Dual color pH probes made from silica and polystyrene nanoparticles and their performance in cell studies

Ratiometric green–red fluorescent nanosensors for fluorometrically monitoring pH in the acidic range were designed from 80 nm-sized polystyrene (PS) and silica (SiO2) nanoparticles (NPs), red emissive reference dyes, and a green emissive naphthalimide pH probe, analytically and spectroscopically characterized, and compared regarding their sensing performance in aqueous dispersion and in cellular uptake studies. Preparation of these optical probes, which are excitable by 405 nm laser or LED light sources, involved the encapsulation of the pH-inert red-fluorescent dye Nile Red (NR) in the core of self-made carboxylated PSNPs by a simple swelling procedure and the fabrication of rhodamine B (RhB)-stained SiO2-NPs from a silane derivative of pH-insensitive RhB. Subsequently, the custom-made naphthalimide pH probe, that utilizes a protonation-controlled photoinduced electron transfer process, was covalently attached to the carboxylic acid groups at the surface of both types of NPs. Fluorescence microscopy studies with the molecular and nanoscale optical probes and A549 lung cancer cells confirmed the cellular uptake of all probes and their penetration into acidic cell compartments, i.e., the lysosomes, indicated by the switching ON of the green naphthalimide fluorescence. This underlines their suitability for intracellular pH sensing, with the SiO2-based nanosensor revealing the best performance regarding uptake speed and stability.

To study the pH signaling mechanism of 3 in more detail, pH titration by NMR measurements in D2O was performed. The pH probe was dissolved in D2O and measured by addition of varying amounts of HCl, to change the pH from 8.5 to 2.5 ( Figure S 11).         Synthesis of reference dye RhB-APTES. The synthesis of RhB-APTES was adopted from the literature (Fehler! Verweisquelle konnte nicht gefunden werden. b)). 2 9.97 mg (0.019 mmol) of RITC was dissolved in 1 mL of ethanol (absolute), followed by the addition of 13.2 µL (0.056 mmol, 2.9 equiv.) of 3-Aminopropyltriethoxysilane (APTES) under a strict argon atmosphere; the resulting mixture was then allowed to stir for 2 d at room temperature (r.t.). Finally, the prepared RhB-APTES was stored under light exclusion at 4 °C, to be utilized later in dye embedding experiments.

Loading of SiO2-NP with NR and NR-Silane.
To obtain 80 nm large SiO2-NPs loaded with NR or NR-Silane, the synthesis of the particles was performed as described for RhB-APTES loaded NPs, only in the last regrowth step 0.15 mL (6.4•10 -7 mol) of NR or 0.15 mL (6.4•10 -7 mol) of NR-APTES in DMSO was injected into the aqueous phase 20 min after the addition of TEOS.

Determination of the amount of reference dyes in the particle cores
To determine the amount of loaded reference dye molecules per particle a dissolution method was used. Different concentrations of NR in THF and RhB-APTES in aqueous B-R buffer were dissolved and the emission spectra ( Figure S 25 and Figure S.27) and calibration curves were recorded. In addition, a previously dried amount of PSNP-NR-COOH of known mass was dissolved in 2.5 mL of THF and the emission spectrum was measured ( Figure S 26), while SiO2-RhB-COOH were dissolved in aqueous B-R buffer at pH 12. The amount of reference dye molecules per particle was then calculated from the experimentally determined (average) amount of incorporated dye and the number of particles in the dispersion, using the number-based hydrodynamic diameters and a density of (PS) = 1.06 g/cm 3 and (SiO2) = 2.09 g/cm 3 .    To determine the accessible number of COOH groups on the particle surface a toluidine blue assay was performed. 3

Surface modification of carboxylated PSNPs and SiO2-NPs with 3
Figure S 30. Reaction scheme of the surface modification of plain, carboxylated PSNPs (no red reference dye in the particle core) with 3.

IR measurements
FT-IR measurements were measured on Vertex 70 instrument from Bruker with MCT (N2(l) cooled) detector.
To confirm the binding of the synthesized optical probe 3 on the surface of the particles, PSNP-COOH with only 3 on the particle surface is synthesized as shown in Figure S

Cellular fluorescence microscopy
The human lung cancer cell line A549 were routinely propagated as described in the literature. 4 In short, the , A549 cells were cultivated in DMEM medium, to which 10% fetal calf serum (FCS), 2% glutamine, and penicillin / streptomycin (purchased from PAN Biotech) were added. Cells were disseminated into medium at a concentration of 1 x 10 5 cells/mL, cultivated at 37 °C with 5% CO2, and split 1:5 twice per week. Using a 24-well culture plate prepared with glass cover slips (Sigma Aldrich), cells were disseminated at a concentration of 1 x 10 5 cells/mL in 1 mL of medium and cultured at 37 °C and 5% CO2 for 48 h. Cells were rinsed with sterile PBS, after which cell pH was adjusted following the protocol by Lucien et al. 5  OIL CS2). The fluorescence of DAPI was excited at 405 nm and its emission was recorded in the spectral window of 420 -480 nm. The pH sensor dye was excited at 405 nm and its emission was detected in the spectral window of 520 -560 nm. The reference dye was excited at 560 nm and its emission was detected in the spectral window of 570 -660 nm. All channels were sequentially excited to minimize spectral bleed through. The pixel size of the recorded images was 29.36 nm. A z-stack over the height of the cells with a step size of 0.3 µm was recorded. All images were acquired with the same imaging settings. Adjustments of the intensity histograms of the images to improve the visibility of the fluorescence signal were equally applied to all images.

Figure S 38.
Epifluorescence images of the pH nanosensors PSNP-NR-3 (a-d), particle concentration 100 µg/mL) and SiO2-RhB-3 (e-h), particle concentration 100 µg/mL) by A549 cells measured after incubation times of 30 min, and 24 h, respectively. Prior to the fluorescence microscopy studies, the cells were incubated alive, fixed with 4% paraformaldehyde (PFA), and then co-stained with DAPI (cell nuclei, blue channel). Excitation was carried out with an Osram 50W/ACL1 Cz HBO Mercury vapor short-arc lamp and the fluorescence was monitored with emission filters set to λEm = 470 nm (green channel) and to λEm = 560 nm (red channel). For the detection of the fluorescence of DAPI Leica filter cube A (Em = 340/380 nm (blue channel) was used. All images show a scale bar of 10 µm.