In vitro assessment of transferrin-conjugated liposomes as drug delivery systems for inhalation therapy of lung cancer
Introduction
Carcinoma of the lung is the leading cause of death from cancer in Western countries (Alberg and Samet, 2003). The traditional method of treatment is surgical resection. However, in more advanced tumour stages, chemotherapeutic agents are given by the oral or intravenous route of administration (Giaccone and Smit, 2005, Moreira et al., 2001), but in most cases, severe toxic side effects are a major obstacle of this therapy.
Liposomal drug delivery systems have been studied extensively to increase the therapeutic index of chemotherapy (Andresen et al., 2005). Doxorubicin-loaded liposomes, for example, deliver significantly higher levels of drug to tumours than can be obtained with the free drug alone (Emanuel et al., 1996). The performance of liposomal drug delivery systems might be further improved by active targeting, e.g. by using a ligand coupled to the surface which recognises specific marker molecules on tumour cells (Sapra and Allen, 2003, Fonseca et al., 2005). Different attempts are being made to explore the potentials of ligand–receptor-mediated delivery systems in anticancer therapy. Among others, the transferrin receptor (TfR) has been identified as one of the successful target molecules (Soni et al., 2005, Iinuma et al., 2002), with a density of 10,000–100,000 molecules per cell commonly found on tumour cells, while cell types with normal phenotype express TfR at low or frequently undetectable levels (Singh, 1999). Transferrin (Tf) is an 80 kDa serum glycoprotein that traffics ferric ions into cells. Upon binding to its receptor, Tf is internalised into the cell through receptor-mediated endocytosis (Singh, 1999).
Recently, we reported on the conjugation of Tf to liposomal surfaces using different linker lipids and the assessment of the conjugation by atomic force microscopy (AFM) and conventional methods (Anabousi et al., 2005). In addition, we reported on the stability of these systems during nebulisation and subsequent contact with pulmonary surfactant (Anabousi et al., in press). It is our long-term objective to use aerosols of these Tf-liposomes for a treatment of lung cancer by inhalation, since we speculate that a topical application of anti-cancer drugs might result in a better therapeutic success combined with lesser side-effects. In the present work, we assessed Tf-conjugated liposomes in an in vitro environment, using cell cultures of pulmonary origin for binding-uptake studies and assays of cytotoxicity. Furthermore, by means of fluorescence-assisted cell sorting (FACS) and confocal laser scanning microscopy (CLSM), we measured levels and locations of TfR expression in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells).
TfR were expressed at significantly higher levels in continuously growing (i.e., cells of cancerous or immortalised origin, A549, 16HBE14o-, and Calu-3 cell lines) cells, compared to alveolar epithelial cells in primary culture. These results were supported by the finding that Tf-conjugated liposomes showed higher binding and uptake rates into cancer cells, compared to their healthy counterparts. Moreover, healthy cells showed a higher resistance to the cytotoxic effects of encapsulated doxorubicin (DOX). Therefore, Tf-conjugated liposomes appear as promising drug delivery systems for an aerosol chemotherapy against lung cancer.
Section snippets
Primary culture of human pneumocytes
Fresh human alveolar epithelial cells (hAEpC) were isolated from non-tumourous lung tissue, which was obtained from patients undergoing lung resection. The use of human material for isolation of primary cells was reviewed and approved by the local ethical committees (Saarland State Medical Board, Germany). Isolation of primary human type II pneumocytes (ATII) was performed according to a previously published protocol (Elbert et al., 1999, Ehrhardt et al., 2005). Briefly, finely minced lung
Characterisation of liposomes
Physicochemical properties of the different liposomal preparations are given in Table 1. The ammonium sulphate gradient method for encapsulation of doxorubicin resulted in an average encapsulation efficiency of 96.7 ± 5.0% (n = 6).
Transferrin receptor levels and location
Flow cytometry of transferrin receptor (TfR) expression in freshly isolated ATII cells, as well as in ATI-like cells after 8 days in primary culture, revealed very low signals not significantly different from the control values (Fig. 1). The adenocarcinoma-derived A549
Discussion
Inhalational therapy for malignancies involving the lung is a considerably new area of research; nevertheless, a number of studies using immune therapy with interleukins and interferons, and chemotherapy with aerosols of free 5-fluorouracil and 9-nitrocamptothecin, resulted in encouraging outcomes (Rao et al., 2003). It is our aim to develop targeted drug delivery systems for local aerosol therapy of lung cancer, since we believe that such therapy could further increase the effectiveness of
Conclusions
In conclusion, the conjugation of transferrin to liposomes as homing device resulted in increased uptake rates into tumour cells via transferrin-receptor mediated endocytosis. Therefore, Tf-modified liposomes might be promising candidates for an aerosol therapy of lung cancer. However, further studies are necessary to test these liposomal systems in an animal xenograft model of lung cancer to obtain important in vivo data.
Acknowledgement
This work was supported by personal fellowships from the DAAD (SA).
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