Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating

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Abstract

The purpose of our work was to compare the biodistribution of liposomes with different surface properties. Phosphatidylcholine (PC)/cholesterol (Chol) liposomes were prepared containing 6% mol of a charged lipid (stearylamine, SA; phosphatidic acid, PA; or phosphatidyl serine, PS) and/or polyethylene glycol (PEG)-PE of different MW (750 and 5000). ζ-Potentials and liposome clearance in mice were investigated. In vitro, the attachment of PEG in a similar fashion neutralizes the effect of any charged component. In vivo, the chemical nature of a charged lipid becomes important. Both short PEG750 and longer PEG5000 inhibit the clearance of positively charged SA-liposomes, while only longer PEG5000 inhibits the clearance of negatively charged PA-liposomes and none of the PEGs inhibit the clearance of negatively charged PS-liposomes. The opsonins with different molecular size may be involved in the clearance of liposomes containing different charged lipids.

Introduction

The use of liposomes as drug carriers requires the liposomal preparations with various clearance rates and biodistribution patterns to better fit the specifics of each particular application. Liposome charge and liposome coating with different polymers, such as PEG, are among the parameters known to strongly affect biological properties of liposomes. The effect of charge and PEG on biological behavior is well investigated (Gabizon and Papahadjopoulos, 1992, Lasic and Martin, 1995). It was repeatedly demonstrated that the incorporation of charged phospholipids into liposomes accelerates their clearance (Lee et al., 1992, Liu and Liu, 1996), while grafting liposomes with PEG and similar polymers makes liposomes long-circulating (Klibanov et al., 1990, Torchilin and Trubetskoy, 1995).

Thus, the incorporation of PS or dicetyl phosphate (DCP) into PC/Chol liposomes dramatically enhances liposome uptake by the perfused mouse liver (Liu and Liu, 1996). The fact that the negative charge strongly increases the clearance of liposomes was also shown by Gabizon and Papahadjopoulos (1992). Negatively charged PS was found to abolish the longevity of liposomes prepared of a lipid composition resembling that of erythrocyte membrane (Allen et al., 1988). The major mechanism behind the charge-facilitated liposome clearance is an interaction of charged phospholipid head-groups with certain opsonizing proteins. The components of the complement system (fragments of C3) are involved in the clearance of PS-containing liposomes in the guinea pig and rat (Liu et al., 1995a, Huong et al., 2001). Liposomes containing PA, phosphatidyl glycerol and DCP compete in different degrees for serum components, which mediate the liver uptake of PS-containing liposomes (Liu et al., 1995a). Both negative and positive phospholipids (such as phosphatidyl ethanolamine (PE)) are strongly opsonized (for review, see Moghimi and Hunter, 2001), however, certain groups, such as PS, are especially efficient binding centers for plasma proteins (Chiu et al., 2001). Generally speaking, liposomes of different composition and charge exhibit different protein-binding properties and bind a different spectrum of proteins (Chonn et al., 1992, Liu et al., 1995b). In vitro, binding and endocytosis of liposomes by cells are mediated by specific lipid head-groups and surface charge density (Lee et al., 1992). It is interesting that in mice, the liver uptake of liposomes does not involve any specific opsonins, while the liposome uptake by the rat liver strongly depend on serum opsonins (Liu et al., 1995b). In addition, within the same species different ligands can interact with different opsonins.

The mechanisms of the protective effect of PEG on liposomes include an electrostatic and steric repulsion between PEG-grafted bilayers (Needham et al., 1992, Kenworthy et al., 1995) and the formation of protecting polymeric layer by flexible polymeric chain on the surface of liposomes (Torchilin et al., 1994). Liposome-grafted PEG prevents liposome clearance by neutralizing the surface charge of liposomes and shielding various opsonins. While PEG-PE itself demonstrates slight electronegativity, with shorter PEG producing a more electronegative product, a larger PEG can effectively shield the charge of a phospholipid block as well as the charge of the whole liposome (Needham et al., 1992, Shimada et al., 1995). This means that even if the surface potential of a PEG-liposome is negative, the net ζ-potential of such a liposome is close to neutral (Moribe et al., 1997). Thus, it was shown that the increase in the quantity of liposome-attached PEG from 0 to 10% sharply decreases ζ-potential of the liposome (Arnold et al., 1990). PEG (and other similar polymers) inhibits the attraction of opsonins because opsonins cannot bind immobilized water on the surface of liposomes and the thickness of polymer layer decreases the uptake of PEG-liposomes by macrophages (Zeisig et al., 1996), Above a certain level, an attached polymer can completely eliminate the effect of the liposome-incorporated charged and opsonin-binding groups, as was shown for PS-containing liposomes with 15% PEG-PE (Chiu et al., 2001).

An interesting question arises—does PEG provide the same effect for liposomes of all compositions or does the protective effect (and liposome clearance) depend not only on the thickness of the layer of protecting polymer on the liposome surface (i.e. on PEG concentration and MW), but also on the type of a charged and opsonin-attracting site? Assuming that different ‘attraction’ sites (PS, PA, PE, etc.) may predominantly interact with certain specific opsonins (Cullis et al., 1998, Moghimi and Hunter, 2001), differently sized opsonins, varying from 17.5 kDa (Kelly et al., 1992) to 80 kDa (Thornqvist et al., 1994) and up through all the intermediate sizes (Yang and Yoshino, 1990, Thornqvist et al., 1994 and see Cullis et al., 1998, for review), may demonstrate different degrees of interaction with liposomes of different compositions, especially at low and moderate concentrations of a liposome-attached polymer or at low MW of this polymer. Moreover, as a result, the shielding effect of the liposome-attached PEG may be seen differently in vitro and in vivo.

Here, we compare the biodistribution of liposomes with negative or positive surface charge additionally coated with a similar molar quantity of PEG with different MW, in order to investigate the relative role of the liposome charge and the length of the liposome-attached PEG chains on the liposome circulation time and liver accumulation in mice.

Section snippets

Materials

PC, Chol, PE, PS, PA, PEG750-PE and PEG5000-PE were from Avanti Polar Lipids. Triethylamine (TEA), SA and diethylene triamine pentaacetic acid (DTPA) anhydride are products of Sigma-Aldrich. 111–InCl3 was obtained from NEN. All solvents and components of buffer solutions were analytical grade products.

Liposome preparation by extrusion

Liposomes were composed of PC and Chol in a molar ratio of 7:3. When necessary, the initial lipid mixture was supplemented with 6% mol (% mol is the molar fraction of the compound in the mixture

Characterization of liposomes

Liposomes of different phospholipid/polymer composition were prepared having the size of ≈200 nm and rather narrow size distribution. Several typical patterns of liposome size distribution are presented in Fig. 1; see also the corresponding data in Table 1. It is evident that under similar preparative conditions, such parameters as phospholipid composition, charge and presence of protecting polymer only minimally influence liposome size. On the other hand, as one could expect, ζ-potential of

Discussion

In vitro, in good agreement with some earlier reports (Arnold et al., 1990, Shimada et al., 1995, Zeisig et al., 1996), we have clearly observed the shielding effect of the liposome-attached PEG on the net charge of liposome containing any type of positively or negatively charged phospholipid. Electric properties of PEGylated liposomes look rather similar almost independently on the nature of the charged component of liposome (compare the data for PA-and PS-liposomes from Table 1). Does it,

Acknowledgements

The authors thank Dr David Lynn (Department of Chemical Engineering, Massachusetts Institute of Technology) for his assistance with ζ-potential measurements. This study was supported by NIH grant HL55519 to Vladimir Torchilin.

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