Pulmonary circulation-mediated heart targeting for the prevention of heart failure by inhalation of intrinsically bioactive nanoparticles

Abstract

Heart failure is a serious clinical and public health problem. Currently there is an unmet demand for effective therapies for heart failure. Herein we reported noninvasive inhalation delivery of nanotherapies to prevent heart failure. Methods: A reactive oxygen species (ROS)-scavenging material (TPCD) was synthesized, which was processed into antioxidative and anti-inflammatory nanoparticles (i.e., TPCD NP). By decoration with a mitochondrial-targeting moiety, a multilevel targeting nanotherapy TTPCD NP was engineered. Pulmonary accumulation of inhaled TPCD NP and underlying mechanisms were examined in mice. In vivo efficacies of nanotherapies were evaluated in mice with doxorubicin (DOX)-induced cardiomyopathy. Further, an antioxidative, anti-inflammatory, and pro-resolving nanotherapy (i.e., ATTPCD NP) was developed, by packaging a peptide Ac2-26. In vitro and in vivo efficacies of ATTPCD NP were also evaluated. Results: TPCD NP alleviated DOX-induced oxidative stress and cell injury by internalization in cardiomyocytes and scavenging overproduced ROS. Inhaled TPCD NP can accumulate in the heart of mice by transport across the lung epithelial and endothelial barriers. Correspondingly, inhaled TPCD NP effectively inhibited DOX-induced heart failure in mice. TTPCD NP showed considerably enhanced heart targeting capability, cellular uptake efficiency, and mitochondrial localization capacity, thereby potentiating therapeutic effects. Notably, TPCD NP can serve as bioactive and ROS-responsive nanovehicles to achieve combination therapy with Ac2-26, affording further enhanced efficacies. Importantly, inhaled TPCD NP displayed good safety at a dose 5-fold higher than the efficacious dose. Conclusions: Inhalation delivery of nanoparticles is an effective, safe, and noninvasive strategy for targeted treatment of heart diseases. TPCD NP-based nanotherapies are promising drugs for heart failure and other acute/chronic heart diseases associated with oxidative stress.

Keywords: bioactive nanoparticles; cardiac dysfunction; heart failure; inhalation delivery; nanotherapy; targeted therapy.

Figure 1

Pulmonary circulation-mediated targeted therapy of heart failure by inhalation of bioactive nanotherapies. (A) Schematic illustration of in vivo targeting of the injured myocardium via inhalation of a nanotherapy TPCD NP. (B) A sketch showing preparation of a bioactive nanotherapy by nanoprecipitation/self-assembly. (C-E) Typical TEM (C) and SEM (D) images as well as size distribution (E) of TPCD NP. (F-H) Dose-dependent elimination of H₂O₂ (F) and superoxide anion (G) as well as time- and dose-dependent scavenging of DPPH radical (H) by TPCD NP. Scale bars in (C-D): 200 nm. Data in (F-H) are mean ± SD (n = 3).

Figure 2

Cellular uptake of TPCD NP and in vitro biological effects in H9C2 cells. (A) Confocal microscopy images showing time-dependent cellular uptake of Cy5/TPCD NP. (B-C) Flow cytometric curves (left) and quantitative analysis (right) of time-dependent (B) or dose-dependent (C) cellular uptake of Cy5/TPCD NP in H9C2 cells with or without DOX treatment. For time-dependent experiments, the dose of Cy5/TPCD NP was 50 µg/mL, while the incubation time was 2 h for dose-response studies. (D-F) Representative fluorescence images (D) and flow cytometric quantification (E-F) of intracellular ROS generation after stimulation with DOX and treatment with different doses of TPCD NP. (G) Intracellular MDA levels after different treatments. The protein content was measured by the BCA assay. (H-I) Expression levels of cTnI (H) and LDH (I). Scale bars: 40 μm (A, D). Data are mean ± SD (B, C, F, n = 3; G-I, n = 4). Statistical significance was assessed by the unpaired t-test for data in (B-C) and the one-way ANOVA with post-hoc LSD tests for data in (F-I). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Distribution of TPCD NP in the lungs and hearts after inhalation in mice. (A-B) Ex vivo fluorescent images (left) and quantitative analysis (right) of mouse lungs (A) and hearts (B) at various time points after inhalation of Cy7.5/TPCD NP. (C) A typical fluorescence image shows the distribution of Cy5/TPCD NP in a cryosection of the heart at 60 h after inhalation. (D-F) Immunofluorescence analysis of co-localization of Cy5/TPCD NP with EpCAM⁺ lung epithelial cells (D), CD31⁺ lung endothelial cells (E), and CD68⁺ macrophages (F) in lung sections. Scale bars: 40 μm. Data in (A-B) are mean ± SD (n = 3).

