New perspectives in nanomedicine

JPT-06585; No of Pages 10 Pharmacology & Therapeutics xxx (2013) xxx–xxx

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: M. Belvisi

New perspectives in nanomedicine Andrew J. Thorley ⁎, Teresa D. Tetley Lung Cell Biology, National Heart & Lung Institute, Imperial College London, United Kingdom

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a b s t r a c t Recent advances in nanotechnology have revolutionised all aspects of life, from engineering to cosmetics. One of the most exciting areas of development is that of nanomedicine. Due to their size (less than 100 nm in one aspect), nanoparticles exhibit properties that are unlike that of the same material in bulk size. These unique properties are being exploited to create new diagnostics and therapeutics for application in a broad spectrum of organ systems. Indeed, nanoparticles are already being developed as effective carriers of drugs to target regions of the body that were previously hard to access using traditional drug formulation methods. However, in addition to their role as a vehicle for drug delivery, nanoparticles themselves have the potential to have therapeutic benefit. Through manipulation of their elemental composition, size, shape, charge and surface modification or functionalisation it may be possible to target particles to specific organs where they may elicit their therapeutic effect. In this review we will focus on the recent advances in nanotechnology for therapeutic applications with a particular focus on the respiratory system, cancer and vaccinations. In addition we will also address developments in the field of nanotoxicology and the need for concomitant studies in to the toxicity of emerging nanotechnologies. It is possible that the very properties that make nanoparticles a desirable technology for therapeutic intervention may also lead to adverse health effects. It is thus important to determine, and appreciate, the fine balance between the efficacy and toxicity of nanomedicine. © 2013 Elsevier Inc. All rights reserved.

Keywords: Lung Nanotoxicology Nanoparticles Carbon nanotubes Silver Cancer

Contents 1. Introduction . . . . . . . . . . . . . . . 2. Targeting the lung . . . . . . . . . . . . . 3. Treatment of cancer . . . . . . . . . . . . 4. Treatment of chronic lung diseases . . . . . 5. Treatment of systemic diseases by inhalation 6. Vaccines . . . . . . . . . . . . . . . . . 7. Too good to be true? . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction

Abbreviations: ATI, Alveolar type I epithelial; ATII, Alveolar type II epithelial; CNT, Carbon nanotube; COPD, Chronic Obstructive Pulmonary Disease; EGF, Epidermal growth factor; PCL, Periciliary liquid; PEG, Polyethylene glycol; RSV, Respiratory syncytial virus; TB, Tuberculosis; TT1, Transformed Type I. ⁎ Corresponding author at: Lung Cell Biology, National Heart & Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, United Kingdom. Tel.: +44 207 594 2990. E-mail address: [email protected] (A.J. Thorley).

In the last decade there has been a rapid increase in the research and development of new engineered nanomaterials for a wide range of commercial and industrial uses. This emerging field has been driven by the knowledge that when a substance is engineered to be nano-sized (less than 100 nm in one dimension), its properties can differ greatly from its bulk-sized counterpart; differences such as increased conductivity, strength, surface area to volume ratio and optical properties can all be harnessed and taken advantage of. One particular field that nanoparticles have the potential to revolutionise

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Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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is medicine; nanomedicine could provide new technological advances in not only developing new and novel drugs, but also reformulation of already existing drugs to increase their efficacy, improve delivery and lower side effects. 1.1. Designing nanoparticles for therapeutic use “Nanoparticle” and “nanomaterial” are umbrella terms for a diverse range of nanosized structures. Nanoparticles can be synthesised in a wide range of shapes, sizes, and depending on the molecular basis of the structure, can be termed soft or hard (Fig. 1). Generation of nanosized structures for medical applications is not itself a new area, liposomes and dendrimers were developed over 30 years ago and there are a number of nanomedicines already in clinical use using this technology. Where 21st century developments in nanomedicine really add to the field, is in building more complex structures that can be targeted to specific tissues, have mechanisms for controlled release, and evade rapid clearance. Whilst the agreed definition for engineered nanomaterials states that a nanoparticle must be less than 100 nm in one dimension, this is an arbitrary definition and in the field of nanomedicine the term nanoparticle is more flexible and includes particles up to 1 μm. 2. Targeting the lung 2.1. Hitting the right spot The lung is a particularly enticing target for nanomedicine for many reasons, not least its large surface area for drug absorption. Thus, the inhalation route can be used to treat not only pulmonary disease, but also systemic disease. The ability to deliver medicine via the lung is particularly attractive as it is non-invasive, can be selfadministered and avoids first pass metabolism. Inhalable drugs have been available for many years; however, reformulating drugs to be nano-sized, rather than the micron-sized particles that are currently used, offers a number of potential benefits. Nanoparticles are able to penetrate deeper in to the respiratory tree and enter the alveolar

Fig. 1. Different forms of nanoparticles. Nanoparticles have been investigated for drug delivery for more than 30 years. Organic “soft” nanoparticles, such as dendrimers and liposomes, were some of the first nanoparticles developed and allowed effective encapsulation of drugs for delivery. More recently, hard inorganic nanoparticles have been developed for drug delivery. These nanoparticles can be produced in a variety of shapes from a number of substrates, the most common being carbon (e.g. carbon nanotubes), silver, gold and zinc (e.g. spheres, rods and wires). Due to the high surface reactivity of nanoparticles, they can be easily tagged with drugs and molecules that improve delivery or target the particles to the site of disease.

