Lipid nanocapsules for intracellular delivery of microRNA: A first step towards intervertebral disc degeneration therapy.
Int J Pharm
; 624: 121941, 2022 Aug 25.
Article
en En
| MEDLINE
| ID: mdl-35781028
ABSTRACT
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.
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Base de datos:
MEDLINE
Asunto principal:
MicroARNs
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Nanocápsulas
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Degeneración del Disco Intervertebral
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Int J Pharm
Año:
2022
Tipo del documento:
Article