Bismuth Ferrite Harmonic Nanoparticles and Biphotonic Microscopy: Innovative Approach to Track In Vivo Muscle Stem Cells, a Promising Candidate for Cell Therapy of Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked recessive muscle disease that represents the most common form of muscular dystrophy, affecting one in 5,000 male births. It is caused by mutations or deletions in the gene encoding dystrophin leading to the lack of dystrophin protein. This results in muscle fiber degeneration followed by severe endomysial sclerosis, leading to progressive muscle weakness and premature death at the age of 20-30 years. Currently, there is no effective treatment for DMD. In 2011, we showed that a stem cell population (named MuStem cells) isolated from healthy dog skeletal muscle induces long-term muscle repair and striking clinical efficacy after its systemic delivery in the dystrophic dog representing the clinically relevant DMD animal model. During last years, our group isolated the human counterparts (hMuStem) and demonstrated that they share the same phenotypic and in vitro behavioral features of canine cells. Currently, little is known about the homing in the muscle tissue and the whole body distribution of the MuStem cells following a systemic delivery which are nevertheless essential to determine the safety and efficacy aspect of the therapeutic strategy. To address this need, we use bismuth ferrite harmonic nanoparticles (BFO-NP, 100-120 nm range) as probes for the tracking of MuStem cells to study in vivo the hMuStem cell engraftment.

The nanoparticles are very interesting tools because capable to generate both Second Harmonic Generation (SHG) and Third Harmonic Generation (THG) signals. The origin of the signals is related to the crystalline properties of the nanoparticles, in particular its peculiar non-centrosymmetric structure. Cell tracking is therefore possible from the NPs SHG and THG signals detected with a two-photon confocal microscope A1RMP Nikon and this work provides the opportunity for long-term, three-dimensional cell tracking.

Uncoated and poly-ethylene glycol(PEG)-coated BFO-NP were investigated to label hMuStem cells in vitro. The cells were exposed to BFO-NP and BFO-PEG-NP. Localisation and cytotoxicity of the NPs were investigated in vitro during two weeks.BFO-labeled cells were tracked in the mouse Tibialis anterior muscle after their intramuscular injection by using biphotonic microscopy.

Acknowledgements

This research was supported by funds from the FEDER (Fonds Européens de Développement Régional N°37085 and 38436). It was carried out in the context of the IHU-Cesti project that received French government financial support managed by the National Research Agency via the investment for the future programme ANR-10-IBHU-005. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region.

Figures:

Figure 1 : Multiphotonic imaging of labeled cells with harmonic nanoparticles BFO PEG. A: dual excitation 820/1040 nm, red Phalloïdine-alexa555 exc 1040 nm, green nucleus and green SHG exc 1040 nm, blue SHG exc 820 nm B: dual excitation 1040/1300 nm, red Phalloïdine-alexa555 (exc 1040 nm) and red SHG exc 1300 nm, blue THG exc 1300 nm, green SHG exc 1040 nm.

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