Medical nanoparticles for next generation drug delivery to the lungs

Surface modifications

Most particles allow for surface modifications that can positively affect their biodistribution and circulation times. Interestingly, nanocarrier hydrophobicity determines, to a large degree, the level of particle binding to blood components. Indeed, nonsurface-modified hydrophobic particles are known to be cleared rapidly [16]. To increase the blood circulation time of these particles, they can be coated with hydrophilic polymers or surfactants. For example, low molecular weight poly(ethylene glycol) (PEG) chains that decorate the particle in a brush-like fashion (PEGylation) are known to reduce phagocytosis [17]. Importantly for chronic obstructive lung diseases, PEGylation of nanoparticles has also been shown to increase mucous penetration [18]. Chitosan on the other hand is used as surface modification to create more mucoadhesive nanoparticles thus prolonging circulation times [19]. This may be more beneficial for treating nonobstructive lung diseases, such as lung cancer and allergy, since it will allow prolonged residence time at the disease site and thus increased drug uptake and bioavailability. In addition to chemical modifications, the surface of nanoparticles can also be modified in biological fluids by the formation of a so-called protein corona, changing the properties of the particles [20]. Lung surfactant phospholipids are also known to coat inhaled particles [21, 22], and this can drastically change their toxicity and cellular uptake [23]. In addition, this may lead to agglomeration [24], which might result in agglomerates that are large enough to be digested by macrophages. The binding strength and mode of binding of lung surfactants to the particles largely depends on the surface of the particle [25, 26].

Targeting

Nanoparticles also offer the possibility for specific tissue or cell targeting, which has the potential to greatly increase their therapeutic effect and decrease drug toxicity. Both active and passive nanoparticle-based targeting is possible. Passive accumulation of nanoparticles in tumour tissue is observed due to a phenomenon known as the enhanced permeability and retention effect. This is caused by the insufficient drainage and poorly regulated angiogenesis of tumours, resulting in an unstructured and leaky vasculature [27]. Passive targeting can also be achieved by surface charge of nanoparticles. For example, cationic liposomes loaded with paclitaxel (Bristol-Myers Squibb, New York, NY, USA) were able to target endothelial cells in solid tumours in patients with advanced cancer and liver metastasis [28]. Passive uptake of particles by macrophages can also be used for drug delivery, for example in tuberculosis where the bacteria reside inside the macrophages [29].

Active targeting can be achieved by attaching targeting ligands to the surface of nanocarriers that bind to molecules specific for the diseased cell (i.e. cell receptors that are over-expressed or cell type specific). For example, integrins are transmembrane receptors that mediate interactions with the surrounding extracellular matrix and are upregulated in proliferating tumours cells [30]. Integrin targeting liposomes loaded with doxorubicin inhibited tumour progression in a colon carcinoma mouse model, whereas non-targeting liposomes were ineffective [31]. Several other examples of successful integrin-specific binding of nanoparticles exist [32–34]. Another well-known example for targeted drug delivery is the folate receptor that is over-expressed in a variety of tumours [35]. In one study, folate receptor-targeted polymeric micelles loaded with doxorubicin exhibited increased cellular uptake and circulation time, and decreased cardiotoxicity compared to doxorubicin alone in a nude mice xenograft model [36]. Other examples of cell receptors used for nanoparticle-based active drug targeting include the epidermal growth factor receptor (EGFR) and transferrin receptors [34, 37]. Interestingly, cell type-specific targeting can be achieved by conjugating nanoparticles with cell adhesion antibodies [38].

Controlled drug release

Controlled release of the drug from the particle is also essential for therapeutic success. Release from the particles can either be stimuli responsive or sustained. Most polymeric and liposome particles have sustained or continuous release of the drug by either diffusion out of the particle or slow degradation of the particle over time. Stimuli-responsive drug release may result in a more targeted release of the drugs and can be achieved by a change in biological environment, such as reducing environment of the cell, change of pH (e.g. acidic endosomal compartments) or altered levels of disease-specific enzymes. For example, polymeric particles containing pH sensitive linkers have been used for the removal of the outer polymer coat through endocytosis-mediated uptake [39]. The different redox potential between the oxidising extracellular and reducing intracellular environment of tumour cells has also been exploited for polymer biodegradation [40]. Another example of responsive drug release is represented by PEG-peptide-lipid conjugates, where the PEG molecules could be removed from the carriers via cleavage by matrix metalloproteinases that are overexpressed in tumour tissues [41]. Alternatively, external physical stimuli such as light, ultrasound, heat and electric or magnetic fields may also be used to achieve controlled drug release. One example of such an approach is the use of ultrasound-sensitive microbubbles for local release of drugs [42]. Interestingly, microbubbles created from lung surfactants showed a three-fold increase in targeted deposition of the drug compared to common lipid-only microbubbles [43]. Another excellent example is the use of magnetic gradient fields to direct aerosol droplets containing superparamagnetic iron oxide nanoparticles to the desired regions of the lungs in mice [44].

