Finerenone-Loaded PLGA Nanoparticles Alleviate Outflow Tract Stenosis in a Rat Arteriovenous Fistula Model in Thermo-responsive Hydrogels

Arteriovenous Fistula (AVF) failure is a thorny clinical problem, which seriously affects the long-term patency of access. Stenosis is the pathological basis for the failure of AVF and also the primary breakthrough for improving the patency of AVF. At present, neither oral drug treatment nor balloon dilation can achieve satisfactory therapeutic effects. Local release drug therapy can provide higher doses of drugs for AVF while avoiding adverse reactions associated with systemic treatment, and it is a promising treatment approach. This study aims to explore the effect of hydrogel-supported non-steroidal selective mineralocorticoid receptor antagonist Finerenone (Fin) on the outflow tract stenosis of AVF in rats and its possible mechanism of action, providing new therapeutic ideas for improving vascular stenosis of AVF and theoretical support for related clinical treatment.

Nanoparticles of polylactic glycolic acid copolymer loaded with Finerenone  were fabricated by solvent evaporation and loaded onto thermosensitive hydrogel polyoxyethylene polyoxypropylene ether block copolymer (F127) (F127/NPs@Fin). Nanoparticles were characterized in vitro by nanoparticle size analyzer, transmission electron microscope (TEM) and liquid chromatography-mass spectrometry (LCMS). In vitro, the appropriate drug loading concentration was screened through vascular smooth muscle cell (VSMC) and endothelial cell (EC) proliferation and migration experiments. After 21 days of in vivo application of the rat external jugular vein - carotid artery AVF model, the therapeutic effect of local drug application was detected by ultrasound and histology, and histological sequencing was sent to explore the mechanism of action. The correctness of the mechanism was verified by combined in vivo and in vitro gene and drug intervention.

The nanoparticles were prepared, which included NPs, NPs@Fin-1, NPs@Fin-2 and NPs@Fin-3 (1, 2, 3 represent Finerenone dosings of 100, 300, 600, respectively). The average particle sizes of NPs, NPs@Fin-1, NPs@Fin-2, NPs@Fin-3 were 381.44 ± 5.76 nm, 385.72 ± 7.17 nm, 383.86 ± 7.05 nm and 388.44 ± 10.24 nm respectively. There was no difference in particle sizes between NPs and NPs with different Fin loading amounts. The encapsulation rates of NPs@Fin-1, NPs@Fin-2 and NPs@Fin-3 were 69.3 ± 2.59%, 73.8 ± 1.04% and 74.1 ± 1.80%, respectively. In vitro experiments have shown that F127/NPs@Fin-2 can not only inhibit the proliferation and migration of VSMCs, but also promote the migration of ECs without inhibiting their proliferation. Further analysis of the phenotype of SMCs revealed that F127/NPs@Fin-2 treatment could inhibit the synthesis differentiation of SMCs, manifested as an increase in the expression of the contractile marker Calponin and a decrease in the expression of the synthesis marker Osteopontin. In vivo experimental ultrasound results showed that compared with the control group, the use of F127/NPs@Fin-2 increased the lumen diameter and blood flow of the outflow tract. HE results showed that F127/NPs@Fin-2 significantly inhibited neointimal hyperplasia in the outflow tract. The results of immunofluorescence staining and Western Blot indicated that compared with the control group, the expression of the outflow tract contraction marker Calponin increased and the expression of the synthesis marker Osteopontin decreased in the F127/NPs@Fin-2 group. RNA-seq results showed that F127/NPs@Fin-2 treatment improved insulin resistance in the outflow tract by inhibiting Foxo1. In vitro application of lentivirus with overexpression and knockdown of Foxo1 in smooth muscle cells combined with F127/NPs@Fin-2 verified that F127/NPs@Fin-2 improved the phenotypic changes of SMCs by inhibiting Foxo1 expression. Topical application of Foxo1 adenovirus loaded with SMC-specific promoter by hydrogel F127 in vivo combined with F127/NPs@Fin-2 verified that F127/NPs@Fin-2 improved AVF stenosis in rats by inhibiting Foxo1 expression.

The treatment with F127/NPs@Fin-2 may improve the transdifferentiation of SMCs and the hyperplasia of neointima in the outflow tract of AVF in rats by inhibiting the expression of Foxo1, providing a basis for clinical treatment.