GalNAc bio-functionalization of nanoparticles assembled by electrostatic interactions improves siRNA targeting to the liver.

RNA interference (RNAi) has the potential to reversibly silence any gene with high efficiency and specificity. To fulfill the clinical potential of RNAi, delivery vehicles are required to transport the short interfering RNA (siRNA) to the site of action in the cells of target tissues. Here, we describe the features of novel liver-targeted siRNA nanoparticles (NPs), co-assembled due to the complexation of alginate sulfate (AlgS) with siRNA, mediated by calcium ions bridges (AlgS-Ca2+-siRNA NPs) and then bioconjugation of a targeting ligand onto the AlgS upon the NP surface. To gain insight into the complexation process and confirm AlgS accessibility on NP surface, we investigated different schemes for fabrication. All resulting NPs, independently of the component addition order, were of average size of 130-150nm, had surface charge of <-10mV, exhibited a similar atomic composition on their surface, were efficiently uptaken by HepG2 cells and induced approx. ~90% silencing of STAT3 gene. Ca2+ and AlgS concentrations in NPs affected cell uptake and gene silencing. Bioconjugation of N-acetylgalactosamine (GalNAc), a ligand to the asialoglycoprotein receptor (ASGPR) overexpressed on hepatocytes, was validated by XPS analysis and cell uptake by receptor-mediated mechanism. After intravenous (i.v.) injection to BALB/c mice, GalNAc-NPs were targeted to liver by a factor of ~3 with lesser renal clearance compared to non-targeted NPs. We foresee that the combined advantages of site-specific targeting and reversibility of the tri-component NPs as well as the simplicity of their fabrication make them an attractive system for targeted delivery of siRNA.

Surface Analysis of Nanocomplexes by X-ray Photoelectron Spectroscopy (XPS).

Self-assembled nanocomplexes composed of individual molecules that spontaneously connect via noncovalent interactions have recently emerged as versatile alternatives to conventional controlled drug delivery systems because of their unique bioinspired properties (responsiveness, dynamics, etc.). Characterization of such nanocomplexes typically includes their size distribution, surface charge, morphology, drug entrapment efficiency, and verification of the coexistence of labeled components within the nanocomplexes using a colocalization study. Less common is the direct examination of the molecular interactions between the different components in the coassembled nanocomplex, especially in nanocomplexes composed of hygroscopic components, because convenient methods are still lacking. Here, we present a detailed experimental protocol for determining the surface composition and the chemical bonds by X-ray photoelectron spectroscopy (XPS) after drying the deposit hygroscopic sample overnight under UHV. We applied this method to investigate the surface chemistry of binary Ca2+-siRNA nanocomplexes and ternary nanocomplexes of hyaluronan-sulfate (HAS)-Ca2+-siRNA, deposited on a wafer. Notably, we showed that the protocol can be implemented to study the surface composition and interactions of the deposited nanocomplexes with a traditional XPS instrument, and it requires only a relatively small amount of the nanocomplex suspension.

A bridge to silencing: Co-assembling anionic nanoparticles of siRNA and hyaluronan sulfate via calcium ion bridges.

Therapeutic implementation of RNA interference (RNAi) through delivery of short interfering RNA (siRNA) is still facing several critical hurdles, which mostly can be solved through the use of an efficient delivery system. We hereby introduce anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. The NPs have an average size of 130nm and a mild (-10mV) negative surface charge. Transmission electron microscopy (TEM) using gold-labeled components and X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. Efficient cellular uptake of the anionic siRNA NPs, associated with potent gene silencing (>80%), was observed across multiple cell types, including murine primary peritoneal macrophages and human hepatocellular carcinoma cells. In a clinically-relevant model of acute inflammatory response in IL-6-stimulated human hepatocytes, STAT3 silencing induced by HAS-Ca(2+)-siRNA NPs resulted in marked decrease in the total and activated STAT3 protein levels, as well as in the expression levels of downstream acute phase response genes. Collectively, anionic NPs prove to be an efficient and cytocompatible delivery system for siRNA.

Calcium-siRNA nanocomplexes: what reversibility is all about.

Gene silencing using small interfering RNA (siRNA) relies on the critical need for a safe and effective carrier, capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. We hypothesized that a delivery platform based on the eletrostatic interactions of siRNA with calcium ions in solution would fulfill these needs, ultimately leading to effective gene silencing. Physical characterization of the calcium-siRNA complexes, using high resolution microscopy and dynamic light scattering (DLS), showed the formation of stable nanosized complexes ~80nm in diameter, bearing mild (~-7mV) negative surface charge. The complexes were extremely stable in the presence of serum proteins or high concentrations of heparin; they maintained their nanosized features in suspension for days; and effectively protected the siRNA from enzymatic degradation. The Ca-siRNA complexes were disintegrated in the presence of Ca-chelating ion exchange resin, thus proving their reversibility. Excellent cytocompatibility of calcium-siRNA complexes was achieved using physiological calcium ion concentrations. The calcium-siRNA complexes successfully induced a very high (~80%) level of gene silencing in several cell types, at both mRNA and protein levels, associated with efficient cellular uptake. Collectively, our results show that the developed delivery platform based on reversible calcium-siRNA interactions offers a simple and versatile method for enhancing the therapeutic efficiency of siRNA.