Pharmaceutical nanotechnologyEvaluation of intracellular trafficking and clearance from HeLa cells of doxorubicin-bound block copolymers
Graphical abstract
Introduction
Recently, genomic drug discovery techniques, organic synthesis, and screening technologies have been used to develop molecularly targeted medicines, some of which are already being used clinically (Hopkins and Groom, 2002, Hughes, 2009). However, these new technologies do not necessarily lead to the introduction of new treatments because even when promising compounds are discovered by genomic drug discovery techniques, they often have harmful properties or are difficult to deliver to the target because they are relatively insoluble (Hopkins and Groom, 2002, Lipinski et al., 2001). New formulation technologies are being developed to enhance the effectiveness and safety of pharmaceutical products by focusing on improving the release, targeting, and stability of drugs within the body, so that the location and timing of their action in the living body can be controlled.
Nanotechnological advances have contributed to the development of new drug delivery system (DDS) products such as polymeric micelles and liposomes that range in size from several tens of nanometers to 100 nm (Ferrari, 2005). Some of these DDS products are already being marketed as innovative medical treatments (O’Brien et al., 2004), and the number being used in clinical trials has risen impressively in recent years (Hamaguchi et al., 2007, Kuroda et al., 2009, Matsumura et al., 2004). These nanoparticulates possess several unique advantages for drug delivery, including high drug-loading capacity, controlled drug release, and small size, which allows the drug to accumulate in pathological tissues such as tumors, which have increased vascular permeability (Nishiyama and Kataoka, 2006).
Polymeric micelles have received considerable attention recently as promising macromolecular carrier systems (Allen et al., 1999, Kataoka et al., 1993, Kataoka et al., 2001, Lavasanifar et al., 2002, Torchilin, 2002, Torchilin et al., 2003). Polymeric micelles are amphipathic systems in which a hydrophobic core is covered with an outer shell consisting of hydrophilic macromolecules such as polyethylene glycol (PEG) chains. Polymeric micelles can both encapsulate medicine of high density and evade the foreign body recognition mechanism within the reticuloendothelial system (RES), and they show excellent retention in the blood (Illum et al., 1987). In addition, accurate size control of the nanoparticulates enables them to accumulate in cancerous tissue, owing to the increased permeability of tumor vessels due to the enhanced permeability and retention (EPR) effect (Matsumura and Maeda, 1986).
To maximize the efficacy and safety of DDS products, it is important to deliver these products to specific target cells and subcellular compartments. In the experiments reported here, we used confocal microscopy to study the intracellular trafficking of polymeric nanoparticulate carriers. The use of covalently bound fluorescent reagents as probes is gradually clarifying the internalization pathways and intracellular localizations of polymeric nanoparticulate carriers (Lee and Kim, 2005, Manunta et al., 2007, Murakami et al., 2011, Rejman et al., 2005, Richardson et al., 2008, Sahay et al., 2008, Savić et al., 2003). However, the excretion of the polymers from target cells after they have released the incorporated drugs has not yet been clarified in detail, although information about the clearance of carriers from cells is important from the perspective of safety. In this study, we examined the trafficking of a polymeric nanoparticulate carrier in detail, including the efflux of the polymers from cells to medium, by direct measurement of doxorubicin (Dox) covalently bound to the block copolymer. This technique avoids the necessity of considering the effects of exogenously tagged fluorescent probes on the intracellular trafficking.
Dox is one of the most effective available anticancer drugs in spite of its severe toxic effects, especially cardiotoxicity (Olson et al., 1988). As the carrier we used a PEG-poly(aspartic acid) block copolymer with covalently bound Dox (Fig. 1) (Yokoyama et al., 1999), because Dox has relevant hydrophobicity to form globular micelles by means of the hydrophobic interactions, and inherent fluorescence to investigate the intracellular trafficking of the carrier itself. Dox is partially covalently bound to the side chain of the aspartic acid (about 45% of aspartic acids), so that prepared Dox-conjugated block copolymers show good Dox entrapment efficiency possibly due to the π–π interaction between conjugated and incorporated Dox molecules (Bae and Kataoka, 2009, Nakanishi et al., 2001). Therefore, in this carrier system, there are two kinds of Dox; one is Dox covalently bound to block copolymers, and the other is free Dox which is incorporated in the inner core and has a pharmacological activity by its release from the inner core. The inner core of the micelles is greatly hydrophobic owing to the conjugated Dox, while the PEG of the outer layer prevents uptake by the RES. The resulting micelle effectively accumulates in tumor tissue by the EPR effect and shows much stronger activity than free Dox (Nakanishi et al., 2001). Because the block copolymer can form globular micelles by means of hydrophobic interactions with the conjugated Dox, as shown in Section 3.1, we used a carrier without incorporated free Dox to investigate the intracellular trafficking of the carrier itself. Furthermore, by quantifying directly the amount of Dox covalently bound to the polymers, we could measure the intracellular amount of the polymers.
Section snippets
Cells and micelles
HeLa cells (Health Science Research Resources Bank, Osaka, Japan) were kept in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan) and 100 U/mL penicillin/streptomycin (Invitrogen). Cells were grown in a humidified incubator at 37 °C under 5% CO2. Dox-bound polymeric micelles and fluorescent dye (DBD)-labeled PEG-polyaspartate block copolymers partially modified with
Physicochemical properties of Dox-bound micelles
The micelle carrier (Fig. 1) consisted of a block copolymer of PEG (molecular weight about 5000) and poly(aspartic acid) (polymerization degree, 30). To increase the hydrophobicity of the inner core, Dox was partially conjugated (about 45%) to the side chain of the aspartic acid. Because particle size affects the intracellular uptake of nanoparticulate formulations, we first examined the particle size of the micelles without free Dox. The Dox-bound micelles had a hydrodynamic diameter of about
Conclusion
We investigated the intracellular trafficking of Dox-bound polymers. The polymers are internalized into cells by endocytosis, then transported to endosomal/lysosomal compartments, followed by partial distribution to the ER, or transported directly to the ER. The active excretion of the polymers from the cells may be mediated by the ABCB1 transporter. It is surprising that cells utilize their endogenous transport system for intracellular trafficking of this artificial drug carrier. Our results
Acknowledgements
The authors are grateful for support from Research on Publicly Essential Drugs and Medical Devices (Japan Health Sciences Foundation), a Health Labor Sciences Research Grant, and the Global COE Program for the Center for Medical System Innovation, MEXT, KAKENHI (21790046), and Nippon Kayaku Co. Ltd. We thank Mr. R. Nakamura (Nikon Corp.) for technical assistance.
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