Targeted delivery via avidin fusion protein: Intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes
Graphical abstract
Introduction
The poor efficiency and severe adverse effects of cancer treatment are mainly due to systemic drug distribution and the multidrug resistance mechanisms appearing in cancer cells. These undesirable properties could be largely avoided by targeting chemotherapeutics to the cancer tissue. Targeting is intended to increase the drug concentration in the tumor tissue while reducing the concentration elsewhere in the body i.e. improving the efficacy of the treatment and minimizing detrimental side effects.
Nanocarriers, such as polymers, nanoshells and liposomes, alter the pharmacokinetics and biodistribution of anticancer drugs. The nanocarriers used for targeting purposes are generally coated with hydrophilic polyethylene glycol (PEG) chains, which hinder the opsonization process and subsequently reduce the plasma clearance (Klibanov et al., 1990). Due to their prolonged circulation time and the enhanced permeability and retention (EPR) effect, nanocarriers are efficiently accumulated in the tumor tissue (Maeda, 2001). The specificity against the tumor tissue can be further enhanced by active targeting approaches that utilize antigens and receptors, which are over-expressed on the target cell membrane (Peer et al., 2007). With these approaches, the active targeting moiety, such as a ligand or a monoclonal antibody, can be coupled directly to a drug molecule or in most cases to the distal end of the PEG chain on the surface of the nanoparticle. The ligand need to possess the appropriate conformation and high affinity towards the corresponding receptor in order to evoke receptor-mediated endocytosis. On the other hand, the expression of the target receptor should be cell specific, high (density 104–105 copies per cell) (Lopes de Menezes et al., 1998, Park et al., 2002), stable and homogenous in the target tissue to mediate the active targeting. Some naturally over-expressed receptors such as epidermal growth factor (EGF) receptor, folate receptors (FRs) and transferrin (Tf) receptors, are commonly used in targeted cancer therapies (Daniels et al., 2006, Hynes and Lane, 2005, Low and Kularatne, 2009, Nicholson et al., 2001). However, in many cases, these same target receptors are also expressed, albeit at lower levels, in fast-growing healthy cells such as fibroblasts, epithelial and endothelial cells (Ekblom et al., 1983, Peer et al., 2007). Moreover, the expression level of the endogenous receptors may vary between individuals. Actively targeted nanocarriers have been studied extensively and, at the moment, there are multiple clinical trials in progress. However, despite huge efforts in the field only a few actively targeted formulations have been approved for clinical use. Therefore, there is still the need to develop new strategies for use in targeted drug therapies.
Avidin, isolated originally from chicken eggs, has an extremely high affinity for the coenzyme biotin (affinity constant 7 × 1014 M−1) (Green and Toms, 1973, Green, 1990). This feature has made the avidin–biotin technology popular for wide-range of biotechnological applications such as imaging and drug targeting. In multi-step targeting approaches, avidin is coupled to antibodies or used as a linker between a biotinylated antibody and a biotinylated therapeutic agent (radionuclide, drug, etc.) (Lesch et al., 2010). In addition, biotin can be covalently linked to almost any kind of molecule via its relatively inert side chain without affecting the avidin binding properties (Richards, 1990, Wilchek and Bayer, 1988). Previously, we have described an avidin fusion protein expression system utilizing avidin–biotin technology (Lehtolainen et al., 2002, Lehtolainen et al., 2003). The approach is based on using a virus-mediated transgene vector that leads to the expression of the avidin fusion protein receptor on the surface of the target cell. This protein can selectively bind biotinylated compounds and subsequently trigger endocytosis. The advantage of the avidin fusion protein receptor is its ability to be expressed in any type of tumor tissue (Lehtolainen et al., 2003), which enables the usage of one single ligand instead of requiring different ligands for each tumor type. Previously, we have shown that the avidin fusion protein can be expressed in the target tissue (Lehtolainen et al., 2002, Lehtolainen et al., 2003, Lesch et al., 2009), and the functionality of the receptor to bind the biotinylated compounds has been demonstrated under both in vitro and in vivo conditions (Lehtolainen et al., 2002, Lehtolainen et al., 2003). In addition, preliminary results of the potential usage of the avidin fusion protein for therapeutic purposes revealed that biotinylated nanoparticles containing paclitaxel were more cytotoxic towards transduced rat glioma (BT4C) cells than non-targeted nanoparticles or paclitaxel alone. However, a similar trend was observed in the control cells, and the effect seemed to be concentration dependent (Lesch et al., 2009). Thus, avidin fusion protein technology can be considered to be as a promising tool for the active targeting of biotinylated therapeutic compounds even for other applications in addition to cancer.
