A specifically designed nanoconstruct associates, internalizes, traffics in cardiovascular cells, and accumulates in failing myocardium: a new strategy for heart failure diagnostics and therapeutics

Abstract

Aims: Ongoing inflammation and endothelial dysfunction occurs within the local microenvironment of heart failure, creating an appropriate scenario for successful use and delivery of nanovectors. This study sought to investigate whether cardiovascular cells associate, internalize, and traffic a nanoplatform called mesoporous silicon vector (MSV), and determine its intravenous accumulation in cardiac tissue in a murine model of heart failure.

Methods and results: In vitro cellular uptake and intracellular trafficking of MSVs was examined by scanning electron microscopy, confocal microscopy, time-lapse microscopy, and flow cytometry in cardiac myocytes, fibroblasts, smooth muscle cells, and endothelial cells. The MSVs were internalized within the first hours, and trafficked to perinuclear regions in all the cell lines. Cytotoxicity was investigated by annexin V and cell cycle assays. No significant evidence of toxicity was found. In vivo intravenous cardiac accumulation of MSVs was examined by high content fluorescence and confocal microscopy, with results showing increased accumulation of particles in failing hearts compared with normal hearts. Similar to observations in vitro, MSVs were able to associate, internalize, and traffic to the perinuclear region of cardiomyocytes in vivo.

Conclusions: Results show that MSVs associate, internalize, and traffic in cardiovascular cells without any significant toxicity. Furthermore, MSVs accumulate in failing myocardium after intravenous administration, reaching intracellular regions of the cardiomyocytes. These findings represent a novel avenue to develop nanotechnology-based therapeutics and diagnostics in heart failure.

Keywords: Cardiomyopathy; Heart failure; Nanoconstructs; Nanomedicine; Nanoparticles; Nanotechnology.

Figure 1

Cellular association and internalization of MSVs. (A) Scanning electron micrographs of cardiovascular cells incubated with mesoporous silicon vectors (MSVs) (1:25, Cell:MSV ratio) demonstrating particle adhesion to the cellular membrane after 15 min of administration; bars, 1 μm. (B) Extensive cellular membrane rearrangement and engulfment of MSVs following 2 h of incubation; bars, 1 μm. (C) Z-stack analysis at 24 h via confocal microscopy demonstrating internalization and co-localization of MSVs beneath the cellular membrane. Actin cytoskeleton was labelled red, nuclear structures blue, and particles are visualized in green; bars, 10 μm.

Figure 2

Mesoporous silicon vector (MSV) uptake dynamics. (A) Confocal micrographs showing scarce association of MSVs after 1 h of incubation; bars, 100 μm. (B) After 120 h in human cardiovascular cell lines and 24 h in rat cardiomyocytes, a gradual increase of nanoconstructs accumulation was observed; bars, 100 μm. Actin cytoskeleton was labelled red, nuclear structures blue, and particles are visualized in green. (C) MSV uptake dynamics of cardiovascular cells were measured by flow cytometry at predetermined time-points for human cell types (1, 3, 6, 12, 24, 48, 72, and 120 h) and rat adult cardiomyocytes (1, 12, and 24 h). Results shown are the mean of three independent experiments.

Figure 3

Mesoporous silicon vector (MSV) intracellular trafficking. High-magnification confocal micrographs of cardiovascular cells were obtained and (A) at the first hour MSVs were randomly distributed through the cellular membrane and underwent phagocytosis; bars, 40 μm. (B) Over time, MSVs trafficked to the perinuclear region of the cells through the microtubules; bars, 40 μm. Actin cytoskeleton was labelled red, nuclear structures blue, and particles are visualized in green.

Figure 4

Accumulation of mesoporous silicon vectors (MSVs) in control normal and failing hearts. Twenty-four hours after administration, normal and failing hearts were sectioned in transverse and sagittal planes, and imaged using high-content fluorescent microscopy. (A) Control normal cardiac tissue shows slight amount of fluorescence emitted from the MSVs (green). (B) In contrast, a significant increase of homogeneously distributed fluorescence associated with the nanoconstructs was detected in the failing heart. Insets show microscopic magnifications of myocardial tissue (nanoconstructs are visualized in green). (C) MSVs were quantified by measuring area fraction, surface area in millimetres and automatic particle counting by the microscopy software. A 14-fold increase of MSVs uptake was detected in the failing heart.

Figure 5

In vivo cellular and subcellular localization of mesoporous silicon vectors (MSVs). To evaluate the extravasation of MSVs (green) in the failing heart, sarcomeric actinin of myocardial cells was labelled in red, CD-31-positive vasculature in yellow, and nuclear structures in blue. (A) MSVs extravasation across the endothelial barrier was observed and endothelial cells showed minimal accumulation. In contrast, high levels of nanoconstructs were homogeneously co-localized within the cardiomyocytes. Particle fluorescence (green) was extracted from the micrograph and can be observed next to the image; bars, 100 μm. (B) High magnification confocal micrograph demonstrating MSV localization within the cardiomyocytes, particle fluorescence (green) was extracted from the micrograph and can be observed next to the image; bars, 40 μm. (C) Z-stack and tridimensional rendering analysis was performed to demonstrate the internalization and high density of MSV distribution within the tissue; bar, 40 μm. (D) High magnification z-stack micrographs of cardiomyocytes presenting MSVs internalization beneath the cellular membrane and intracellular microtubule trafficking to the perinuclear region of the cell; bars, 10 μm.

Figure 6

Schematic presentation of the mechanism of accumulation and intravascular transport dynamics of mesoporous silicon vector (MSV) to the failing heart. After intravenous injection, the rationally designed MSVs transport through the circulatory system and, as a result of their size, shape, and chemical modifications, avoid reticuloendothelial system uptake and undergo vascular margination and extravasation from dysfunctional fenestrated capillaries present in the failing heart, where they reach the cardiomyocytes.