Toxicity of doxorubicin entrapped within long-circulating liposomes

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Abstract

We studied the effect of doxorubicin entrapped within long-circulating liposomes (Dox-LCL) on the phagocytic capacity and bacterial blood clearance capacity of rat liver macrophages. Dox-LCL (125 nm in diameter) were composed of egg phosphatidylcholine (PC), cholesterol (CH) and poly(ethyleneglycol)distearoylphosphatidylethanolamine (PEG-PE) (55:45:5 molar ratio; MW PEG 1900), and loaded with doxorubicin by means of a trans-membrane pH gradient. The doxorubicin/lipid ratio was 0.36:1 (mol/mol). At different time-points after one, two or three intravenous injections of Dox-LCL, radiolabeled negatively charged test liposomes (egg PC, CH, and phosphatidylserine in a 4:5:1 molar ratio) were injected. After 2 h, liver macrophages were isolated and the amount of macrophage-associated radioactivity was determined. Twenty-four hours after a single injection of 5 mg/kg Dox-LCL, no significant effect was observed. However, 48 h after injection, phagocytic activity was reduced significantly (49%). Recovery of phagocytic capacity of the liver macrophages took 8 days after two injections of Dox-LCL (2×5 mg/kg). Seventy-two hours after the last of two injections of Dox-LCL, bacterial blood clearance was significantly reduced as compared to clearance in control rats and in rats injected twice with doxorubicin combined with placebo liposomes. When comparing these Dox-LCL data with previous data on the effects of Dox-L, Dox-LCL appears less toxic than Dox-L for the liver macrophage population following i.v. administration both with respect to specific phagocytic activity and cell numbers. Due to the delay in onset of toxic effects and the faster recovery from Dox-LCL treatment as compared to Dox-L treatment, it is conceivable that therapeutic protocols can be designed with Dox-LCL that circumvent long-term impairment of the liver macrophage population.

Introduction

In animal studies, it was shown that encapsulation of the chemotherapeutic agent doxorubicin in liposomes decreases dose-limiting toxic side effects such as cardiotoxicity and nephrotoxicity while maintaining or even increasing the antitumor effect 1, 2. However, due to an altered biodistribution of doxorubicin after administration in a liposome-encapsulated form, toxicity may occur in other organs. As short-circulating liposomes are cleared rapidly from the circulation and predominantly taken up by macrophages in the liver, toxicity in the liver and, more specifically, in the liver macrophages, due to release of the entrapped drug within the cell, needs to be considered.

In a previous study [3]we showed that, at dose levels lower than those required for a therapeutic effect in animal studies (i.e. 3–5 times 10–20 mg/kg, weekly or 5 times 2 mg/kg, daily) 4, 5, 6, Dox-L in vivo not only strongly reduces phagocytic capacity of the liver macrophages but also eliminates these cells from the liver. Concomitantly, Dox-L treatment severely impairs bacterial blood clearance capacity.

Immunocompromised patients, including patients treated with cytostatic drugs, often develop septicemia from local infections which may readily result in generalization of the infection if cells belonging to the mononuclear phagocyte system (MPS) are eliminated. As shown before, depletion of macrophages indeed resulted in an increased susceptibility to infections [7]but also in impaired eradication of metastases (8, 9, Daemen et al., unpublished observation). Thus, treatment with Dox-L may not only increase the risk of the generalization of (lethal) infections but also the risk of metastatic spread, particularly to the liver.

The liposomes used in our previous study were short-circulating liposomes (210 nm in diameter) that are rapidly cleared from the circulation by the MPS. In recent clinical studies with doxorubicin-containing liposomes, however, so-called long-circulating liposomes have been used 10, 11. By incorporation of, for example, the ganglioside GM1 or a lipid-anchored derivative of poly(ethylene glycol) (PEG) into the bilayer, surface properties of liposomes are changed such that there is a reduction in the degree of recognition and the rate of uptake by the MPS [12].

We, therefore, considered it important to determine whether doxorubicin-containing liposomes with a prolonged circulation-time are less toxic/harmful for liver macrophages than short-circulating doxorubicin-containing liposomes. To this end, we measured phagocytic capacity and bacterial blood clearance capacity of the macrophages following Dox-LCL administration and compared the results with our previous results with Dox-L.

Section snippets

Animals

Male Wag/Rij rats (specified pathogen-free, 8–10 weeks of age, weighing approximately 185–225 g; ITRI-TNO Rijswijk, The Netherlands) were used in the phagocytosis assays.

Female R strain albino rats (specified pathogen-free, 18–25 weeks of age, weighing approximately 185–225 g; ITRI-TNO Rijswijk, The Netherlands) were used in the bacterial blood clearance studies.

Materials

Egg phosphatidylcholine (l-α-lecithin from egg yolk), phosphatidylserine (from bovine brain) and

Phagocytic capacity of liver macrophages after administration of Dox-LCL; effect of number of injections

Rats received a single injection, or 2–3 injections at 3-day intervals, of 5 mg/kg Dox-LCL (25 μmol liposomal lipid/kg body wt., per injection). At 24 h after the last injection, [3H]-CO containing test liposomes were injected. After an additional 2 h, liver macrophages were isolated and the amount of cell-associated [3H]-label was determined (Table 1). Whereas 24 h after a single injection of Dox-L uptake of labeled liposomes by cells was decreased by 50–70% [3], a single injection of Dox-LCL

Discussion

In the present study we show that, similar to the effects observed after doxorubicin in short-circulating liposomes, doxorubicin in long-circulating liposomes affects liver macrophages. The effects are however, less acute and slightly less severe, as will be discussed below.

A severe reduction in phagocytic capacity of liver macrophages after Dox-L was already observed 24 h after a single injection of 5 mg/kg. With Dox-LCL, no effect was observed 24 h after a single injection. Since these

Acknowledgements

The authors thank Bert Dontje and Jan Wijbenga for expert technical assistance.

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