Research ArticleNanomedicine

Inhalation of peptide-loaded nanoparticles improves heart failure

See allHide authors and affiliations

Science Translational Medicine  17 Jan 2018:
Vol. 10, Issue 424, eaan6205
DOI: 10.1126/scitranslmed.aan6205
  • Fig. 1 Inhalation delivers CaPs to the heart.

    (A) Schematic representation of CaP heart targeting via the inhalation route. 1, nanoparticle inhalation; 2, nanoparticle deposition in lungs and translocation through the air-blood pulmonary barrier; 3, heart targeting and drug release into the heart. (B) Quantification of Cy7 fluorescence signals from heart tissue of mice treated with CaP-Cy7 via gavage, intraperitoneal (ip), intravenous (iv), and inhalation administration. Data are means ± SEM. *P < 0.05 and ***P < 0.001 compared to gavage [one-way analysis of variance (ANOVA) test] (n = 5). (C) Time-course quantification of Cy7 fluorescence signals from heart and lung tissue of mice treated with CaP-Cy7 via inhalation administration. Data are means ± SEM. *P < 0.05 and **P < 0.01 compared to the 10-min time point (two-way ANOVA test) (n = 5). R.F.U., relative fluorescence units. (D) Whole-body optical in vivo imaging to evaluate the distribution of CaP-Cy7 and Cy7 in CD1 nude mice at different time points after inhalation. Data are representative of at least six independent experiments. Values are expressed as radiance efficiency (×109). (E) 3D FMT imaging of mice as presented in (D) and analyzed 60 min after CaP-Cy7 and Cy7 administration. T, trachea; H, heart; L, lung. (F) Fluorescence imaging of explanted hearts from perfused mice analyzed 60 min after inhalation of 50 and 100 μl of CaP-Cy7. NT, heart from nontreated mouse. Values are expressed as radiance efficiency. Scale values: heart, 1.04 × 107 (maximum) to 3.56 × 106 (minimum); lung, 7.78 × 107 (maximum) to 6.17 × 106.9 (minimum) (n = 4).

  • Fig. 2 STED microscopy of isolated cardiomyocytes from mice treated with MP-rhodamine–loaded CaPs.

    (A) STED microscopy on an isolated cardiomyocyte from mice treated with MP-rhodamine–loaded CaPs. Green, LTCC; red, MP-rhodamine. Representative of 10 different acquisitions. Original magnification, ×100; scale bars, 15 μm. (B) Enlargement of yellow dotted line square in (A). Scale bars, 5 μm. (C) Orthogonal view of isolated cardiomyocyte in (A) and (B). Original magnification, ×100; scale bars, 5 μm.

  • Fig. 3 Inhaled CaP-MPs restore cardiac function in a mouse model of diabetic cardiomyopathy.

    (A) Design of the study. ECO, echocardiography. (B) LV fractional shortening and ejection fraction (indices of cardiac contractile function) as determined by echocardiographic analysis on STZ-treated mice treated as indicated. Data are means ± SEM. ***P < 0.001 compared to NT mice (two-way ANOVA test) (n = 10). (C) Western blot analysis for the LTCC pore unit (Cavα1.2) on adult cardiomyocytes isolated from treated mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Average peak of LTCC current density as a function of voltage command measured in adult cardiomyocytes isolated from treated mice. All values are means ± SD for n cells. *P < 0.02 for NT compared to MP and CaP-HA mice (n = 16 to 21). (E) Contractility of adult cardiomyocytes isolated from treated mice. Data are means ± SEM. *P < 0.05 and **P < 0.01 compared to NT mice (two-way ANOVA test) (n = 20). (F) Z-stack confocal laser scanning microscopy images showing HA peptide in myocardial tissue from mouse treated by CaP-HA inhalation. Immunofluorescence staining for HA (red), LTCC (green), and cell nuclei [4′,6-diamidino-2-phenylindole (DAPI); blue]. Scale bar, 20 μm (n = 3).

  • Fig. 4 CaP-HA inhalation in landrace pigs.

    (A) Design of the study. (B) Systemic, pulmonary, and LV hemodynamics (n = 6; data are means ± SEM). Steady-state data before and after CaP inhalation were compared by one-way ANOVA for repeated measurements. Pressure-volume relationships were compared by analysis of covariance. Post hoc testing was performed by Tukey’s test. P < 0.05 was considered significant. (C) Original registrations from one representative animal of LV pressure-volume loops before (pre, top) and after (post, bottom) CaP-HA inhalation. All registrations were recorded at spontaneous heart rate. ESPVR, end-systolic pressure-volume relationship; EDPVR, end-diastolic pressure-volume relationship. (D) Kinetics of inhaled CaP-HA cardiac targeting as examined by dot blot analysis from biopsies obtained at the indicated times after inhalation. IB, immunoblotting. (E) Western blot analysis for HA on tissues of treated pigs. (F) Z-stack confocal laser scanning microscopy images showing HA peptide in myocardial tissue from pigs treated by CaP-HA inhalation. Immunofluorescence staining for HA (red), LTCC (green), and cell nuclei (DAPI; blue). Scale bar, 20 μm (n = 6).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/424/eaan6205/DC1

    Materials and Methods

    Fig. S1. Size stability of CaPs and CaPs functionalized with MP and HA in protein-free and protein-enriched aqueous medium, and surface charge of CaP-Cy7 and CaPs with Cy7 adsorbed on their surface.

