Focused ultrasound-induced targeted delivery of miR-155-5p inhibitor employing biomimetic microbubbles reduces NADPH oxidase expression and inflammation in atherosclerotic ApoE-/- mice
Authors: Manea A , Vlad ML , Lazar AG , Manea SA 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): UEFISCDI

Background
MicroRNAs are important biomarkers and potential therapeutic targets in atherosclerosis. MiR-155-5p amplifies inflammation in atherosclerosis by targeting negative regulators of NF-kB signaling pathway. Inhibition of pro-atherogenic miRNAs or mimicking the function of selected anti-atherogenic miRNAs have emerged as prospective oligonucleotide-base therapeutic interventions in order to correct the expression of key genes that are mechanistically linked to atherosclerotic lesion formation.

Purpose
The aim of this study was to develop a strategy to reduce oxidative stress and inflammation in atherosclerotic mice employing sterically stabilized biomimetic microbubbles (MB) as focused ultrasound-responsive targeted delivery systems of miR-155-5p inhibitor.

Methods
ApoE-/- mice were fed a normal (control) or a high-fat, cholesterol-rich diet for 14 weeks to accelerate de development of aortic atherosclerotic lesions. FAM-labelled miRNA negative control inhibitor, miRNA negative control inhibitor or miR-155-5p inhibitor were charge-coupled to VCAM1-targeted MB. Targeted MB were delivered to mice in a single dose via retro-orbital injection. Ultrasound imaging interrogation of atherosclerotic lesions in ApoE-/- mice was performed using a conventional 30 MHz transducer (Vevo 2100 System). The Sonidel SP100 sonoporator was used for ultrasound-induced targeted release of miRNA inhibitors in mice. The biodistribution of MB was monitored by high-resolution fluorescence imaging. Atherosclerotic lesions were assessed by Oil Red O staining. Real time PCR and Western blot were employed to determine the expression of NADPH oxidase (Nox) subtypes, a major source of oxidative stress, and markers of oxidative stress inflammation (4-HNE-protein adducts, inducible nitric oxide synthase/NOS2, CD68).

Results
Epi-fluorescence analysis demonstrated the enhanced uptake of FAM-labelled miRNA control inhibitor by lungs and the atherosclerotic aorta of ApoE-/- mice following ultrasound wave exposure (1 MHz, 2.0 w/cm2, 50% duty cycles, 15 minutes). The mRNA/protein levels of Nox1-4 subtypes, 4-HNE-protein adducts, NOS2, and macrophage infiltration marker CD68 were found significantly elevated in the atherosclerotic aorta of ApoE-/- mice. The expression levels of Nox isoforms, 4-HNE-protein adducts, NOS2, and CD68 were significantly decreased in the atherosclerotic aorta of ApoE-/- mice 1-week after administration of targeted MB carrying miR-155-5p inhibitor as compared with atherosclerotic ApoE-/- mice treated with targeted MB carrying miRNA negative control inhibitor.

Conclusion
Systemic administration of miR-155-5p inhibitor conjugated with VCAM1-targeted MB reduces the expression of oxidative stress and inflammation-related markers in experimental atherosclerosis. Ultrasound-induced targeted delivery of miRNA-oriented oligonucleotides could become an important theranostic strategy combining imaging and treatment procedures in clinical atherosclerosis.