Effects of nanomaterials on human buccal epithelium

EFFECTS OF NANOMATERIALS ON HUMAN BUCCAL EPITHELIUM FANG WANRU (B.Sc., NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________________________ FANG WANRU 23 AUGUST 2012 II Acknowledgements First and foremost, I would like to express my heartfelt gratitude to my supervisor Assistant Professor Catherine Hong Hsu Ling for her mentorship and the opportunity to work under her. Her suggestions and critical comments have made this thesis possible. I would like to extend my sincere thanks to my co-supervisor Dr Sum Chee Peng, for his advice and encouragement throughout the course of study. I am greatly indebted to Assistant Professor David Leong Tai Wei for allowing me to carry out this project in his lab. Without his support, guidance and constructive criticisms, this M.Sc. would not be possible. Special thanks go to my co-workers, Inggrid, Marie, Ming Han, Samantha and Sing Ling for their support and invaluable advices. You have accompanied me through the ups and downs of lab work and made my stay in the lab enjoyable. Last, but not least, sincere thanks to my family and friends for their constant support and patience throughout my M.Sc. studies. III Table of Contents Declaration .................................................................................................................... II Acknowledgements ...................................................................................................... III Table of Contents .........................................................................................................IV Abstract ...................................................................................................................... VII List of Tables ...............................................................................................................IX List of Figures ............................................................................................................... X Abbreviations ...............................................................................................................XI Chapter 1: Introduction .................................................................................................. 1 1.1 Nanotechnology and Nanomaterials ............................................................... 2 1.2 Nanotechnology – a friend or foe? .................................................................. 2 1.2.1 Nanotechnology – a friend ....................................................................... 2 1.2.2 Nanotechnology – a foe ........................................................................... 5 1.3 Nanomaterials in oral care products ................................................................ 8 1.3.1 Hydroxyapatite NM ................................................................................. 9 1.3.2 Silicon dioxide NM ................................................................................ 10 1.3.3 Titanium dioxide NM ............................................................................ 12 1.3.4 Silver NM............................................................................................... 13 1.4 Human oral mucosa and nanomaterials ........................................................ 13 1.5 Experimental approach and study rationale .................................................. 15 Chapter 2: Materials and Methods ............................................................................... 17 2.1 Identification of inorganic NM in commercially available toothpastes ........ 18 2.1.1. Extraction of inorganic compounds in toothpastes ................................ 18 2.1.2. Identification of elements present in toothpaste extracts ....................... 18 2.1.3. Analysis of surface chemistry of toothpaste extracts ............................. 19 2.1.4. Determination of hydrodynamic size distribution ................................. 19 IV 2.1.5. Quantification of DNA .......................................................................... 19 2.1.6. Determination of physiologically relevant concentrations of pristine NM ................................................................................................................ 20 2.2 Pristine NM ................................................................................................... 21 2.2.1 Tagging of Pristine NM with FITC ....................................................... 21 2.2.2 Characterization of pristine NM ............................................................ 22 2.3 TR146 Human buccal epithelial cells culture ............................................... 22 2.4 Internalization of NM into cells .................................................................... 23 2.4.1 Confocal microscopy imaging ............................................................... 23 2.4.2 Quantification of cellular uptake of NM ................................................ 23 2.4.3 Membrane and cytosolic fractions ......................................................... 24 2.5 Evaluation of cytotoxicity ............................................................................. 24 2.6 Evaluation of inflammatory response ........................................................... 26 2.6.1 Intracellular Reactive Oxygen Species (ROS) level measurement........ 26 2.6.2 Inflammatory genes expression profile using Quantitative Real Time PCR (qPCR) ........................................................................................... 