Gene Therapy (2002) 9, 850–858  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Lipoplex-induced hemagglutination: potential involvement in intravenous gene delivery H Eliyahu 1 , N Servel 1 , AJ Domb 2 and Y Barenholz 1 1 Department of Biochemistry, Hebrew University–Hadassah Medical School, Jerusalem, Israel; and 2 Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University–Hadassah Medical School, Jerusalem, Israel We report a study aiming to characterize the interaction of blood and blood components with lipoplexes under con- ditions relevant to in vivo intravenous transfection. In this study we focus on the interaction of lipoplexes with red blood cells (RBC). It was found that no significant hemolysis occurred during several hours’ incubation using lipoplex compositions and lipoplex/red blood cell ratios in the range commonly used for in vivo transfection. However, the inter- action of RBC with lipoplexes resulted in massive aggluti- nation, which occurs irrespective of the type of cationic lipid or helper lipid. Agglutination was also induced by polyplexes (such as dendrimer/DNA complexes) and lipoplexes in the presence of spermidine or protamine sulfate (the latter induced hemagglutination by itself). DSPE-PEG 2000 inserted into the lipoplexes inhibits hemagglutination somewhat. In order to understand the effect of serum on the agglutination better, plasma was separated into its high molecular weight components (HMWC, >14 kDa) and its low molecular weight components (LMWC, 14 kDa). These fractions were characterized for their level of proteins, primary amino Keywords: cationic lipids; transfection; serum components Introduction Cationic liposome–DNA complexes (lipoplexes) 1 are used as the most favorable non-viral nucleic acid delivery sys- tem in vitro and in vivo. Several lipoplexes based on vari- ous formulations of cationic lipids are serving in clinical trials in cases of cystic fibrosis 2 or cancer. 3 In vitro trans- fection using lipoplex-mediated gene delivery in cultured cells showed relatively high expression of a reporter gene. 4–6 However, these lipoplexes show very poor expression in vivo. For in vivo transfection, lipoplexes are required to reach the target cells; therefore the first obstacle is their interaction with the biological milieu at the site of injection (ie blood, in the case of intravenous administration). Successful transfection in vitro was car- ried out under conditions of net positive charge of lipo- plexes due to excess of cationic lipid positive charges over DNA negative charges. 7,8 Recent experiments sug- gest that in vivo, and especially for i.v. delivery, this Correspondence: Y Barenholz, Hebrew University–Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel Received 23 August 2001; accepted 15 February 2002 groups, osmotic pressure, and electrical conductivity, and compared with saline (0.15 M NaCl). It was found that both LMWC and HMWC inhibit agglutination by themselves, although whole serum demonstrates better hemagglutin- ation inhibition than each fraction separately. The inhibitory effect of the serum (or plasma) is explained by its effect on the electrostatics of the lipoplexes, reducing their positive charge, as was demonstrated using fluorescein-phosphatid- ylethanolamine-labeled lipoplexes. The effect of LMWC was related to ionic strength and was equal to the effect of 0.15 M NaCl. The level of agglutination was reduced with increas- ing lipoplex DNA - /cationic lipid + (DNA - /L + ) ratio. However, at the low DNA - /L + ratio needed to achieve significant in vivo transfection after i.v. administration, massive agglutination occurred. These data suggest that i.v. administration of lipo- plexes and polyplexes may lead to RBC agglutination, and the agglutinates formed may explain the localization of lipo- plexes and expression of their transgenes in the lungs. Gene Therapy (2002) 9, 850–858. doi:10.1038/sj.gt.3301705 excess of positive charge has to be larger than in vitro. 9 Since the development of early transfection kits, 10 it became clear that for many of them the biological milieu, such as serum, inhibits transfection efficiency, and the higher the serum (or plasma) percent the larger is the inhibition. 11–13 Preliminary results 7 indicate that these results are related to physical changes occurring in lipo- plexes in the biological milieu. Various approaches were recently introduced in order to overcome this discrep- ancy between successful in vitro transfection and the much less successful in vivo transfection. Since lipoplexes have not been as efficient as viral vectors for in vivo gene delivery, much effort has been devoted to synthesis of new cationic lipids that are more serum resistant; 12,14,15 changing helper lipids; 16,17 stabilization of the complexes by DNA-condensing agents such as polyamines and protamine sulfate; and steric stabilization of lipoplexes by poly (ethylene glycol) (PEG) phospholipid conjugates. 16,18–20 These studies make it clear that for improved in vivo gene delivery it is necessary to understand the interaction of lipoplexes with blood components. Indeed, the interac- tion of complexes with plasma proteins has been studied to some degree, 21,22 but almost no information is available