Purpose Calcium (Ca2+) plays a major role in various cellular processes and variations in its concentration are detected by calcium sensor proteins, which change their conformation to regulate molecular targets. Calcium sensors like Guanylate Cyclase Activating Proteins are involved in different retinal diseases like retinopathies and cone dystrophies. In this study CaF2 nanoparticles and nanovesicles with lipid composition similar to that of membranes of rod outer segments were probed as biocompatible carriers of Ca2+-sensors by assessing the structural and functional effects of the interaction with the nanodevice. In particular, Recoverin (Rec) and Calmodulin (CaM) were incubated with 20-25 nm CaF2 nanoparticles or 70-80 nm nanovescicles and their structure-function was investigated. Methods Circular dichroism spectroscopy was employed to investigate changes in protein secondary and tertiary structure and in thermal stability, both in the presence and in the absence of free Ca2+. Isothermal titration calorimetry was used to estimate the stoichiometry and thermodynamics of binding. Variations in hydrodynamic radius of the nanodevices upon protein binding were monitored by dynamic light scattering. The residual functionality of the Ca2+ sensor was investigated using fluorescence spectroscopy, by monitoring the exposure of aromatic residues upon Ca2+ or nanoparticle binding. Results Binding of Rec on lipid nanovesicles was reversible without significant perturbation of either structure or function. Results for CaF2 nanoparticles were instead protein-dependent. While Rec did not significantly change its structure in the presence of CaF2 nanoparticles and the binding was only partially reversible, CaM preserved both its secondary and tertiary structure, and the binding to the nanoparticle was fully reversible depending on the level of free Ca2+. Conclusion Lipid nanovesicles are widely used as carriers for encapsulated proteins, but our data show that the high area-to-volume ratio conferred from the nanoscale can be proficiently used to carry high concentrations of Rec on their surface for targeted delivery. CaF2 nanoparticles on the other hand are promising tools for biomedical purposes, but the ability to carry proteins with preserved function is system-dependent. Our study sets the basis for a potentially successful delivery of recombinant proteins in eye disease and equally in cases where a limited administration frequency and high efficiency are highly recommended.

CaF2 nanoparticles and lipid nanovesicles as novel carriers of calcium sensors for targeted retinal therapy

MARINO, VALERIO;ASTEGNO, Alessandra;Maresi, Elena;PEDRONI, Marco;PICCINELLI, FABIO;DELL'ORCO, Daniele
2014-01-01

Abstract

Purpose Calcium (Ca2+) plays a major role in various cellular processes and variations in its concentration are detected by calcium sensor proteins, which change their conformation to regulate molecular targets. Calcium sensors like Guanylate Cyclase Activating Proteins are involved in different retinal diseases like retinopathies and cone dystrophies. In this study CaF2 nanoparticles and nanovesicles with lipid composition similar to that of membranes of rod outer segments were probed as biocompatible carriers of Ca2+-sensors by assessing the structural and functional effects of the interaction with the nanodevice. In particular, Recoverin (Rec) and Calmodulin (CaM) were incubated with 20-25 nm CaF2 nanoparticles or 70-80 nm nanovescicles and their structure-function was investigated. Methods Circular dichroism spectroscopy was employed to investigate changes in protein secondary and tertiary structure and in thermal stability, both in the presence and in the absence of free Ca2+. Isothermal titration calorimetry was used to estimate the stoichiometry and thermodynamics of binding. Variations in hydrodynamic radius of the nanodevices upon protein binding were monitored by dynamic light scattering. The residual functionality of the Ca2+ sensor was investigated using fluorescence spectroscopy, by monitoring the exposure of aromatic residues upon Ca2+ or nanoparticle binding. Results Binding of Rec on lipid nanovesicles was reversible without significant perturbation of either structure or function. Results for CaF2 nanoparticles were instead protein-dependent. While Rec did not significantly change its structure in the presence of CaF2 nanoparticles and the binding was only partially reversible, CaM preserved both its secondary and tertiary structure, and the binding to the nanoparticle was fully reversible depending on the level of free Ca2+. Conclusion Lipid nanovesicles are widely used as carriers for encapsulated proteins, but our data show that the high area-to-volume ratio conferred from the nanoscale can be proficiently used to carry high concentrations of Rec on their surface for targeted delivery. CaF2 nanoparticles on the other hand are promising tools for biomedical purposes, but the ability to carry proteins with preserved function is system-dependent. Our study sets the basis for a potentially successful delivery of recombinant proteins in eye disease and equally in cases where a limited administration frequency and high efficiency are highly recommended.
calcium, sensors, nanodevices
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/936096
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