Critical Review in Designing Plant-Based Anticancer Nanoparticles against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), accounting for 85% of liver cancer cases, continues to be the third leading cause of cancer-related deaths worldwide. Although various forms of chemotherapy and immunotherapy have been investigated in clinics, patients continue to suffer from high toxicity and undesirable side effects. Medicinal plants contain novel critical bioactives that can target multimodal oncogenic pathways; however, their clinical translation is often challenged due to poor aqueous solubility, low cellular uptake, and poor bioavailability. Nanoparticle-based drug delivery presents great opportunities in HCC therapy by increasing selectivity and transferring sufficient doses of bioactives to tumor areas with minimal damage to adjacent healthy cells. In fact, many phytochemicals encapsulated in FDA-approved nanocarriers have demonstrated the ability to modulate the tumor microenvironment. In this review, information about the mechanisms of promising plant bioactives against HCC is discussed and compared. Their benefits and risks as future nanotherapeutics are underscored. Nanocarriers that have been employed to encapsulate both pure bioactives and crude extracts for application in various HCC models are examined and compared. Finally, the current limitations in nanocarrier design, challenges related to the HCC microenvironment, and future opportunities are also discussed for the clinical translation of plant-based nanomedicines from bench to bedside.

Multifunctional Zn and Ag co-doped bioactive glass nanoparticles for bone therapeutic and regeneration.

Bone cancer has traditionally been treated using surgery, radiotherapy, and/or chemotherapy. The nonspecific distribution of chemotherapy and implantable infections are significant risk factors for the failure of the bone to heal. Multifunctional zinc and silver co-doped bioactive glass nanoparticles (yAg-xZn-BGNPs) with a diameter of 150 ± 30 nm were successfully synthesized using modified sol-gel and two-step post-functionalization processes, tailored to provide antibacterial and anticancer activity whilst maintaining osteogenesis ability. Co-doped BGNPs with Zn and Ag did not significantly alter physicochemical properties, including size, morphology, glass network, and amorphous nature. Apatite-like layer was observed on the surface of yAg-xZn-BGNPs and resorbed in the simulated body fluid solution, which could increase their bioactivity. Human fetal osteoblast cell line (hFOB 1.19) treated with particles showed calcified tissue formation and alkaline phosphatase activity in the absence of osteogenic supplements in vitro, especially with 0.5Ag-1Zn-BGNPs. Moreover, these particles preferentially disrupted the metabolic activity of bone cancer cells (MG-63) and had an antibacterial effect against B. subtilis, E. coli, and S. aureus via the disc diffusion method. This novel 0.5Ag-1Zn-BGNP and 1Ag-1Zn-BGNPs, with wide-ranging ability to stimulate bone regeneration, to inhibit bone cancer cell proliferation, and to prevent bacterial growth properties, may provide a feasible strategy for bone cancer treatment. The 0.5Ag-1Zn-BGNPs and 1Ag-1Zn-BGNPs can be applied for the preparation of scaffolds or filler composites using in bone tissue engineering.

Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro.

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

Effect of silver nanospheres and nanowires on human airway smooth muscle cells: role of sulfidation.