region. Furthermore, nanoparticles may be able to evade clearance by macrophages and enter the respiratory epithelium more easily than larger-sized particles. A number of studies have sought to determine the effect of particle size on distribution and deposition of inhaled particles in the lung; this is complex and relies on mathematical models. These studies have been invaluable in suggesting likely patterns of distribution; however they are unable to fully model the complex physiological parameters of the lung, and the predicted distribution versus reallife distribution may differ. One of the first studies to systematically address the effect of particle size on distribution, suggested that particle size had a critical effect on regional deposition in the lung (ICRP, 1994). The data indicated that particles in the 1–100 nm range preferentially deposit in the alveolar region of the lung, whereas particles in the micron range largely deposit in the upper airways. This observation is particularly significant when considering current formulations of inhaled drugs and formulation of future drugs for either pulmonary or systemic targeting. Many dry powder inhalers, such as those used for the treatment of asthma, produce particles in the micron-sized range; studies using β2-agonist inhalers have shown that a large proportion of inhaled particles greater than 5 μm in diameter deposit in the oropharyngeal region. A study on the effect of particle size on the distribution and clinical efficacy of an inhaled β2-agonist by Usmani et al. demonstrated that, as particle size decreased, the deeper the penetration of the drug in to the lungs and the better the deposition rate (Usmani et al., 2005). Using technetium-99 (Tc-99 m)-labelled albuterol, this study demonstrated that 1.5 μm particles preferentially deposited in the small airways and bronchioles, whereas 6 μm particles deposited in the oropharynx and central and intermediate regions. Furthermore, the smaller sized particles had a significantly greater total lung deposition rate than the larger particles. Despite this, 6 μm particles had a greater effect on improving FEV1 (forced expiratory volume in one second) in asthmatic lungs compared to the smaller sized particles, demonstrating that there is a clear balance between reducing particle size to improve distribution and deposition and ensuring that the particle is the right size to target the desired region of the lung where its effect is required. Whilst the improved distribution and deposition rate of the 1.5 μm particles did not result in improved lung function compared to the 6 μm particles over the 90 minute time frame used in this experiment, the authors hypothesised that deposition of 1.5 μm particles in the peripheral lung would avoid rapid clearance by the mucociliary escalator (discussed later) and thus may be more effective over longer time periods compared to larger particles and requires further investigation. As yet, there are few studies that have examined pulmonary deposition of particles in the nano-range. A study by Bhavna et al. compared the distribution of inhaled Tc-99 m-labelled nano-sized (approximately 60 nm) salbutamol with that of micronised salbutamol (approximately 3 μm) (Bhavna et al., 2009). They demonstrated that a significantly greater amount of the inhaled nano-salbutamol deposited in the lungs compared to the micronised salbutamol (64.1% vs 28.3%) and that nano-salbutamol distributed more evenly through the lung, reaching the peripheral lung, whereas the micronised salbutamol deposited almost entirely in the central airways. Furthermore, it was demonstrated that nano-salbutamol was cleared back up the respiratory tract over 2– 4 h. The authors postulate that nano-salbutamol may be a more effective formulation as, unlike micronised salbutamol, it can penetrate deep enough to elicit effects on the small airways and is slowly cleared via the large airways, back up the respiratory tract, therefore prolonging the effect of a single dose in comparison with micronised salbutamol. However, this study used healthy normal subjects, rather than asthmatics, and thus did not compare clinical efficacy of the two formulations or the possible effects of pre-existing airways disease on distribution and clearance. A number of groups have investigated novel methods for effective delivery of nanoparticles to the large airways, where they would not

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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normally deposit due to their nanosize. One strategy is to generate “cluster bombs” of nanoparticles. This approach encapsulates nanoparticles within a micron-sized structure so that it deposits in the proximal airways before releasing the nanoparticles into the local milieu. Recently, dexamethasone loaded nanoparticles within gelatin microparticles were generated in different sizes by altering the pressure and flow rate parameters of reagents during synthesis (Joshi et al., 2010). In acellular in vitro tests, it was demonstrated that there was an initial rapid release of dexamethasone from the particles at physiological pH which was followed by a sustained steady state release for up to 4 days. Similarly, Kaye et al. produced antibody-loaded “nano-in-micro” particles; these particles measured 1–5 μm but rapidly dissolved to release 400–600 nm nanoparticles in an aqueous environment (Kaye et al., 2009). Furthermore, the nanoparticles were able to release intact IgG at physiological pH (7.0) as well as under acidic conditions (pH 2.5) suggesting that release could happen even if they were internalised by cells and directed to the acidic lysosome. The authors suggested that this approach may be beneficial in avoidance of clearance by macrophages, by releasing particles too small to be recognised (discussed later).

2.2. Evading capture and clearance Once in the lung, there are a number of obstacles inhaled particles must overcome in order to elicit a therapeutic effect; by virtue of its constant exposure to the external environment, the lung has evolved a number of protective mechanisms to rapidly clear inhaled foreign material. The primary barrier nanoparticles must first cross in order to access the epithelium is the fluid that lines the entire respiratory tree. In the large airways (Fig. 2), it is estimated that the airway surface liquid layer is approximately 8 μm in depth (Song et al., 2009), and consists of a viscous air-facing mucus layer and an underlying periciliary liquid (PCL) layer that facilitates the co-ordinated beating of the epithelial cilia. In the healthy lung, it remains unclear whether the mucus layer is continuous or not (Knowles & Boucher, 2002).

Fig. 2. Delivery of nanoparticles to the bronchial epithelium. For effective delivery of nanomedicine to the large airways, a number of barriers must be overcome. 1) Only a small proportion of inhaled nanosized particles deposit in the large airways; to improve delivery, nanoparticles can be encapsulated in larger micron-sized structures which preferentially deposit in this region of the lung. Once deposited in the large airways, the micron capsules can release the nanoparticles for drug delivery. 2) The bronchial epithelium is protected by a mucus layer that traps and removes inhaled particles via the mucociliary escalator; in disease this layer can become thickened and continuous, further hindering drug delivery to the underlying epithelial layer. Coating particles with low molecular weight PEG can neutralise particle charge and improve penetration through the mucus layer. 3) The cilia of the bronchial epithelium are rich in tethered mucopolysaccharides and macromolecules which can act as a barrier to particle penetration into the periciliary liquid layer. Thus, for particles to pass through they must be neutral charged and nanosized.

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However, in diseases such as cystic fibrosis and chronic obstructive pulmonary disease, which are characterised by increased mucus production and thicker mucus, it is likely that this layer forms a continuous and more impervious barrier to inhaled particles, thereby hindering the therapeutic potential of inhaled drugs. Once deposited on the mucus layer, the hydrophobic and electrostatic forces within the layer are highly effective in trapping particles and preventing escape. In addition, it has recently been postulated that the PCL layer can act as a second barrier to particles due to the high concentration of mucopolysaccharides and macromolecules tethered to the cilia, microvilli and surface of bronchial epithelial cells (Button et al., 2012). In this study it was demonstrated that fluorescently labelled dextrans greater than 40 nm in size were excluded from the PCL layer and remained in the mucus layer. Thus, particles trapped within these layers would be cleared rapidly via the mucociliary escalator and cough before they could elicit any therapeutic effect. Thus, particle delivery to the underlying bronchial epithelium could be particularly difficult. However, dextrans are charged molecules (Chen et al., 2007a) which can be trapped in the mucus layer due to hydrophobic interactions. Studies using positively and negatively charged nanoparticles have also demonstrated that a charged surface significantly reduces transport across mucus barriers (Suk et al., 2011). For effective delivery of a nanomedicine to the large airways it is clear that a number of hurdles must be overcome. To avoid the effect of particle charge, evidence suggests that coating particles with a high density of low molecular weight polyethylene glycol (PEG), thus rendering the surface of the particle neutrally charged, significantly improves transport of particles across the mucus layer (Lai et al., 2009; Suk et al., 2011). In the peripheral lung, the alveolar epithelium (Fig. 3) consists of two cell types, type I epithelial (ATI) cells and type II epithelial (ATII) cells. These cells form tight junctions and lie on a thin fused basement membrane that separates them from the capillary endothelium. Whilst ATII cells slightly outnumber ATI cells, they only occupy