27 2.6.3 Secreted embryonic alkaline phosphatase (SEAP) assay ...................... 28 2.7 Wound healing assay..................................................................................... 28 2.7.1 2.8 Blocking of clathrin-mediated endocytosis ........................................... 29 Statistical analysis ......................................................................................... 29 Chapter 3: Results ........................................................................................................ 30 3.1 Determination of NM found in commercially available toothpastes ............ 31 3.1.1 Characterization of NM found in commercially available toothpastes . 31 3.1.2 Effect of toothpaste extracts on cell proliferation rate ........................... 33 3.1.3 Calculation of physiologically relevant concentrations of NM ............. 34 3.2 Characterization of pristine NM .................................................................... 35 3.3 Effects of pristine NM on human buccal epithelium .................................... 37 V 3.3.1 Internalization and bio-distribution of NM into TR146 human buccal epithelial cells ........................................................................................ 37 3.3.2 Uptake of NM by human buccal epithelial cells and interaction with membrane and cytosolic proteins........................................................... 41 3.3.3 HA, TiO2 and SiO2 NM are non-cytotoxic ............................................. 45 3.3.4 Nanomaterials induced mild inflammatory response ............................ 46 3.3.5 NM impaired wound healing ................................................................. 51 3.3.6 Uptake of NM into human buccal epithelial cells via clathrin mediated endocytosis ............................................................................................. 55 Chapter 4: Discussion .................................................................................................. 59 4.1 Characterization of NM in commercially available toothpastes ................... 60 4.1.1 Effect of inorganic toothpaste extracts on cell proliferation rate ........... 61 4.2 Characterization of pristine NM .................................................................... 61 4.3 Effects of pristine NM ................................................................................... 62 4.3.1 Internalization and bio-distribution of pristine NM ............................... 62 4.3.2 Interaction of HA, SiO2 and TiO2 NM with membrane and cytosolic proteins ................................................................................................... 64 4.3.3 Cell cycle progression and apoptosis ..................................................... 65 4.3.4 Inflammatory response........................................................................... 67 4.3.5 NM impaired wound healing and their route of uptake into cells ......... 70 Chapter 5: Conclusion and Future Work ..................................................................... 72 5.1 Conclusion..................................................................................................... 73 5.2 Future work ................................................................................................... 74 Chapter 6: References .................................................................................................. 76 VI Abstract Nanotechnology has tremendous potential to change the quality and effectiveness of manufactured products. The rapid growth of nanotechnology has thus led to a high prevalence of nanomaterials (NM) in consumer products such as cosmetics, sun cream and even toothpastes. Due to their widespread usage, it is important to understand the potential impact NM exert on human health. Presently, there is limited information on the impact of NM on human buccal epithelium; even though NM are commonly found in oral care products. We sought to assess the impact of NM found in commercially available toothpastes using an in vitro human buccal epithelial cell model. By understanding the effects of NM exposure on human health, we may be able to design NM that possess the same desired properties for commercial use, but with improved safety. In this study, we characterized the inorganic NM present in four commercially available toothpastes using X-ray photoelectron spectroscopy and ICP-MS. The effect of these inorganic toothpaste extracts on the proliferation rate of monolayer human buccal epithelial cells was monitored from the amount of DNA content present in the cells. However, the effects of the individual NM on human oral buccal epithelium cannot be accurately quantified because of the complex mixture of inorganic substances present in the toothpastes extracts. Hence, commercially available pristine NM were employed to elucidate the potential toxic effects. The internalization and bio-distribution of pristine NM in human buccal epithelium were determined with confocal microscopy. The mechanism of NM uptake into human buccal epithelium was investigated using a specific endocytosis inhibitor – monodansylcadaverine VII (MDC). Via immunoblotting, we further investigated the impact of pristine NM on cell cycle progression and apoptotic pathway. The oxidative stress level and inflammatory responses induced by NM were assessed by measuring reactive oxygen species (ROS) production and expression levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and nuclear factor kappa B (NFκB) respectively. The effect of NM on wound healing was determined by performing an in vitro scratch assay. We identified the presence of HA, SiO2 and TiO2 NM in commercially available toothpastes. All pristine NM tested were internalized into the cells, regardless of the chemistry and size. Clathrin-mediated endocytosis was the main route of entry for the NM tested. The NM uptake led to elevated ROS level and increase expressions of TNF-α, IL-6 and NFκB genes. However, pristine NM did not affect protein expressions involved in cell cycle progression and apoptotic events suggesting the existence of an adaptive mechanism to counteract NM induced oxidative stress. The cells’ wound healing capacity was impaired by pristine HA, SiO2 and TiO2 NM. This finding was confirmed by the significant recovery of wound healing when internalization of NM were prevented by blocking clathrin-mediated endocytosis. VIII List of Tables Table 1 | Elemental composition detected in the surface chemistry of inorganic extracts from commercially available toothpastes. ...................................................... 32 Table 2 | Identification and concentration of metal oxide-based nanomaterials present in inorganic extracts of commercially available toothpastes via ICP-M/S .................. 32 Table 3 | Amount of HA, SiO2 and TiO2 present in 0.25 gms of commercially available toothpastes. ................................................................................................... 35 Table 4 | Size characterization of pristine NM and their surface charges.................... 36 IX List of Figures Figure 1 | Identification of elements present in inorganic extracts of commercially available toothpastes. ................................................................................................... 31 Figure 2 | Distribution of particle size present in toothpaste extracts. ......................... 33 Figure 3 | Toothpaste extracts reduced TR146 human buccal epithelial cells proliferation rate........................................................................................................... 34 Figure 4 | Physical characterization of pristine NM.. .................................................. 36 Figure 5 | Internalization of FITC-HA NM by TR146 human buccal epithelial cells . 38 Figure 6 | Internalization of FITC-SiO2 NM by TR146 human buccal epithelial cells ...................................................................................................................................... 39 Figure 7 | Internalization of FITC-TiO2 NM by TR146 human buccal epithelial cells ...................................................................................................................................... 40 Figure 8 | Tracking of NM uptake by TR146 human buccal epithelial cells ............... 42 Figure 9 | Internalization of NM and interaction of NM with membrane and cytosolic proteins ......................................................................................................................... 44 Figure 10 | Exposure to NM did not affect cell cycle progression or activated apoptotic pathway ........................................................................................................ 46 Figure 11 | NM induced elevated ROS expression level in TR146 human buccal epithelial cells .............................................................................................................. 47 Figure 12 | NM induced dose dependent elevation of IL-6 expression ....................... 48 Figure 13 | NM induced dose dependent elevation of TNF-α expression ................... 49 Figure 14 | NM induced dose dependent elevation of NFκB ...................................... 50 Figure 15 | HA NM impaired wound healing .............................................................. 52 Figure 16 | SiO2 NM hindered wound healing............................................................. 53 Figure 17 | TiO2 NM impaired wound healing ............................................................ 54 Figure 18 | HA NM internalized into cells via clathrin-mediated pathway ................. 56 Figure 19 | SiO2 NM internalized into cells via clathrin-mediated pathway ............... 57 Figure 20 | TiO2 NM internalized into cells via clathrin-mediated pathway ............... 58 X Abbreviations Ag - Silver APTES - Aminopropyltriethoxysilane BSA - Bovine Serum Albumin C - Carbon CDK - Cyclin Dependent Kinases Cu - Copper DCFH-DA - Dichlorofluorescin Diacetate DMEM/F12 - Dulbecco’s Modified Eagle’s Media : nutrient mixture F-12 DLS - Dynamic Light Scattering ELISA - Enzyme-Linked Immunosorbent Assay FBS - Fetal Bovine Serum G1 - gap1 G2/M - gap2/mitosis GI - Gastro-Intestinal HA - Hydroxyapatite HRP - Horseradish Peroxidase hTBP - Human TATA-box Binding Protein IL-6 - Interleukin-6 MDC - Monodansylcadaverine NFκB - Nuclear Factor Kappa B NM - Nanomaterials nm - Nanometer NO - Nitric Oxide NTA - Nanomaterials Tracking Analysis XI O2- - Superoxide Anion ONOO- - Peroxynitrite PBS - Phosphate Buffered Saline PEN - Project on Emerging Nanotechnology PDI - Polydispersity Index qPCR - Quantitative Real Time PCR ROS - Reactive Oxygen Species SEAP - Secreted Embryonic Alkaline Phosphatase SiO2 - Silicon Dioxide SLS - Sodium Laurel Sulphate TEM - Transmission Electron Microscopy TiO2 - Titanium Dioxide TNF-α - Tumor Necrosis Factor-Alpha UVA - Ultraviolet A UVB - Ultraviolet B XPS - X-ray Photoelectron Spectroscopy ZnO - Zinc Oxide XII Chapter 1: Introduction 1.1 Nanotechnology and Nanomaterials Nanotechnology is a fast growing research niche that has led to significant breakthroughs, allowing the manipulation of materials at atomic level to create nanosized materials [1, 2]. Nanomaterials (NM) refer to materials with at least one dimension in the range of nanometer (nm) [3]. The properties of NM often differ from their bulk materials due to factors such as size, shape, surface chemistry, reactivity and purity [4, 5]. The NM are usually used in their pristine forms or can be modified by the addition of surface functional groups to achieve certain desired properties [6, 7]. 