In this study, the avidin–biotin technology was applied in a two-step targeting approach. First the avidin fusion protein receptor was expressed on the cell membrane and second, transfected cells were treated with either the biotinylated doxorubicin (B-DOX) (Allart et al., 2003) or sterically stabilized pH-sensitive liposomes encapsulated with doxorubicin (BL-DOX). The cytotoxicity of B-DOX was analyzed in BT4C, and the intracellular distribution was evaluated by fluorescence microscopy. Then, the cytotoxicity of BL-DOX was evaluated in several cancer cell lines in vitro, which were characterized in terms of efflux protein expression at the RNA level. The cellular uptake kinetics of BL-DOX was also measured quantitatively by high performance liquid chromatography mass spectrometry (HPLC-MS/MS) in BT4C cells. Furthermore, the in vivo biodistribution of the BL-DOX in nude mice bearing subcutaneous glioma tumor stably expressing the avidin fusion protein was determined by HPLC-MS/MS.
Section snippets
Cell lines and culture conditions
Human glioblastoma–astrocytoma (U-87 MG; American Type Culture Collection, Manassas, VA, USA), human glioblastoma (U-118 MG; American Type Culture Collection) and rat glioma (BT4C) (Tyynela et al., 2002) cell lines were maintained in Dulbecco´s Modified Eagle´s Medium (DMEM; Sigma–Aldrich®, St. Louis, MO, USA) and human renal clear carcinoma (Caki-2; American Type Culture Collection) cell line in McCoy’s 5A+GlutaMAXTM-I medium (GIBCO®, invitrogen™, Grand Island, NY, USA). The growth media were
Part 1. Inefficient cell uptake and trapping inside the endosomal vesicles reduce the cytotoxicity of the biotinylated doxorubicin derivative
In the first approach, biotin was directly covalently linked with doxorubicin which has been earlier shown to decrease binding to topoisomerase II by 10–30-fold (Allart et al., 2003). The cytotoxicity of the conjugated (B-DOX) and free DOX towards BT4C cells was determined by the MTT assay, and the results showed that B-DOX was evidently less toxic towards both control and transduced cells than free DOX. Thus, the IC50 values for B-DOX were 100 and 78-fold higher than for free DOX in the
Conclusion
Multifunctional drug carrier systems are attractive tools for targeted cancer therapies. This is the first study to combine the avidin–biotin technology with the avidin fusion protein receptor expression system, a biotinylated drug conjugate and a liposomal nanocarrier. The results indicate that biotinylation induced the cell internalization of the conjugate but nuclei entry was hindered due to the trapping of the drug in the endo/lysosomal compartments. Liposomal doxorubicin restored the
Disclosures
The authors Minna U. Kaikkonen, Hanna P. Lesch and Ann-Marie Määttä are employees of Ark Therapeutics. No competing financial interests exist for Suvi K. Soininen, Pauliina Dalkilic-Lehtolainen, Tanja Karppinen, Tiina Puustinen, Galina Dragneva, Marjo Jauhiainen, Brigitte Allart, David L. Selwood, Thomas Wirth, Jukka Mönkkönen, Seppo Ylä-Herttuala and Marika Ruponen.
Acknowledgements
The authors thank Lea Pirskanen, Heini Koukkari, Aila Erkinheimo, Anne Martikainen and Mervi Nieminen for technical assistance. This study was supported by European Regional Development Fund, The Finnish Funding Agency for Technology and Innovation, and Ark Therapeutics Ltd.
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