Toxicity

Although the utilization of nano-scaled medications entails numerous advantages, including new material properties, an increased surface capability due to enlarged surface-volume ratio, a shorter transport time, the potential of selective targeting and the minimized exposure of healthy tissue to the incorporated drug [364], the miniaturization of systems always involves the danger of arousing toxicity. To determine a safe dose within the therapeutic window as well as the lethal dose of drug-loaded nanoparticles, it is essential to perform toxicological testing in vitro, ex vivo and in vivo in cell lines, tissue and animal models before starting clinical trials with humans.

Although it is not possible to convey the data gained from those experiments directly to the conditions of an actual patient, it is crucial to reduce the risk of toxic effects and possible adverse reaction as much as possible beforehand.

Several assays to ascertain the toxicity of nano-based drug formulations have already been established and can employ different cellular targets, like mitochondria, lysosomal activity, cell membrane integrity or DNA ladder assays to determine cell death mechanisms. The standard testing method for cell viability after treatment with nanomaterials in a great range of cell lines is the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Other tests resting upon the principle of the metabolic activity of mitochondria involve tetrazolium salts or resazurin. To improve statistical validity and minimize the error chance, it is recommended to combine multiple experiments. For example, the simultaneous performance of MTT and resazurin assay can be rational, as they both utilize a similar principle of testing. [365] Studies revolving around the specific disease instances of asthmatic lungs should take into consideration the correspondent conditions,

pre-eminently airway hyperresponsiveness, mucus hypersecretion and the influx of inflammatory cells as well as their cytokines. [294] In order to detect possible immune-related and inflammatory responses, variations in activation levels of cytokines, e.g.

TNF-α, interleukins and prostaglandin should be monitored.

Particular materials have to be chosen to reduce toxic effects contingent upon the desired target region. While polyamidoamine (PAMAM), for example, indeed exhibited favorable characteristics as nanocarriers in several studies, it was shown to foster acute lung injury by inducing autophagic cell death via the Akt-TSC2-mTOR signaling pathway [366]. Card et al. reviewed different imaging, diagnostic and therapeutic applications of engineered nanoparticles in the lung and identified whole groups of nanomaterials that can have negative repercussions on the pulmonary structure and function. According to their findings, nonbiodegradable substances such as carbon nanotubes, carbon black, fullerenes, silica, metals and metal oxides can generate inflammation and/or fibrosis in the lung after inhalation, intranasal or oropharyngeal aspiration as well as systemic administration [367]. Biodegradable nanoparticles, e.g. made of PEG-PLA (polyethylene glycol-polylactide) [363] or PLGA (poly(lactic-co-glycolic acid)) [368], on the contrary, have been proven to be useful pulmonary drug carriers.

Different studies in humans have been performed to investigate the deposition of inhaled nanoparticles in healthy and diseased lungs [369, 370]. An increased pulmonary deposition and retention in constricted airways was predicted by computational models [371] and demonstrated in obstructive lung disease [372] and asthma [373] patients.

Regarding the latter, the exposure of subjects with mild to moderate asthma with ultrafine carbon particles during spontaneous breathing led to an increased fraction of deposited particles compared to healthy individuals. Pietropaoli et al., nevertheless, did not ascertain any differences in respiratory parameters between healthy and asthmatic subjects after inhalation of respective particles. No airway inflammation was observed in either group, but an exposure of healthy individuals to a higher concentration of particles resulted in a decreased midexpiratory flow rate and carbon monoxide diffusing capacity, indicating that nanoparticles may influence respiratory function and gas exchange [374]. Moreover, several investigations have been conducted to test the translocation of nanoparticles from the lung to the systemic circulation after inhalation, as cardiovascular effects similar to the impact of urban air pollution were apprehended.

Most findings indicate that the tested 99mtechneticum-labeled carbon nanoparticles are not detected outside the lungs in appreciable concentrations [370, 375, 376].

