Pulmonary administration of a doxorubicin-conjugated dendrimer enhances drug exposure to lung metastases and improves cancer therapy
Graphical abstract
Introduction
Primary lung cancer is one of the leading causes of death in developed countries and is responsible for 23% of all cancer-related deaths [1]. Primary lung cancers are mainly of epithelial origin and most commonly result from inhaled exposure to cigarette smoke (the main cause of primary lung cancer) and environmental contaminants such as asbestos and radon. The lungs are also a major site of metastasis for other cancers including those of the breast, prostate and colon. These cancers typically metastasise towards the lungs by invading into the blood stream, from where they lodge within the narrow pulmonary capillaries, invade into the surrounding tissue and establish solid secondary tumours. From here, they may metastasise further to thoracic lymph nodes or elsewhere in the body. Despite their prevalence, lung-resident cancers are difficult to treat, and mortality rates with conventional intravenous chemotherapy are high (approximately 85% within 5Ā years) [2]. The lack of efficacy of intravenous chemotherapy against lung tumours stems from a combination of (typically) late diagnosis, and poor drug access to lung tissue after intravenous administration [2], [3], [4], [5]. Inhaled drug delivery results in improved access to lung tissue and has shown superior activity when compared to intravenous administration in both animals and humans [6], [7], [8], [9], [10]. Clinical studies, however, suggest that inhaled delivery of cytotoxic drug alone, leads to high local drug concentrations and unacceptable lung-related toxicity [9], [11], [12].
The realisation that pulmonary delivery of cytotoxic drug may enhance therapy, but is associated with unacceptable toxicity, has stimulated increasing interest in the development of inhaled delivery systems that improve drug exposure to lung-resident tumours, but limit the toxic effects of the drug. Thus, a number of nanoparticulate and liposomal formulations of doxorubicin have been evaluated as potential delivery systems for the improved treatment of lung-resident cancers after inhaled administration [2], [10], [13], [14]. These studies provide promising indications of pre-clinical utility, with reduced lung-related side effects compared to the pulmonary administration of drug alone, and reduced systemic toxicity compared to intravenous drug delivery [2], [10], [13], [14].
The nanoparticulate carriers evaluated to this point, however, have been relatively large (54 to >Ā 600Ā nm) [10], [15], [16], [17], [18], [19], [20]. Absorption across the pulmonary epithelia or diffusion into solid lung tumours is therefore expected to be limited [21], [22], [23], [24]. For colloidal carriers, such as liposomes (reviewed in [25]), drug release is also usually non-specific, providing limited control over the kinetics of drug release [2], [26]. Finally, for non-biodegradable particles that are retained in the lungs for extended periods of time, there are increasing risks of toxicity via the promotion of localised infiltration of alveolar macrophages and inflammation in response to the presence of the particles [27], [28].
In contrast, dendritic polymers (dendrimers) [29], [30] are approximately one order of magnitude smaller (approx 4ā20Ā nm) than the majority of nanoparticles and liposomes, and therefore provide possible advantage with respect to the efficiency of interstitial diffusion, the extent of absorption and the degree of tumour penetration [31]. Drug conjugation to the dendrimer scaffold via linkers that are cleaved selectively in the environment of a tumour also provides a greater level of control over the location and kinetics of drug release [32]. Dendrimers based on poly-amino acid structures are also biodegradable, reducing the potential for immune stimulation. The kinetics of dendrimer clearance from the lungs of rats were therefore recently evaluated using PEGylated systems based on a poly-L-lysine scaffold. Small PEGylated dendrimers (<Ā 20Ā kDa) were absorbed after pulmonary instillation as low molecular weight fragments following initial degradation in the lungs. In contrast, increasing the molecular weight of the PEGylated dendrimers led to increased metabolic stability and significant lung retention [33]. PEGylated dendrimer based delivery systems therefore have the potential to provide controlled and site specific drug delivery to lung resident tumours. For these systems, programmed drug liberation followed by metabolic breakdown is expected to provide advantage over other colloidal or particulate vehicles as inhalable drug delivery systems for chemotherapeutic drugs.
In the current study, we have explored whether a 56Ā kDa biodegradable PEGylated polylysine dendrimer, conjugated with doxorubicin via an acid labile linker (Fig.Ā 1, described previously [34]) provides therapeutic benefit against lung-resident cancers when compared to the inhaled or intravenous administration of solution formulations of drug alone. Since a lack of understanding of the mechanisms by which nanomedicines are cleared from the lungs is also a significant current limitation to the translation of inhalable nanomedicine technologies into human clinical trials, the mechanisms and time course of dendrimer (and associated doxorubicin) clearance from the lungs have also been evaluated in detail.
Section snippets
Materials and reagents
Doxorubicin, thiazolyl blue formazan (MTT) and heptanesulphonic acid were purchased from Sigma-Aldrich (Sydney, Australia). RPMI media, hanks balanced salt solution (HBSS), penicillin/streptomycin, fetal bovine serum and glutamax were obtained from Gibco (NY, USA). Soluene-350 and IRGASafe scintillant were from Packard Biosciences (Meriden, CT). Cell culture flasks and microplates were from Corning (NY, USA). Polyethylene tubing (0.96Ā ĆĀ 0.58Ā mm external and internal diameter) was purchased from
Pharmacokinetics and biodistribution of D-DOX and doxorubicin after pulmonary instillation to rats
Pulmonary instillation of doxorubicin alone led to rapid and essentially complete absorption (Fabs 96%, Fig.Ā 2A, TableĀ 1). Following the peak in plasma concentrations 5Ā min after dosing, plasma concentrations of doxorubicin remained higher than in rats administered IV doxorubicin due to the continual absorption of drug from the lungs (Fig.Ā 2A). However, after 24Ā h, only 5% of the initial pulmonary dose of doxorubicin was quantifiable in the lungs (Fig.Ā 2D, E), and this was mainly associated with
Discussion
Pulmonary administration of cytotoxic drugs can improve chemotherapy against lung-resident cancers, but is usually associated with significant toxicity due to the exposure of non-cancerous lung tissue to high localised concentrations of the cytotoxic agent [9]. Interest has therefore increased in the development of inhalable drug delivery systems that enable the prolonged and controlled exposure of lung-resident cancers to chemotherapeutic drugs, and that simultaneously limit lung-associated
Acknowledgments
The authors would like to thank Erica Sloan for her assistance in transfecting the cells. This work was supported by an Australian Research Council Linkage grant. LMK was supported by an NHMRC CDF.
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