Measurement of Microenvironmental pH Changes in Pharmaceutical Formulations Using pH-Sensitive Nanosensors

Abstract

Pharmaceutical drug development is a long, costly, and high-risk process, often taking over a decade and requiring billions in investment to bring a single compound to market. A major source of failure arises not from biological inefficacy, but from fundamental physicochemical limitations, particularly poor stability, that compromise drug performance and delay regulatory approval. Stability testing is therefore a critical component throughout development, yet many challenges remain, especially in detecting and managing microenvironmental pH (μpH) within complex dosage forms. pH shifts, especially under acidic or humid conditions, can drive chemical degradation, salt disproportionation, and solid-state transformations that are difficult to detect using conventional methods. Traditional pH measurement techniques lack the spatial resolution, non-invasiveness, and in situ applicability required to monitor μpH inside solid or polymeric formulations.

To address this gap, this thesis develops and applies pH-sensitive fluorescent nanosensors based on polyacrylamide hydrogels for real-time, spatially resolved μpH monitoring in pharmaceutical systems. The work is structured into three experimental chapters. First, a new nanosensor design is optimised for low-pH detection by incorporating difluoro-Oregon Green (DFOG) and replacing 5-(and-6) – carboxytetramethylrhodamine TAMRA with ATTO 655 as a more stable reference fluorophore. The resulting ratiometric configuration extends the functional detection range to pH 2.5 (p < 0.01) using spectrofluorometry and to pH 3.0 using fluorescence microscopy. These nanosensors are characterised using spectrofluorometry, fluorescence microscopy, dynamic light scattering (DLS), and zeta potential analysis.

The second phase applies the nanosensors to solid-state dosage forms, allowing non-invasive tracking of internal pH shifts during storage. Spatial μpH mapping reveals how formulation components influence internal stability, and findings are supported by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) to confirm solid-state transformations such as salt disproportionation. In the third application, nanosensors are integrated into alginate-based hydrogel particles for gastro-resistant drug delivery. Real-time imaging demonstrates how pH-modifying excipients and chitosan coating affect internal μpH and enhance omeprazole stability during simulated gastrointestinal transit.

Overall, the research presented in this thesis has demonstrated that internal μpH plays a critical role in the chemical stability and performance of pharmaceutical formulations, particularly in systems containing acid-labile drugs or salts prone to disproportionation. Formulation factors such as the pHmax of the API, excipient selection, and environmental exposure conditions were shown to significantly influence internal pH behaviour and, in turn, drug stability. The use of pH-sensitive fluorescent nanosensors provided a non-invasive, spatially resolved, and real-time method for μpH measurement within intact dosage forms and hydrogel carriers. Unlike traditional analytical approaches, this method enabled in situ dynamic tracking of internal pH changes without the need for additional sample preparation or destructive testing. These findings highlight the value of fluorescence-based nanosensors as a practical and adaptable analytical tool for studying pH-related phenomena in drug delivery systems. Ultimately, this work supports the development of more stable, effective, and well-characterised pharmaceutical formulations.