Fig. 3. Translocation of nanoparticles in the alveolar region. Inhaled nanoparticles preferentially deposit in the alveolar region, making nanomedicine particularly attractive for treatment of peripheral lung diseases as well as systemic diseases. 1) The alveolar surface is coated by a thin layer of surfactant which can draw nanoparticles towards the epithelial layer by wetting forces; surfactant proteins and phospholipids, released from type II epithelial cell lamellar bodies following release onto the alveolar surface, may coat the surface of the particles and promote particle agglomeration or uptake by the underlying epithelium. 2) Macrophages maintain the sterility of the alveolar region by phagocytosing inhaled particulate matter. However, nanoparticles are below the size range of particles that macrophages can efficiently clear. Thus for effective delivery to the epithelium, it is essential that nanoparticles remain well dispersed and not agglomerate to form structures large enough for macrophages to recognise and phagocytose. 3) The mechanisms underlying uptake of nanoparticles by the alveolar epithelium remain unclear. Particles may be internalised via passive diffusion or by endocytosis; studies in our laboratory have demonstrated that ATI cells, which cover 95% of the alveolar surface, avidly internalise nanoparticles, whereas ATII cell, which cover the remaining 5%, do not. 4) For treatment of systemic diseases, nanoparticles must be able to enter the underlying capillary network. Studies suggest that the proportion of inhaled nanoparticles translocating into the circulation is low (b3%), however inhaled therapies for diabetes have been successfully developed.

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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5% of the alveolar surface, only a small projection of the cell can be seen between the large, thin, flattened ATI cells that cover 95% of the alveolar surface. ATII cells play two major roles in the lung, synthesis and release of surfactant and as a progenitor cell for ATI cells. The surfactant released by ATII cells is essential for lowering surface tension in the lung and preventing alveolar collapse upon exhalation. Furthermore, components of surfactant are also important in the immune response, surfactant proteins A and D can opsonise inhaled microbes and particles, marking them for phagocytosis and clearance by macrophages. It is likely that any inhaled nanomedicines that enter and deposit in the alveoli would also adsorb surfactant components which may affect their cellular uptake by macrophages or even the underlying epithelium. The airway surface liquid lining the alveoli is much thinner (b0.1–0.2 μm) than that lining the bronchial tree, and thus represents less of a physical hindrance to particle uptake and transport. Indeed, studies have demonstrated that rather than a barrier, the surfactant layer may actually promote interaction with the underlying epithelium through wetting forces that draw the particles in to the surfactant towards the alveolar wall (Geiser et al., 2003; Mijailovich et al., 2010). In vivo studies using rodents have demonstrated that inhaled puffball spores are deposited in the alveolar region and are fully immersed in the liquid lining layer and drawn towards the epithelial layer to the extent that they cause indentations in the epithelial surface (Geiser et al., 2000). The lack of mucociliary escalator in the peripheral lung means that inhaled particles must be cleared by alternative methods; the primary mechanism for particle clearance in the alveoli is phagocytosis by alveolar macrophages, highly specialised cells whose main role is to maintain the sterility of the peripheral lung. The exact clearance mechanism of particle-laden macrophages from the alveolar region is poorly understood but it is thought that they migrate up the respiratory tree and are cleared by the mucociliary escalator; macrophages have a long life span in the lung, which would facilitate the relatively slow clearance by migration to the larger airways. Alveolar macrophages are highly efficient at phagocytosing particles in the 1–3 μm range (Kawaguchi et al., 1986; Ayhan et al., 1995; Champion et al., 2008) but this reduces drastically for particles falling outside of this size range, suggesting that inhaled nanoparticles may be able to effectively evade clearance by this route. However, particle chemistry and interactions with endogenous factors such as surfactant phospholipids and surfactant proteins may alter this. A number of in vitro studies using rodent macrophages and in vivo studies have demonstrated that nanoparticles adsorb surfactant proteins and phospholipids to varying degrees based on their composition and that this can modulate uptake by alveolar macrophages (Ruge et al., 2011; Kapralov et al., 2012; Ruge et al., 2012). It has previously been demonstrated that adsorption of surfactant proteins to the surface of particles can promote agglomeration (Kendall et al., 2004; Kendall et al., 2012); this process may therefore promote uptake by creating structures large enough for macrophages to recognise and phagocytose. Thus, for effective delivery and translocation of nano-sized particles to the peripheral lung tissue, it is essential that particles are designed to be small enough to deposit in the alveolar region and to remain well dispersed so that they are not recognised and phagocytosed by macrophages.

