Targeted Therapy for Acute Autoimmune Myocarditis with Nano-Sized Liposomal FK506 in Rats

Immunosuppressive agents are used for the treatment of immune-mediated myocarditis; however, the need to develop a more effective therapeutic approach remains. Nano-sized liposomes may accumulate in and selectively deliver drugs to an inflammatory lesion with enhanced vascular permeability. The aims of this study were to investigate the distribution of liposomal FK506, an immunosuppressive drug encapsulated within liposomes, and the drug’s effects on cardiac function in a rat experimental autoimmune myocarditis (EAM) model. We prepared polyethylene glycol-modified liposomal FK506 (mean diameter: 109.5 ± 4.4 nm). We induced EAM by immunization with porcine myosin and assessed the tissue distribution of the nano-sized beads and liposomal FK506 in this model. After liposomal or free FK506 was administered on days 14 and 17 after immunization, the cytokine expression in the rat hearts along with the histological findings and hemodynamic parameters were determined on day 21. Ex vivo fluorescent imaging revealed that intravenously administered fluorescent-labeled nano-sized beads had accumulated in myocarditic but not normal hearts on day 14 after immunization and thereafter. Compared to the administration of free FK506, FK506 levels were increased in both the plasma and hearts of EAM rats when liposomal FK506 was administered. The administration of liposomal FK506 markedly suppressed the expression of cytokines, such as interferon-γ and tumor necrosis factor-α, and reduced inflammation and fibrosis in the myocardium on day 21 compared to free FK506. The administration of liposomal FK506 also markedly ameliorated cardiac dysfunction on day 21 compared to free FK506. Nano-sized liposomes may be a promising drug delivery system for targeting myocarditic hearts with cardioprotective agents

Effects of free and liposomal FK506 on histological findings in the myocarditic hearts.

<p>Representative transverse sections in ventricles stained with hematoxylin and eosin (upper panels) and Masson’s trichrome (lower panels) at low magnification (A) and high magnification (C). (B) Quantitative analysis of areas of cell infiltration and fibrosis (N = 4–12 in each group). Scale bars indicate 5mm and 500 μm in (A) and (C), respectively. Data are expressed as the mean ± SEM. * P < 0.05 versus the saline treated group. # P < 0.05 versus the free FK506 treated group.</p

Effects of free and liposomal FK506 on inflammatory cytokines in the hearts of EAM rats.

<p>Cytokine expression on day 21 after immunization was evaluated using real-time quantitative PCR. Relative expression of IFN-γ (A), IL-17 (B), TNF-α (C), IL-1β (D), IL-10 (E), IL-4 (F) and TGF-β (G) were normalized to GAPDH (N = 4–9 in each group). Data are expressed as the mean ± SEM. * P < 0.05 versus the saline treated group.</p

Effects of free and liposomal FK506 on hemodynamic parameters in the rat EAM model.

<p>Quantitative data of hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), max dP/dt and minimum dP/dt. EAM rats were treated with free or liposomal FK506 (0.035 mg/kg) on days 14 and 17 after immunization (N = 6 and 9, respectively). Data are expressed as the mean ± SEM. * P < 0.05 versus the free FK506 treated group.</p

Time-course changes in vascular permeability in myocarditic hearts in the rat EAM model.

<p>The representative brightfield and fluorescent images of the myocarditic hearts of rats that received fluorescent-labeled nano-sized beads (100 nm) are shown in the upper and lower panels, respectively. Hearts were excised on days 0, 10, 14, 17 and 21 after immunization. In each time point, 2–3 rats were checked. Bar indicates 10 mm.</p

Tissue distribution of free and liposomal FK506 in the rat EAM model.

<p>Tissue distributions of [³H]-labeled FK506 in the rat EAM model. Radioisotope activity in tissues was measured 2 hours after the single intravenous administration of free or liposomal [³H]-labeled FK506 on day 17 after immunization (N = 4 in each group). Data are expressed as the mean ± SEM. * P < 0.05 versus the free FK506 treated group.</p

Effects of free FK506 on hemodynamic parameters in the rat EAM model.

<p>Quantitative data of hemodynamic parameters, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), max dP/dt and minimum dP/dt, on day 21 after immunization. Drugs were intravenously administered on days 14 and 17 after immunization. Rats were divided into Normal, EAM with saline treatment and EAM with free FK506 (0.035, 0.17 or 0.35 mg/kg) treatment groups (N = 5–15 in each group). Data are expressed as the mean ± SEM. * P < 0.05 versus the saline treated group.</p