Figure 4

Flow cytometric analysis of the distribution of Cy5/TPCD NP in different pulmonary cells and blood cells. (A) The gating strategy used for flow cytometry analysis of lung cells at 24 h after inhalation of Cy5/TPCD NP. Biomarkers for lung epithelial cells (EpCAM⁺), lung endothelial cells (CD31⁺), and macrophages (F4/80⁺ and CD11b⁺) were separately used to distinguish different sub-types. (B) Typical dot plots show Cy5 distribution in control and Cy5/TPCD NP-treated mouse lung tissues. (C-E) Quantified Cy5⁺ cell proportions in pulmonary endothelial cells (C), epithelial cells (D), and macrophages (E). (F) The gating strategy used for flow cytometry analysis of blood cells at 12 h after inhalation of Cy5/TPCD NP. i, lymphocytes; ii, neutrophils; iii, Ly6C^low monocytes; iv, Ly6C^high monocytes; v, dendritic cells; and vi, macrophages. (G) Typical dot plots show Cy5 distribution in different blood cells. (H) Quantified Cy5⁺ cell proportions in lymphocytes, neutrophils, Ly6C^low monocytes, Ly6C^high monocytes, dendritic cells, and macrophages. Data in (C-E,H) are mean ± SD (n = 4). Statistical significance was assessed by the unpaired t-test. ***P < 0.001.

Figure 5

Therapeutic effects of inhaled TPCD NP on DOX-induced heart failure in mice. (A) Schematic illustration of the treatment protocols. (B) Typical digital photos show hearts isolated from mice in different groups. (C) The heart weight of different groups. (D) Ratios of HW/TL for different groups. (E) Representative M-mode echocardiography images of mouse hearts after different treatments. (F-G) Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) quantified by echocardiography. (H-I) The levels of MDA (H) and H₂O₂ (I) in tissue homogenates of hearts from mice treated with different formulations. The total protein content in heart homogenates was measured by the BCA assay. (J-K) Fluorescence images (J) and quantitative analysis (K) of DHE-stained heart cryosections for mice subjected to different treatments. (L-M) Serum levels of cTnI (L) and CK-MB (M). (N) H&E-stained histological sections of hearts. Control, healthy mice treated with saline; DOX, mice treated with DOX and saline. In different TPCD NP groups, diseased mice were treated with different doses of TPCD NP. Scale bars: 3 mm (B), 500 μm (J), 20 μm (N). Data in (C-D,F-I,K-M) are mean ± SD (n = 5). Statistical significance was assessed by the one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Comparison of therapeutic effects of dexrazoxane (DEX) and TPCD NP in mice with DOX-induced heart failure. (A) Schematic illustration of the treatment protocols. (B) The heart weight of different groups. (C) Ratios of HW/TL for different groups. (D) Representative M-mode echocardiography images of mouse hearts after different treatments. (E-F) LVEF and LVFS quantified by echocardiography. (G-H) The levels of MDA (G) and H₂O₂ (H) in tissue homogenates of hearts isolated from mice treated with different formulations. The total protein content in heart homogenates was measured by the BCA assay. (I-J) Serum levels of cTnI (I) and CK (J). Control, healthy mice treated with saline; DOX, mice treated with DOX and saline; DEX, mice treated with DOX and DEX at 200 mg/kg; TPCD NP, mice treated with DOX and TPCD NP at 25 mg/kg. Data in (B-C,E-J) are mean ± SD (n = 5). Statistical significance was assessed by the one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Design, engineering, and biological effects of a mitochondrial-targeting nanotherapy TTPCD NP. (A) Schematic of TPP-decorated TPCD NP (TTPCD NP). (B-E) TEM (B) and SEM (C) images as well as size distribution (D) and zeta-potential (E) of TTPCD NP. (F-G) Confocal microscopy images showing the mitochondrial localization of Cy5/TPCD NP (F) and Cy5/TTPCD NP (G) in H9C2 cells. Images in the lower panels show quantitative analysis of fluorescence intensities along the yellow lines on single cells in indicated images. (H) Flow cytometric curves (left) and quantitative analysis (right) of time-dependent cellular uptake of Cy5/TPCD NP and Cy5/TTPCD NP at 50 µg/mL in H9C2 cells stimulated with DOX. (I) The co-localization ratios of Cy5/TPCD NP or Cy5/TTPCD NP with mitochondria. (J) Intracellular MDA levels after DOX stimulation and treatment with TPCD NP or TTPCD NP at 50 µg/mL. The protein content was measured by the BCA assay. (K-L) Expression levels of cTnI (K) and LDH (L) by H9C2 cells after stimulation with DOX and treatment with TPCD NP or TTPCD NP. Scale bars: 200 nm (B-C), 20 μm (F-G). Data are mean ± SD (E,H-I, n = 3; J-L, n = 4). Statistical significance was assessed by the unpaired t-test for data in (H-I) and the one-way ANOVA with post-hoc LSD tests for data in (J-L). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