Advances in nanomedicine hope to overcome this by creating formulations that can target tumour cells specifically, thereby avoiding systemic side effects. One of the primary benefits of formulating chemotherapy agents at the nanoscale is that it may take advantage of the disordered development of the microenvironment of solid tumours. Tumours are known to have insufficient lymphatic drainage and poorly regulated angiogenesis, leading to formation of an unstructured and leaky vasculature. These two factors cause a phenomenon known as enhanced permeability and retention (EPR), a physiological bottle neck that allows nanoparticles to passively accumulate in tumour tissue (Fig. 4). EPR was discovered over 25 years ago when it was noted that macromolecules greater than 40 kDa selectively accumulated in tumour tissue in mice (Matsumura & Maeda, 1986). Subsequent studies have demonstrated that the endothelial layer of tumour blood vessels is poorly structured and contains intercellular gaps approximately 0.5– 2.5 μm wide (Yuan et al., 1995; Hashizume et al., 2000), a feature that is absent in normal healthy vasculature. There are already nanoformulations of chemotherapy agents on the market and many more in development. Abraxane is a nanoparticle formulation of the commonly used chemotherapy agent paclitaxel, bonded to albumin, and has been shown to be more effective and have fewer side effects than standard paclitaxel formulations (Gradishar et al., 2005); whilst most commonly used for treatment of breast cancer, the FDA recently approved it as a first line therapy for the treatment of non-small cell lung cancer. The reduction in side effects observed with Abraxane is due to its formulation into albumin nanoparticles, thus removing the need for cremaphor and ethanol as excipients, which are found in standard paclitaxel formulations and exhibit toxic effects. Other researchers have built on the success of this approach and extended it to other chemotherapy agents. Ernsting et al. created a nanoformulation of docetaxel, a drug used to treat breast, prostate and non-small cell lung cancer, and carboxymethylcellulose (Cellax) and compared its efficacy to Abraxane in a murine in vivo tumour model (Ernsting et al., 2012). They demonstrated that Cellax selectively accumulated in the tumour tissue to a greater extent than Abraxane and exhibited 40-fold greater half-life and thus was more effective at inhibiting tumour growth and preventing metastases. The authors suggested that this increased efficacy was due to a number of characteristics incorporated in to the formulation; the use of a polymeric nanoparticle as the carrier can improve drug solubility and minimise premature release of the drug in the circulation before it has accumulated in the tumour tissue. Furthermore, Cellax utilises the enzyme carboxylesterase, present at high concentrations

3. Treatment of cancer It is estimated that 1 in 3 people will develop cancer at some point in their lifetime. Lung cancer accounts for approximately 20% of all deaths from cancer and has a poor five year survival rate. Many chemotherapy agents are used to treat a wide variety of different cancers, however, a key hurdle in the efficacy of current therapies is the ability to achieve high concentrations of chemotherapy agent in the tumour without causing severe side effects, this is particularly difficult as most chemotherapy agents are administered systemically by intravenous infusion.

Fig. 4. Selective accumulation of nanoparticles in tumour tissue. Due to the rapid and poorly regulated growth of tumour tissue, the vasculature supplying the tumour is often leaky due to loss of tight cell–cell contact between endothelial cells lining the capillaries, which is not found in healthy blood vessels. These gaps (0.5–2.5 μm) allow circulating nanoparticles to exit the capillary and selectively accumulate in the tumour tissue. In addition, there is poor lymphatic drainage surrounding tumour tissue, which impedes clearance of nanoparticles. Thus tumour tissue represents a physiological bottleneck for circulating therapeutic nanoparticles.

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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in tumours, to release the drug from the carboxymethylcellulose nanoparticle ensuring improved selective delivery at the tumour site. Reformulating standard therapies at the nanoscale also has the potential to overcome tumour resistance. It is a well-known that tumours are able to become resistant to chemotherapy agents by up-regulating a number of cellular mechanisms, such as DNA repair processes, that repair the damage done by chemotherapy agents, as well as multi-drug transporter channels, responsible for efflux of drugs from the cell cytosol (Persidis, 1999; Luqmani, 2005). Sengupta et al. synthesised cholesterol nanoparticles containing cisplatin, a drug commonly used to treat lung, testicular, ovarian and stomach cancers, and demonstrated that they were significantly more cytotoxic in in vitro tumour cell viability assays than standard cisplatin or carboplatin; the nanoformulation of cisplatin was also able to induce significant cell death in a cisplatin-resistant cell line with an IC50 of 3.02 ± 0.013 μM (compared to 42.84 ± 0.04 μM using standard cisplatin) (Sengupta et al., 2012). The cholesterol-tethered cisplatinum was also more effective at reducing tumour size and increasing survival in in vivo murine tumour models. It was also shown that the nanoparticles selectively accumulated in the tumour tissue and avoided elimination via the kidney, resulting in prolonged and targeted treatment with minimal nephrotoxicity, a common side-effect of cisplatin treatment. These studies, demonstrating selective accumulation of nanoformulations of chemotherapeutic agents in the tumour with reduced systemic toxicity, suggest that higher doses may be tolerated using nanoformulations. Furthermore, the ability of nanoformulations to overcome chemotherapy resistance in tumour cells suggests that reformulation of already existing drugs may improve their efficacy in tumours with high expression of cellular drug resistance pathways. In addition to passive accumulation of drugs in tumours, numerous studies have investigated the possibility of active accumulation by coating nanoparticles with ligands that target receptors that are highly expressed on tumour cells. Integrins are receptors found on the surface of cells that mediate its interaction with the surrounding extracellular matrix and have been shown to be up-regulated on proliferating tumour cells and associated vascular endothelium (Costantini et al., 1990; Bandyopadhyay & Raghavan, 2009). Studies using nanoliposomes loaded with doxorubicin, a chemotherapy agent used to treat a broad spectrum of cancers, and tagged with a ligand for the integrin α5β1 have demonstrated that this formulation is significantly more effective than doxorubicin alone at reducing tumour size and increasing survival in in vivo murine tumour models (Dai et al., 2012). Similarly, the epidermal growth factor (EGF) receptor is highly up-regulated on non-small cell lung cancer tumours and is a potential target for improving selective delivery of chemotherapy agents (Rusch et al., 1997). A recent study using a murine model of lung cancer demonstrated that, following inhalation, cisplatin levels in the lung were maintained for longer following treatment with EGF-tagged cisplatin-containing gelatin nanoparticles compared to untagged cisplatin nanoparticles and free cisplatin (Tseng et al., 2009). Furthermore, the EGF-tagged nanoparticles had the greatest effect on reduction of tumour volume. The majority of current drug delivery nanoformulations are “soft” nanoparticles e.g. organic polymers and liposomes; however numerous studies have also investigated the use of “hard” inorganic nanoparticles. Perhaps one of the most well-known classes of nanoparticle, carbon nanotubes, is a potentially desirable vehicle for drug delivery due to its hollow structure and ease of surface modification, allowing cell specific targeting and drug loading. Bhirde et al. recently showed that EGF-tagged cisplatin-containing single walled CNTs were more effective at inhibiting tumour growth in a murine cancer model than non-tagged CNTs (Bhirde et al., 2009). Furthermore, the addition of a PEG coating improved the efficacy of the drug, and reduced toxic side effects (Bhirde et al., 2010); however, it should be noted that these were acute studies in a limited number of animals so further investigation is required. Similarly, using a rat tumour

