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Lipid Nanoparticles Vectorized with NFL-TBS.40-63 Peptide Target Oligodendrocytes and Promote Neurotrophin-3 Effects After Demyelination In Vitro

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Abstract

Promoting remyelination in multiple sclerosis is important to prevent axon degeneration, given the lack of curative treatment. Although some growth factors improve this repair, unspecific delivery to cells and potential side effects limit their therapeutic use. Thus, NFL-TBS.40-63 peptide (NFL)—known to enter specifically myelinating oligodendrocytes (OL)—was used to vectorize 100 nm diameter lipid nanoparticles (LNC), and the ability of NFL-LNC to specifically target OL from newborn rat brain was assessed in vitro. Specific uptake of DiD-labeled NFL-LNC by OL characterized by CNP and myelin basic protein was observed by confocal microscopy, as well as DiD colocalization with NFL and with Rab5—a marker of early endosomes. Unvectorized LNC did not significantly penetrate OL and there was no uptake of NFL-LNC by astrocytes. Canonical maturation of OL which extended compacted myelin-like membranes was observed by transmission electron microscopy in cells grown up to 9 days with NFL-LNC. Endocytosis of NFL-LNC appeared to depend on several pathways, as demonstrated by inhibitors. In addition, vectorized NFL-LNC adsorbed on neurotrophin-3 (NT-3) potentiated the proremyelinating effects of NT-3 after demyelination by lysophosphatidyl choline, allowing noticeably decreasing NT-3 concentration. Our results if they were confirmed in vivo suggest that NFL-vectorized LNC appear safe and could be considered as putative carriers for specific drug delivery to OL in order to increase remyelination.

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Acknowledgements

The authors thank Dr R. Perrot for expert technical assistance with confocal microscopy, and Mrs F. Manero for electron microscopy techniques.

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Designed study: CF. Performed experiments: CF, OT, AMU. Analyzed data: CF. Wrote paper: CF, PS.

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Correspondence to Catherine Fressinaud.

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11064_2020_3122_MOESM1_ESM.tif

Supplementary file1 (TIF 22200 kb). Supplementary Fig. 1 Transmission electron microscopy of 100 nm LNC adsorbed on NFL Scramble peptide (NFL-SCR) - composed of the same aminoacids as NFL-TBS peptide although in random order -. This peptide is inactive on oligodendrocytes. Note the filament bundles formed by NFL-SCR. Pure LNC were diluted 1/10 (v/v); NFL-SCR 0.027 µM final concentration (same concentration as NFL-TBS, see Fig. 1). Uranyl acetate negative stain. Scale bar 200 nm as indicated

11064_2020_3122_MOESM2_ESM.tif

Supplementary file2 (TIF 40345 kb). Supplementary Fig. 2 Long term treatment (9 days) with NFL-LNC-DiD does not alter OL characteristics. TEM analyses of control (left) and NFL-LNC treated cultures (right) after 9 days demonstrate numerous microtubules in OL processes. Scale bars = 200 nm

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Fressinaud, C., Thomas, O., Umerska, A.M. et al. Lipid Nanoparticles Vectorized with NFL-TBS.40-63 Peptide Target Oligodendrocytes and Promote Neurotrophin-3 Effects After Demyelination In Vitro. Neurochem Res 45, 2732–2748 (2020). https://doi.org/10.1007/s11064-020-03122-y

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