In vivo heart targeting and therapeutic effects of a mitochondrial-targeting nanotherapy TTPCD NP in mice. (A) Ex vivo fluorescence images (left) and quantitative analysis (right) of hearts isolated from mice after inhalation of Cy7.5/TPCD NP or Cy7.5/TTPCD NP. (B) The heart weight of different groups. (C) Ratios of HW/TL for mice with DOX-induced heart failure after treatment with TPCD NP or TTPCD NP at 25 mg/kg via inhalation. (D) Representative M-mode echocardiography images of mouse hearts after different treatments. (E-F) LVEF and LVFS quantified by echocardiography. (G-H) The levels of MDA (G) and H₂O₂ (H) in heart homogenates of mice treated with different formulations. The total protein content in heart homogenates was measured by the BCA assay. (I-J) Serum levels of cTnI (I) and CK (J). Data are mean ± SD (A, n = 3; B-C,E-J, n = 5). Statistical significance was assessed by the one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

Design, engineering, and biological effects of peptide nanotherapies derived from TPCD NP. (A) Schematic of a peptide Ac2-26 nanotherapy (ATPCD NP) based on TPCD NP. (B-D) TEM (B) and SEM (C) images as well as size distribution (D) of ATPCD NP. (E) In vitro release profiles of FITC-Ac2-26 from ATPCD NP in 0.01 M PBS at pH 7.4 with or without 1.0 mM H₂O_2. (F) A sketch showing the Ac2-26 nanotherapy (ATTPCD NP) based on mitochondrial-targeting TPCD NP. (G-I) TEM (G) and SEM (H) images as well as size distribution (I) of ATTPCD NP. (J) Intracellular MDA levels in H9C2 cells after different treatments. Protein contents were measured by the BCA assay. (K-M) Expression levels of cTnI (K), LDH (L), and TNF-α (M) by H9C2 cells subjected to different treatments. Scale bars in (B-C,G-H): 200 μm. Data are mean ± SD (E, n = 3; J-M, n = 4). Statistical significance was assessed by the one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10

Therapeutic effects of the mitochondrial-targeting peptide nanotherapy ATTPCD NP after inhalation delivery in mice with DOX-induced heart failure. (A) The heart weight of different groups. (B) The ratios of HW/TL for different groups. (C-F) The levels of MDA (C), H₂O₂ (D), TNF-α (E), and MPO (F) in tissue homogenates of hearts isolated from mice treated with different formulations. Total protein contents in heart homogenates were measured by the BCA assay. (G-H) Serum levels of cTnI (G) and CK (H). (I) Representative M-mode echocardiography images of mouse hearts after different treatments. (J-K) LVEF and LVFS quantified by echocardiography. Data are mean ± SD (n = 5). Statistical significance was assessed by the one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001.