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model, multi-walled CNTs loaded with doxorubicin and tagged with the tumour-targeting ligand hyaluronate were more effective at reducing tumour size compared to free doxorubicin (Datir et al., 2012). In addition, the nanotube formulation reduced the cardiotoxic side effects associated with standard doxorubicin treatment. Gold has long been used in medicine as a treatment for rheumatic diseases and diagnostic imaging. In recent years, there has been a growing interest in the use of gold nanoparticles as a carrier for chemotherapy agents. Studies of a variety of tumour cell lines and an in vivo rodent tumour model demonstrated that coating gold nanoparticles with methotrexate, a drug that is used to treat a wide variety of different cancers, significantly improved uptake of the drug into cells and resulted in a significant increase in its chemotherapeutic effect (Chen et al., 2007b). More recently, tumour necrosis factoralpha (TNFα)-labelled gold nanoparticles have entered Phase I clinical trials where TNFα conjugated gold nanoparticles selectively accumulated in tumour tissue and allowed for higher doses of TNFα to be given without any of the toxicity associated with administration of free TNFα (Libutti et al., 2010). 4. Treatment of chronic lung diseases As already mentioned, reformulation of current asthma drugs at the nanoscale could be beneficial in increasing the distribution and residency time of inhaled drug particles in the lung. Similar approaches could also benefit other lung diseases such as chronic obstructive pulmonary disease (COPD). COPD is the culmination of three pathological disorders, chronic bronchitis, small airways disease and emphysema, all of which can exist separately or in combination. The latter two components involve a chronic cycle of inflammation and remodelling in the peripheral lung, which is difficult to target and for which there is currently no effective therapy; the disease is classed as steroid insensitive and is thus unresponsive to traditional inhaled anti-inflammatory steroid therapies. As novel therapies are developed in the future, the field of nanomedicine could be of great help as formulation at the nano-size would most effectively deliver the drug directly to the site of injury. Local delivery would also hopefully avoid any serious systemic side effects as many of the anti-inflammatory therapies in development target widely expressed mediators of the inflammatory response (Barnes, 2010) and thus could be immunosuppressive if given orally. Tuberculosis (TB) is predominantly a respiratory disease characterised by invasion of alveolar macrophages by Mycobacterium tuberculosis. The bacterium resides inside phagosomes within the macrophage and survives by preventing fusion and formation of the phagolysosome which would normally kill bacteria. TB is one of the largest burdens on public health globally. In their 2011 report, WHO stated that there were nearly 9 million new cases of TB and 1.4 million deaths worldwide. At present, first line therapy involves a 6 month program of multi-drug treatment with 3–4 different antibiotics. This intensive regimen often leads to poor patient compliance resulting in ineffectual treatment and development of multi-drug resistant strains. Recent studies have investigated the potential for inhaled nanoformulations to revolutionise the treatment of this disease; inhaled preparations would deliver the drug to the primary site of infection, unlike current oral therapy, and could allow for administration of all the drugs at once rather than in separate tablets as is currently necessary. A recent study in a rodent model of tuberculosis compared the bioavailability and efficacy of a combined inhaled nanoformulation against traditional oral therapy (Pandey et al., 2003). Isoniazid, rifampicin and pyrazinamide were encapsulated within polymer nanoparticles and nebulised into M. tuberculosis-infected guinea pigs. Inhaled therapy resulted in significantly longer bioavailability of all three drugs compared to traditional oral therapy; thus inhaled therapy maintained plasma levels for as long as 192 h whereas following oral dosing, plasma levels of all three drugs returned to baseline within 12 h. Furthermore, dosing every 10 days by inhalation was as effective as daily dosing via

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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the oral route. Other similar studies investigating nanoformulations of tuberculosis therapies administered via the inhalation and oral routes have shown beneficial effects over traditional therapies (Sosnik et al., 2010); this suggests that in the future nanomedicine has the potential to overcome the current difficulties associated with treatment of tuberculosis and improve global health. 5. Treatment of systemic diseases by inhalation As already mentioned, inhalable nanomedicines could also be beneficial for treatment of systemic diseases, not just pulmonary diseases. The alveolar gas exchange region of the lung offers a large surface area for drug deposition and absorption. Furthermore, the alveoli are surrounded by a dense network of capillaries; the barrier between the alveolar air space and the underlying capillary circulation is less than one micron in depth, suggesting that administration of drugs by inhalation could allow for rapid absorption of drug in to the systemic circulation. Development of nanomedicines for administration via inhalation is particularly desirable for delivery of peptide and protein-based drugs that cannot be given via the oral route and are thus given by injection. At present the evidence for translocation of nanoparticles across the air/blood barrier is unclear; a number of in vivo studies have demonstrated that pulmonary delivered nanoparticles enter the systemic circulation, however the exact mechanisms remain unclear. In vivo rodent studies using 11–31 nm isotopically labelled gold nanoparticles have suggested that translocation of pulmonary delivered nanoparticles is low (Lipka et al., 2010; Schleh et al., 2013); both studies found that the majority of nanoparticles remained in the lungs, only 0.5–3% of the amount delivered to the lungs translocated into the blood between up to 24 h post exposure. Due to the complexity and diversity in nanomaterials, it is difficult to make assumptions about nanoparticle behaviour based on one physicochemical property alone; it is likely that surface modifications and elemental composition may drastically alter the ability of nanoparticles to interact with and traverse biological barriers. Indeed, a study investigating the pulmonary translocation of a range of organic and inorganic nanoparticles of varying sizes, found that size and surface chemistry played a significant role in systemic distribution of intratracheally instilled nanoparticles (Choi et al., 2010), with low translocation of nanoparticles into the blood within the first hour post-exposure (approximately 0.1–0.4%). Interestingly, nanoparticles less than 35 nm, regardless of whether they were organic or inorganic, were rapidly translocated into regional lymph nodes and the bladder within 30 min whereas particles larger than this were not. Furthermore, when the effect of surface modification, and thus charge, was examined, neutral and negative charged nanoparticles were rapidly translocated whereas cationic nanoparticles were not. It was hypothesised that the cationic charge promoted uptake and sequestration of the nanoparticles by alveolar macrophages and epithelial cells, preventing them from translocating out of the lung. In vivo studies of the distribution of inhaled gold nanoparticles in the lungs of rats demonstrated significant uptake by ATI cells, and to a lesser extent, ATII cells (Takenaka et al., 2006). Other in vivo rodent studies have also demonstrated that ATI cells internalise nanoparticles, and have suggested that translocation across ATI cells may lead to accumulation of nanoparticles in the interstitium (Ferin & Oberdorster, 1992). The study of the mechanisms underlying uptake of nanoparticles by the alveolar epithelium has been hampered by the lack of robust in vitro models of the human alveolar epithelium. Many studies utilise the “ATII cell-like” A549 carcinoma-derived cell line, which we have shown does not behave like primary human ATII cells (Thorley et al., 2011). As already stated, ATI cells cover most of the alveolar surface, making them a primary target for inhaled nanoparticles. ATI cells are important in regulating fluid and protein transport in the peripheral lung and as such possess a number of pathways for transcytosis of molecules at the alveolar surface, in particular clathrin and caveolin-mediated