1.2 Nanotechnology – a friend or foe? 1.2.1 Nanotechnology – a friend Nanotechnology has been hailed to have tremendous potential to change the quality and effectiveness of manufactured products. This has brought about improvements to numerous sectors of the economy such as healthcare, consumer products, energy and information technology. As of July 2012, the inventory list managed by US Project on Emerging Nanotechnology (PEN) listed at least 1317 globally sold consumer products containing NM. Some of the consumer products which frequently contain engineered NM include personal care products, textiles, and sports equipments [8-10]. The number of consumer products with NM additives is projected to exceed 3000 products by 2020 [11]. Silver (Ag), carbon (C), titanium oxide (TiO2), silicon dioxide (SiO2) and zinc oxide (ZnO) are the top 5 NM commonly added in consumer products [11]. 2 SiO2, TiO2 and ZnO NM are commonly added to personal care products such as deodorants, face creams and toothpastes [12] for their anti-irritant properties [13, 14]. For instance, SiO2 NM are added to sunscreen to coat the organic components to prevent the direct contact of the organic components with the human skin, reducing the potential of local irritation [15]. TiO2 and ZnO NM are known for their ability to absorb ultraviolet B (UVB; 290-320 nm) and scatter ultraviolet A (UVA; 320-400 nm) [16]. As such, these metal oxide NM are frequently found in sunscreens [17, 18]. Additionally, because of their small size, TiO2 and ZnO NM scatter very little visible light and do not form an opaque layer when applied on skin, a desirable property among consumers [17, 18]. NM such as HA and SiO2 have also been explored as drug delivery vehicles or diagnostic probes in the medical field [19, 20] mainly due to their high surface area to volume ratio which is a function of their small sizes. The small sizes of NM are also thought to increase specificity of drug delivery to target sites [21, 22] as the large functional surfaces of the NM allow them to bind or adsorb compounds such as drugs, probes and proteins more efficiently. Additionally, the drug can be stored in its matrix form for sustained release of the medication via diffusion or degradation of the matrix, reducing the need for frequent dosing regimens [19, 23]. The small size of NM is also useful in the food industry [24]; one such example is the addition of SiO2 NM to Nanoceuticals™ Slim Shake Chocolate. The manufacturer claims that the addition of NM enhances the chocolate flavour of the product by 3 increasing the surface area of the product that comes into contact with the taste buds [24]. The anti-microbial properties of NM allow them to be increasingly utilized during the food production process [25-28] and in the medical equipment industry [29, 30]. These NM are used to coat surfaces to create nano-scale topography to discourage bacterial adherence. TiO2 and SiO2 NM are used frequently to prevent growth of biofilms on the surfaces of steel and glass in the food processing industry [31-33], while Ag and copper (Cu) NM are widely used to coat surfaces of medical equipments or dental implants [29, 30]. The photocatalytic anti-microbial property of NM, in particular TiO2 NM is also harnessed in various industries. TiO2 NM are added to food products to increase the quality and shelf life of food product [25-28]. The photocatalytic property of TiO2 NM is also utilized in treatment of waste water to aid removal of organic pollutants [34, 35] and/or to allow ease of removal of arsenate [36, 37]. Metallic based NM, such as Cu, Ag, TiO2, and ZnO are incorporated in food packaging material to enhance the light-proofing properties by strengthening mechanical and barrier properties of the polymers in the packing material and, thereby preventing the photo-degradation of plastics [24, 38]. 4 1.2.2 Nanotechnology – a foe While the positive impacts of nanotechnology are widely publicized, recent studies have documented the toxic properties of NM [39-41]. Concerns have been raised because of the ability of the NM to enter the cells, accumulate in various locations in the body and potentially trigger downstream biological effect [42]. At nanometer dimensions, inert elements can become highly active due to the quantum size effect [43]. In conventional bulk materials, the toxicity is determined by three key factors – chemical composition, dosage and exposure route. As the size decreases, the surface area to volume ratio increases exponentially, bringing about unique physico-chemical properties. Therefore, at the nanoscale level, more factors need to be considered when evaluating the toxicity of the NM [44]. In addition to the size and surface chemistry, the structure of NM also plays an important role in determining the toxicity of materials. The properties are further affected by the aggregation of NM that could change the reactivity of NM and result in detrimental effects. 