Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.

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Purpose: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4V-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol^coated (pegylated) liposomes. Experimental Design: MMC was released from the MMC lipid^based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of f90 nm.This formulation was used for in vitro and in vivo tests in rodents. Results: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was f3-fold less toxic than free MMC.The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype. Conclusions: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.

BACKGROUND Stealth liposomes hold promise as a mode of delivering cytotoxic agents selectively to tumors in cancer patients. The objective of this study was to determine whether stealth liposomal doxorubicin accumulates selectively in bone metastases based on clinical material obtained from two patients with breast carcinoma. METHODS Tumor tissue was obtained from two women (ages 33 years and 41 years, respectively) with metastatic breast carcinoma who responded to treatment with stealth liposomal doxorubicin and later underwent a surgical fixation procedure to treat a pathologic fracture of the femur. Drug levels in the tumor and adjacent muscle were examined by high performance liquid chromatography analysis in both patients and by fluorescence microscopy in one of the patients. RESULTS Bone tumor fragments obtained during surgery performed 6 days after the administration of the 12th course of stealth liposomal doxorubicin in 1 patient and 12 days after the administration of the 16th course of stealth liposomal doxorubicin in the second patient had a 10‐fold greater concentration of liposomal doxorubicin than tumor free muscle. Doxorubicin fluorescence and specific nuclear staining showed good colocalization, thus confirming the presence of the liposome‐delivered drug in the nuclei of tumor cells. CONCLUSIONS Using skeletal muscle as a comparator, stealth liposomal doxorubicin accumulates selectively in metastatic breast carcinoma cells within bone. Cancer 1999;86:72–8. © 1999 American Cancer Society.