Development of optical pH nanosensors for biological insights into the intracellular trafficking of nanomedicines

The field of nanomedicine has progressed to a stage where a diverse set of materials are available for controlling how a drug is delivered in the body. Although these materials can be engineered to overcome many of the obstacles associated with drug delivery, the complexity of cellular trafficking mechanisms means controlling intracellular delivery remains a major challenge. The primary portal for the cellular internalisation of nanomedicines is endocytosis, which involves transport through a network of highly complex intracellular compartments undergoing a dynamic process of acidification. As a result, nanoparticle-based pH sensors offer a new perspective from which to investigate this process. In this study, ratiometric polyacrylamide pH nanosensors were utilised to probe fundamental aspects of intracellular trafficking with the view of developing biological insights to aid the rational design of nanomedicines. Nanosensors were fabricated with a dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm. Endocytic uptake of nanosensors was induced in four different cell types (HeLa, 3T3, MRC-5 and JAWS II) by increasing the surface charge on the nanosensor. Dynamic pH measurements were found to be highly sensitive to experimental methodology for performing ratiometric measurements, particularly image analysis. Consequently an optimised procedure for performing ratiometric measurements was developed, and subsequently validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM). Application of pH nanosensors in studies investigating fundamental aspects of intracellular trafficking resulted in three key findings: 1) HeLa, 3T3 and JAWS II cells process material in different ways with respect to the extent and rate of acidification in endocytic organelles, 2) surface charge does not affect the final intracellular location of polyacrylamide nanoparticles internalised by endocytosis, and 3) lipid-mediated transfection of siRNA is associated with a greater degree of lysosomal disruption compared to cationic polymer-mediated transfection, with the former observed to show increased toxicity. These findings represent biological insights, which can be utilised to provide a rational basis for tailoring the response of pH-sensitive nanomedicines to a specific cell type, tuning the physicochemical properties of a material for more efficient intracellular trafficking and optimising siRNA formulations for endo-lysosomal release