In a recent article published in Scientific Reports, researchers presented the development of a novel pH nanosensor utilizing polystyrene nanoparticles functionalized with a pH-responsive dye and wheat germ agglutinin (WGA) for cell targeting.
Screening of targeting capabilities of WGA as a targeting moiety. CLSM images of the lung cell line A549, skin cell line HaCaT and placenta cell line BeWo incubated with 5 µg/mL WGA-FITC (green) and Hoechst for nuclei stain (blue). Image Credit: https://www.nature.com/articles/s41598-024-62976-2
Background
Accurate monitoring of extracellular pH is essential for understanding cellular physiology and disease mechanisms, as pH plays a critical role in various biological processes, including cell signaling, metabolism, and proliferation.
Traditional pH measurement techniques often lack the spatial and temporal resolution required to capture dynamic pH changes in live cells. Therefore, the development of non-invasive and cell-compatible pH nanosensors is crucial for real-time pH monitoring in diverse cell types.
The Current Study
The fabrication of the pH nanosensor involved a two-step strategy. Initially, the dye Nile red (NR) was incorporated into polystyrene nanoparticles (PS NPs) using a well-established swelling method. The selection of PS NPs as the platform for this nanosensor was influenced by their availability in various surface functionalizations. Specifically, PS NPs featuring carboxylic surface groups were chosen for their hydrophilic properties, which facilitate the conjugation of dye molecules and proteins.
In the second step of the fabrication process, wheat germ agglutinin (WGA) labeled with fluorescein isothiocyanate (FITC), a pH-responsive dye, was covalently attached to the carboxylic groups on the surface of the polystyrene nanoparticles (PS NPs) via carbodiimide coupling. WGA, a lectin known for its affinity for N-acetylglucosamine and sialic acid residues, was specifically chosen to enable the active targeting of cell membranes by the pH nanosensor.
To assess the effectiveness of WGA as a targeting agent for cell membranes, a cell binding assay was performed. This involved comparing WGA-conjugated nanosensors with precursor nanosensors lacking WGA, serving as non-targeted control nanoparticles. The quantification of nanoparticles bound to cells was measured through the fluorescence signal from Nile red, and these values were correlated to the total amount of nanoparticles present. This assay provided critical insights into the specificity and efficiency of WGA in targeting various cell lines' membranes.
Furthermore, the biocompatibility and non-toxic nature of the pH nanosensor were evaluated through viability assays on A549 cells exposed to various concentrations of the nanosensor over different time periods, compared to controls without the nanosensor. These assessments utilized standard protocols and colorimetric viability assay kits, such as WST-1, to determine the impact of the nanosensor on cell health and proliferation.
Additionally, scanning electron microscopy (SEM) was employed to visualize the interactions between the pH nanosensors and cells, specifically A549 and HaCaT cells. Samples treated with the nanosensor were compared against untreated controls to observe any morphological changes or interactions at the cellular surface, further elucidating the physical engagement of the nanosensors with target cells.
Results and Discussion
The imaging of the pH nanosensor with various cell lines in buffers of known pH values demonstrated its effective targeting of cell membranes. The observed fluorescence signals of the nanosensor localized at the cell membranes, facilitating direct sensing of extracellular pH on the cell surface. The co-localization of FITC and NR fluorescence signals indicated stable dye conjugation, enabling ratiometric determination of extracellular pH.
The fluorescence intensity (FI) of FITC varied with pH changes, showing increased FI in basic conditions and decreased FI in acidic conditions, affirming the pH-responsive nature of the nanosensor. The overlay of FITC and NR fluorescence channels resulted in a visible color shift from red to yellow as pH values moved from acidic to basic, underscoring the nanosensor’s high sensitivity in detecting pH fluctuations.
Furthermore, the cell binding assay validated the successful targeting of cell membranes by the WGA-conjugated nanosensor. This was evidenced by quantifying the nanoparticles bound to the cells through the NR fluorescence signal. The correlation between the amount of NP bound and the total NPs provided insights into the effectiveness of WGA in facilitating the binding of the nanosensor to various cell types.
SEM analysis of A549 and HaCaT cells treated with and without the pH nanosensor highlighted distinct morphological changes and interactions at the cell surface. Cells treated with the nanosensor showed features indicative of nanosensor binding, emphasizing the localization of the nanosensor on the cell membranes.
This SEM analysis further supported the effective targeting of cell surfaces by the pH nanosensor, validating its potential for precise extracellular pH measurements in cellular environments. This ability to accurately monitor extracellular pH holds significant implications for understanding cellular behavior in various physiological and pathological conditions.
Conclusion
In conclusion, the successful application of the pH nanosensor in different cell lines underscores its versatility and potential for studying extracellular pH dynamics in various biological contexts. The nanosensor's high sensitivity, quick response times, and universal applicability make it a valuable tool for real-time monitoring of extracellular pH changes, offering insights into cellular physiology, disease mechanisms, and targeted therapeutic interventions.
Future research may explore the nanosensor’s utility in investigating pH-related phenomena in complex biological systems and advancing our understanding of cellular responses to pH alterations.
Journal Reference
Kromer, C., Katz, A., Feldmann, I. et al. (2024) A targeted fluorescent nanosensor for ratiometric pH sensing at the cell surface. Scientific Reports 14, 12302. https://doi.org/10.1038/s41598-024-62976-2, https://www.nature.com/articles/s41598-024-62976-2