endocytosis; it is likely that these pathways may also contribute to uptake of nanoparticles and their possible translocation into the bloodstream (Maynard et al., 2012). Due to their fragile nature, it is not possible to isolate human ATI cells in primary culture. However, we have recently transdifferentiated primary human ATII cells into ATI cells, as can occur in vivo, and immortalised them to create a unique cell line (TT1). Using primary cultures of human ATII cells and the TT1 cell line, recent studies in our laboratory have demonstrated that human ATI cells rapidly and avidly internalise nanoparticles, significantly more so than ATII cells (Kemp et al., 2008). This finding suggests that transcytosis of nanoparticles across the epithelial barrier could occur; whether particles trafficking across the epithelial layer are sequestered into the underlying basement membrane and/or enter the circulation in significant quantities remains to be established. For effective drug delivery in to the circulation, biodegradable nanoparticles may be a more desirable vector, as dissolution of the particle following delivery to the peripheral lung may avoid any sequestration or limiting steps in translocation in to the bloodstream that may occur with solid nanoparticles. Indeed, this approach has already been successfully developed for the delivery of insulin. Interest in delivery of insulin by inhalation began in the late 1990s when it was demonstrated that nebulised or intratracheal delivery of insulin-loaded polymer nanospheres significantly reduced blood glucose levels in rodents (Kawashima et al., 1999; Zhang et al., 2001). Subsequently, Exubera, a dry powder inhaler formulation of insulin made it to market in 2006. Despite receiving FDA approval and UK licensing, the product never gained any traction in the market due to its poor cost effectiveness and minimal therapeutic benefit over traditional injected insulin; despite the hope that it would make insulin therapy easier for the patient, the dosing regimen was complicated and required the use of a conversion table to calculate the correct dose and the delivery device was large and cumbersome. Due to its lack of success, Exubera was removed from the market in 2007 and as a result, other companies halted development of inhalable insulin preparations; there is however one inhalable dry powder microsphere formulation (Affrexa) currently in Phase III trials and under FDA review (Neumiller & Campbell, 2010). Affrexa overcomes a number of issues associated with Exubera, in particular the complicated dosing; it has also been formulated for use in a pocket sized inhaler device. Studies are still on-going to develop other formulations of inhalable insulin, numerous studies continue to demonstrate that nano-sized particles achieve excellent pulmonary deposition and distribution in conjunction with a rapid onset of effect (Bailey et al., 2008; Liu et al., 2008; Zhao et al., 2012), however a direct comparison to investigate whether a nanoformulation is better than micron-sized formulations already in development has yet to be done. 6. Vaccines The development of inhalable vaccines could have a significant impact on world health. One major drawback of current injection-based vaccines is that they need to be stored refrigerated, require a sterile environment for administration, trained personnel to administer the drug and means of disposing of used needles safely and securely, all of which are limiting factors in maintaining good public health vaccination programmes, particularly in developing countries. There are currently a number of inhalable vaccines in development, targeting both pulmonary and systemic diseases. Respiratory syncytial virus (RSV) is a significant cause of hospitalisation in infants (Hall et al., 2009) and there is growing evidence for its role in chronic lung diseases such as COPD (Sikkel et al., 2008) and asthma (Sigurs et al., 2000). As yet there is no vaccine available for RSV, the only vaccine to be released was withdrawn as it worsened subsequent RSV infections; this was due to immunopotentiation induced by the alum adjuvant. A recent study comparing the effect of a nanoemulsionadjuvanted inactivated RSV vaccine against an alum-adjuvanted RSV

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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vaccine showed that, following intranasal administration, the nanoemulsion vaccine effectively immunised against RSV infection and did not induce the Th2 mediated-immunopotentiation response associated with the alum based vaccine (Lindell et al., 2011). The nanoemulsion preparation was also stable at room temperature and thus represents a potential non-invasive vaccine that does not require special storage. The development of vaccine nanoformulations could also improve on currently available vaccines; studies in to nanoformulated alternatives to the standard BCG vaccine for tuberculosis (TB) are particularly promising and may overcome a significant shortfall in BCG efficacy. At present the BCG vaccine is most effective in the first 10 years of life, however its efficacy in adult life is highly variable; this is of particular concern given the evolution of drug resistant strains of the disease. In a recent study, the TB antigen Ag85B, commonly used in subunit vaccines, was conjugated to 30 nm polypropylene nanoparticles and its efficacy compared with the Ag85B antigen alone. In addition, the effect of route of delivery was examined, comparing pulmonary and intradermal delivery. Using a murine model of tuberculosis, they demonstrated that nanoparticle conjugation significantly increased the efficacy of the vaccine compared to antigen alone and that pulmonary administration was much more effective than intradermal administration in conferring immunity to tuberculosis infection (Ballester et al., 2011). Similar to the RSV studies described earlier, the nanoparticles had a significant adjuvant effect; thus it would appear that nanoparticles may not only improve delivery of a vaccine but also provide an alternative to currently used adjuvants which often elicit adverse effects.