Background: The toxicity of inhaled silver nanoparticles on contractile and pro-inflammatory airway smooth muscle cells (ASMCs) that control airway calibre is unknown. We explored the oxidative activities and sulfidation processes of the toxic-inflammatory response. Method: Silver nanospheres (AgNSs) of 20 nm and 50 nm diameter and silver nanowires (AgNWs), short S-AgNWs, 1.5 µm and long L-AgNWs, 10 µm, both 72 nm in diameter were manufactured. We measured their effects on cell proliferation, mitochondrial reactive oxygen species (ROS) release and membrane potential, and also performed electron microscopic studies. Main results and findings: The greatest effects were observed for the smallest particles with the highest specific surface area and greatest solubility that were avidly internalised. ASMCs exposed to 20 nm AgNSs (25 µg mL-1) for 72 hours exhibited a significant decrease in DNA incorporation (-72.4%; p < 0.05), whereas neither the 50 nm AgNSs nor the s-AgNWs altered DNA synthesis or viability. There was a small reduction in ASMC proliferation for the smaller AgNS, although Ag+ at 25 µL mL-1 reduced DNA synthesis by 93.3% (p < 0.001). Mitochondrial potential was reduced by both Ag+ (25 µg mL-1) by 47.1% and 20 nm Ag NSs (25 µg mL-1) by 40.1% (*both at p < 0.05), but was not affected by 50 nm AgNSs and the AgNWs. None of the samples showed a change in ROS toxicity. However, malondialdehyde release, associated with greater total ROS, was observed for all AgNPs, to an extent following the geometric size (20 nm AgNS: 213%, p < 0.01; 50 nm AgNS: 179.5%, p < 0.01 and L-AgNWs by 156.2%, p < 0.05). The antioxidant, N-acetylcysteine, prevented the reduction in mitochondrial potential caused by 20 nm AgNSs. The smaller nanostructures were internalised and dissolved within the ASMCs with the formation of non-reactive silver sulphide (Ag2S) on their surface, but with very little uptake of L-AgNWs. When ASMCs were incubated with H2S-producing enzyme inhibitors, the spatial extent of Ag2S formation was much greater. Conclusion: The intracellular toxicity of AgNPs in ASMCs is determined by the solubility of Ag+ released and the sulfidation process, effects related to particle size and geometry. Passivation through sulfidation driven by biogenic H2S can outcompete dissolution, thus reducing the toxicity of the smaller intracellular Ag nanostructures.

Label-Free Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Sulfur-Producing Enzymes inside Microglia Cells following Exposure to Silver Nanowires.

There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.

Frizzled-7-targeted delivery of zinc oxide nanoparticles to drug-resistant breast cancer cells.

There is a need for novel strategies to treat aggressive breast cancer subtypes and overcome drug resistance. ZnO nanoparticles (NPs) have potential in cancer therapy due to their ability to potently and selectively induce cancer cell apoptosis. Here, we tested the in vitro chemotherapeutic efficacy of ZnONPs loaded via a mesoporous silica nanolayer (MSN) towards drug-sensitive breast cancer cells (MCF-7: estrogen receptor-positive, CAL51: triple-negative) and their drug-resistant counterparts (MCF-7TX, CALDOX). ZnO-MSNs were coated on to gold nanostars (AuNSs) for future imaging capabilities in the NIR-II range. Electron and confocal microscopy showed that MSN-ZnO-AuNSs accumulated close to the plasma membrane and were internalized by cells. High-resolution electron microscopy showed that MSN coating degraded outside the cells, releasing ZnONPs that interacted with cell membranes. MSN-ZnO-AuNSs efficiently reduced the viability of all cell lines, and CAL51/CALDOX cells were more susceptible than MCF7/MCF-7-TX cells. MSN-ZnO-AuNSs were then conjugated with the antibody to Frizzled-7 (FZD-7), the receptor upregulated by several breast cancer cells. We used the disulphide (S-S) linker that could be cleaved with a high concentration of glutathione normally observed within cancer cells, releasing Zn2+ into the cytoplasm. FZD-7 targeting resulted in approximately three-fold amplified toxicity of MSN-ZnO-AuNSs towards the MCF-7TX drug-resistant cell line with the highest FZD-7 expression. This study shows that ZnO-MSs are promising tools to treat triple-negative and drug-resistant breast cancers and highlights the potential clinical utility of FZD-7 for delivery of nanomedicines and imaging probes specifically to these cancer types.

Laparoscopic fluorescence image-guided photothermal therapy enhances cancer diagnosis and treatment.

