Mechanistic study of the nucleation and conformational changes of polyamines in presence of phosphate ions

Polyamine Phosphate Nanoparticles (PANs) have great potential for the delivery of large therapeutics, such as plasmids and/or siRNAs. The formation of PANs by complexation of Poly(allylamine hydrochloride) (PAH) and phosphate ions from Phosphate Buffer (PB) was studied here, and how it is affected by the presence of phosphate ions from PB and ionic strength. From Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) the critical PB concentration for PANs formation was determined. Below this critical point, Small Angle X-ray Scattering (SAXS) studies revealed that small PAH-phosphate aggregates coexist with not complexed or weakly complexed polymer chains in solution and that the presence of the phosphate ions increases the Kuhn length of the polymer chains until that only spherical aggregates are present in solution. TEM, DLS and SAXS showed the increase of PANs size with ionic strength up to 250 mM NaCl. At higher NaCl concentrations, PANs disassemble into smaller aggregates. Isothermal Titration Calorimetry (ITC) showed that PAN formation is an exothermic process and the association of phosphates below the critical PB concentration is entropically controlled.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Technical tip: high-resolution isolation of nanoparticle–protein corona complexes from physiological fluids

Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP–protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches

Mechanistic study of the nucleation and conformational changes of polyamines in presence of phosphate ions

Polyamine Phosphate Nanoparticles (PANs) have great potential for the delivery of large therapeutics, such as plasmids and/or siRNAs. The formation of PANs by complexation of Poly(allylamine hydrochloride) (PAH) and phosphate ions from Phosphate Buffer (PB) was studied here, and how it is affected by the presence of phosphate ions from PB and ionic strength. From Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) the critical PB concentration for PANs formation was determined. Below this critical point, Small Angle X-ray Scattering (SAXS) studies revealed that small PAH-phosphate aggregates coexist with not complexed or weakly complexed polymer chains in solution and that the presence of the phosphate ions increases the Kuhn length of the polymer chains until that only spherical aggregates are present in solution. TEM, DLS and SAXS showed the increase of PANs size with ionic strength up to 250 mM NaCl. At higher NaCl concentrations, PANs disassemble into smaller aggregates. Isothermal Titration Calorimetry (ITC) showed that PAN formation is an exothermic process and the association of phosphates below the critical PB concentration is entropically controlled.Fil: Andreozzi, Patrizia. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Ricci, Caterina. Università Politecnica Delle Marche; ItaliaFil: Martinez Porcel, Joaquin Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Moretti, Paolo. Università Politecnica Delle Marche; ItaliaFil: Di Silvio, Desirè. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Amenitsch, Heinz. Graz University Of Technology.; AustriaFil: Ortore, Maria Grazia. Università Politecnica Delle Marche; ItaliaFil: Moya, Sergio Enrique. Centro de Investigacion Cooperativa En Biomateriales.; Españ

Self-assembly of poly(allylamine)/siRNA nanoparticles, their intracellular fate and siRNA delivery

Silencing RNA (siRNA) technologies attract significant interest as a therapeutic tool for a large number of diseases. However, the medical translation of this technology is hampered by the lack of effective delivery vehicles for siRNAs in cytosol that prevent their degradation in the bloodstream. The use of molecular complexes based on polyamines have great potential for siRNA delivery as polyamines can protect the siRNA during circulation and at the same time favor siRNA translocation in cytosol. Here, nanoparticles are prepared by complexation of poly(allylamine hydrochloride) (PAH) and siRNA varying the ratio of nitrogen groups from PAH to phosphate groups from siRNA (N/P ratio). Nanoparticles are characterized by transmission electron microscopy and dynamic light scattering. The stability of complexes of green rhodamine labelled PAH (G-PAH) and Cy5 labelled siRNA (R-siRNA) at different pHs and in cell media is studied by fluorescence cross-correlation spectroscopy (FCCS). FCCS studies show that the nanoparticles are stable at physiological pH and in cell media but they disassemble at acidic pH. An optimal N/P ratio of 2 is identified in terms of stability in media, degradation at endosomal pH and toxicity. The intracellular fate of the complexes is studied following uptake in A549 cells. The cross-correlation between G-PAH and R-siRNA decreases substantially 24 h after uptake, while diffusion times of siRNA decrease indicating that the complexes disassemble, liberating the siRNAs. The release of siRNAs into the cytosol is confirmed with parallel confocal laser scanning microscopy. Flow cytometry studies show that PAH/siRNA nanoparticles are effective at silencing green fluorescent protein expression at low N/P ratios at which polyethylenimine/siRNA shows no significant silencing.Fil: Di Silvio, Desirè. No especifíca;Fil: Martínez Moro, Marta. No especifíca;Fil: Salvador, Cristian. No especifíca;Fil: Ramirez, Maria de Los Angeles. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caceres Velez, Paolin Rocio. No especifíca;Fil: Ortore, Maria Grazia. No especifíca;Fil: Dupin, Damien. No especifíca;Fil: Andreozzi, Patrizia. No especifíca;Fil: Moya, Sergio E.. No especifíca