Olga Kryukov

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 (&gt...

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.

Polysaccharides have emerged as important functional materials because of their unique properties such as biocompatibility, biodegradability, and availability of reactive sites for chemical modifications to optimize their properties. The overwhelming majority of the methods to modify polysaccharides employ random chemical modifications, which often improve certain properties while compromising others. On the other hand, the employed methods for selective modifications often require excess of coupling partners, long reaction times and are limited in their scope and wide applicability. To circumvent these drawbacks, aniline-catalyzed oxime formation is developed for selective modification of a variety of polysaccharides through their reducing end. Notably, it is found that for efficient oxime formation, different conditions are required depending on the composition of the specific polysaccharide. It is also shown how our strategy can be applied to improve the physical and functional properties of alginate hydrogels, which are widely used in tissue engineering and regenerative medicine applications. While the randomly and selectively modified alginate exhibits similar viscoelastic properties, the latter forms significantly more stable hydrogel and superior cell adhesive and functional properties. Our results show that the developed conjugation reaction is robust and should open new opportunities for preparing polysaccharide-based functional materials with unique properties.

Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank=0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (P<0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.

The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.