Endoscopy is the gold standard investigation in the diagnosis of gastrointestinal cancers and the management of early and pre-malignant lesions either by resection or ablation. Recently gold nanoparticles have shown promise in cancer diagnosis and therapeutics (theranostics). The combination of multifunctional gold nanoparticles with near infrared fluorescence endoscopy for accurate mapping of early or pre-malignant lesions can potentially enhance diagnostic efficiency while precisely directing endoscopic near infrared photothermal therapy for established cancers. The integration of endoscopy with near infrared fluorescence imaging and photothermal therapy was aided by the accumulation of our multifunctionalized PEG-GNR-Cy5.5-anti-EGFR-antibody gold nanorods within gastrointestinal tumor xenografts in BALB/c mice. Control mice (with tumors) received either gold nanorods or photothermal therapy, while study mice received both treatment modalities. Local (tumor-centric) and systemic effects were examined for 30 days. Clear endoscopic near infrared fluorescence signals were observed emanating specifically from tumor sites and these corresponded precisely to the tumor margins. Endoscopic fluorescence-guided near infrared photothermal therapy successfully induced tumor ablations in all 20 mice studied, with complete histological clearance and minimal collateral damage. Multi-source analysis from histology, electron microscopy, mass spectrometry, blood, clinical evaluation, psychosocial and weight monitoring demonstrated the inherent safety of this technology. The combination of this innovative nanotechnology with gold standard clinical practice will be of value in enhancing the early optical detection of gastrointestinal cancers and a useful adjunct for its therapy.

Towards multiplexed near-infrared cellular imaging using gold nanostar arrays with tunable fluorescence enhancement.

Sensitive detection of disease biomarkers expressed by human cells is critical to the development of novel diagnostic and therapeutic methods. Here we report that plasmonic arrays based on gold nanostar (AuNS) monolayers enable up to 19-fold fluorescence enhancement for cellular imaging in the near-infrared (NIR) biological window, allowing the application of low quantum yield fluorophores for sensitive cellular imaging. The high fluorescence enhancement together with low autofluorescence interference in this wavelength range enable higher signal-to-noise ratio compared to other diagnostic modalities. Using AuNSs of different geometries and therefore controllable electric field enhancement, cellular imaging with tunable enhancement factors is achieved, which may be useful for the development of multicolour and multiplexed platforms for a panel of biomarkers, allowing to distinguish different subcell populations at the single cell level. Finally, the uptake of AuNSs within HeLa cells and their high biocompatibility, pave the way for novel high-performance in vitro and in vivo diagnostic platforms.

Gold nanorod reshaping in vitro and in vivo using a continuous wave laser.

Gold nanorods (GNRs) are increasingly being investigated for cancer theranostics as they possess features which lend themselves in equal measures as contrast agents and catalysts for photothermal therapy. Their optical absorption spectral peak wavelength is determined by their size and shape. Photothermal therapy using GNRs is typically established using near infrared light as this allows sufficient penetration into the tumour matrix. Continuous wave (CW) lasers are the most commonly applied source of near infrared irradiation on GNRs for tumour photothermal therapy. It is perceived that large tumours may require fractionated or prolonged irradiation. However the true efficacy of repeated or protracted CW irradiation on tumour sites using the original sample of GNRs remains unclear. In this study spectroscopy and transmission electron microscopy are used to demonstrate that GNRs reshape both in vitro and in vivo after CW irradiation, which reduces their absorption efficiency. These changes were sustained throughout and beyond the initial period of irradiation, resulting from a spectral blue-shift and a considerable diminution in the absorption peak of GNRs. Solid subcutaneous tumours in immunodeficient BALB/c mice were subjected to GNRs and analysed with electron microscopy pre- and post-CW laser irradiation. This phenomenon of thermally induced GNR reshaping can occur at relatively low bulk temperatures, well below the bulk melting point of gold. Photoacoustic monitoring of GNR reshaping is also evaluated as a potential clinical aid to determine GNR absorption and reshaping during photothermal therapy. Aggregation of particles was coincidentally observed following CW irradiation, which would further diminish the subsequent optical absorption capacity of irradiated GNRs. It is thus established that sequential or prolonged applications of CW laser will not confer any additional photothermal effect on tumours due to significant attenuations in the peak optical absorption properties of GNRs following primary laser irradiation.

Silver nanoparticles reduce brain inflammation and related neurotoxicity through induction of H2S-synthesizing enzymes.

