Microscopic evaluation of two-photon activated molecular nanomachines for next generation targeted cancer therapeutics

Abstract

Unidirectional molecular nanomachines are small organic molecules consisting of two distinct halves; a stator, and rotor, connected by a sterically overcrowded carbon-carbon double bond. When excited with specific wavelengths of light, commonly used 355 and 365 nm, a cis-trans isomerisation occurs, resulting in a unidirectional 360° rotary mechanism capable of overcoming Brownian motion. This has previously been exploited to mechanically damage the outer membranes of cells, triggering an acceleration of irreversible necrotic cell death.
Herein a series of systematic live-cell fluorescence microscopy studies are reported evaluating the capability of new functionalised molecular nanomachines to induce premature cell death. Specifically, work has been carried out to develop methodology and instrumentation capable of activating these molecules with more biologically favourable NIR wavelengths by way of two-photon activation - utilising pulsed (fs) laser light. In addition, experiments aimed at assessing newly developed families of molecular nanomachines capable of crossing the phospholipid bilayer and activating confined and controlled rotary mechanism from within the cell are presented.
Key findings are presented showing how functionalisation of molecular nanomachines, with diamine moieties of various levels of methyl substitution, is able to induce a bathochromic shift in activation wavelength. Specifically, a greater level of amine substitution by electron donating alkyl chains is shown to increase this effect when attached to the rotor half of the nanomachine. Activation of polyethylene glycol functionalised nanomachines by way of a two-photon process is also illustrated both when in solution and, importantly, from within the cell. This is also extended to nanomachines functionalised with the mitochondrial targeted triphenylphosphine addend.
These developments are then combined. Using attempts to activate internalised molecular nanomachines functionalised with triphenylphosphine from withinside cells. Specifically using therapeutically favourable, less phototoxic, 710 nm NIR wavelengths to induce more biologically compatible routes towards cell death, such as apoptosis. Miscopy images are presented illustrating that with the combination of these techniques, with short precisely designed windows of laser exposure, it is possible to observe clear morphological features of cell death without the addition of fluorescent signs of necrosis.
These results represent a significant step forward towards the development of next generation targeted cancer therapeutics, but also leave a clear avenue for further work within the field. Suggestions are presented on how to extend this research, specifically on methods to measure the multiphoton cross section of photomechanical compounds, such as molecular nanomachines, more accurately. As well as methods for measuring apoptosis caused by nanomechanical damage, and importantly, how to distinguish this from necrotic processes.