Streo laser writing on water – SLOW2

Task 1: Synthesis of engineered fluorophores (Task leader Partner 2). Various 2-Photon excited (TPE) fluorophores will be synthesised, bearing two different functional groups, to allow their incorporation in the branches of the dendrimers. An important task is the photo-physical study of the TPE fluorophores, in particular their 2P absorption response.
Task 2: Synthesis of water-soluble fluorescent dendrimers (Task leader Partner 1). Dendrimers should fulfilling three requirements: several TPE-fluorophores in the structure (issued from Task 1), solubility in water, and aggregation properties under 2P irradiation. A structure/activity relationship study will be carried out to determine the most efficient dendrimer(s).
Task 3: Characterisation of the stereo-(nano)-lithography process (Task leader Partner 1). This Task is scheduled for characterising the aggregation phenomenon, using physical, chemical, theoretical, and biological tools available in the laboratories of the three Partners, on the dendrimers synthesised in Task 2.
Task 4: Understanding the stereo-(nano)-lithography process (Task leader Partner 3). All the experiments carried out in Task 3 by the joint efforts of the three Partners should afford clear tracks pointing towards the causes that induce the aggregation process. In addition, molecular dynamics (MD) simulations will allow to understand the detailed dendrimer structure in the presence of explicit solvent molecules, then the dendrimer aggregation will be studied.
Task 5: Labelling of cells by stereo-(nano)-lithography. Tissue patterning? (Task leader Partner 3). Two-photon confocal microscopy will be used as an efficient and non-invasive tool to first induce nanoprinting and then to visualise labelled individual cells in 2D and 3D (spheroids) cultures. Attempts will be carried out for tissue engineering.

T1: Four original fluorophores, with strong 2-photon absorption and emitting respectively in blue, green, lemon-yellow, and red, were synthesized and characterized by Partner 2. A comprehensive photo-physical study was conducted. These fluorophores have been doubly functionalized, to be used as branches of dendrimers.
T2: A second-generation dendrimer, incorporating the blue emitting fluorophore in the branches, and surface water-solubilizing functions, was synthesized and characterized by Partner 1. This is the product with which the properties of stereo-nanolithography had been observed, but whose method of synthesis had to be modified. It will be necessary to incorporate the other fluorophores into the branches of the dendrimers, to vary their location, and the type of terminal functions (in progress).
T3: The blue fluorescent dendrimer was assigned to Partner 2 by Partner 1 for photo-physical studies that are underway, in particular for a study of the influence of the environment on the association of dendrimers. This study will provide valuable insights to better understand the conditions in which the phenomenon of association inducing stereo-nanolithography occurs.
T4: Partner 3 has performed several molecular dynamics (MD) simulations on the blue fluorescent dendrimer in water. The first MD 100 ns showed that the molecule folds, with the core on one side and all the water-solubilizing chains on the other. Simulations in water incorporating DMF showed that folding is as in the previous case, but is delayed by interactions with DMF molecules, some of which are found within the dendrimer structure. MD trials about the aggregation of two dendrimers in water have shown their rapid association.

The first attempts to diversify the structure of fluorescent dendrimers (types of fluorophores, size of the dendrimer, and types of terminal functions) have been carried out, but are largely to be developed, to produce a library of compounds allowing a structure / activity relationship on aggregation performance.
The phenomenon of aggregation of fluorescent dendrimers capable of absorbing 2 photons under the influence of a laser is still largely to characterize, to analyze and above all, to understand. When it will be understood and proven, we plan to study the possibility of patenting, then a publication in an excellent general scientific journal, followed by other publications, the presentation in various congresses, and a communication towards the ANR, our institutions, and the general public.
Application of the nano-imprinting process for labeling of individual cells has to be carried out both in 2-dimensions (classical cell cultures) and in 3-dimensions (spheroids). Stability, toxicity and localisation of the printing (on the surface or inside the cells) have to be studied. This process could provide a tool-kit for a simple, non-invasive and highly innovative technology for marking single cells, which could be developed by a start-up. In addition, a derivative of this technology could provide a new technology for tissue engineering, even if this is highly speculative.