Silver nanoparticles (AgNP) are known to penetrate into the brain and cause neuronal death. However, there is a paucity in studies examining the effect of AgNP on the resident immune cells of the brain, microglia. Given microglia are implicated in neurodegenerative disorders such as Parkinson's disease (PD), it is important to examine how AgNPs affect microglial inflammation to fully assess AgNP neurotoxicity. In addition, understanding AgNP processing by microglia will allow better prediction of their long term bioreactivity. In the present study, the in vitro uptake and intracellular transformation of citrate-capped AgNPs by microglia, as well as their effects on microglial inflammation and related neurotoxicity were examined. Analytical microscopy demonstrated internalization and dissolution of AgNPs within microglia and formation of non-reactive silver sulphide (Ag2S) on the surface of AgNPs. Furthermore, AgNP-treatment up-regulated microglial expression of the hydrogen sulphide (H2S)-synthesizing enzyme cystathionine-γ-lyase (CSE). In addition, AgNPs showed significant anti-inflammatory effects, reducing lipopolysaccharide (LPS)-stimulated ROS, nitric oxide and TNFα production, which translated into reduced microglial toxicity towards dopaminergic neurons. Hence, the present results indicate that intracellular Ag2S formation, resulting from CSE-mediated H2S production in microglia, sequesters Ag+ ions released from AgNPs, significantly limiting their toxicity, concomitantly reducing microglial inflammation and related neurotoxicity.

Effect of pulmonary surfactant on the dissolution, stability and uptake of zinc oxide nanowires by human respiratory epithelial cells.

Inhaled nanoparticles (NPs) have high-deposition rates in the alveolar region of the lung but the effects of pulmonary surfactant (PS) on nanoparticle bioreactivity are unclear. Here, the impact of PS on the stability and dissolution of ZnO nanowires (ZnONWs) was investigated, and linked with their bioreactivity in vitro with human alveolar epithelial type 1-like cells (TT1). Pre-incubation of ZnONWs with Curosurf® (a natural porcine PS) decreased their dissolution at acidic pH, through the formation of a phospholipid corona. Confocal live cell microscopy confirmed that Curosurf® lowered intracellular dissolution, thus delaying the onset of cell death compared to bare ZnONWs. Despite reducing dissolution, Curosurf® significantly increased the uptake of ZnONWs within TT1 cells, ultimately increasing their toxicity after 24 h. Although serum improved ZnONW dispersion in suspension similar to Curosurf®, it had no effect on ZnONW internalization and toxicity, indicating a unique role of PS in promoting particle uptake. In the absence of PS, ZnONW length had no effect on dissolution kinetics or degree of cellular toxicity, indicating a less important role of length in determining ZnONW bioreactivity. This work provides unique findings on the effects of PS on the stability and toxicity of ZnONWs, which could be important in the study of pulmonary toxicity and epithelial-endothelial translocation of nanoparticles in general.

Translocation of Functionalized Multi-Walled Carbon Nanotubes across Human Pulmonary Alveolar Epithelium: Dominant Role of Epithelial Type 1 Cells.

Uptake and translocation of short functionalized multi-walled carbon nanotubes (short-fMWCNTs) through the pulmonary respiratory epithelial barrier depend on physicochemical property and cell type. Two monoculture models, immortalized human alveolar epithelial type 1 (TT1) cells and primary human alveolar epithelial type 2 cells (AT2), which constitute the alveolar epithelial barrier, were employed to investigate the uptake and transport of 300 and 700 nm in length, poly(4-vinylpyridine)-functionalized, multi-walled carbon nanotubes (p(4VP)-MWCNTs) using quantitative imaging and spectroscopy techniques. The p(4VP)-MWCNT exhibited no toxicity on TT1 and AT2 cells, but significantly decreased barrier integrity (*p < 0.01). Uptake of p(4VP)-MWCNTs was observed in 70% of TT1 cells, correlating with compromised barrier integrity and basolateral p(4VP)-MWCNT translocation. There was a small but significantly greater uptake of 300 nm p(4VP)-MWCNTs than 700 nm p(4VP)-MWCNTs by TT1 cells. Up to 3% of both the 300 and 700 nm p(4VP)-MWCNTs reach the basal chamber; this relatively low amount arose because the supporting transwell membrane minimized the amount of p(4VP)-MWCNT translocating to the basal chamber, seen trapped between the basolateral cell membrane and the membrane. Only 8% of AT2 cells internalized p(4VP)-MWCNT, accounting for 17% of applied p(4VP)-MWCNT), with transient effects on barrier function, which initially fell then returned to normal; there was no MWCNT basolateral translocation. The transport rate was MWCNT length modulated. The comparatively lower p(4VP)-MWCNT uptake by AT2 cells is proposed to reflect a primary barrier effect of type 2 cell secretions and the functional differences between the type 1 and type 2 alveolar epithelial cells.

