BIOCOMPATIBLE AND PHOTOSTABLE PHOTOACOUSTIC CONTRAST AGENTS BASED ON BODIPY-SCAFFOLD AND POLYLACTIDE POLYMERS: SYNTHESIS, FORMULATION, PHOTOACOUSTIC CHARACTERIZATION AND IN VIVO EVALUATION

Jean-Baptiste Bodin^1,2, Jérôme Gateau³, Justine Coïs⁴, Théotim Lucas³, Flora Lefebvre², Laurence Moine², Magali Noiray², Catherine Cailleau², Stéphanie Denis², Gilles Clavier⁴, Nicolas Tsapis², Rachel Méallet-Renault¹
1 Université Paris-Saclay, CNRS, Institut des Sciences Moléculaires d’Orsay, 91405, Orsay, France.
2 Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 92296, Châtenay-Malabry, France.
3 Sorbonne Université, CNRS, INSERM, Laboratoire d’Imagerie Biomédicale, LIB, F-75006, Paris, France
4 Université Paris-Saclay, ENS Paris-Saclay, CNRS, PPSM, 91190, Gif-sur-Yvette, France.

Introduction
Imaging the biodistribution of a drug nanovector (NVs) at a targeted site could greatly improve the prediction of its therapeutic efficiency. To label NVs for photoacoustic (PA) imaging , we have synthesized a new Bodipy scaffold with the following properties: 1) absorption in the NIR region, 700-900 nm, where biological tissues absorb and scatter less favoring the penetration depth for PA imaging, 2) a high extinction coefficient, 3) a low fluorescence quantum yield to increase the PA generation efficiency. This PA Bodipy was covalently linked to a polymer and formulated into NVs.

Methods
The PA Bodipy was obtained with the three-step one-pot protocol to obtain the main core of the Bodipy with a pentafluorophenyl ring, further reacted with 2-mercaptoethan-1-ol and finally labelled with two 4-(N,N-dimethylamino)styryl groups. The PA-Bodipy was then used to initiate ring opening polymerization of lactide and yield polylactide-Bodipy (PLA-Bodipy). The PLA-Bodipy was formulated into PEGylated NVs by mixing with PLA-PEG at different mass ratios. NVs were characterized by absorption and fluorescence spectroscopies and their PA efficiency determined as a function of their PLA-Bodipy content. The photostability of PLA-Bodipy NVs was compared to PLA-cyanine 7 NVs. Finally, the ability to detect NVs in vivo was assessed in healthy mice using a commercial PA imaging system.

Results/Discussion
PA-Bodipy was obtained with a good yield and successfully initiated the ring-opening polymerization of lactide (MW ≈14 500 g.mol-1, narrow dispersity of 1.6). PLA-Bodipy had an absorption peak at 753 nm, a low quantum yield (0.052) and a fluorescence lifetime of 1.05 ns. NVs at mass percentages of PLA-Bodipy of 5, 10, 25 and 50% were obtained with diameters from 90 nm up to 140 nm. NVs exhibited a good photoacoustic signal even for 5% PLA-Bodipy NVs thanks to their molar extinction coefficient of 1.5x107 L.mol-1.cm-1 at 757 nm and 88% PA efficiency. In addition, PLA-Bodipy NVs presented excellent photostability as compared with PLA-cyanine 7 NVs. After intravenous injection of PLA-Bodipy NVs to mice, we observed an abrupt increase of the PA amplitude at 750 nm in blood vessels. Spectral unmixing of PA images acquired at different optical wavelengths in the kidney region demonstrated the absence of NPs signal pre-injection and their detection with a high contrast post injection.

Conclusion
A novel Bodipy absorbing around 750 nm was synthesized. After covalent linking to PLA, it was formulated into NVs of about 100 nm. The PA efficiency of the NVs allows to position them in between molecular PA absorbers and gold nanoparticles. Our Bodipy label presents a much better photostability than cyanine. The easy detection of PLA-Bodipy NVs in healthy animals paves the way for the evaluation of the distribution of PLA NVs in animal models.