ABSTRACT
Poly(2-dimethylamino)ethyl methacrylate (PDMAEMA) is an attractive polycation frequently proposed as a non-viral vector for gene therapy. As expected for other cationic carriers, intravenous administration of PDMAEMA can result in its ionic complexation with various negatively charged domains found within the blood. To gain more insight into this polycation hemoreactivity, we followed the binding kinetics of a free form (FF) of fluorescein labelled PDMAEMA (Mn below 15 kDa) in normal human blood using flow cytometry. This in vitro study highlighted that platelets display higher affinity for this polycation compared to red blood cells (RBCs), with an adsorption isotherm characteristics of a specific saturable binding site. PDMAEMA (1-20 µg/mL) exerted a concentration dependent proaggregant effect with a biphasic aggregation of washed platelets. Activation of platelets was also noticed in whole blood with the expression of P-selectin and fibrinogen on platelet surface. Although additional studies would be needed in order to elucidate the mechanism of PDMAEMA mediated activation of platelets, our manuscript provides important information on the hemoreactivity of FF PDMAEMA.
Subject(s)
Blood Platelets/drug effects , Erythrocytes/drug effects , Methacrylates/pharmacology , Nylons/pharmacology , Blood Platelets/physiology , Cells, Cultured , Erythrocytes/physiology , Fibrinogen/metabolism , Humans , P-Selectin/metabolism , Platelet Activation/drug effects , Platelet Aggregation/drug effectsABSTRACT
Poly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA. In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein. The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood-brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p<0.05) inhibition (40%) in presence of 2-dexoxy-D-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones. As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.
Subject(s)
Fluorescein , Fluorescent Dyes , Human Umbilical Vein Endothelial Cells/drug effects , Methacrylates , Nylons , Biological Transport , Cell Line , Cell Survival/drug effects , Fluorescein/chemistry , Fluorescein/pharmacology , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Methacrylates/chemistry , Methacrylates/pharmacology , Molecular Weight , Nylons/chemistry , Nylons/pharmacologyABSTRACT
Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA) is a cationic polymer when dissolved in a 7.4 pH fluid. Owing to its ionic nature, this polycation interacts with the negatively charged cell membrane surface of red blood cells (RBCs). The electrostatic self-assembly of PDMAEMA on RBCs membrane can be employed for inducing the formation of a polymeric shield camouflaging blood group antigens on RBCs as a valuable strategy for developing "universal RBCs" for blood transfusion. The purpose of this research was to evaluate the camouflaging ability of PDMAEMA homopolymers and PDMAEMA-co-poly(ethylene glycol) copolymers differing in molecular weight and architecture. Surprisingly, the PDMAEMAs caused a partially masking, no masking, and sensitization of the same RBCs population. The MW and architecture of the polymers as well as temperature of PDMAEMA-RBCs treatment influenced the results observed. Herein, the very particular reactivity of PDMAEMAs and RBCs is analyzed and discussed.