High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells.

Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic, quantitative and multi-scale techniques in understanding the different cellular processing routes of functionalised nanomaterials. This correlative approach has wide implications for assessing the biopersistence of MWNT aggregates elsewhere in the body, in particular their interaction with macrophages in the lung.

Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells.

BACKGROUND: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo. METHODS: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1-100 µg/ml (1.25-125 µg/cm(2); 0 µg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy). RESULTS: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP. CONCLUSION: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

Functional consequences for primary human alveolar macrophages following treatment with long, but not short, multiwalled carbon nanotubes.

PURPOSE: Multiwalled carbon nanotubes (MWCNTs) are a potential human health hazard, primarily via inhalation. In the lung, alveolar macrophages (AMs) provide the first line of immune cellular defense against inhaled materials. We hypothesized that, 1 and 5 days after treating AMs with short (0.6 µm in length; MWCNT-0.6 µm) and long (20 µm in length; MWCNT-20 µm) MWCNTs for 24 hours, AMs would exhibit increased markers of adverse bioreactivity (cytokine release and reactive oxygen species generation) while also having a modified functional ability (phagocytosis and migration). METHODS: Primary human AMs were treated with short and long MWCNTs for 24 hours, 1 and 5 days after which toxicity end points, including cell death, reactive oxygen species generation, and inflammatory mediator release, were measured. AM functional end points involving phagocytic ability and migratory capacity were also measured. RESULTS: AM viability was significantly decreased at 1 and 5 days after treatment with MWCNT-20 µm, while superoxide levels and inflammatory mediator release were significantly increased. At the same time, there was reduced phagocytosis and migratory capacity alongside increased expression of MARCO; this coincided with frustrated phagocytosis observed by scanning electron microscopy. In contrast, the adverse bioreactivity of the shorter MWCNT-0.6 µm with AMs (and any resulting reduction in AM functional ability) was substantially less marked or absent altogether. CONCLUSION: This study shows that after 24-hour treatment with long, but not short, MWCNTs, AM function is severely affected up to 5 days after the initial exposure. This has potentially significant pathophysiological consequences for individuals who may be intentionally (via therapeutic applications) or unintentionally exposed to these nanomaterials.

Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: Investigating crystalline phase as a critical determinant.

There can be significant variability between bioreactivity studies of nanomaterials that are apparently the same, possibly reflecting differences in the models used and differing sources of experimental material. In this study, we have generated two crystal forms of titanium dioxide nanoparticles (nano-TiO2), pure anatase and pure rutile to address the hypothesis that the bioreactivity of these nanoparticles with human alveolar epithelium will depend on their crystal phase. We used a human alveolar type-I-like epithelial cell model (TT1; generated in-house from primary human alveolar epithelial type II cells); these cells cover 95% of the alveolar epithelial surface area and are an important target cell for inhaled nanomaterials. Using literature as a guide, we hypothesised that pure anatase nano-TiO2 would display greater bioreactivity with TT1 cells in comparison to pure rutile nano-TiO2. However, we found the profile and pattern of inflammatory mediator release was similar between these two nano-TiO2 formats, although pure rutile treatment caused a small, but consistently greater, response for IL-6, IL-8 and MCP-1. Interestingly, the temporal induction of oxidative stress (increased reactive oxygen species levels and depleted glutathione) varied markedly between the different nano-TiO2 formats. We have shown that a combination of using nanomaterials synthesised specifically for toxicological study and the use of a highly relevant, reproducible human lung cell model, offers a useful approach to delineating the physicochemical properties of nanomaterials that may be important in their cellular reactivity.

