NANOPARTICLE-MEDIATED SELECTIVE SFRP-1 SILENCING ENHANCES BONE DENSITY IN VIVO IN OSTEOPOROTIC MICE BY THE STIMULATION OF THE CANONICAL WNT/β-CATENIN PATHWAY

Jul 1, 2022, 11:40 AM
10m
Room: S3 A

Room: S3 A

Speaker

Diaz-Rodriguez, Patricia (Universidade de Santiago de Compostela )

Description

"Introduction: Osteoporosis (OP) is characterized by a loss in bone mass and mineral density1. The stimulation of the canonical Wnt/β-catenin pathway has been reported to promote bone formation by increasing the osteogenic potential of mesenchymal stem cells (MSC)2. This pathway is controlled by several regulators as secreted frizzled-related protein-1 (Sfrp-1) that acts as an antagonist3. Thus, Sfrp-1 silencing therapies could be suitable for enhancing bone formation. However, the stimulation of this pathway at a systemic level has been correlated with adverse cardiovascular events. The use of nanoparticles (NPs) for oligonucleotide targeted delivery could be effective avoiding these undesirable effects. Aptamers (Apt) can recognize and bind specific structures based on their three-dimensional conformation. This work hypothesizes the systemic administration of lipid-polymer NPs (LPNPs) functionalized with a MSC specific Apt and carrying an SFRP1 silencing GapmeR, could favor bone formation in OP.
Methodology: Different pegylated-LPNPs formulations were prepared and the effect of pH, GapmeR encapsulation and aptamer functionalization on their physicochemical properties was evaluated by DLS and TEM. The oligonucleotide encapsulation efficiency and its in vitro release at variable temperatures were evaluated using fluorescently-labeled GapmeR. Adequate SFRP1 GapmeR-loaded Apt-LPNPs (Apt-LPNPs-SFRP1) were then evaluated in terms of cytocompatibility and gene silencing efficiency. Finally, the developed systems were administered in vivo in osteoporotic mice and their biodistribution and bone induction capacity was evaluated using radiolabeled LPNPs or by the femurs’ histological, histomorphometric and immunohistochemistry assays after three months, respectively.
Results: The developed LPNPs show an adequate average diameter of approximately 160 nm independently of the GapmeR encapsulation. Moreover, these formulations presented low polydispersity indexes (< 0.3). The incorporation of the aptamer in the nanoparticles surface, as expected, led to a decrease in the ζ-potential. Both LPNPs-SFRP1 and Apt-LPNPs-SFRP1 exhibited a spherical core-shell structure, characteristic of LPNPs. Their GapmeR encapsulation efficiency was 64 ± 4.32 % for both LPNPs showing a biphasic release pattern. The treatment of MSCs with LPNPs at variable concentrations did not show any toxicity. On the other hand, the treatment of MSCs with LPNPs-SFRP1 decreased the sfrp1 expression but less than the positive control (Damafect). Nevertheless, for cells treated with Apt-LPNPs-SFRP1, sfrp1 expression levels were similar to positive control. Moreover, the aptamer functionalization modified the LPNPs biodistribution profile showing a four-fold increase in the bone accumulation and a ten-fold decrease in the hepatic accumulation compared to naked LPNPs. The femurs histological evaluation revealed evident changes in bone structure and microarchitecture observing a more compact trabecular bone and a cortical bone thickness increase, in the Apt-LPNPs-SFRP1 treated mice compared to control (saline solution). Moreover, the immunohistochemical analysis of Col-I and OCN, revealed increased immunoreactivity for both markers in the Apt-LPNPs-SFRP1 treated mice.
Conclusions: Aptamer functionalized LPNPs loaded with SFRP1 silencing GapmeR showed adequate properties and biodistribution profiles leading to an enhancement on the bone density of osteoporotic mice.
1. Yadav V.K. et al., Nat. Med. 16(3), 308-12 (2010).
2. Houschyar K.S et al., Front. Cell Dev. Biol. 6, 170(2018).
3. Bodine P.V. et al., J. Mol. Endocrinol. 18(5), 1222-37 (2004)."

20941856088

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