ABSTRACT
Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.
Subject(s)
Nanoparticles , Tissue Distribution , Animals , Cell Line, Tumor , Doxorubicin/toxicity , HeLa Cells , Humans , Maleates/toxicity , Mice , Nanoparticles/toxicityABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.