Periodic Reporting for period 1 - NanoFUNC (Functionalisation of ultra-bright all organic nanoparticles for super resolution imaging in intact brain preparations)
Berichtszeitraum: 2020-01-01 bis 2021-12-31
Being able to observe the dynamics of biomolecules in their native biological environment is key to understanding the molecular mechanisms at play in health and disease. Fluorescence imaging is a major technology to allow such observation, which has provided a wealth of information in various fields including cancer research and neuroscience since its earliest developments. This technology requires bright, stable, small, spectrally relevant, targetable, and non-toxic fluorescence emitters. Nanoparticles (NPs) are powerful and versatile platforms developed by chemists and physicists to enable safe and efficient bioimaging, but bringing all these features together into a single emitter has not been achieved to date. The objective of the NanoFUNC project was thus to develop the next generation of NPs displaying all the above properties into a single NP. In the past 2 years, we have succeeded in synthesising, characterising and using in biological tissues ultra-bright, size tuneable (down to 10 nm, i.e. the size of endogenous proteins such as antibodies), red-emitting (i.e. in the biological transparency window), all-organic nanoparticles as well as explored strategies for functionalisation to render them targetable to cellular targets of interest.
Optimisation of two main features have driven the work performed throughout this project: i) enhancing the luminescence properties of NPs and ii) making them usable in biological systems. To do so, we have worked with dye-based Fluorescent Organic Nanoparticles (dFONs), a type of NPs made from the self-aggregation of fluorescent organic dyes into spontaneously water soluble NPs. We have studied the structure property relationships between the chemical structure of tailor-made dyes and the emerging properties of the resulting dFONs.
I. Enhancing the luminescence properties of dFONs: Fluorescent NPs aimed for bioimaging should absorb and emit red to near infrared light (above 650 nm), in the so-called biological transparency window where blood and fatty tissues absorb and scatter photons the least. However, improving this property at the molecular level is often detrimental to the brightness of a fluorescence emitter. We have therefore synthesised two series of dyes whose molecular structures were designed with the aim of combining these two properties. Starting from a common polarisable, bulky template designed to maintain dye brightness upon confinement into dFONs, we investigated two strategies to red-shift the absorbance and emission of dFONs. One increased the length and flexibility of the dyes, the second increased their polarity. Both series of dyes and their dFONs were extensively characterised and the results were published in two special issues: the "125 Years of The Journal of Physical Chemistry" (2021) and the "Recent Advances in Luminescent Materials" special issue of the open-access journal Molecules (2022). The first strategy using long flexible dyes was particularly efficient with a dye of the series yielding dFONs surpassing the state of the art in terms of brightness and another yielding near-infrared emitting dFONs.
II. Usability in biological systems: Nano-bio interactions are extremely difficult to predict and a lot of research is still required before we understand all the intricacies of the events occurring at this scale. In particular, whether NPs will stay outside of cells or get inside when put in contact with biological tissues is critical depending on the application. Entering the cells is important for drug delivery systems but staying outside is essential when imaging the extracellular space or membrane bound biomolecules. Most NPs devoted to staying outside of cells are coated with polymers at their surface to prevent their internalisation. However, this procedure complexifies their preparation and their toxicological profile. We have found that the dye template used throughout this project spontaneously yields dFONs of tuneable stealth character without the need for a coating polymer. This feature was examined on model dissociated cells in the two special issue articles cited above as well as with green emitting dFONs made from dyes of related chemical structure in the Proceedings of a SPIE Neurophotonics conference held in 2020. Moreover, this stealth character was exploited with dFONs made from the brightest dye variant for single particle tracking in the extra-cellular space of rat brain slices. This technological feat was published in the high impact journal Advanced Materials in 2021.
Taken together, the various aspects of this work have been published in 3 peer-reviewed journals and presented at 6 conferences, including 4 international conferences.