Nanotoxicology

To fully exploit the potential of nanoparticles for medicinal application, a detailed understanding of biocompatibility, biodistribution and degradation of nanomaterials is required. The physicochemical properties, such as their size, surface charge and shape, will modulate their biological response [45]. For example, rod-shaped nanoparticles are known to be more toxic and harmful than sphere-shaped nanoparticles [46]. In addition, long fibres cannot always be fully engulfed by macrophages, preventing their clearance from the system causing inflammatory responses [47]. Assessing the nanotoxicology of new materials is further complicated by the ability to modify the surface of nanoparticles which affects their bioresponse. For instance, carbon nanotubes are considered to be biopersistant, but it has been shown that functionalised nanotubes can infer water solubility and are rapidly cleared via renal excretion [48]. Due to the infinite possibilities in manipulating particle size, shape and surface chemistry, and because small changes in the physicochemical properties can have a large impact on their biological response, it is necessary to assess the safety of each material separately.

Lung cancer

Accounting for >30% of all cancer deaths, lung cancer is by far the leading cause of cancer deaths in the western world (http://globocan.iarc.fr/). A more tumour-targeted and local delivery of chemotherapeutic drugs to the lungs may improve the outcome and management of this disease. There are many nanoparticle chemotherapeutic formulations under development and several are already on the market. For example abraxane (Abraxis BioScience, Los Angeles, CA, USA), a nano-formulation of paclitaxel and albumin, was shown to be more effective and results in fewer side-effects than using the standard paclitaxel formulation in a phase III study [49]. While mainly used to treat breast cancer, it has recently been approved to treat nonsmall cell lung cancer. Similarly, nano-formulations of docetaxel, a drug commonly used for treatment of nonsmall cell lung cancer, preferentially accumulated in mouse tumour tissue and was more efficient in inhibiting tumour growth and metastatic spread compared to the drug alone or abraxane [50]. Encouragingly, in a recent phase I study, liposomal formulations of cisplatin have successfully been delivered to the lungs via aerosol delivery with higher drug deposition at the tumour site and minimal systemic toxicity [51]. The pulmonary delivery of liposomal 9-nitrocamptothecin, doxorubicin and paxlitaxel has been explored in similar approaches [52–54].

Active targeting strategies for nanoparticle-based drug delivery in the lungs have been limited due to lack of ligands specific to lung cancer cells. Encouragingly, several reports on aptamers (molecules created by selection from a large random sequence pool that bind to a specific target molecule), which can recognise specific small cell lung cancer cell-surface molecular markers with high affinity and specificity, represent a promising strategy to overcome this problem [55, 56].

The EGFR has been exploited for targeted delivery to lung cancer cells because it is highly expressed in nonsmall cell lung cancer [34]. For example, gelatin-based nano-formulations with EGFR targeting ligands have been used to target cisplatin to the cancer tissue following inhalation in a subcutaneous mouse model. These EGFR targeting particles were able to reduce tumour volume more effectively, as well as increase the bioavailability of cisplatin in the lung compared to the free drug [57]. Similarly, epidermal growth factor (EGF)-tagged carbon nanotubes containing cisplatin efficiently reduced tumour volume compared to non-EGF labelled tubes [58]. Moreover, liposomes for gene therapy using an anisamide ligand for targeting lung cancer cells overexpressing sigma transmembrane receptors showed therapeutic effectiveness in H1299 lung cancer cells and in in vivo xenograft models [59].