Publications and presentations acknowledging the ANR:
1 publication in common for two Partners:
Fluorescent phosphorus dendrimers excited by two photons: synthesis, two-photon absorption properties and biological uses. Anne-Marie Caminade,* Artem Zibarov, Eduardo Cueto Diaz, Aurélien Hameau, Maxime Klausen, Kathleen Moineau-Chane Ching, Jean-Pierre Majoral, Jean-Baptiste Verlhac, Olivier Mongin, Mireille Blanchard-Desce. Beilstein Journal of Organic Chemistry (accepted July 4, 2019)
1 invited conference in common for the 3 Partners:
1. Fluorescent dendrimers for biology. How Two-photon absorption properties can help? Caminade A.M.,* Majoral J.P., Hameau A., Zibarov A., Blanchard-Desce M, Verlhac J.B., Teissié J., Golzio M. International Dendrimer Symposium (IDS-11), 14/18 juillet 2019, Funchal, Madère, Portugal.
1 publication for a single partner:
1. Fluorescent Phosphorus Dendrimers: Towards Material and Biological Applications. Qiu J., Hameau A., Shi X., Mignani S., Majoral J.P., Caminade A.M.*, ChemPlusChem 2019, 84, 1070-1080
1 oral communication for a single Partner:
1. Synthesis of fluorescent dendrimers soluble in water. A. Zibarov, A. Hameau, K. Moineau-Chane Ching, A.M. Caminade. LCC Young Investigators Seminar, LCC Toulouse, 28 juin 2019.

We have discovered by serendipity that a fluorescent, two-photon absorbing and water-soluble dendrimer (hyper-branched macromolecule, having a perfectly defined structure) can induce a new type of “stereo-nano-lithography” (3-D) in a volume of water under the influence of a focused laser beam. This process is very different from classical stereolithography, as it is not a polymerisation reaction, it is slowly reversible with time, it occurs at a low concentration of dendrimer in water, and it can be controlled down to the 200 nm scale. There is no comparable technology to date, and it is difficult to conceive how else the same result could be obtained. This is definitely a break with all that is known in the field of 3-D printing, concerning the nature of the media, the process involved, and the spatial resolution of the imprinted structures. From this first unpublished result, we must now determine what are the parameters which affect this phenomenon, by performing a real chemical engineering of the structure of the dendrimers, by varying the nature of the fluorophore subunit, its two-photon absorption efficiency, its absorption and emission wavelengths, the level at which it must be located within dendrimers structure, the number, the nature of the functions of the dendrimers surface, and the size/generation of the dendrimers. In brief, we will take advantage of the chemical modularity of the structure of dendrimers at all levels. We also have to analyse and understand, by using several techniques, including molecular dynamics modelling (MD), the unprecedented physical and chemical phenomena involved in this stereo-nano-lithography in a volume of water, as well as the conditions for its persistence or its vanishing with time. Our main goals are to discover the reasons for this original phenomenon and determine its scope and limitations. It's a real fundamental challenge.
The phenomenon which we have observed is more stable in the vicinity of a surface, but was obtained in bulk water. It could be used for soft flexible surface labelling. We indeed got the proof of this concept, by marking the membrane surface of a single cell B16F10 type (murine melanoma) among dozens of others, under the influence of the laser of a two-photon microscope, in the presence of a water-soluble dendrimer, which fluoresces after two-photon excitation. This specific labelling is not due to a specific interaction with a receptor on the cell surface, and can potentially be applied to any type of cell. Nevertheless, we need to study the persistence over time of the label in various physiological conditions, then checking that the labelling does not alter the viability and the functional biological behaviour of the cell, and testing the process on different cell types. Thus, it could be used potentially for cell targeting. Moreover, these printed structures could be used as a scaffold for 3-D cellular sheets in tissue engineering, in combination or not with the grafting of specific ligands on the dendrimer surface.
This project is a true challenge at the fundamental level, but it could lead to unprecedented technologies for labelling and targeting of a single cell from hundreds, and at the translational level for the guided-construction of tissues (bioprinting). To meet this challenge, we are three teams with unique and complementary knowledge and know-hows, and internationally recognised in their field: molecular multiphotonics, synthetic chemistry, molecular dynamics, bio-imaging and cell engineering. This project will require a permanent sharing of information between the three teams. Two of the teams are located on the same campus in Toulouse (chemist, and biologist-biophysicist, theoretician for MD), a third in Bordeaux (physical organic chemist). It is certain that this geographical proximity will allow many and fruitful exchanges.