1.2.2.1 Route of entry for nanomaterials The main routes of entry of NM into the body are by inhalation, topical absorption/penetration and ingestion [39]. When inhaled, most bulk sized particles will be trapped and cleared by the respiratory tract. In contrast, NM might not be efficiently phagocytosed due to their small size and therefore can accumulate in the highly vascularized alveoli regions of the lungs [40, 45], leading to inflammatory cytokines production and ensuing inflammation [46, 47]. For instance, deposition of NM in the alveoli have been reported to inhibit normal respiratory function, cause chronic inflammation and lead to pulmonary fibrosis [48]. The accumulation of NM 5 in the alveoli could also lead to cell death [49]. In addition to its local effect, NM could enter the circulatory system and translocate to vital organs [39]. Inhaled Ag NM can be deposited in the liver and kidneys [50] and cause organ damage. Local organ damage is thought to be caused by oxygen radicals which is produced when the macrophages in the kidneys or liver attempt to remove the accumulated NM via phagocytosis [51]. Animal studies have demonstrated that Ag NM deposited in the liver can inhibit mitochondria activity and reduce the amount of available energy for the cells [52, 53]. The constant low level of energy available for the cells could result in impaired antioxidant defence and/or weak immunity response [54]. Furthermore, the presence of Ag NM reduces the concentration of glutathione which is necessary to protect the cells against damage caused by ROS [51, 55]. The association of low levels of glutathione [56] and auto-immune and degenerative diseases (i.e. Alzheimer's disease, Parkinson's disease) has been reported which suggests that repeated exposure to Ag NM may put individuals at risk for development of these conditions. The skin is the largest organ in the human body that is constantly exposed to the environment and therefore is potentially a major route for NM to enter the body. The ability of NM to penetrate the intact skin is debatable [57-59]. However, studies have shown that significant absorption of NM can occur when the skin is damaged [60, 61]. Since NM is a frequent component in wound dressings for their anti-bacterial properties [62, 63], NM can penetrate through the broken skin into the bloodstream and subsequently translocate to the various organs. 6 Due to the increasing trend of incorporating NM in food and beverages or food packaging, the possibility of ingestion and absorption of NM through the gastrointestinal (GI) tract is elevated. In an animal study where the mice were fed with Zn NM, the mice developed diarrhoea, vomiting, anorexia and subsequently expired due to intestinal blockage [64]. The authors proposed that the pH in the GI tract increased NM agglomeration, which caused obstruction of the GI tract and consequently death [64]. Another recent animal study found that nano-sized polystyrene, a FDA-approved NM in food additives, disrupted iron absorption from the gut epithelium when fed to chickens [65]. This is an important health hazard as iron is essential for red blood cells production. Similarly, in two other in vivo rat studies, ingestion of Ag NM led to elevated alkaline phosphatase and cholesterol levels, indicating bile duct obstruction and liver damage [50, 66]. From the animal studies, it is well documented that NM can enter the circulatory system via various portals (e.g., skin, GI etc) and be distributed to various locations in the body such as the liver, spleen and lungs [67]. As the NM come into contact with the cells, they may trigger endocytosis by binding to specific receptors on the cellular membrane and be internalized into the cells. Some of the suggested mechanisms for the NM uptake into the cells include passive diffusion [68], lipid rafts [61] and/or active endocytosis pathways such as clathrin and caveolin mediated endocytosis [6971]. Since different types of NM display variability in toxic potency and cellular uptake [72], it is unlikely that the same mechanisms are applicable for all types of NM. 7 The effects of internalisation, prolonged NM exposure and the intracellular targets of NM are still largely unknown in humans. Reports have suggested that the internalized NM by the cells may mimic ligands and bind to specific receptors resulting in oxidative stress which triggers inflammatory response and DNA mutation [68]. Other detrimental effects observed include structural damage to mitochondria and cell death [73, 74]. Several studies have refuted the toxicity profile of NM and their ability to induce apoptosis [75-77]. However, the absence of apoptosis does not necessarily confirm the benign nature of NM. NM-induced oxidative stress has been demonstrated to increase the risk of certain neurological and auto-immune diseases, and cancers [4, 39]. Furthermore, the internalized NM have been shown to disrupt cell cycle progression [78-80], though these effects induced by the NM have not yet been studied exhaustively. Although, nanotechnology has dramatically improved various sectors of the economy, studies have also highlighted the potential health risks following NM exposure. As the different properties of NM may result in them having unique toxicity profile, there is a need to ascertain the safety of NM prior to human application. 