There is currently significant worldwide effort to develop, fabricate and characterize novel nanoscale materials for a variety of novel applications. In the present work, we have developed procedures to prepare novel biocompatible MNPs that possessed suitable properties for biomedical applications. Biocompatibility of the developed MNPs was characterized in vitro (the influence of MNPs was assessed in terms of cell viability, cellular and nuclear morphology, and observations of actin cytoskeleton) and in vivo (the influence of MNPs on glutathione and IL-6 secretion in mice). The developed MNPs were successfully loaded with a promising anti-cancer drug quercetin. Further, in this study we described a novel method of drug-loaded nanoparticle delivery to lung cancer using aerosols. The optimised proof-of-concept nanoplatform documented in the present study can further be exploited to load functionalities onto the MNP surfaces via various mechanisms with broad implications for pharmacotherapies, drug delivery and molecular imaging.
A targeted drug delivery system requires the design of carriers capable of selectively releasing their payloads at specific sites in the body. Although a number of nanosize materials are being exploited for drug delivery purposes including for example PLGA nanoparticles [34, 35], MNPs represent a highly promising option for selective drug targeting as they exhibit a wide variety of desirable attributes. In particular, they can be concentrated and held in position with the aid of an external magnetic field. The deposition, accumulation, and retention of drug-conjugated MNPs in target tissue can thus be enhanced by magnetic guidance. Such magnetic targeting allows very concentrated drug doses to be delivered to specific area while minimizing the exposure of healthy tissues to uncontrollable highly toxic therapeutic substances; e.g. chemotherapeutic agents. Moreover, the superparamagnetic behaviour of MNPs provides multifunctional effects such as controlled heating capability under an alternating magnetic field, which has demonstrated tremendous promise as theranostics for the detection and treatment of cancer [7, 9, 36, 37]. In addition, iron oxides occur naturally in human heart, spleen and liver , which supports the biocompatibility and non-toxicity of MNPs at a physiological concentration. Due to the above-mentioned favorable features and versatility, in our opinion, Fe2O3 MNPs would serve as an excellent core material for a nano carrier system particularly suitable for the controlled aerosol drug delivery.
To date a wide variety of MNPs have been developed by several researchers, differing in size and type of coating materials used [7–13, 39–42]. Some preparations are currently in preclinical or clinical use in intracellular hyperthermia treatments and MRI contrast agents [7, 9]. It is important to note that in order to improve the size distribution of MNPs and prevent their aggregation in aqueous solution these nanoparticles have to be coated with materials that keep particles apart. However, there is quite contradictory information on the effect of magnetic nanoparticles - biopolymer core-shell structures on cytotoxicity. It has been suggested that upon internalization, the coating shell on the MNPs may be broken down yielding particle chains and aggregates, which may influence biological processes [42, 43]. In this study, we modified the surface of Fe3O4 MNPs by coating them with a biocompatible polymeric material PLGA, which has been proven to be beneficial for nanoparticle coating purposes with no measurable toxicity reported [42, 44].
In order to evaluate the biocompatibility of developed MNPs, we performed a series of in vitro assays using a human lung alveolar epithelial cell line A549 and in vivo studies using normal Balb/c mice. The carcinoma-derived A549 cells are a well-characterised in vitro lung epithelial model and have been extensively used for assessing cytotoxicity, including nanomaterials-induced cytotoxicity [45–47]. Additionally, A549 cells display similar uptake and toxicity of nanoparticles as compared to normal primary lung epithelial cells, although both cell types respond differentially for the release of cytokines involved in inflammatory reactions . Based on these reports and our data from in vitro as well as in vivo experiments presented here, we expect that the developed MNPs will have similar effect(s) on normal lung epithelial cells in terms of their cytocompatibility. However, a detailed characterization of MNPs on normal lung cells should be performed before their potential clinical applications in drug delivery.
We employed the use of HCS in combination with an impedance-based assay for the biocompatibility analysis of MNP preparations. The HCS assay utilizes a novel quantitative imaging technique and offers rapid analysis of toxicity (if any) at cellular level [46, 47]; whereas, impedance sensing allows a kinetic profile of cytotoxicity (if any), and maps the processes that cells undergo when challenged with nanoparticles such as MNPs [49, 50]. Since the insulating properties of cells are based on whole cell structure, cellular responses such as cell death, proliferation, spreading and attachment can be detected by impedance measurements [49, 50]. This cell-based label-free non-invasive detection method thus not only provides toxicity data, but also can identify a time-frame during which further targeted analysis can be performed. Both HCS and impedance measurement assays confirmed that MNPs developed in the present work were not toxic to A549 cells up to a concentration of 100 μg/ml, although a high concentration of 250 μg/ml were moderately toxic.
