Elsevier

Journal of Controlled Release

Volume 100, Issue 1, 5 November 2004, Pages 135-144
Journal of Controlled Release

Tumor-targeted liposomes: doxorubicin-loaded long-circulating liposomes modified with anti-cancer antibody

https://doi.org/10.1016/j.jconrel.2004.08.007Get rights and content

Abstract

Commercially available doxorubicin-loaded long-circulating liposomes (Doxil®, Alza Pharmaceuticals) were modified with the monoclonal nucleosome (NS)-specific 2C5 antibody (mAb 2C5) that recognizes a broad variety of tumors via the tumor cell surface-bound NSs. For incorporation into liposomes, mAb 2C5 was modified with poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEG-PE) with the free PEG terminus activated with the p-nitrophenylcarbonyl group (pNP-PEG-PE). Derivatives of mAb 2C5 containing a variable number of PEG-PE residues (10–32) per protein molecule were prepared with a reasonably good preservation of the antibody specific activity even at the highest degree of modification. PEG-PE-modified antibody quantitatively incorporated into the liposomal membrane of doxorubicin-loaded liposomes with a loss of not more than 20% of the encapsulated doxorubicin. 2C5-targeted Doxil® liposomes acquired the ability to recognize NSs and specifically bind to various tumor cells. Doxorubicin-loaded long-circulating liposomes modified with the mAb 2C5 kill various tumor cells in vitro with the efficiency higher than non-targeted doxorubicin-loaded liposomes.

Introduction

Long-circulating liposomes with a decreased uptake by the cells of the reticulo-endothelial system (RES) are usually prepared by surface modification of liposomes with a flexible hydrophilic polymer [1], [2], such as poly(ethylene glycol) [3], [4], [5]. Long-circulating liposomes, similar to macromolecules [6], [7], are capable of spontaneous accumulating in solid tumors via the enhanced solubility and retention (EPR) effect [8]. An additional benefit of using liposome-based formulations is that the encapsulation into liposomes prevents the passive diffusion of a drug into non-target cells typical for low-molecular-weight therapeutics, thus increasing the circulation time and decreasing the accumulation of the drug in non-targeted tissues. Encapsulated drug is also protected from often-aggressive in vivo environment.

The development of long-circulating liposomes allowed for the creation of Doxil®—doxorubicin, incorporated into long-circulating PEG-coated liposomes [9]. Compared to doxorubicin, Doxil® demonstrates a superior efficiency in tumor therapy and diminished toxic side effects [10], [11]. Doxil® is currently approved for the treatment of AIDS-related Kaposi's sarcoma, recurrent ovarian cancer, and metastatic breast cancer, and undergoes clinical trials against other malignancies [9], [12].

Several attempts have been made to further improve the anticancer efficiency of Doxil® by targeting it with vector molecules specific to receptors typical for cancer cells. Thus, an attachment of the folate residue to the liposomal surface improved the ability of liposomes to selectively recognize cancer cells that over-express folate receptors [13]. Folate-targeted liposomes loaded with doxorubicin demonstrated a substantial increase in cytotoxicity towards target cells in vitro. Modification with folate promoted the internalization and nuclear delivery of liposomes and allowed to overcome the multiple drug resistance [14]. Similar results were obtained with doxorubicin-loaded long-circulating liposomes modified with a peptide, growth factor antagonist [15]. Using a small cell lung cancer cell line, it was shown that targeted liposomes were internalized much faster, delivered doxorubicin to the cell nuclei more efficiently, and were more cytotoxic compared to non-targeted liposomes. The modification of Doxil® with anti-HER2 monoclonal antibody fragments resulted in a formulation that demonstrated anticancer efficiency against tumor lines over-expressing HER2 far superior to that of control non-targeted liposomes both in cell culture and in in vivo models [16].

Vector molecules described above are specific to certain types of tumors. Unlike these molecules, certain antinuclear autoantibodies (ANAs) with NS-restricted specificity possess an ability to recognize a broad variety of tumors. The monoclonal non-pathogenic ANAs, such as mAb 2C5, recognize the surface of numerous tumor, but not normal, cells [17], [18]. Apparently, live tumor cell surface-bound intact NSs originating from apoptotically dying neighboring tumor cells are the targets for these antibodies on the surface of the variety of tumor cells [18], [19]. Here, the results are presented on the preparation of doxorubicin-loaded long-circulating liposomes modified with mAb 2C5, and investigation of their anticancer properties in various cell culture models.

Section snippets

Materials

Doxil® was purchased from Pharmaceutics (West Roxbury, MA). All lipids were from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Doxorubicin was purchased from Sigma (St. Louis, MO). Cell culture media, DMEM, EMEM and RPMI 1640, fetal bovine serum (FBS) and concentrated solutions of Na-pyruvate, non-essential amino acids, l-glutamine and penicillin/streptomycin stock solutions were purchased from CellGro (Kansas City, MO). All solvents and other chemicals were

Results and discussion

Earlier, a convenient procedure for the attachment of amino group-containing ligands to the liposome surface using the lipid-conjugated PEG with its distal terminus activated with p-nitrophenylcarbonyl group (pNP) was reported [20]. In the original design, the activated PEG-PE (pNP-PEG-PE) was first incorporated into liposomes, which then were reacted with ligands of interest [20]. In our current work, an alternative design was used, when a protein was first modified with an activated lipid

Acknowledgement

This study was supported by the NIH grant RO1 HL-55519 to Vladimir Torchilin.

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1

Current address: Department of Pharmaceutics, University of Washington, Seattle, WA 98195, USA.

2

ANL and TAE equally contributed to this study.

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