Imaging tumor microscopic viscosity in vivo using molecular rotors

A fluid mosaic model is presented for the gross organization and structure of the proteins and lipids of biological membranes. The model is consistent with the restrictions imposed by thermodynamics. In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid. The bulk of the phospholipid is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent. Recent experiments with a wide variety of techniqes and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model. It therefore seems appropriate to suggest possible mechanisms for various membrane functions and membrane-mediated phenomena in the light of the model. As examples, experimentally testable mechanisms are suggested for cell surface changes in malignant transformation, and for cooperative effects exhibited in the interactions of membranes with some specific ligands. Note added in proof: Since this article was written, we have obtained electron microscopic evidence (69) that the concanavalin A binding sites on the membranes of SV40 virus-transformed mouse fibroblasts (3T3 cells) are more clustered than the sites on the membranes of normal cells, as predicted by the hypothesis represented in Fig. 7B. T-here has also appeared a study by Taylor et al. (70) showing the remarkable effects produced on lymphocytes by the addition of antibodies directed to their surface immunoglobulin molecules. The antibodies induce a redistribution and pinocytosis of these surface immunoglobulins, so that within about 30 minutes at 37 degrees C the surface immunoglobulins are completely swept out of the membrane. These effects do not occur, however, if the bivalent antibodies are replaced by their univalent Fab fragments or if the antibody experiments are carried out at 0 degrees C instead of 37 degrees C. These and related results strongly indicate that the bivalent antibodies produce an aggregation of the surface immunoglobulin molecules in the plane of the membrane, which can occur only if the immunoglobulin molecules are free to diffuse in the membrane. This aggregation then appears to trigger off the pinocytosis of the membrane components by some unknown mechanism. Such membrane transformations may be of crucial importance in the induction of an antibody response to an antigen, as well as iv other processes of cell differentiation.

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport.

Viscosity is a measure of the resistance of a fluid against gradients in flow (shear rate). Both flow and viscosity play an important role in all biological systems from the microscopic (e.g., cellular) to the systemic level. Many methods to measure viscosity and flow have drawbacks, such as the tedious and time-consuming measurement process, expensive instrumentation, or the restriction to bulk sample sizes. Fluorescent environment-sensitive dyes are known to show high sensitivity and high spatial and temporal resolution. Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation and therefore exhibit two competing deexcitation pathways: fluorescence emission and non-radiative deexcitation from the TICT state. Since TICT formation is viscosity-dependent, the emission intensity of molecular rotors depends on the solvent's viscosity. Furthermore, shear-stress dependency of the emission intensity was recently described. Although the photophysical processes are widely explored, the practical application of molecular rotors as sensors for viscosity and the fluid flow introduce additional challenges. Intensity-based measurements are influenced by fluid optical properties and dye concentration, and solvent-dye interaction requires calibration of the measurement system to a specific solvent. Ratiometric dyes and measurement systems help solve these challenges. In addition, the combination of molecular rotors with specific recognition groups allows them to target specific sites, for example the cell membrane or cytoplasm. Molecular rotors are therefore emerging as new biosensors for both bulk and local microviscosity, and for flow and fluid shear stress on a microscopic scale and with real-time response.