Critical determinants of uptake and translocation of nanoparticles by the human pulmonary alveolar epithelium.

The ability to manipulate the size and surface properties of nanomaterials makes them a promising vector for improving drug delivery and efficacy. Inhalation is a desirable route of administration as nanomaterials preferentially deposit in the alveolar region, a large surface area for drug absorption. However, as yet, the mechanisms by which particles translocate across the alveolar epithelial layer are poorly understood. Here we show that human alveolar type I epithelial cells internalize nanoparticles, whereas alveolar type II epithelial cells do not, and that nanoparticles translocate across the epithelial monolayer but are unable to penetrate the tight junctions between cells, ruling out paracellular translocation. Furthermore, using siRNA, we demonstrate that 50 nm nanoparticles enter largely by passive diffusion and are found in the cytoplasm, whereas 100 nm nanoparticles enter primarily via clathrin- and also caveolin-mediated endocytosis and are found in endosomes. Functionalization of nanoparticles increases their uptake and enhances binding of surfactant which further promotes uptake. Thus, we demonstrate that uptake and translocation across the pulmonary epithelium is controlled by alveolar type I epithelial cells, and furthermore, we highlight a number of factors that should be considered when designing new nanomedicines in order to improve drug delivery to the lung.

Aqueous cationic, anionic and non-ionic multi-walled carbon nanotubes, functionalised with minimal framework damage, for biomedical application.

The use of a thermochemical grafting approach provides a versatile means to functionalise as-synthesised, bulk multi-walled carbon nanotubes (MWNTs) without altering their inherent structure. The associated retention of properties is desirable for a wide range of commercial applications, including for drug delivery and medical purposes; it is also pertinent to studies of intrinsic toxicology. A systematic series of water-compatible MWNTs, with diameter around 12 nm have been prepared, to provide structurally-equivalent samples predominantly stabilised by anionic, cationic, or non-ionic groups. The surface charge of MWNTs was controlled by varying the grafting reagents and subsequent post-functionalisation modifications. The degree of grafting was established by thermal analysis (TGA). High resolution transmission electron microscope (HRTEM) and Raman measurements confirmed that the structural framework of the MWNTs was unaffected by the thermochemical treatment, in contrast to a conventional acid-oxidised control which was severely damaged. The effectiveness of the surface modification was demonstrated by significantly improved solubility and stability in both water and cell culture medium, and further quantified by zeta-potential analysis. The grafted MWNTs exhibited relatively low bioreactivity on transformed human alveolar epithelial type 1-like cells (TT1) following 24 h exposure as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase release (LDH) assays. The exposure of TT1 cells to MWNTs suppressed the release of the inflammatory mediators, interleukin 6 (IL-6) and interleukin 8 (IL-8). TEM cell uptake studies indicated efficient cellular entry of MWNTs into TT1 cells, via a range of mechanisms. Cationic MWNTs showed a more substantial interaction with TT1 cell membranes than anionic MWNTs, demonstrating a surface charge effect on cell uptake.

Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy.

Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle-cell interactions.

Sulfidation of silver nanowires inside human alveolar epithelial cells: a potential detoxification mechanism.

Silver nanowires (AgNWs) are being developed for use in optoelectronics. However before widespread usage, it is crucial to determine their potential effects on human health. It is accepted that Ag nanoparticles (AgNPs) exert toxic effects by releasing Ag(+) ions, but much less is known about whether Ag(+) reacts with compounds, or any downstream bioactive effects of transformed AgNPs. Analytical high-resolution transmission electron microscopy has been employed to elucidate cellular uptake and reactivity of AgNWs inside human alveolar epithelial type 1-like cells. AgNWs were observed in the cytoplasm and membrane-bound vesicles, and precipitation of Ag2S within the cell occurred after 1 h exposure. Cell viability studies showed no evidence of cytotoxicity and reactive oxygen species were not observed on exposure of cells to AgNWs. We suggest that Ag2S formation acts as a 'trap' for free Ag(+), significantly limiting short-term toxicological effects - with important consequences for the safety of Ag-nanomaterials to human health.