614. Delivery of Micro-RNA Loaded Plasmid Through Nanoparticles Efficiently Modulate Transforming Growth Factor-Beta1 Expression in Healing Flexor Tendons: An In Vitro and In Vivo Study

Abstract

Aims: Formation of adhesion around the healing tendon is a major cause of poor function of the extremities. Adhesions developed around healing digital flexor tendons are extremely harmful to active hand movement, and modulating its gene expression may be a promising approach to prevent adhesion formations. Because transforming growth factor (TGF)-beta1 is highly expressed in the adhesion tissues and is considered responsible for adhesion and scar formation, we propose to deliver micro-RNAs (miRNAs) to down-regulate expression of this key gene in order to prevent adhesions in the healing digital flexor tendon.Methods: Nanoparticles made of polylactic-co-glycolic acid were prepared and were then incorporated with plasmids expressing enhanced green fluorescence protein and miRNAs for inhibiting the transforming growth factor-beta1 gene expression. The cultured tenocytes from chicken toes were used to test efficiency of transfection of the cells by plasmid released from nanoparticles. Then 30 long toes from bilateral feet of 15 chickens were used because of their structural similarity to human digital flexor tendons. The toes were divided into 6 groups (5 tendons per group) according to injected nanoparticles and timing of harvesting samples after surgery. The 6 groups were 2-, 4- and 6-weeks after nanoparticle-plasmid (mock miRNA)-injected groups and 2-, 4- and 6-week nanoparticle-plasmid (miRNA TGF-beta1)-injected groups. Expression levels of TGF-beta1 in the tendons in different groups were evaluated with Western blotting. Tendon healing status was also evaluated.Results: In the in vitro setting, the tenocytes could be effectively transfected by the nanoparticle-plasmid complex. The expression of TGF-beta1 is significantly down-regulated in healing chicken flexor tendon treated with nanoparticle-plasmid at all three time-points after surgery (p < 0.05 or p < 0.01). Histology analysis did not demonstrate any significant inflammation or necrosis in tendons injected with nanoparticle-plasmid after surgery. The tendon healing status was not affected by use of nanoparticle-plasmid.Conclusions: Delivery of micro-RNA loaded plasmid through nanoparticles is a promising gene therapy of the injured digital flexor tendon. Our results show that this approach is a non-viral delivery method, and can efficiently modulate genes associated with adhesion formation, which may be a promising method to limit adhesion formations.