PACA nanoparticles target and deliver sildenafil to rejuvenate aged mouse liver sinusoidal endothelial cells

Abstract

Ageing is established as the most significant risk factor for disease. About 75% of people over 75 years have diabetes or pre-diabetes and/or hyperlipidaemia which are established risk factors for cardiovascular outcomes, and risk factors for age-related conditions such as dementia, sarcopenia, frailty and osteoporosis. Age-related changes in the liver microcirculation, in particular relating to the cells lining the blood vessels, the liver sinusoidal endothelial cells (LSEC), are a potential cause for dyslipidaemia and insulin resistance in old age. There is also loss of LSEC mediated waste clearance functions essential for homeostasis. Finding ways to reverse these age-related changes in the LSEC will fill a significant gap in therapeutic options available for the treatment of ageing disorders. Such therapies may also benefit patients with fibrotic livers, since LSEC changes in this disease resemble those seen in the ageing LSEC in many aspects. Nanoparticles that access systemic circulation frequently accumulate in the liver. This could be utilized as a promising strategy for targeted drug delivery to the liver. The present study assessed if poly(alkyl-cyanoacrylate) nanoparticles (PACA NPs) are a suitable vector for the targeted transport of such therapeutics to LSEC, to reverse age-related changes such as fenestration/porosity loss. Mice were co-injected with PACA NPs and formaldehyde denatured serum albumin (FSA) and their livers were then examined by microscopy. PACA and FSA co-localised to LSEC, including at the sub-cellular level in endocytic vesicles. Isolated LSEC were challenged with Nile Red (NR668) labelled PACA NPs, which were rapidly internalized. HEK293 cells overexpressing stabilin-2 internalized PACA NPs, suggesting that stabilin-2 mediates PACA uptake on LSEC. Cultured LSEC from aged mice were challenged with PACA NPs containing sildenafil and examined with scanning electron microscopy to determine effects on fenestrations. Sildenafil PACA reversed age-related changes LSEC fenestration frequency and porosity at 3-fold lower sildenafil concentrations than sildenafil alone. If sildenafil PACA induces similar changes in vivo, age-related reduction of LSEC porosity could be reversed by the targeted delivery of sildenafil via PACA NPs.

Keywords: LSEC; PACA; ageing; liver sinusoidal endothelial cell; nanoparticle; sildenafil; targeted delivery.

Figure 1

Uptake of PACA NPs loaded with NR668 dye (PACA-NR668) in mouse liver sinusoidal endothelial cells (LSEC): (A) in vivo in a liver section and (B) in vitro in cultured mouse LSEC. (A) Liver section from one mouse injected with formaldehyde treated serum albumin-AlexaFluor488 (FSA-AF488) (2.5 mg/kg) and PACA-NR668 (225 mg/kg). Liver sinusoids appear diffuse green, indicating uptake of FSA-AF488, arrows in inset indicate colocalization (yellow) of PACA-NR668 (red) and FSA-AF488 (green) in LSEC when administered in vivo. The sample was stained with DAPI (blue). Selected cross-section shows uptake of PACA-NR668 and its intracellular perinuclear localisation. (B) Uptake of PACA-NR668 (red) in cultured (young) mouse LSEC (n = 1). Localization of PACA-NR668 indicated in z-stack (red arrows). The sample was stained with CellMask™ Green (green) and DAPI (blue).

Figure 2

Stabilin-2 in HEK cells enables more uptake of PACA NPs than stabilin-1. HEK293 cells were treated with PACA-NR668 (20 µg/mL) for 90 min, in serum (5% FBS). (A) Uptake of PACA-NR668 NPs in stabilin-1, stabilin-2 or vector-plasmid control transfected HEK293 cells. Left panels: merge of phase-contrast and red channel; right panels: NR668 channel alone. Stab1 = stabilin-1 transfected HEK293 cells, Stab2 = stabilin-2 transfected HEK293 cells, VC = vector-plasmid control transfected HEK293 cells. (B) Inhibition of PACA nanoparticle uptake by the stabilin-1/2 ligand FSA (200 µg/mL, unlabelled) in HEK293 cells expressing stabilin-2. Left panels: PACA uptake in the absence of inhibiting concentration of FSA (200 µg/mL, unlabelled), Right panels: PACA uptake in the presence of inhibiting concentration of FSA (200 µg/mL, unlabelled). (C) Uptake of PACA nanoparticle co-incubated with FSA-AF488 (20 µg/mL) ± unlabelled FSA (200 µg/mL). PACA = PACA NPs, phaco. = phase contrast, FSA-AF488 = AlexaFluor488 labelled FSA. Top left and right panels: merged FSA-AF488, PACA-NR668 and phaco channels; middle left and right panels: FSA-AF488 channel alone; bottom left and right panels PACA-NR668 channel alone. All scale bars are 100 µm.

Figure 3

Representative images (A) of LSEC from aged mice in the various treatment groups. Effect of PACA NPs on (B) fenestration frequency (number of fenestrations/µm²) and (C) porosity (percentage of cell surface covered by fenestrations) of cultured LSEC. LSEC were challenged with either control cell media, vector control PACA or sildenafil PACA NPs for 1 h. Fenestration frequency (B) and porosity (C) are increased in cells treated with 0.1 µg/mL and 1 µg/mL sildenafil PACA NPs, compared to the untreated control and the matched concentrations of vector control PACA NPs. There is no significant difference in fenestration frequency or porosity between the untreated control cells and cells treated with vector control PACA. The data is presented as individual points representing single cells from 3 biological replicates (4 for the untreated control group), with a line representing the mean, and is based on 8⁠-21 images per biological replicate per treatment group. *, ** and **** indicate statistically significant differences (p<0.05; p<0.002; p<0.0001, respectively) between the sildenafil PACA and untreated control/vector control PACA groups. VC PACA = vector control PACA, Sild PACA = sildenafil PACA. All scale bars are 5 µm.

Figure 4

Distribution of the fenestration diameter of liver sinusoidal endothelial cell (LSEC) isolated from aged mice. Each histogram represents equivalent diameter measurements of fenestrations from 3 biological replicates (4 for the untreated control group), with 8-21 images per replicate for each treatment group. First row shows untreated control group, followed by 0.1 µg/mL (blue) and 1 µg/mL (red) of vector control PACA (plain color) and sildenafil PACA (oblique square pattern). The dashed lines represent fitted Gaussian curves. The centers of each Gaussian distribution are noted in the legend for each treatment group separately.

Figure 5

Viability and toxicity assays on cultured liver sinusoidal endothelial cells (LSEC) from aged mice challenged with control media, and with matching concentrations of sildenafil-loaded- and vector control PACA NPs. Lactose dehydrogenase cytotoxicity assay (LDH) (A) shows the effect on LSEC following 1 h challenge with PACA NPs. LDH data is based on 4 biological replicates and presented as means with standard deviations. There is no significant difference in LDH release between PACA-treated groups and the untreated control. For the alamarBlue cell viability assay (B), LSEC were pre-treated with PACA NPs for 1 h followed by a 3 h incubation with 10% alamarBlue reagent. There was no significant difference in cell viability between the untreated control group and 0.1 and 1 µg/mL PACA NPs, but ≥5 µg/mL of both sildenafil PACA and vector control PACA NPs significantly reduces cell viability. The alamarBlue data is based on 3 biological replicates and presented as means with standard deviation.