Nevertheless, it remains uncertain whether other nanoparticles behave similarly, and the possibility of particles influencing the vasculature is still not excluded. Besides that, all studies used single inhalation exposure protocols so that further investigations on the repercussions of repeated exposure, stronger pulmonary accumulation and, therefore, translocation of greater particle quantities are urgently needed. [367]

In a recent study, it was examined whether intratracheal instillation studies can be used for evaluating any harmful effects of inhaled nanoparticles. Therefore, rats were exposed to nanoparticles composed of Nickel oxide and titanium dioxide as high and low toxicity examples. Among others, increases in neutrophils in BALF (bronchoalveolar lavage fluid) and concentration of cytokine-induced neutrophil chemoattractants were compared after single intratracheal installations and inhalations over 4 weeks, and results suggest that intratracheal studies can be a useful tool in ranking adverse influences of nanoparticles. [377]

5.1.2. In vivo Pharmacokinetics, Administration and Metabolism

The lung and its large surface area with a high vascularization as well as a thin air-blood-barrier, on the one hand, displays an ideal location for the absorption of agents [31]. On the other hand, several physicochemical and biological barriers await the nanotherapeutics in the pulmonary system, making it essential to thoroughly track their routes and deposition in the body. Figure 2 illustrates the different defense mechanisms nanoscale particles have to encounter in the lung.

Figure 2. Lung-intrinsic barriers to efficient pulmonary siRNA delivery. Reprinted with permission from [20].

The most important parameter influencing the deposition of particles in the different areas of the lung is their size. Depending on that, three different mechanisms of allocation are possible: impaction, sedimentation and Brownian diffusion. Particles with a mass median aerodynamic diameter (MMAD) greater than 5 µm pass through the oropharynx and upper respiratory passage with a higher pace, collide with the respiratory wall due to the centrifugal force, and are deposited in the mouth and pharyngeal regions [20]. This so-called impaction usually occurs with dry powder inhalation (DPI) and metered dose inhalators (MDI). Deposition of drugs formulated as DPIs is especially dependent on the inspiratory effort of the patient: an insufficient force of inhalation leads to aggradations of the particles in the upper airways. Nevertheless, large and aggregated particles can also become subject to this process when MDIs are used, despite the higher speed of the generated aerosol. As gravitational forces

preponderantly condition the sedimentation process, particles with an MMAD between 1-5 µm are slowly deposited in the smaller airways and bronchioles, whereas Brownian motion is the prevalent mechanism in the lower alveolar sections. The molecules surrounding the aqueous lung surfactant underlie the Brownian motion itself and induce a random moving of the particles. The dissolution of the therapeutic agents in the lung surfactant, depending on the concentration gradient, influences this process as well. Particles with a size smaller than 1 µm deposit in the alveolar region or can be exhaled. Therefore, sedimentation is the preferably achieved process for therapeutic nanosystems in order for them to stay in the bronchiolar area for a long time and to result in the desired effects. In addition to these particle and target surface depending characteristics, breathing patterns, the holding of breaths and tidal volume, but also air velocity and humidity are factors influencing the deposition and hence need to be considered. [378]

The inside of the upper airways is covered with a film of mucus that is responsible for trapping and purging invading particles. Before those can reach lower sections of the lung through coughing or swallowing, they are often cleared by mucociliary movements.

Consequentially, nano-sized drugs should be able to cross the mucus layer and reach the sol that covers the stratum below the gel coat. [379] As PEG nanoparticles were repeatedly shown to be capable of permeating across mucus [380-382], PEGylation is a possible approach to avoid bronchial clearance of nanomedicines. In the alveolar regions, the alveolar lining consisting of various proteins and lipids. Additionally, the existing tight junctions hamper the transport of molecules. According to the structure of the nanomedicine, active transport or passive diffusion through those transporter proteins are possible. Larger particles are, furthermore, prone to be cleared through phagocytosis by alveolar macrophages. [383]

Once arrived inside the peripheral lung, the particles have to dissolve, and the incorporated drug has to diffuse through the epithelial barrier in order to reach the blood. There are still some deficiencies in the exact understanding of the process of cell-uptake and how the particles are transported and reach the systemic circulation.

Despite the existence of in vitro models for studying the uptake and permeation, the precise behavior of the cells under disease conditions has yet to be examined further.

[365]