Increment in molecular weight of poly (dimethylamino-ethylmethacrylate) based polymers cause strong red blood cell aggregation but not hemolytic response.

summary Polycations are frequently reported to tailor drug delivery systems, there is still a need to better correlate their macromolecular features with their biocompatibility, in particular with blood. In this view, we have analysed the RBC aggregation and hemolysis response caused by poly (dimethylamino-ethylmethacrylate) or poly(ethyleneimine) under a free form, i.e. without their preliminary association with polyanionic drug. Our results highlight that the molecular weight of PDMAEMA significantly affects the RBC aggregation without inducing any hemolysis. Introduction Positively charged polymers, commonly named polycations, are widely used as carriers in drug delivery research. However, if cationic nanocarriers are intravenously injected, they may interact with several blood elements [1]. Their hemocompatibility have been reported by several authors, evaluating their ability to induce RBC aggregation and hemolysis.These tests have been particularly reported for two of the most common polycations explored for gene delivery: Poly(ethylenimine) (PEI) and poly(dimethylamino-ethylmethacrylate) (PDMAEMA). However, until now most of these tests have been usually applied considering polyelectrolyte complexes and not the free form of these polycations [2-3]. Considering that these complexes could potentially dissociate either in the blood itself or later on, it was also important to better analyze their biological behavior when diluted in the blood under a free form. Interaction between negatively charged red blood cells (RBC) and polycations is especially important because it may provoke RBC aggregation or hemolytic responses [2]. Although the decrease in Zeta potential resulting from the electrostatic adsorption of the polycations to the erythrocyte surface can explain such responses, other factors depending of the molecular weight and free polymer concentration could also affect this reactivity [4]. In this respect, RBC aggregation and hemolytic response were evaluated in human whole blood considering three PDMAEMA with Mw ranging from 10,000 to 40,000, one block copolymer of PDMAEMA-b-Poly(ethylene glycol) (PEG) copolymer (Mw 30,700) and a PEI (Mw : 10,000). Experimental methods Three linear PDMAEMA homopolymers of distinct Mw (Table 1) and one linear PDMAEMA-b-PEG copolymer were synthesized by living radical polymerization [2]. These polymers were extensively purified after preparation in view to eliminate any solvent, catalyst and monomer residues. Branched PEI was purchased from Polysciences Europe. Polymer solutions in isotonic PBS (pH 7.4) were diluted in whole blood (1/10 volume) in cylindrical Eppendorf tubes and incubated at 37 °C during 15 to 240 min under roller agitation. After incubation, RBC aggregation was evaluated by inverted optical microscope (25x) analyzing at least 3 fields to have a statistic overview of the samples. Percentage of hemolysis was measured adopting ASTM method (F 756-00). Saponine (0.8 mg/mL) was adopted as positive control and PBS as negative control. Published in : Journal of Controlled Release (2010), vol. 148, pp. e30-e31. Status : Postprint auhtor Table 1 PDMAEMA based polymers and PEI from commercial origin. Polymer Code Mn, Mw Polymer concentration (μg/mL) PDMAEMA P1 10,000 10, 100, 200, 500, 1000 PDMAEMA P2 26,400 PDMAEMA P3 40,000 PDMAEMA-b-PEG (FDMAEMA = 95%) P4 30,700 PEI P5 10,000 Table 2 Score of red blood cell aggregation: (—) single cells; (+R) single cells, doublets, triplets and rouleaux; (++) slight aggregation with presence of single cells; ( +++) strong aggregation with presence of single cells; (++++) strong aggregation without presence of single cells; (+++++) strong aggregation in all the visual fields. 15 minutes incubation 60 minutes incubation 120 minutes incubation 240 minutes incubation Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml) 10 100 200 50