Pulmonary artery-targeted low-dose metformin-loaded nanocapsules safely improve pulmonary arterial hypertension in rats

Abstract

Introduction: Pulmonary arterial hypertension (PAH) remains a challenge to tackle despite various available medications. Metformin, although promising, has major adverse effects; the use of an appropriate drug delivery method may improve its efficacy and safety. The aim of this study was to develop a novel treatment for PAH using metformin. We developed a novel approach of using low-dose metformin encapsulated in pulmonary artery-targeted nanocapsules to alleviate PAH while avoiding adverse effects.

Methods: Metformin-loaded lung-targeted nanocapsules (MET nanocapsules) were created using a specific lipid composition, including cationic lipids. Their uptake and effects on cell viability were assessed in human pulmonary arterial smooth muscle cells (hPASMCs) from healthy individuals and patients with PAH. Their therapeutic effects were assessed in a PAH rat model. The safety of MET nanocapsules was confirmed using rat serum biochemical tests.

Results: We successfully prepared MET nanocapsules and demonstrated their effectiveness in inhibiting PASMC proliferation. In PAH model rats, MET nanocapsule treatment led to improved hemodynamics, right ventricular hypertrophy, and pulmonary arterial medial thickening. The nanocapsules effectively accumulated in the lungs of PAH model rats.

Conclusion: Intravenous administration of MET nanocapsules is a safe and innovative therapeutic approach for PAH. This method could improve PAH treatment outcomes while minimizing adverse effects, with potential applications in other types of pulmonary hypertension.

Keywords: drug delivery system; liposome; metformin; nanocapsule; pulmonary arterial hypertension.

FIGURE 1

Details of the pulmonary artery-targeted MET-loaded nanocapsules. (A) Schematic representation of the nanocapsules. (B) Particle size distribution of the empty nanocapsules and MET nanocapsules. (C) Storage stability of the nanocapsules. (D) Average particle size and zeta potential of the empty nanocapsules and MET nanocapsules (N = 3). Statistical analyses in (D) involved unpaired t-test. DOPE, dioleoyl phosphatidylethanolamine; DOCP, dioleoyl phosphatidylcholine; DCP, diocetyl phosphate; MET, metformin; ns, not significant.

FIGURE 2

Effects of pulmonary artery-targeted MET nanocapsules on hPASMCs. (A) Distribution of fluorescently labeled nanocapsules in hPASMCs. Light blue: Hoechst 33342 (fluorescence wavelength: 510–540 nm); red: rhodamine-PE (fluorescence wavelength: 578 nm). (B) Administration protocol of nanocapsules or metformin alone to control hPASMCs and hPASMCs from patients with heritable PAH. (C) Cell viability assessment using WST-8. N = 5/group. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses in (C) involved the one-way analysis of variance followed by Dunnett’s test. hPASMC, human pulmonary arterial smooth muscle cell; MET, metformin; PAH, pulmonary arterial hypertension.

FIGURE 3

Effects of pulmonary artery-targeted MET nanocapsules in PAH model rats. (A) Protocol of administration of nanocapsules or metformin alone to rats in each group. (B) Results of cardiac catheterization (systolic right ventricular pressure/systolic aortic pressure. control group N = 6, PAH group N = 5, PAH + MET group N = 6, PAH + Empty nanocapsules group N = 5, PAH + MET nanocapsules N = 7) and the Fulton index (right ventricular weight/left ventricle + ventricular septum weight. control group N = 6, PAH group N = 5, PAH + MET group N = 7, PAH + empty nanocapsules group N = 6, PAH + MET nanocapsules N = 8). (C) Western blotting for AMPK and pAMPK expression in the lungs of rats from each group. (D) Expression of phosphorylated AMPK is shown relative to that of total AMPK. The values represent mean ± standard deviation from independent experiments (N = 3 per group). *P < 0.05, ***P < 0.001, ****P < 0.0001. Statistical analyses in (B) and (D) involved the one-way analysis of variance followed by Dunnett’s test. hPASMC, human pulmonary arterial smooth muscle cell; MET, metformin; PAH, pulmonary arterial hypertension; sAop, systolic aortic pressure; sRVp, systolic right ventricular pressure; pAMPK, phosphorylated AMPK; tAMPK, total AMPK; ns, not significant.

FIGURE 4

Immunostaining and statistical analysis of the lungs in each rat group. (A) Immunostaining of lung sections from rats. (B) Number of PCNA-positive cells per 10 small pulmonary arteries, ratio of medial wall thickness to the minor diameter of the small pulmonary arteries, and percentage of fully muscularized small pulmonary arteries per 10 small pulmonary arteries (control group N = 6, PAH group N = 5, PAH + MET group N = 6, PAH + Empty nanocapsules group N = 7, PAH + MET nanocapsules N = 8). *P < 0.05, **P < 0.01. ***P < 0.001, ****P < 0.0001. Statistical analyses in (B) involved one-way analysis of variance followed by Dunnett’s test. PCNA, proliferating cell nuclear antigen; αSMA, alpha smooth muscle actin; EM, Elastica–Masson staining; MET, metformin; PAH, pulmonary arterial hypertension.

FIGURE 5

In-vivo distribution of pulmonary artery-targeted nanocapsules. (A) Protocol for the administration of rhodamine-PE-labeled nanocapsules to control (N = 5) and PAH model rats (N = 5). (B) Fluorescent staining demonstrating the distribution of fluorescently labeled nanocapsules in five organs. (C) Ratio of the rhodamine-positive to 4′,6-diamidino-2-phenylindole-stained areas in each organ. ***P < 0.001. Statistical analyses in (C) involved unpaired t-tests. PE, phosphatidylethanolamine.