Knockdown of Gene Expression in Macrophages by microRNA Mimic-Containing Poly (Lactic-co-glycolic Acid) Microparticles.

Paul Mckiernan,Sally Cryan,Joanne Ramsey,Patrick Lynch,Catherine Greene

doi:10.3390/medicines5040133

Abstract

Background: microRNA (miRNA) regulate target gene expression through translational repression and/or mRNA degradation and are involved in the regulation of inflammation. Macrophages are key inflammatory cells that are important in chronic inflammatory lung diseases such as cystic fibrosis (CF). Macrophage-expressed miRNA represent therapeutic drug targets, yet delivery of nucleic acids to macrophages has proved challenging. Methods: miRNAs were encapsulated in poly (lactic-co-glycolic acid) (PLGA)-based microparticles using double emulsion solvent evaporation and characterised for physicochemical features. Phorbol myristic acetate (PMA)-differentiated U937 macrophages were transfected with empty PLGA microparticles or those encapsulating a premiR-19b-3p or scrambled control miRNA mimic. miRNA internalisation and knockdown of a miR-19b-3p target gene, secretory leucoprotease inhibitor (SLPI), were determined by qRT-PCR. Results: Microparticle formulations were consistently found to be 2–3μm and all had a negative ζ potential (−5 mV to −14 mV). Encapsulation efficiency of premiR-19b-3p was 37.6 ± 13.4%. Levels of mature miR-19b-3p were higher in macrophages after delivery of premiR-19b-3p microparticles compared to empty or scrambled control miRNA-containing microparticles. Significant SLPI knockdown was achieved 72 hours post-delivery of premiR-19b-3p microparticles compared to controls. Conclusions: miRNA-encapsulating PLGA microparticles offer a new treatment paradigm for delivery to macrophages that could potentially be administered to CF lungs via inhalation.

Highlights

Cystic fibrosis (CF) is a clinically complex, autosomal recessive genetic disease that occurs due to mutation of a single gene, the cystic fibrosis conductance regulator gene (CFTR)
PLGA microparticles of 2–3 μm were previously determined as an optimal size for alveolar macrophage uptake [34]
Size was determined for each microparticle formulation and mean diameters were found to be between 2 μm and 3 μm (Figure 1), which is within the size range for effective pulmonary delivery and macrophage uptake

Summary

Introduction

Cystic fibrosis (CF) is a clinically complex, autosomal recessive genetic disease that occurs due to mutation of a single gene, the cystic fibrosis conductance regulator gene (CFTR). The major pulmonary pathology is characterised by inadequate homeostasis of the airway surface layer and impaired mucociliary clearance. This leads to increased mucus viscosity and chronic bacterial infection. MicroRNA (miRNA) regulate target gene expression through translational repression and/or mRNA degradation and are involved in the regulation of inflammation. Macrophage-expressed miRNA represent therapeutic drug targets, yet delivery of nucleic acids to macrophages has proved challenging. Methods: miRNAs were encapsulated in poly (lactic-co-glycolic acid) (PLGA)-based microparticles using double emulsion solvent evaporation and characterised for physicochemical features. Phorbol myristic acetate (PMA)-differentiated U937 macrophages were transfected with empty PLGA microparticles or those encapsulating a premiR-19b-3p or scrambled control miRNA mimic.

Methods

Results

Conclusion