7. Too good to be true? There is little doubt that nanotechnology has the potential to revolutionise how we treat a range of diseases, however, as with all medicines, the need for thorough testing and regulation of nanomedicines is essential. A particular concern with nanomedicine is the reformulation of existing drugs or the use of inorganic materials that are safe in their micron size format; due to the unique properties acquired by particles when they are formulated at the nanoscale, unforeseen adverse and toxicological effects may be seen that cannot be predicted by studies on the equivalent micron-sized particles. Much of the concern surrounding the use of nanoparticles in consumer products arose from epidemiological studies investigating the link between adverse health events and ambient particulate air pollution. A study of 20 US cities demonstrated that a 10 μg/m3 increase in levels of ambient particulate matter measuring less than 10 μm in diameter (PM10) was associated with a 0.68% increase in death from respiratory and cardiovascular causes (Samet et al., 2000). More startlingly, a study of 500,000 people residing across 50 states of the USA showed that a 10 μg/m3 increase in nanoparticle-containing PM2.5 (particulate matter less than 2.5 μm) was associated with a 6% increase in cardiopulmonary mortality (Pope et al., 2002). Another major concern regarding the physical shape of nanoparticles has been highlighted by the effect of asbestos inhalation on the lung, Asbestos is a naturally occurring fibre that has been used in construction as far back as the 19th century but is now banned due to its ability to induce pulmonary fibrosis and mesothelioma, even in subjects exposed to very low levels for short periods of time (Becklake et al., 2007). The harmful effects of asbestos are related to their physical properties i.e. their shape, length and biopersistence. The most hazardous class of asbestos, amphibole, forms long, rigid, needle like structures; they may be less than one micron in diameter but many microns in length. Due to their size and shape they are unable to be cleared by alveolar macrophages and thus persist in the lung for decades, where they can initiate pro-fibrotic and tumorigenic pathways. It is for this reason that cases of

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mesothelioma can typically take anywhere between 30 and 40 years to present (Lin et al., 2007). Due to the diverse range of sizes, shapes and elemental composition of nanoparticles, there are an almost infinite number of potential formats. At present, the two most common elements used in commercial nano-containing products are carbon and silver; both exhibit beneficial properties at the nano-scale that could be harnessed for medical purposes, however concerns over their usage in certain physicochemical formats have arisen in recent years. In addition, the safety of nanoparticles is further confounded by the ability to modify the surface of nanoparticles with moieties that can change their behaviour and properties. 7.1. Carbon nanotubes As already discussed, nanowires and nanotubes are potentially attractive structures for drug delivery and therapeutics; however, due to their structural similarity with asbestos there is concern that prolonged exposure to, or failure to degrade and clear, these nanostructures could result in development of fibrosis and mesothelioma in much the same way as asbestos. A number of studies have already been carried out which begin to address this concern. In vivo studies, where carbon nanotubes have been directly applied to the pleural and peritoneal mesothelium, have demonstrated fibrosis and granuloma formation (Poland et al., 2008; Murphy et al., 2011) and one study demonstrated development of peritoneal mesothelioma after intrascrotal injection of carbon nanotubes in rats (Sakamoto et al., 2009). However, it should be highlighted that these effects occurred only when using high doses of CNT or very long CNTs and only following direct application to the mesothelial surface. It is likely that CNTs and other nanofibres used for medical purposes will be significantly shorter than the CNTs identified as pathogenic in these studies. Other studies have shown that longer term, repeated inhalation exposure to carbon nanotubes causes similar toxicological endpoints. 3 month exposure to poly-dispersed multi-walled CNTs (0.1–10 μm), resulted in lung inflammation and granuloma formation (Ma-Hock et al., 2009). However, other studies suggest that this was due to agglomerated bundles of CNTs that were not well dispersed (Shvedova et al., 2005; Pauluhn, 2010). Due to the sheer diversity in the length, diameter, number of walls and surface functionalisations of carbon nanotubes used in the studies to date, it is not yet possible to make a firm statement on what, if anything, makes a “safe” CNT; more systematic investigations in to the effect of specific physicochemical properties of CNTs on their toxicity are urgently needed. 7.2. Silver Silver is the most commonly used metal nanoparticle in consumer products at present (The Project on Emerging Nanotechnologies: Consumer Product Inventory, 2008). It has been used for millennia for its antimicrobial effects; it is said that Hippocrates extolled the virtues of silver for its wound healing properties and many ancient civilisations used silver vessels for preserving food and water (Alexander, 2009). The antimicrobial effects of silver rely on ionic silver dissolving from its substrate and interfering with bacterial DNA, cell wall integrity and respiration (Lara et al., 2011). In the 20th century silver was incorporated in to a number of different formulations including a silver sulfadiazine cream formulation to treat burns (Inman et al., 1984) and has been investigated as a catheter coating to prevent infection (Johnson et al., 1990). Most recently, there has been a move towards using nanosized silver particles for slow, sustained release of ionic silver. In 1998, Acticoat™ wound dressings were launched which contained nanocrystalline silver, these dressings were shown to be more effective than silver sulfadiazine

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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cream, accelerating wound healing and decreasing odour (Fong & Wood, 2006; Huang et al., 2007). Despite these beneficial effects, prolonged exposure to high levels of ionic silver is associated with undesirable side effects such as argyria where the skin turns an irreversible bluish grey; similarly, prolonged ingestion of silver can lead to accumulation in other vital organs. At present most cases of argyria are associated with chronic intake of colloidal silver as an alternative to antibiotics; this is not recommended by medical authorities but is widely available from homeopathic medicine vendors. Whilst argyria is purely cosmetic, there are increasing concerns that nanosilver may have long term toxic effects; with respect to pulmonary health, this is particularly concerning in light of silver nanoparticles being incorporated in to face masks and antibacterial sprays, and the unregulated use of nebulised colloidal silver as a homeopathic treatment for respiratory infections. Numerous in vitro studies using cell lines have demonstrated that silver nanoparticles have the potential to be toxic depending on their physicochemical properties. Exposure of the glioblastoma cell line U251 and the pulmonary fibroblast cell line IMR-90 to polydispersed 6–20 nm silver nanoparticles caused significant dose-dependent cytotoxicity, oxidative stress, cell cycle arrest and genotoxicity (AshaRani et al., 2008). Furthermore, transcriptomic analysis of the A549 pulmonary adenocarcinoma cell line following 24 hour exposure to 16 nm silver nanoparticles demonstrated altered regulation of over 1000 genes; the most significant changes in gene expression were associated with the cell cycle pathway and suggested induction of cell cycle arrest. In addition, up-regulation of gene expression for metallothioneins and heat shock proteins suggested that cellular and oxidative stress pathways are activated (Foldbjerg et al., 2012). As yet there are few studies which have systematically addressed the effect of size and shape on the pulmonary toxicity of silver nanoparticles. One study, using A549 cells, compared the effect of size and shape on cytotoxicity using silver microparticles, 30 nm silver nanoparticles and a range of silver nanowires with diameters of 100–160 nm and a length of 1.5–25 μm and demonstrated that spherical nano- and micro-particles were not toxic whereas nanowires were highly toxic; interestingly it was not possible to differentiate the degree of toxicity of the differing lengths of the nanowires, which was very similar (Stoehr et al., 2011). The lack of toxicity of the nanoparticles in this study may be attributable to the use of PVP as a capping agent, which may make the particles less reactive. It has also been demonstrated using a number of cell lines that cytoxicity was size dependent, with 5 nm nanoparticles being significantly more toxic than 20 nm and 50 nm nanoparticles (Liu et al., 2010). Furthermore, uptake of silver nanoparticles by the HepG2 liver cell line was also size dependent, 5 nm nanoparticles were internalised significantly more than 20 nm and 50 nm. Interestingly, despite the difference in toxicity and uptake, the level of oxidative stress observed was similar between all three nanoparticles, suggesting that nanoparticles are able to elicit cellular effects without being internalised or inducing cell death. Silver nanoparticles can also elicit toxic and inflammatory effects on the lung in vivo. A single intratracheal instillation of 240 nm silver nanoparticles in mice was shown to induce an acute infiltration of leukocytes within 24 h and increased pro-inflammatory cytokine levels in the lavage fluid and serum for up to 28 days after administration (Park et al., 2011). There are very few studies investigating the effect of repeat chronic exposure to inhaled nanoparticles. One recent study investigated the effect of exposure to inhaled 18 nm silver nanoparticles for 6 h/day, 5 days/week for 13 weeks on lung function and inflammatory markers in Sprague Dawley rats, which impaired lung function and induced inflammatory lesions in the lung (Sung et al., 2008). Subsequently, the same group showed that lung function was impaired for up to 12 weeks post-exposure to 3 × 106 particles/cm2 of silver and that silver was slowly cleared from the lungs during the recovery period. Levels of silver were also measured in other organs and were found to persist in the liver and spleen up to

