Effective Delivery of Hypertrophic miRNA Inhibitor by Cholesterol-Containing Nanocarriers for Preventing Pressure Overload Induced Cardiac Hypertrophy

Abstract

Persistent cardiac hypertrophy causes heart failure and sudden death. Gene therapy is a promising intervention for this disease, but is limited by the lack of effective delivery systems. Herein, it is reported that CHO-PGEA (cholesterol (CHO)-terminated ethanolamine-aminated poly(glycidyl methacrylate)) can efficiently condense small RNAs into nanosystems for preventing cardiac hypertrophy. CHO-PGEA contains two features: 1) lipophilic cholesterol groups enhance transfection efficiency in cardiomyocytes, 2) abundant hydrophilic hydroxyl groups benefit biocompatibility. miR-182, which is known to downregulate forkhead box O3, is selected as an intervention target and can be blocked by synthetic small RNA inhibitor of miR-182 (miR-182-in). CHO-PGEA can efficiently deliver miR-182-in into hearts. In the mice with aortic coarctation, CHO-PEGA/miR-182-in significantly suppresses cardiac hypertrophy without organ injury. This work demonstrates that CHO-PGEA/miRNA nanosystems are very promising for RNA-based therapeutics to treat heart diseases.

Keywords: cardiac hypertrophy; cardiomyocyte transfection; gene therapy; miRNA delivery; nanocarriers.

Figure 1

Schematic diagram illustrating the preparation of CHO‐PGEA/miRNA complexes and the resultant gene therapy in hypertrophic cardiomyocytes (CMs).

Figure 2

A) Electrophoretic mobility retardation assay of polycation/miRNA complexes at various N/P ratios. B) Particle size and ζ‐potential of the polycation/miRNA complexes at various N/P ratios. C) AFM images of polycation/miRNA complexes at the typical N/P ratio of 10 (scale bar: 500 nm). D) Qualification of hemolysis ratio and E) images of red blood cells (RBCs) treated with PEI and CHO‐PGEA at the concentration of 1 mg mL⁻¹ (scale bar: 5 µm), where PBS was used as the control. ***P < 0.001; Data are from 3 independent experiments.

Figure 3

Cellular internalization of polycation/miRNA complexes in CMs or heart. A) Cellular internalization assay by MD where PEI/miR‐Cy3 was performed at the N/P ratio of 10. B) Confocal fluorescence images of H9C2 cells treated with miR‐Cy3 or polycation/miR‐Cy3(red) at the N/P ratio of 10 (scale bar: 50 µm). C) Image and relative radiant efficiency of mice hearts treated with saline, free miR‐Cy3, PEI/miR‐Cy3 or CHO‐PGEA/miR‐Cy3. D) Fluorescence images of heart section in four groups (scale bar: 100 μm). *P < 0.05, **P < 0.01, and ***P < 0.001; Data are from 3 independent experiments.

Figure 4

miRNA was effectively delivered into CMs by CHO‐PGEA and preform biology function. A) Alignment of mmu‐miR‐182 with putative 3′UTR target sites: 2 sites in FOXO3. miR‐182 seed sequence and the corresponding target sites were indicated in green box. Complementary bases were shown in red color. B) qRT‐PCR shows relative expression of miR‐182 and FOXO3 mRNA in cardiomyocytes after PE stimulation for 48 h. C) Representative FOXO3 expression after overexpressing miR‐182 in heart by western blot (WB). D) qRT‐PCR shows relative expression levels of miR‐182 in H9C2 cells treated with CHO‐PGEA/miR‐182 complexes after 24 h. E) Protein level of FOXO3 in H9C2 cells administered with polycation/miRNA. F) Relative expression of miR‐182 in mouse hearts after intravenous (IV) injection of CHO‐PGEA/miR‐182 complexes (5 nmol) or miR‐182 agomir (5 nmol), (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t‐test or one‐way ANOVO.

Figure 5

CHO‐PGEA/miR‐182‐in complex prevents hypertrophy. A) Gross phenotypic pattern captured by digital camera and heart weight (HW)/tibia length (TL) ratio at the end of treatment. B) Images of wheat germ agglutinin (WGA) stained hearts and quantification of myocyte cross section areas (scale bar: 50 µm). C) Left ventricle (LV) M‐mode images and D) echocardiographic data of mice hearts administered with CHO‐PGEA/miRNA‐in complexes from the 3rd week to the 9th week after TAC surgery, including ejection fraction (EF), FS, and LV mass. E) FOXO3 protein levels in mice heart after TAC by WB. *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 5 per group).

Figure 6

CHO‐PGEA/miRNA complexes toxic effect on organs. A) Representative photographs of H&E staining of the liver, kidney, lung, and spleen in mice treated with CHO‐PGEA/miRNA‐in after TAC 9 weeks (bar: 100 µm). B) Plasma biochemical measurement for aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CRE), and creatine kinase (CK) in mice with saline, agomir or CHO‐PGEA/miRNA for 24 h (n = 3 per group).