.05% NaCl (c,d) and MEM α‐10% ASF with MC3T3‐E1 cells (g,h) (inset, higher magnification), respectively. The green (c) and magenta (g) dots indicate relatively low FRET ratio in 0.05% NaCl and high FRET ratio in the medium, respectively. The purple (d) and red (h) dots indicate relatively low osmotic pressure in 0.05% NaCl and high osmotic pressure in the medium, respectively.