Chronic obstructive lung diseases

Mucus hypersecretion and severe inflammation characterise chronic obstructive airway diseases including asthma, CF and COPD. Therefore, the main treatment strategy of these diseases comprises the use of anti-inflammatory drugs (e.g. corticosteroids and antibiotics). The use of nanoparticles as drug delivery vehicles in these diseases may prove beneficial as they could provide sustained drug release and overcome airway defences but also target diseased cells or tissues. Indeed, liposomes encapsulating budesonide, a potent corticosteroid, was shown to control the drug release rate and allowed therapeutic concentrations to be maintained in rat lungs for longer periods while reducing systemic toxicity [60]. Notably, nanoparticles also allow for combination treatment of drugs. For example, liposomes containing beclomethasone (glucocorticoid steroid) and formoterol (β₂-selective receptor agonist) showed that beclomethasone maintained its long-lasting effect while formoterol enhanced lung function and peripheral lung deposition without a significant effect on the mucociliary escalator [61, 62].

The mucus in obstructive airway diseases is not only much thicker, but is also much less viscoelastic. A major additional challenge of using nanoparticles for treating chronic obstructive lung diseases is the penetration of the mucus layer. Studies on that subject suggest that particles with a charged surface are transported across the mucus barrier significantly less [63]. Apart from charge, size also seems to be important for mucociliary clearance of particles. A study by Rytting et al. [64] showed that small polystyrene particles (diameter ∼120 nm) moved efficiently through the sputum of CF patients while larger particles (diameter 270–560 nm) had significantly less mobility. In addition to size, surface modifications such as PEGylation, rendering the particles more neutrally charged, have been shown to enhance transport across the mucosal barrier [65], and also improve bronchial clearance of particles [63, 66]. Particle uptake and localisation is also dependent on disease type. Inhaled gold particles were taken up less by surface macrophages but at a higher rate by alveolar type I epithelial cells in Scnn1b-transgenic COPD mice compared to wild-type mice [67].

Inhaled steroids are the treatment of choice for controlling asthma, a major public health burden, but their pharmacological effect is relatively short. Encouragingly, polymeric steroid nano-formulations have shown to accumulate and have higher benefits at the site of airway inflammation compared to free steroids [68]. Similarly, polymeric micelles containing budesonide were able to significantly reduce inflammatory cell counts in bronchoalveolar lavage fluid in an asthmatic/COPD rat model [69]. Another approach was reported by Joshi and Misra [70] who used liposomes to effectively deliver ketofin fumarate (a mast cell stabiliser known to control the inflammatory response in asthma) to the desired locations in rat lungs using dry powder inhalers [71].

CF is a monogenic disease characterised by mutation(s) in the CF transmembrane conductance regulator (CFTR) gene that causes exaggerated inflammatory response and decreased mucociliary clearance. Due to this inability to clear mucus and consequently also bacteria and fungi, CF lungs are very susceptible to infections. Therefore, the targeted delivery of antimicrobials by inhalation has received much interest in the past years. Indeed, some liposomal formulations of antibiotics for aerosolised delivery to the lungs are currently in phase II clinical trials [72, 73]. In particular, the liposomal amikacin (Bristol-Myers Squibb, New York, NY, USA) for inhalation therapy showed sustained improvement in lung function and significant reduction of bacterial density in CF patients with chronic pseudomonas lung infections [74]. Similarly, the antifungal drug amphotericin B formulated in liposomes showed reduced side-effects and increased pulmonary deposition and retention in animal models of aspergillosis compared to non-liposomal amphotericin B [75], and was well tolerated in immunocompromised patients [76]. In another approach by Vij et al. [77], a polymer-based nano-delivery system showed delivery of a proteasome inhibitor to the lungs of mice in a controlled and sustained manner, which could rescue Pseudomonas aeruginosa-lipopolysaccharide induced CF lung disease.

In addition to anti-inflammatory drugs to combat chronic obstructive lung diseases, another promising approach using nanoparticles is gene therapy. Indeed, some nanoparticle vectors could achieve significant levels of transgene expression in the lungs [78]. Several reports have been published on developing cationic liposomes for pulmonary gene delivery [79]. Interestingly, liposomes conjugated to cell-penetrating peptides (e.g. antennapedia octargenine) increased cellular uptake of liposomes to airway cells [80]. Since CF is a monogenic disease, gene therapy seems to be a particularly attractive approach to treat CF [81]. This approach has already shown promise with adenoviruses that could partial correct CF in clinical studies [82]. Unfortunately, transgene expression subsided within weeks and repeating doses triggered adverse immune response. Some clinical trials with cationic lipid formulations have also been studied for their CFTR gene transfer efficiency, but no CF improvement could be observed [83]. α₁-Antitrypsin deficiency is another monogenic lung disease that would be a good candidate for nanoparticle-based gene therapy [84].