    Fig. S2. In vivo and ex vivo characterization of CaPs.

    Fig. S3. Echocardiographic analysis of CaP-treated mice.

    Fig. S4. Stability, loading, degradation, and structural characterization of CaPs and CaPs functionalized with MP and HA.

    Fig. S5. STED microscopy of isolated cardiomyocytes from mice treated with unloaded CaPs.

    Fig. S6. MP-CaP treatment of diabetic mice.

    Table S1. Echocardiographic analysis of 10-week-old male wild-type mice treated with CaP.

    Table S2. Excitability and refractoriness parameters obtained by epicardial multiple lead recording in untreated rats or rats treated with CaP.

    Table S3. CaP-MP loading.

    Table S4. Echocardiographic analysis of 10-week-old male wild-type and STZ mice treated with MP, CaP-HA, and CaP-MP.

    Table S5. Characterization of LTCC current during rectangular double-pulse protocols and membrane capacities measured in adult cardiomyocytes isolated from treated mice.

    Table S6. Thiobarbituric acid–reactive substance detection in mice treated with MP, CaP-HA, and CaP-MP.

    Movie S1. 3D FMT imaging of the cardiopulmonary area from Cy7-treated mice.

    Movie S2. 3D FMT imaging of the cardiopulmonary area from CaP-Cy7–treated mice.

    Movie S3. STED microscopy of isolated cardiomyocytes from mice treated with MP-rhodamine–loaded CaPs.

  • Supplementary Material for:

    Inhalation of peptide-loaded nanoparticles improves heart failure

    Michele Miragoli,* Paola Ceriotti, Michele Iafisco, Marco Vacchiano, Nicolò Salvarani, Alessio Alogna, Pierluigi Carullo, Gloria Belén Ramirez-Rodríguez, Tatiana Patrício, Lorenzo Degli Esposti, Francesca Rossi, Francesca Ravanetti, Silvana Pinelli, Rossella Alinovi, Marco Erreni, Stefano Rossi, Gianluigi Condorelli, Heiner Post, Anna Tampieri, Daniele Catalucci*

    *Corresponding author. Email: michele.miragoli{at}humanitasresearch.it (M.M.); daniele.catalucci{at}cnr.it (D.C.)

    Published 17 January 2018, Sci. Transl. Med. 10, eaan6205 (2018)
    DOI: 10.1126/scitranslmed.aan6205

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Size stability of CaPs and CaPs functionalized with MP and HA in protein-free and protein-enriched aqueous medium, and surface charge of CaP-Cy7 and CaPs with Cy7 adsorbed on their surface.
    • Fig. S2. In vivo and ex vivo characterization of CaPs.
    • Fig. S3. Echocardiographic analysis of CaP-treated mice.
    • Fig. S4. Stability, loading, degradation, and structural characterization of CaPs and CaPs functionalized with MP and HA.
    • Fig. S5. STED microscopy of isolated cardiomyocytes from mice treated with unloaded CaPs.
    • Fig. S6. MP-CaP treatment of diabetic mice.
    • Table S1. Echocardiographic analysis of 10-week-old male wild-type mice treated with CaP.
    • Table S2. Excitability and refractoriness parameters obtained by epicardial multiple lead recording in untreated rats or rats treated with CaP.
    • Table S3. CaP-MP loading.
    • Table S4. Echocardiographic analysis of 10-week-old male wild-type and STZ mice treated with MP, CaP-HA, and CaP-MP.
    • Table S5. Characterization of LTCC current during rectangular double-pulse protocols and membrane capacities measured in adult cardiomyocytes isolated from treated mice.
    • Table S6. Thiobarbituric acid–reactive substance detection in mice treated with MP, CaP-HA, and CaP-MP.
    • Legends for movies S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). 3D FMT imaging of the cardiopulmonary area from Cy7-treated mice.
    • Movie S2 (.mov format). 3D FMT imaging of the cardiopulmonary area from CaP-Cy7–treated mice.
    • Movie S3 (.mov format). STED microscopy of isolated cardiomyocytes from mice treated with MP-rhodamine–loaded CaPs.

Navigate This Article