RESUMEN
Approximately 40% of cases of lower back pain are caused by disc degeneration disease (DDD). It is well established that microRNA (miR) dysregulation is a key player in various diseases, and its impact on DDD has recently been highlighted. RNAi (miR in particular) is increasingly being considered as a novel therapeutic tool. However, free miR is degraded rapidly in vivo, and its protection is thus a prerequisite. Nanoparticular platforms, such as lipid nanocapsules (LNC), could be specifically adapted for miR delivery, allowing the transfer and release of miR in the cell cytoplasm. The objective of the current study was to formulate and characterize miR-loaded LNC to establish their in vitro potential (cell internalization, bioactivity) as well as to determine the safety and feasibility of in situ intervertebral disc (IVD) injection of miR LNC in a healthy sheep model. Using a miR library, miR-155 was clearly identified as being involved in the DDD process and was selected for further assessment. miR-155-loaded LNC (miR-155 LNC) were successfully formulated using a phase inversion process, with the addition of lipoplexes in the cooling step. Following purification, miR-155 LNC were fully characterized, and the optimized formulation had an average diameter of 75 nm, a polydispersity index below 0.1, and a positive zeta potential. By fluorescence spectroscopy, an encapsulation efficiency (EE) of 75.6% and a drug loading (DL) of 0.6% were obtained, corresponding to a sufficient amount of miR per mL of LNC to potentially have a biological effect. The sustained release of miR-155 from LNC was demonstrated compared with free miR-155: only 22% was released after 2 h and 58% after 24 h. miR-155 protection against endonuclease degradation by LNC was confirmed by gel electrophoresis, a sine qua non condition for it to be administered in vivo. Cell viability assays were performed on human adipose stromal cells (hASCs) and ovine Nucleus pulposus cells (oNP), and a cytotoxicity of <30% was obtained at the considered concentrations. Additionally, miR-155 LNC cell internalization was demonstrated by flow cytometry and confocal imaging. Moreover, downregulation of total ERK1/2 in hASCs and oNP cells, after miR-155 LNC treatment, was demonstrated by Western blot and quantitative reverse-transcription PCR (qRT-PCR), thus confirming maintenance of its bioactivity after formulation and internalization. Finally, the feasibility and safety of miR-155 LNC in situ injection (compared to control groups: blank LNC and sham condition) was demonstrated in healthy sheep by imaging (MRI and T2wsi measurement) and histology (Boos' scoring) analysis. T2wsi was measured, and no significant difference was observed three months after the injection between the different conditions. No histological impact was observed, with no significant difference in Boos' scoring between the different conditions. All these results suggest LNC may be a potent strategy for the encapsulation and delivery of miR (particularly miR-155) and can be considered as a first step towards IVD regenerative medicine.
Asunto(s)
Degeneración del Disco Intervertebral , MicroARNs , Nanocápsulas , Animales , Regulación hacia Abajo , Humanos , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/patología , Lípidos/química , Nanocápsulas/química , OvinosRESUMEN
Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long-term (6 months) follow-up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham-injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM-injected IVDs showed significantly higher BAX/BCL2 ratio vs sham-injected IVDs suggestive of an anti-apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 707-714, 2017.