1.3 Nanomaterials in oral care products NM are frequently added in oral hygiene products due to their desirable anti-bacterial and anti-irritant properties. In recent years, nanotechnology has been identified as one 8 of the novel strategies in the management of bacterial biofilms and remineralization in tooth decay [81]. Examples of NM frequently added to oral care products include Ag, hydroxyapatite (HA), SiO2 and TiO2 NM. Currently, most studies have concentrated on the anti-microbial ability of NM in oral care products [30, 82-84]. However, the toxic profile of these NM is still unknown. Presently, there is no authority regulating the use of NM in personal care products. Given the rising trend for incorporation of NM into personal care products and uncontrolled human exposure to such products, it is important to systematically assess the potential health hazards of their use. This begins with an understanding of the biological and molecular mechanisms so as to better predict exposure effects. This knowledge will aid in devising strategies to minimize NM related health risks as well as to design NM that possess the same useful properties but without the deleterious effects. 1.3.1 Hydroxyapatite NM Hydroxyapatite (HA) is an inorganic mineral composed of calcium and phosphate [Ca10(PO4)6(OH)2] and is a major inorganic component found in mammalian bones and teeth. HA’s structure resembles tooth enamel [85] and is added to toothpaste because of its capability to restore the enamel layer, treat hypersensitivity and increase the tooth’s resistance to dental decay [86-90]. Due to their biocompatibility and bioactivity, HA NM are also extensively used as a bone defect filling material [91] and being tested as a delivery vehicle for various therapeutic agents such as 9 growth factors, anticancer drugs, enzymes, antibiotics and antigens for slow release vaccination [92]. There are conflicting views with regards to the cellular response to HA NM. Some studies have reported that HA NM inflict minimal adverse effects on the cells [93, 94], while others have proposed that HA NM can affect cell morphology, inhibit cell proliferation, induce cytokines production and apoptosis [91, 92, 95]. Currently, most of the research studies conducted in the field of dentistry are focused on the ability of HA NM to manage biofilm growth and re-mineralize the tooth surface [87, 88, 9698]. There remains limited information on the cellular effects of HA NM on the human buccal mucosa despite the widespread use of HA NM in oral care products [99, 100]. 1.3.2 Silicon dioxide NM Due to their unique biochemical properties, SiO2 NM are also widely used in biomedical applications such as drug delivery systems, cells labelling, cancer therapy, medical diagnostics, DNA delivery and tissue engineering [101]. Other uses include additives in cosmetics, paints and as a food or animal feed ingredient [102, 103]. SiO2 NM are also frequently found in toothpastes for their abrasive property which aids in the removal of food stains and plaque [104]. As with HA NM experiments, most of the research efforts on SiO2 NM have focused on SiO2 NM’s ability to control oral biofilm. SiO2 NM have been shown to remove adherent bacterial more effectively compared to conventional polishing toothpastes [105]. However, it is still unclear if SiO2 NM can effectively inhibit mineralization of the polished teeth 10 surfaces and reduce plaque formation [30]. Additionally, SiO2 NM have also been employed to coat surfaces to create nanoscale surface topography in order to reduce adherence and growth of C. albicans [106]. The ability of SiO2 NM to induce toxicity is largely dependent on the surface modification. Mesoporous SiO2 NM exhibit high biocompatibility and are widely used for therapeutic or diagnostic purposes [107, 108]. However, SiO2 NM with modified surface properties may be potentially harmful. For instance, the surface chemistry of SiO2 NM have been altered to release nitric oxide (NO) in order to increase their efficacy against biofilm bacteria [109, 110]. NO is a reactive and unstable radical, which can react with superoxide anion (O2-) to produce peroxynitrite (ONOO-), which is capable of inducing oxidative stress [111, 112]. The induction of oxidative stress is a concern as it can potentially induce numerous detrimental effects such as DNA damage, inhibit cell cycle progression and tumour progression [113, 114]. The excessive release of NO has been implicated in the development of neurodegenerative and inflammatory diseases [115, 116]. Even without modification of the surface chemistry, SiO2 NM can elicit an inflammatory response [117, 118]. The mechanism is thought to be due to the generation of ROS by the surface silanol group of SiO2 NM, which can damage the cell membrane and cause apoptosis [119]. SiO2 NM have also been shown to induce morphological changes to the cells, reduce cell migration and proliferation [102, 120, 121]. An in vivo study further showed that SiO2 NM used as food additive could accumulate in the gut epithelium [122] and not be excreted as one would expect. 11 Although several mechanisms have been proposed, the exact mechanism of the SiO2 NM induced toxicity is still unclear. 