I. Enhancing the luminescence properties of dFONs: Fluorescent NPs aimed for bioimaging should absorb and emit red to near infrared light (above 650 nm), in the so-called biological transparency window where blood and fatty tissues absorb and scatter photons the least. However, improving this property at the molecular level is often detrimental to the brightness of a fluorescence emitter. We have therefore synthesised two series of dyes whose molecular structures were designed with the aim of combining these two properties. Starting from a common polarisable, bulky template designed to maintain dye brightness upon confinement into dFONs, we investigated two strategies to red-shift the absorbance and emission of dFONs. One increased the length and flexibility of the dyes, the second increased their polarity. Both series of dyes and their dFONs were extensively characterised and the results were published in two special issues: the "125 Years of The Journal of Physical Chemistry" (2021) and the "Recent Advances in Luminescent Materials" special issue of the open-access journal Molecules (2022). The first strategy using long flexible dyes was particularly efficient with a dye of the series yielding dFONs surpassing the state of the art in terms of brightness and another yielding near-infrared emitting dFONs.
II. Usability in biological systems: Nano-bio interactions are extremely difficult to predict and a lot of research is still required before we understand all the intricacies of the events occurring at this scale. In particular, whether NPs will stay outside of cells or get inside when put in contact with biological tissues is critical depending on the application. Entering the cells is important for drug delivery systems but staying outside is essential when imaging the extracellular space or membrane bound biomolecules. Most NPs devoted to staying outside of cells are coated with polymers at their surface to prevent their internalisation. However, this procedure complexifies their preparation and their toxicological profile. We have found that the dye template used throughout this project spontaneously yields dFONs of tuneable stealth character without the need for a coating polymer. This feature was examined on model dissociated cells in the two special issue articles cited above as well as with green emitting dFONs made from dyes of related chemical structure in the Proceedings of a SPIE Neurophotonics conference held in 2020. Moreover, this stealth character was exploited with dFONs made from the brightest dye variant for single particle tracking in the extra-cellular space of rat brain slices. This technological feat was published in the high impact journal Advanced Materials in 2021.
Taken together, the various aspects of this work have been published in 3 peer-reviewed journals and presented at 6 conferences, including 4 international conferences.
Exploring the extra-cellular space of the brain is a hot topic in neuro-imaging research as this compartment varies in size and composition with sleep, age and disease. However, the stringent criteria necessary to make it possible are such that so far, only inorganic NPs possessed the required luminescence properties. It had thus only been possible using polymer-coated heavy-metal containing nanocrystals or micrometer sized carbon nanotubes. Our work is therefore the first observation of single component fluorescence emitters deep in living biological tissues using all-organic NPs. In this sense, the studies performed during this project open the way to safe bio-imaging. Now that we have obtained bright, stable, small, spectrally relevant and potentially non-toxic fluorescence emitters, we have started investigating a number of strategies to make them targetable to biomolecules of interest. This work has not been published yet and is still ongoing. The target molecules considered are two membrane-bound receptors: the EGF receptor, over-expressed in cancer cells as compared to healthy cells and the NMDA receptor, involved in neuro-transmission, learning and memory and neuropsychiatric diseases including schizophrenia. The dFONs developed in this project thus have a strong technological impact, with the obtention of bright all-organic red fluorescence emitters, a conceptual impact, being the first nanotools investigating a bottom-up approach to rationalise the link between chemical composition of NPs and stealthiness towards biological membranes, and a societal impact with the use of functionalised dFONs targeted against known therapeutic targets.
Science communication to the general public has also been highly considered throughout the duration of this project with the experienced researcher participating in 3 communication events including the European Researchers’ Night and an online event for the promotion of women in science organised by the RESET European initiative (Redesigning Equality and Scientific Excellence Together). All peer-reviewed publications have also been communicated to the public through social media and/or institutional communication channels.
Finally, this project has been central to the career development of the experienced researcher benefiting from it who is now short-listed for the final round of selection for a permanent position at the CNRS (Centre National de la Recherche Scientifique).
Science communication to the general public has also been highly considered throughout the duration of this project with the experienced researcher participating in 3 communication events including the European Researchers’ Night and an online event for the promotion of women in science organised by the RESET European initiative (Redesigning Equality and Scientific Excellence Together). All peer-reviewed publications have also been communicated to the public through social media and/or institutional communication channels.
Finally, this project has been central to the career development of the experienced researcher benefiting from it who is now short-listed for the final round of selection for a permanent position at the CNRS (Centre National de la Recherche Scientifique).