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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.

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.

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.