Osteoarthritis (OA) is a progressive osteo-arthritis that causes significant disability and pain and for which there are limited treatment options
August 9, 2020
Osteoarthritis (OA) is a progressive osteo-arthritis that causes significant disability and pain and for which there are limited treatment options. lubricin production and decreased chondrocyte apoptosis. This is a proof-of-concept study showing that mRNA can be efficiently delivered to articular cartilage, an avascular tissue that is poorly accessible even when drugs are intra-articularly (IA) administered. The ability to accommodate a wide range of oligonucleotides suggests that this platform may find use in a broad range of clinical applications. 0.05. Open in a separate window Figure 2 Cy3-labeled HA-NPs (~55 nm) were incubated with 5 mm2 cartilage discs from human OA knee. After 48 h, cartilage explants were washed, processed, and examined for depth of NPs penetration by confocal microscopy. The HA-NPs deeply penetrated the cartilage explants (up to ~1 mm in depth). 2.2. p5RHH-mRNA NP Preparation and Characterization Next, we prepared the peptide-mRNA NPs by mixing a set Erg amount of p5RHH peptide (10 mol) with increasing concentrations of WNT16 mRNA (~1100 nucleotides, nt). The combining of 10 mol p5RHH with 1 g of WNT16 mRNA (peptide:mRNA percentage 3500:1) yielded a NP of ~65 nm after software of the HA layer, as assessed by transmitting electron microscopy (TEM, Shape 3), and a zeta potential of ~30 mV by powerful light scattering (DLS, Desk 1). Raising the focus of mRNA led to a significantly improved particle size ( 200 nm by TEM at an mRNA focus of 4 g and a peptide:mRNA percentage of 875:1, Shape 1A) and designated heterogeneity in the sizes from the NPs. The bigger NP size assessed by DLS (Desk 1) suggests aggregates from smaller sized contaminants, which is backed from the TEM pictures (Shape 3, right -panel). While DLS can be a computation that suits the light scattering data for an algorithm predicated on Mies scattering theory, TEM permits immediate visualization from the transfective exclusion and contaminants of the bigger aggregates through the computation, which we realize, aren’t transfective from prior function [17,18]. Open up in another window Shape 3 HA-coated p5RHH-WNT16 mRNA NPs had been generated by combining 10 mol of p5RHH with 1 g mRNA (peptide:mRNA percentage 3500:1), 2 g of mRNA (peptide:mRNA percentage of 1750:1), or 4 g of mRNA (peptide:mRNA percentage 875:1). Inset (in the remaining panel) displays the NP at an increased magnification. Desk 1 Features of NPs at different peptide:mRNA ratios. 0.001. 2.4. Delivery of WNT16 mRNA in Cartilage Explants We following examined the delivery of WNT16 mRNA. The purified WNT16 mRNA create was created commercially and included the correct endcaps and poly-A tail. HA-coated p5RHH-WNT16 mRNA NPs had been ready using 3 different concentrations of mRNA: 1 g, 2 g, or 4 g (as comprehensive in Section 2.2). The self-assembled NPs had been incubated with human being cartilage explants for 48 h after that analyzed for protein manifestation of WNT16, -catenin, and WNT3a. We discovered that manifestation of WNT16 was considerably enhanced using the delivery of mRNA at 1 g and 2 g, however, not at 4 g (Shape 5A,B), most likely as the size from the self-assembled NPs as of this focus (4 g) of mRNA was too large for effective cartilage penetration. Improved WNT16 manifestation was followed by reduced -catenin (Figure 5C,D) and WNT3a (Figure 5E,F). Open in a separate window Figure 5 HA-coated p5RHH-WNT16 mRNA NPs generated at the indicated concentrations of mRNA Wortmannin inhibitor and p5RHH were incubated with 5 mm2 cartilage discs derived from human OA knee joints. After 48 h, cartilage explants were washed, processed, and examined for WNT16 (A,B), Beta-catenin (C,D), and WNT3a (E,F) expression. Immunohistochemistry (IHC) photomicrographs were derived from cartilage Wortmannin inhibitor discs transfected with 1 g of peptide-mRNA NPs. Scale bar = 100 m. The numbers of WNT16+, beta-catenin+, and WNT3a+ cells/cartilage section were enumerated. Values represent mean SEM. Data were derived from 6 to 8 8 cartilage sections, from 4C6 independent human cartilage Wortmannin inhibitor explants. * 0.05, ** 0.01, *** 0.001, n.s. = not significant. 2.5. Effect of WNT16 Overexpression on Cartilage Homeostasis We examined the downstream effects following the delivery of the NPs. We chose the NPs formulated with 1 g of WNT16 mRNA, since this concentration resulted in the highest expression WNT16 (Figure 5A). We assessed the expression of lubricin, an essential joint lubricant that protects against chondrocyte apoptosis and cartilage deterioration . We observed that WNT16 mRNA delivery led to a significant upregulation of lubricin-expressing cells and lubricin in the superficial level from the cartilage explants (Body 6A,B), which resulted in suppression of chondrocyte apoptosis, as evidenced with a decrease in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)+ cells (Body 6C,D). Open up in another window Body 6 HA-coated p5RHH-WNT16 mRNA NPs (generated at 1.