Fluorescence lifetime nanoscopy of Onvyde(®) liposomal formulation: from synthetic identity to biological function

This research project aims to leverage optical techniques on cutting-edge pharmaceuticals for applications in both preclinical experimentation and post-market analysis within the pharmaceutical sector. Specifically, we employ the Fluorescence Lifetime Imaging (FLIM) technique, allowing for the analysis of the supramolecular organization of luminescent active principles within the nanoformulation, hereafter referred to as the drug composition. FLIM enables the study of its stability under various experimental conditions of interest. In contrast to concurrent characterization techniques such as TEM, SEM, CryoEM, and HPLC, which are semi-quantitative and involve high costs, long processing times, and complex instrumentation, our approach, based on Francesco Cardarelli's patented method 1, permits rapid (seconds), cost-effective analysis of the drug in its natural solvent without manipulation or labelling. Furthermore, it is crucial to note that this project is conducted in collaboration with Chiesi Farmaceutici and FLIM LABS S.r.l., an innovative Italian startup specializing in the development of systems, components, and software for applications requiring fluorescence decay time measurements. To underscore the significance of this approach, our research focuses on Onivyde, an FDA-approved orphan drug of the latest generation, with significant implications in the treatment of metastatic Pancreatic adenocarcinoma (mPDAC). Onivyde was chosen for its medical relevance and distinguished by the encapsulation of irinotecan. Irinotecan is a luminescent molecule, making the study of its dynamics and alterations with fluorescence techniques label-free. It's essential to highlight the advantages of this technology:      It is a label-free procedure, eliminating the need for chemical modification of the drug formulation to introduce fluorescent probes, relying instead on intrinsic signals. This provides a quantitative description of the drug formulation in its native configuration.      It avoids the chemical fixation of samples, as seen in Electron Microscopy, and, when visible light is applicable, allows the analysis of the drug formulation dissolved directly in its optimal dilution solution.      It offers nanoscale sensitivity (molecular organization) in a standard optical microscopy configuration, limited by diffraction, without the need for super-resolution strategies. This relies on the exquisite sensitivity of the fluorescence lifetime parameter to the environment and/or nanoscale organization of the fluorophore.      It relies on fast, robust, adaptation-free, and entirely graphical data analysis procedures.      It is highly flexible in terms of application range, from cuvette tests to in-vivo experiments.      It can be applied to any molecule/drug/compound capable of absorbing photons at a certain wavelength and emitting photons with detectable lifetimes.      When combined with a complete spectroscopic characterization of emitting species (e.g., absorption/fluorescence spectroscopy to derive the absorption spectrum and quantum yield of all emitting species in their isolated form), the described procedure leads to the calculation of fractional populations (e.g., stoichiometry) of emitting species within the formulation.      It can detect changes in the physical state of the encapsulated drug induced by interaction with bodily fluids (e.g., human serum). This is relevant for mimicking in vivo conditions, contributing to an understanding of the drug's behavior in the human body.      Depending on the specific wavelengths used, it can detect changes in the physical state of the encapsulated drug in compartments of living cells (cytosol, nucleus, etc.) where particle characterization techniques cannot be applied Of note, the case study on Onivyde is not limiting, given that among the nanomedicinal drugs approved to date, estimated to be around 50 by the European Technology Platform2, 35% of these consist of luminescent molecules. In a context where contrast agents, devices, and over 400 drugs are undergoing clinical trials, the reference market is the pharmaceutical sector, particularly in the field of nanomedicine. Based on what said, it may appear clear that a fast analytical procedure capable to characterize the supramolecular organization of an encapsulated drug, Doxorubicin in the case-study selected here, would represent a significant step forward in the field. In this regard, it is interestingly to note that many FDA-approved molecules are characterized, in terms of chemical structure, by the presence of aromatic rings, a structural feature that makes them very similar to natural fluorophores3.