As described, we selected quercetin as a model drug. Quercetin is one of the most prevalent as well as thoroughly studied dietary flavonoids with several biological and pharmacological properties. Evidence indicates that quercetin has a variety of anti-cancer mechanisms, including anti-proliferative, pro-apoptotic, cell signalling effects, and growth factor suppression, as well as potential synergism with some chemotherapeutic agents [32, 51]. Quercetin also exhibits anti-inflammatory, anti-oxidant, and anti-viral activities . Moreover, it has a role in reversing drug resistance, re-sensitizing cancer cells to some chemotherapeutic agents and in potentiating the effectiveness of some chemotherapeutic agents . However, realizing the therapeutic benefits of quercetin in the clinical setting is hampered by its low solubility (~ 2%) in aqueous medium and poor absorption in the body. Thus, the low bioavailability and poor solubility in aqueous medium are major concerns associated with the therapeutic application of quercetin [51, 52]. Similar limitations apply to experimental evaluation of quercetin’s effect on cultured human cells in biological medium, and therefore quercetin alone could not be used for comparison in the present study. The MNP carrier system developed in the present study was appropriate in this regard; and therefore, the ability of quercetin to inhibit lung cancer cell growth was evaluated in comparative analysis of non-functionalized and drug-loaded PLGA-MNPs.
Administration of the MNPs resulted in elevated levels of GSH in lung tissue, an indicator of oxidative stress , but this was not observed to be consistently elevated over the follow-up period of 7 days unlike the LPS control. IL-6, which acts as both a pro-inflammatory and anti-inflammatory cytokine , and is secreted by T-cells and macrophages to stimulate an immune response during infection and after tissue trauma was also investigated as a marker of immune response. The LPS caused a significant increase in IL-6 levels in BAL samples 1 day after treatment as expected, but this was not replicated in the case of the MNPs. In fact the IL-6 levels in BAL samples from mice exposed to MNP or PLGA-MNP were comparable to those observed after administration of normal saline solution in control groups. IL-6 levels in blood plasma (data not shown) were of a much lower level and more variable indicating the localized nature of the response in the pulmonary tissue. This is in agreement with the in vitro results discussed above. Previously it has been shown that intranasal delivery of iron nanoparticles can lead to an increase in inflammatory markers including IL-6 . PLGA particles themselves have been shown to have a low propensity to cause immune responses when delivered directly to the lung . The biocompatible MNPs developed in this work may also be potentially exploited for targeting using external magnetic fields as demonstrated recently in nebulized mice .
Regional chemotherapy has been proposed as a treatment modality in a number of disease situations in order to increase exposure of the target tissues to the drug, while minimising systemic side-effects. Administration of drugs directly via inhalation allows localized drug delivery to the lungs and airways with smaller doses and minimal systemic toxicity . An additional reason for maximising total deposition and targeting drugs to their desired location is to improve the cost effectiveness of drug delivery . There is now increasing evidence to support the role of inhalation therapeutics in the treatment of various lung diseases. For example in lung cancer, nebulization therapeutics could be useful in 1) unresectable bronchioloalveolar carcinoma or main bronchus carcinoma with limited invasion, 2) endobronchial tumour relapse after surgery, 3) in situ carcinoma or synchronous, or 4) metachronous lesions in patients where a lesion has already been detected. However, few studies have documented the feasibility of applying nanotechnology for inhalation delivery of anticancer agents . Therefore, new aerosol delivery technologies are currently being developed to meet these goals of improved targeting, reduced waste and improved patient compliance. Vibrating mesh-based nebulizers (e.g., Aerogen nebulizer) can allow for sensitive tracking of flows or pressures during breathing manoeuvres and offer the potential for high efficiency delivery of aerosolized medications. These nebulizers have been used for breath actuated high efficiency aerosol delivery during mechanical ventilation of humans and rodents [61, 62]. Appropriate aerosol actuation during defined portions of the breath, allow for aerosol-free intervals, if required, thus avoiding drug deposition in the dead space of the patient interface, and even targeting of specific potions of the lung, e.g., introducing the aerosol in a small bolus at the end of inspiration to target the upper airways. Although the customizable vibrating mesh-type nebulizers have not been applied clinically to deliver MNP-based cancer therapeutics to the lung heretofore, the present study provides proof of principle for such targeting.