12 weeks in both male and female rats and in the eyes of male rats (Song et al., 2012). 7.3. Surface functionalisation It is not just the basic structure and elemental composition of nanoparticles that can determine their safety or toxicity. As mentioned earlier, surface functionalisation can improve a particle's ability to traverse biological barriers such as mucus and the cell membrane. However, these moieties also have the potential to make particles more toxic. Many nanoparticles for drug delivery are cationic which is thought to improve delivery by facilitating the interaction of the nanoparticle with the anionic cell membrane. However, studies by us and others have demonstrated that the addition of cationic amine groups to the surface of a nanoparticle may significantly increase its toxicity. 3–4 nm cerium nanoparticles, positively charged with an aminated poly acrylic coating, were shown to induce significant cytotoxicity in a variety of cell lines compared to neutral charged or negatively charged cerium nanoparticles of the same size (Asati et al., 2010). Similarly, in studies comparing positively and negatively charged CdSe quantum dots, it was shown that a positive charge induced more cellular necrosis in primary human bronchial epithelial cells than a negative charge, while positively charged but not negatively charged quantum dots induced expression of a number of genes involved in inflammation and apoptosis (Nagy et al., 2012). The diversity of applications for which CNTs can be used for is greatly increased by the addition of surface functionalisations; addition of functional groups to the surface can alter the solubility, dispersion and cellular uptake of CNTs (Antonelli et al., 2010; Chen et al., 2011; Kraszewski et al., 2012). However, alteration of the surface chemistry of CNTs may also impact on their toxicity. Studies in human monocyte-derived macrophages investigating the effect of CNT surface functionalisations have demonstrated that the addition of carboxyl or amine groups induced greater oxidative stress compared to unmodified CNTs. Furthermore, functionalisation with carboxyl groups induced greater apoptosis compared to unmodified CNTS (Gasser et al., 2012). Similar findings were demonstrated in a murine pulmonary cell line; acid treatment of CNTs (which adds carboxyl functional groups) significantly increased cytotoxicity and release of pro-inflammatory mediators compared to untreated CNTs (Saxena et al., 2007). In both studies the increased toxicity was hypothesised to be linked to the agglomeration status of the CNTs; the addition of carboxyl groups to CNTs reduces agglomeration by making the CNTS less hydrophobic and may thus make it easier for CNTs to interact with cells. More recently, the effect of oropharyngeally aspirated acid-treated CNTS on cardiopulmonary toxicity was investigated in a murine model (Tong et al., 2009). Results demonstrated that acid-treated CNTS induced significantly more neutrophilia than untreated CNTs. Interestingly, pulmonary exposure to acid-treated CNTs was also associated with systemic effects; after 24 h there was evidence of myocardial degeneration, whether this was a result of CNT translocation or the local release of mediators in to the circulation was not determined and requires further investigation. Studies in our laboratory have investigated how the combination of size and surface functionalisation affects cytotoxicity. In a recent study we demonstrated that an amine modification alone does not necessarily confer cytotoxicity to a nanoparticle (Ruenraroengsak et al., 2012). Using the unique alveolar type I epithelial cell line, TT1, generated in out laboratory, we compared the effect of size and surface functionalisation on cell viability, caspase activity and membrane integrity. Our results showed that 50 nm amine-modified polystyrene nanoparticles were highly cytotoxic and induced caspase 3/7, 8 and 9 significantly more than carboxyl-modified or unmodified polystyrene nanoparticles. Furthermore, using hopping probe ion conductance microscopy, we demonstrated that 50 nm amine-modified nanoparticles created holes in the cell membrane, unlike the other nanoparticles tested. Interestingly the cytotoxic effect of the amine-

Please cite this article as: Thorley, A.J., & Tetley, T.D., New perspectives in nanomedicine, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/ j.pharmthera.2013.06.008

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modification was lost when the particle size was increased to 100 nm; at this size there was significantly less cytotoxicity and there was no difference in the toxicity of three modifications, suggesting that the combination of size and surface modification is critical in determining the toxicity of a nanoparticle.

8. Summary Despite the health concerns surrounding exposure to engineered nanoparticles, the rapid increase in the research, development and use of nanotechnologies in the 21st century in a wide range of industries suggests that they are set to filter in to all aspects of daily life. There is no doubt that developments in the field of nanomedicine could improve the treatment of a wide variety of diseases, and huge inroads have already been made; as such is it essential that the health effects of nanoparticle exposure are fully assessed. Due to the almost limitless possibilities in manipulating particle size, shape and surface chemistry, it is evident that particles will need to be assessed on a case by case basis; evidence thus far suggests that even small changes to the physicochemical properties of a particle can have a significant impact on their bioreactivity, making predictive toxicology impossible.

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