Elsevier

Journal of Controlled Release

Volume 115, Issue 3, 27 October 2006, Pages 243-250
Journal of Controlled Release

Research paper
Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes

https://doi.org/10.1016/j.jconrel.2006.08.001Get rights and content

Abstract

It is well known that steric stabilization of the surface of liposomes by a polyethyleneglycol (PEG) conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended circulation times of the liposomes (t1/2  20 h in rat). Recently, we reported on the “accelerated blood clearance (ABC) phenomenon”, causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. We also reported that abundant binding of IgM, secreted into the blood stream after the first dose and, to PEGylated liposomes, plays an essential role in the induction of the ABC phenomenon. Spleen is well known to play a central role in the immune reaction and to produce IgM following a bacterial infection. The aim of the present study was to determine whether spleen contributes to the induction of the ABC phenomenon and to unravel its role in the phenomenon. In rats that were splenectomized (surgical removal of spleen) prior to the first injection of liposomes (0.001 μmol phospholipids/kg), the ABC phenomenon was totally abolished. In these rats serum IgM concentrations as well as the amounts of IgM bound to PEGylated liposomes were substantially reduced. Splenectomy attenuated the ABC phenomenon when performed until 3 days post-first injection. Removal of the spleen 4 days post-first injection left the ABC phenomenon unchanged. This finding indicates that the immune reaction in the spleen against the PEGylated liposomes occurs during at least 2–3 days following the first administration and then IgM reactive to PEGylated liposomes is produced. The present study proves that the spleen plays a critical role in the induction phase of the ABC phenomenon. For effective clinical application, many liposomal drug formulations will require multiple injections. The ABC phenomenon described in this and several preceding papers therefore has important implications for the development and evaluation of therapeutically useful liposomal formulations requiring multiple-dose administration.

Introduction

PEGylated liposomes, i.e. liposomes of which the surface is sterically stabilized by the incorporation of a polyethyleneglycol (PEG)-conjugated lipid, are widely used in the liposomal drug delivery field [1], [2], [3]. It is hypothesized that the presence of PEG on the liposomal surface attracts a water shell, resulting in reduced adsorption of plasma proteins such as opsonins and, as a consequence, impaired recognition of the liposomes by the cells of the mononuclear phagocyte system (MPS) following intravenous administration. This, in turn, is believed to lead to extended blood circulation times of these liposomes [4], [5], [6]. In recent years, however, evidence has accumulated suggesting that PEGylated liposomes are prone to rapid clearance from the circulation under certain conditions [7], [8], [9].

We and others reported that upon repeated injection, circulation time of PEGylated liposomes dramatically decreases while their uptake by the liver increases concomitantly [10], [11], [12], [13], [14], [15], [16], [17]. This so-called “accelerated blood clearance (ABC) phenomenon” was observed in rat, mice, rabbit and Rhesus monkey. These reports clearly indicate that, under conditions in which the ABC phenomenon is induced, PEGylated liposomes can still react with the biological milieu, despite their established steric stabilization.

The mechanism causing the ABC phenomenon is still not entirely clear. Recently, we reported that the production of IgM reactive to PEG in response to a first dose of PEGylated liposomes is responsible for the induction of the phenomenon [17]. Subsequent complement activation induced by IgM bound on PEGylated liposomes likely triggers significant uptake of the liposomes by the Kupffer cells, since IgM by itself does not have the ability to promote such uptake directly. We therefore envisage the following model to explain the ABC phenomenon: IgM, induced by the pre-injection of PEGylated liposomes, selectively binds to the PEG present on the surface of the second dose of liposomes and subsequently activates the complement system, which in turn leads to opsonization by C3 fragments [17]. As a consequence, accelerated blood clearance of a second dose of PEGylated liposomes occurs, despite the steric barrier of the PEG-coating.

The spleen is a highly organized secondary lymphoid organ that plays a central role in the primary defense against all types of antigens that appear in the circulation, and is a major site of antibody production [18], [19]. Absence of the spleen causes an increased susceptibility to systemic infections by encapsulated bacteria [20]. Various hypotheses have been proposed to account for this increased infection risk, including lack of opsonins or marginal zone macrophages, altered T cell function, impaired clearance by the cells of MPS, and mechanical filtration of pathogens by the spleen [21]. Interestingly, some reports described a significant reduction in the concentrations of serum IgM, C3 and Factor B with normal IgG, IgA and C4 values in splenectomized patients [22], [23].

Here we address the issue of whether the spleen plays an important role in the induction of the ABC phenomenon by examining the effect of splenectomy on the development of the ABC phenomenon.

Section snippets

Materials

Hydrogenated egg phosphatidylcholine (HEPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) were generously donated by Nippon Oil and Fat (Tokyo, Japan). Cholesterol (CHOL) was of analytical grade (Wako Pure Chemical, Osaka, Japan). All lipids were used without further purification. Sepharose 4 Fast Flow was purchased from Amersham-Pharmacia Biotech (Upsala, Sweden). Rhodamine-derivatized dihexadecanoylglycerophosphoethanolamine (DHPE)

Effect of splenectomy on the induction of the ABC phenomenon

Our earlier studies demonstrated that the ABC phenomenon reached a maximum level at day 5 following prior injection of a low dose of PEGylated liposomes (0.001 μmol phospholipids/kg) [12], [17]. Therefore, the same experimental condition was employed to address the effect of splenectomy on the induction of the ABC phenomenon. The animals were divided into four groups. Groups 3 and 4 were splenectomized, groups 1 and 2 were not. Groups 2 and 4 received a first injection 5 days prior to the test

Discussion

PEG, a neutral and highly water-soluble polymer, is widely believed to be non-toxic and non-immunogenic. When the biocompatible PEG is attached to compounds such as proteins, oligodeoxynucleotides (ODNs) and DNAs, or liposomes, generally it provides these entities with the property of biocompatibility and “invisibility” that renders them longer circulating in the blood and less immunogenic. However, we and other groups have reported that unexpected changes in the pharmacokinetic behavior of

Acknowledgments

We thank Dr. G.L. Scherphof for his helpful advice in writing the English manuscript. This study was supported, in part, by research grant from The Kurozumi Medical Foundation.

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