1.3.3 Titanium dioxide NM TiO2 is a chemically inert material which is widely used in the pigments, cosmetics, drugs and food production industries. Bulk size TiO2 NM are frequently used in consumer products as a pigment for whiteness due to their brightness and high refractive index [123, 124], whereas, nano-size TiO2 NM are exploited for their antibacterial properties or to provide protection from ultraviolet (UV) rays. In nanometer dimensions, TiO2 NM are easily absorbed and distributed to key organs such as lung, kidney, liver, brain and lymph nodes via the circulatory system [125]. Although the mechanisms involving TiO2-induced toxicity have not been clearly defined, it has been suggested to be due to the ability of TiO2 NM to produce reactive oxygen species (ROS) [126, 127], leading to DNA damage. Due to their photocatalytic property, TiO2 NM in combination with hydrogen peroxide are also being evaluated as a tooth bleaching agent [128]. When activated by light, TiO2 NM will react with hydrogen peroxide to generate hydroxyl radicals to remove dental colorants [129]. The photocatalytic property of TiO2 NM are also harnessed to promote bactericidal activity via peroxidation of the bacteria lipid membrane [130]. The ROS generated by TiO2 NM [131, 132] also aids in their bactericidal property. However, hydroxyl radicals and ROS are well documented to be responsible in numerous cellular disorders such as inflammation [133] and cell death [134]. Thus, this raises the question of whether the amount of hydroxyl radicals 12 and ROS generated during these processes [135] are toxic to the cells in the oral cavity, although this was not further studied. Other studies evaluating the impact of increased level of ROS from exposure to TiO2 NM on other mammalian cells, such as keratinocytes, lung cells and cardiovascular cells, have shown this exposure to be toxic [136-139]. 1.3.4 Silver NM Due to claims of having low toxicity and good biocompatibility [140], the use of silver NM is gaining popularity in oral care products for their potential to control biofilm formation [141]. Ag NM have been proven to reduce biofilm formation by coating the surfaces of dental materials which roughen the surface and thus discourage bacterial adhesion [142-144]. Ag NM are also added in oral care products to prevent fungi and bacteria growth [145-148]. The released Ag ions (Ag+) exert bactericidal effects by the production of ROS which disrupts the bacterial or fungal cell membranes [149, 150]. However, the ROS generated by Ag NM have been shown to inflict toxicity (i.e. chromosomal aberrations, aneuploidy and apoptosis) to mammalian cells [151-153] and in animal models [154, 155]; despite claims of having low toxicity profile. 1.4 Human oral mucosa and nanomaterials The mucous membrane of the oral cavity is made up of various types of mucosa; the masticatory mucosa, lining mucosa and specialized mucosa [156]. The oral mucosa is significantly more permeable than cutaneous skin [157-159] because it is highly vascularized and less keratinized than the skin. The permeability of the oral mucosa 13 displays regional variation with the non-keratinized mucosa being the most permeable [160, 161]. Since the majority of the mucous membrane lining in the oral cavity is made up of non-keratinized mucosa, this increases the possibility for NM to penetrate into and through these cells and into deeper tissues, potentially leading to local cell and tissue damage and possibly systemic exposure. In addition, many oral products often contain sodium laurel sulphate (SLS) and high concentrations of alcohol, both of which can affect the integrity of the oral mucosa [162, 163]. The permeability of oral mucosa is therefore likely to be increased in individuals who are frequent users of oral care products, which would translate to increase penetration of NM through the mucous membrane and uptake into cells. As trauma is extremely common in the oral cavity [164], the frequent break in the integrity of the oral mucosa further increases the chances for the NM in oral care products to penetrate through the barrier and affect cell physiology [39]. Thus far, most studies have focused on the use of NM for oral drug delivery [165167] and the absorption and bio-distribution of NM following oral administration [65, 66, 168]. The high permeability of the oral mucosa amplifies the importance of understanding the impact of NM on the oral mucosa. Yet there are no studies that have evaluated the toxic effects of NM on oral epithelium. With the prevalence of NM found in oral care products, it is imperative to elucidate the effects of NM using an in vitro oral model. A vital function of the epithelium is to form a protective barrier against assault and provide immunological defence [169]. Epithelial wound repair is an important process 14 whereby the viable epithelial cells remodel to maintain the epithelial barrier integrity after an injury. The process involves migration of epithelial cells to the defective area with concomitant cell phenotypic alteration, followed by cell proliferation [170]. Inflammatory cells also migrate to the site of the injury to aid in clearance of invading microbes and cellular debris [171, 172]. However, if the wounds exhibited impaired healing, the continued and sustained presence of inflammatory cells can lead to chronic inflammation [173]. Wound healing is influenced by multiple factors which can be broadly classified into 2 groups; local and systemic factors [173]. Adequate oxygenation to the affected site, local infection and/or presence of foreign body are some of the local factors that can affect wound healing. Systemic factors such as age, gender, obesity, stress level and the presence of systemic diseases (e.g., diabetes mellitus) can also influence healing ability [173]. 