Investigation of layer-by-layer assembly of polyelectrolytes on fully functional human red blood cells in suspension for attenuated immune response.

The encapsulation of live cells with polymeric coat-ings is a versatile approach to modulate or control the response cells to their environment. The layer-by-layer (LbL) self-assembly of nonimmunogenic polyelectrolytes is employed here to attenuate or suppress the binding of antibodies to live red blood cells (RBCs) and, consequently, decrease their inherent immunogenicity toward foreign RBCs. The optimized shell was composed of four bilayers of alginate (AL) and chitosan-graft-phosphorylcholine (CH-PC) surrounded by two bilayers of AL and poly-l-lysine-graft-polyethylene glycol (PLL-PEG). Experimental parameters, including the polyelectrolytes and RBCs concentrations and the cell handling and purification protocols, were optimized to achieve effective encapsulation of live and functional RBCs in suspension. The viability and functionality of coated RBCs were confirmed by a hemolysis assay and by their ability to take up oxygen. The successful immunocamouflage of RBCs was confirmed by observing that the recognition of the ABO/D (Rh) blood group antigens present on the surface of RBCs by their respective antibodies was muted in the case of coated RBCs. The results of this studies mark an important step toward the production of universal RBCs.

Determination of surface-induced platelet activation by applying time-dependency dissipation factor versus frequency using quartz crystal microbalance with dissipation.

Platelet adhesion and activation rates are frequently used to assess the thrombogenicity of biomaterials, which is a crucial step for the development of blood-contacting devices. Until now, electron and confocal microscopes have been used to investigate platelet activation but they failed to characterize this activation quantitatively and in real time. In order to overcome these limitations, quartz crystal microbalance with dissipation (QCM-D) was employed and an explicit time scale introduced in the dissipation versus frequency plots (Df-t) provided us with quantitative data at different stages of platelet activation. The QCM-D chips were coated with thrombogenic and non-thrombogenic model proteins to develop the methodology, further extended to investigate polymer thrombogenicity. Electron microscopy and immunofluorescence labelling were used to validate the QCM-D data and confirmed the relevance of Df-t plots to discriminate the activation rate among protein-modified surfaces. The responses showed the predominant role of surface hydrophobicity and roughness towards platelet activation and thereby towards polymer thrombogenicity. Modelling experimental data obtained with QCM-D with a Matlab code allowed us to define the rate at which mass change occurs (A/B), to obtain an A/B value for each polymer and correlate this value with polymer thrombogenicity.

Silencing red blood cell recognition toward Anti-A antibody by means of polyelectrolyte layer-by-layer assembly in a two-dimensional model system.

Silencing the antigenic response of red blood cells (RBCs) is a prerequisite toward the development of universal blood transfusion. Using a two-dimensional (2D) model whereby nonfixed RBCs are adsorbed on a human fibronectin (HFN)-coated surface, we demonstrate that the layer-by-layer (LbL) assembly technique of biocompatible polyelectrolytes can be employed to achieve the immunocamouflage of RBCs against the Anti-A antibody while maintaining the integrity and viability of the cells. The multilayered film consisted of a protecting shell (P-shell), containing five bilayers of chitosan-graft-phosphorylcholine (CH-PC) and sodium hyaluronate (HA), covered by a camouflage shell (C-shell) made up of five bilayers of poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-PEG) and alginate (AL). Control experiments in which RBCs were coated by (CH-PC/HA)(10) bilayers indicated that the two polyelectrolytes alone did not prevent immunorecognition. The LbL film formation on RBCs and model substrates was monitored by quartz crystal microbalance with dissipation factor (QCM-D) and analyzed through zeta-potential measurements, atomic force microscopy (AFM), and optical microscopy. Antibody interaction with the coated RBCs was investigated by QCM-D, fluorescence microscopy, and hemolysis assays. Results from these measurements demonstrated that the hybrid LbL system built-up with different sets of polyelectrolytes was able to protect the RBCs from hemolysis and recognition by the Anti-A antibody.

Modulating the release kinetics through the control of the permeability of the layer-by-layer assembly: a review.

The layer-by-layer (LbL) self-assembly technique has emerged as a simple and versatile method for coating biological and non-biological templates for various biomedical applications. A promising avenue of this technique lies in the encapsulation of drugs and other biological substances for controlled release. Fundamental studies of LbL assembly on flat surfaces have provided a sound understanding of film deposition theory and its pertinence to ionic and molecular transport and diffusion through polyelectrolyte multilayer (PEM) films. However, there is a lack of information on the permeability of three-dimensional PEM shell systems. In either PEM films or shells, it has been shown that drug release is a function of the ionic strength, pH and/or multilayer thickness. This report aims to provide an overview of the physicochemical parameters affecting the permeability of two- and three-dimensional multilayer shells, including ionic strength, layer number and pH. Furthermore, their synergic effect on loading and release of biologically active molecules from LbL multilayers are discussed.

Characterization of folate-chitosan-DNA nanoparticles for gene therapy.

Gene therapy using polymers such as chitosan shows good biocompatibility, but low transfection efficiency. The mechanism of folic acid (FA) uptake by cells to promote targeting and internalization could improve transfection rates. The objective of this study was to synthesize and characterize FA-chitosan-DNA nanoparticles and evaluate their cytotoxicity in vitro. Chitosan-DNA and FA-Chitosan-DNA nanoparticles were prepared using reductive amidation and a complex coacervation process. The effect of charge ratio on the properties of these nanoparticles was monitored by laser scattering. DNA inclusion and integrity was evaluated by gel electrophoresis. Cell viability was illustrated with the MTT assay. Charge ratio (N/P) controlled the nanoparticles size and their zeta potential. Nanoparticles presented a mean size of 118 nm and 80% cellular viability compared to 30% cell viability using LipofectAMINE2000 controls. Gel electrophoresis showed intact DNA within the carriers. FA-nanoparticles have lower cytoxicity, good DNA condensation, positive zeta potential and particle size around 118 nm, which makes them a promising candidate as a non-viral gene vector.

Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy.

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. However, cationic polymers, like chitosan, have potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. Chitosan is a natural non-toxic polysaccharide, it is biodegradable and biocompatible, and protects DNA against DNase degradation and leads to its condensation. The objective of this paper was to summarize the state of the art in gene therapy and particularly the use of chitosan to improve the transfection efficiency in vivo and in vitro.