No abstract

Purpose: Disregulation of miRNAs, notably the miRNA-155, and their targets has been associated with several diseases including intervertebral disc degeneration (IDD). The relevance of unprotected miRNAs for therapeutic applications suffers from their fast in vivo degradation, requiring iterative injections. Thus, the development of nanocarriers is a prerequisite for miRNA therapeutic use. Therefore, miRNAs protection from nucleases is considered as a prior condition for the development of sustainable systems. Among the approaches proposed, nanoparticulate systems, such as lipid nanocapsules (LNCs), exhibit a suitable strategy thanks to their ability to encapsulate DNA. The purpose of this work was to formulate and fully characterize innovative miR155-LNC for a potential use in DDD treatment. Methods: miR-155-LNCs were formulated by phase inversion process. After purification by size exclusion chromatography, miR155-LNCs were fully characterized (size, polydispersity index (PDI), zeta potential). Encapsulation efficiency (EE) and drug loading (DL) were assessed by fluorescence (Quant-IT dye ® ) quantification. miRNA-155 release and enzymatic protection were investigated by dialysis and gel electrophoresis respectively. miR-155-LNCs internalization and impact on cell viability in human adipose stromal cells (hASC) were assessed by confocal/FACS analysis and MTT assay. Results: miR155-LNCs exhibited a diameter of 75.0 ± 1.3 nm, a PDI of 0.06 ± 0.03 and a positive zeta potential. EE and DL were estimated to 75.2 ± 1.2% and 590 ± 9.3 mg/g of LNC respectively. The release profile showed that LNCs delayed the release of miRNA compared to free miRNAs: at 4 h, 23.3 ± 7.1% was released compared to 94.8 ± 5.4% for free miRNA (Fig. 1). miRNA-endonuclease protection by LNC was confirmed by gel electrophoresis. After 24 h of incubation with miR155-LNCs, hASC viability was of 71.66% ± 4,23% for 5,9 ng/mL of miRNA, a recommended concentration of miRNA for efficient cell transfection (Fig. 2). Finally, internalization of miR-155-LNCs in hASC cells after was demonstrated. Conclusions: LNCs showed promising properties to encapsulate miRNA and to significantly protect the miRNA from degradation, in allowing its delayed release. Thanks to these results, miRNA LNCs seem to be a good strategy to encapsulate and deliver miRNA for a future use in IVD regenerative medicine. In a foreseeable future, biological effects of miRNA LNCs will be studied in vitro. In parallel, modification of the LNC surface in order to specifically target the nucleus pulposus cells could be performed. Finally, further experiments (in vitro and in vivo) will be needed to confirm the interest of this innovative nanoplatform to vectorize new therapeutic in order to counteract DDD. ENDOGENOUS PROGENITOR-LIKE CELLS IN INTERVERTEBRAL DISCSPurpose: Intervertebral disc (IVD) degeneration is the major contributor to discogenic low back pain (LBP). IVD degenerates with natural ageing, but a minor population over 50 years of age do not develop agerelated degeneration. Rece...