Polyethylene Glycol Prevents <i>in Vitro</i> Aggregation of Slightly Negatively-Charged Liposomes Induced by Heparin in the Presence of Bivalent Ions

Abstract

Liposomes are of great interest as drug delivery vehicles, and studies have focused on understanding how the physical and chemical characteristics of liposomes can be modified to improve their in vivo behavior. In a previous study, we found that the slightly negatively-charged liposomes aggregate only in the culture medium of human umbilical vein endothelial cells, whereas the liposomes modified with polyethylene glycol (PEG) (PEGylated) did not aggregate. In the present study, we investigated the underlying mechanism of this phenomenon. Firstly, it was found that heparin in the culture medium is one of the factors that cause aggregation of the non-PEGylated liposomes. Since the addition of ethylenediaminetetraacetic acid (EDTA) prevented the aggregation, metal ions, such as Ca 2 and Mg 2 , in the culture medium could also be important in driving the aggregation. In the presence of heparin, higher concentrations of Ca 2 or Mg 2 increased the particle size of the non-PEGylated liposomes, although no change in the particle size of PEGylated liposomes was observed. Under conditions in which aggregation occurred, we measured the binding and uptake of liposomes by macrophages in vitro. The binding and uptake of non-PEGylated liposomes were significantly increased with increasing Ca 2 concentrations, whereas those of PEGylated liposomes were unchanged. While the formation of aggregations of cationic or anionic liposomes has been reported previously, there are few reports addressing the aggregation of slightly negatively-charged or neutral liposomes. Thus, our data provide useful insights on the effect of PEGylation on liposomal aggregation and in vivo behavior.Key words liposome; heparin; aggregation; polyethylene glycol; Ca 2+Various liposomal products have been developed and applied to clinical treatment. Since methods to control the size of liposome and to improve the in vitro and in vivo stability of liposome had been developed, liposomes have attracted more attention. Incorporation of polyethylene glycol (PEG)-conjugated lipids (PEGylated) into liposomes is known to improve the circulation time of liposomes and prevent their uptake by the reticuloendothelial system (RES).1-4) It was reported that PEGylation inhibits the adsorption of serum proteins in vitro, 5,6) and decreases the uptake of liposomes by cells such as macrophages.7) From these reports, the extension of the circulation time by PEGylation is widely considered to be caused by the formation of a hydration layer, and steric hindrance can also prevent protein (such as opsonin) adsorption following recognition by cells of the RES, such as macrophages. However, the mechanisms producing these effects of PEGylation remain controversial. In particular, as regards protein adsorption, one report suggested that PEGylation can reduce protein adsorption by liposomes, 8) whereas other reports indicated that the total protein adsorption from plasma was not changed or increased by PEG.9,10) In another report, it was indicated that PEG-modification of negatively char...

Development of a bone-targeted pH-sensitive liposomal formulation containing doxorubicin: physicochemical characterization, cytotoxicity, and biodistribution evaluation in a mouse model of bone metastasis

“…The maintenance of the mean diameter over time may have been due to the highly negative surface charge and the presence of PEG chains on the liposome surface, which could have prevented vesicle aggregation through steric repulsion, improving the stability of the formulations. 26,27 These results are in agreement with those previously reported by Lopes et al, using a similar liposomal composition. 28…”

Posing for a picture: vesicle immobilization in agarose gel

“…refs 45 , 46 . Approaches have been reported by which aggregation may be avoided, for example via coating of the liposomes with steric inhibitors, such as polyethylene glycol 47 . This approach, however, might hinder the investigated interaction.…”

Barbigerone-in-hydroxypropyl-β-cyclodextrin-liposomal nanoparticle: preparation, characterization and anti-cancer activities

“…It was generally understood that liposomes with zeta potentials either in excess of ?30 mV or below -30 mV remain stable in the aqueous phase. In addition, it was demonstrated that PEG modification can prevent in vitro aggregation of negatively charged liposomes [28]. Therefore, the stability observed for Bar/HP-b-CD/liposome suggests that the liposomal inclusion complex of Bar meets the requirement for a potentially viable drug delivery system.…”