VCAM-1 Targeted Lipopolyplexes as Vehicles for Efficient Delivery of shRNA-Runx2 to Osteoblast-Differentiated Valvular Interstitial Cells; Implications in Calcific Valve Disease Treatment

Abstract

Calcific aortic valve disease (CAVD) is a progressive inflammatory disorder characterized by extracellular matrix remodeling and valvular interstitial cells (VIC) osteodifferentiation leading to valve leaflets calcification and impairment movement. Runx2, the master transcription factor involved in VIC osteodifferentiation, modulates the expression of other osteogenic molecules. Previously, we have demonstrated that the osteoblastic phenotypic shift of cultured VIC is impeded by Runx2 silencing using fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 (shRunx2) polyplexes. Since the use of polyplexes for in vivo delivery is limited by their instability in the plasma and the non-specific tissue interactions, we designed and obtained targeted, lipid-enveloped polyplexes (lipopolyplexes) suitable for (1) systemic administration and (2) targeted delivery of shRunx2 to osteoblast-differentiated VIC (oVIC). Vascular cell adhesion molecule (VCAM)-1 expressed on the surface of oVIC was used as a target, and a peptide with high affinity for VCAM-1 was coupled to the surface of lipopolyplexes encapsulating C60-PEI/shRunx2 (V-LPP/shRunx2). We report here that V-LPP/shRunx2 lipopolyplexes are cyto- and hemo-compatible and specifically taken up by oVIC. These lipopolyplexes are functional as they downregulate the Runx2 gene and protein expression, and their uptake leads to a significant decrease in the expression of osteogenic molecules (OSP, BSP, BMP-2). These results identify V-LPP/shRunx2 as a new, appropriately directed vehicle that could be instrumental in developing novel strategies for blocking the progression of CAVD using a targeted nanomedicine approach.

Molecular Mechanisms Underlying the Progression of Aortic Valve Stenosis: Bioinformatic Analysis of Signal Pathways and Hub Genes

“…A previous study has shown that VCAM1 is highly expressed in the aortic valves of diabetic/atherosclerotic ApoE-deficient mice and is considered a potential target for nanocarriers developed to block the progression of AS [19]. VCAM1 is a transmembrane sialoglycoprotein that is often used for targeted drug delivery to endothelial cells due to its inducible expression on the cell surface in pathological conditions [20][21][22][23]. Moreover, there is an increased expression of VCAM1 in aortic valve interstitial cells after exposure to IFNγ-and lipopolysaccharide [24].…”

“…Moreover, there is an increased expression of VCAM1 in aortic valve interstitial cells after exposure to IFNγ-and lipopolysaccharide [24]. Efficient drug delivery systems for AS using nanomedicine are currently being examined, with a particular focus on VCAM1-targeted PEGylated lipopolyplexes as a potential encapsulating drug delivery system [23]. However, translating this technology into clinical practice is difficult and may have serious limitations.…”

Biomimetic Nanocarriers of Pro‐Resolving Lipid Mediators for Resolution of Inflammation in Atherosclerosis

“…Negative Staining Transmission Electron Microscopy: Transmission electron microscopy (TEM) was used to investigate the morphology of LN/SPMs, PEI-LN/SPMs, and Bio-LN/SPMs, as previously described. [52] The lipid nanoemulsions were diluted 1:5 in 2% paraformaldehyde (PFA) and deposited on formvar-coated copper grids that had been previously treated for 10 minutes (min) with 1% Alcian blue. The grids were examined using a LaB6 filament-equipped Tecnai G2 Spirit BioTWIN Transmission Electron Microscope (FEI Company, ThermoFisher Scientific, Waltham, MA, USA).…”