1.5 Experimental approach and study rationale As the use of NM become more widespread, it is inevitable that more consumers will come into contact with various types of NM. Currently, there is a lack of knowledge regarding the safety of NM after exposure and existing cytotoxicity data report contradicting views. Furthermore, there is no existing authority to regulate the safe use of NM. Thus, it becomes crucial that more in-depth analysis is carried out to determine the health effects of NM. The oral epithelial cell line (TR146) has been reported to share numerous morphological and functional characteristics to the normal buccal mucosa [174-176]. Due to its morphological similarities and comparable permeability [177], TR146 monolayer cultures were utilized as the in vitro model of the human buccal mucosa in this study 15 The primary aims of this study were to determine the presence of NM in commercially available toothpastes and to assess the impact of these NM on fundamental biological functions of the human buccal epithelium. The hypotheses of this study were firstly; NM found in commercially available toothpastes would not have a toxic impact on the human buccal epithelial cell line in vitro. Secondly, the NM found in commercially available toothpastes would not hinder wound healing. To evaluate the toxicity profile of NM, we first investigated whether the NM were being internalized into human buccal epithelial cells and their bio-distribution in the cell if internalized. Thereafter, we sought to evaluate the cellular responses (i.e presence of cell cycle arrest, evidence of apoptosis and inflammatory response) of human buccal epithelium to the NM - a potential toxicant. Next, we determined the route of uptake of NM and their effects on wound healing. This would provide novel insight of how NM affect the human buccal epithelium and our understanding of how NM might affect the cells’ physiology. 16 Chapter 2: Materials and Methods 17 2.1 Identification of inorganic NM in commercially available toothpastes 2.1.1. Extraction of inorganic compounds in toothpastes Inorganic compounds present in toothpastes were extracted using chloroform (Sigma Aldrich, USA), an organic solvent. In brief, 0.25 gms of toothpaste was dissolved in 1 ml of ultrapure water (MilliQ water). 1 ml of chloroform was then added to the dissolved toothpaste and mixed until homogenous. Samples were centrifuged at 10,000 x g for 10 minutes and the supernatant was subsequently discarded to isolate the inorganic compounds in the toothpastes. The procedure was repeated five times to ensure that all organic materials present in toothpastes were removed. The pellets (inorganic compounds) obtained from each brand of toothpaste were washed thoroughly for five times with ultrapure water to remove any traces of chloroform and dried overnight at room temperature. 2.1.2. Identification of elements present in toothpaste extracts To identify the elements and concentration of the inorganic materials, 5 % nitric acid (10 ml) (Merck, USA) was used to dissolve the inorganic compounds in the pellets. The dissolved toothpaste mixture was then filtered through a 0.45 µm filter to remove presence of impurities. The concentration and type of metal oxide-based NM present in various types of toothpastes was determined via Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (7500 series, Agilent Technologies, USA). 18 2.1.3. Analysis of surface chemistry of toothpaste extracts To analyse the surface chemistry of the dried toothpaste pellets obtained, X-ray Photoelectron Spectroscopy (XPS) (VG ESCALAB MKII spectrometer) was utilized. 2.1.4. Determination of hydrodynamic size distribution Nanosight LM10 (NanoSight, UK) was utilized to evaluate the hydrodynamic size distribution of nano-sized particles found in toothpaste extracts. In brief, the toothpaste pellets were dissolved in 1 % Bovine Serum Albumin (BSA) and dispersed via ultrasonication for 10 minutes to form a colloidal suspension. The hydrodynamic sizes of particles were analysed using nanomaterials tracking analysis (NTA). Each sample was tested in triplicates. 2.1.5. Quantification of DNA TR146 were grown in 12-well plates with plating density of 30,000 cells/cm2 overnight. Media was replaced with media containing 50 µg/ml and 500 µg/ml of toothpaste extracts and further incubated for 24 or 48 hours. Subsequently, cells were washed with cold 1X phosphate buffered solution (PBS) thrice. Next, 500 µL of 0.1 % (v/v) Triton-X100 in Tris-EDTA (TE) Buffer (1st Base, Singapore) was added into each well and incubated for 30 minutes on ice with 80 rpm rocking agitation. Cells were subjected to 5 cycles of freeze (-80 oC) – thawed (37 oC) to ensure complete cell lysis. 5 µL of each sample was added to 95 µL of 1X SYBR green, diluted in 1X TE buffer (Sigma-Aldrich, USA), into 96-well plate. Samples were incubated for 5 minutes at room temperature in the dark before measuring the fluorescence intensity 19 using a microplate reader (Infinite 200, Tecan Inc, Maennedorf, Switzerland) using an excitation and emission wavelength of 485 nm and 535 nm, respectively. The relative percentage of viable cells was obtained by normalizing the fluorescence values of the treated cells to untreated control. Data represented the mean of three independent experiments. 2.1.6. Determination of physiologically relevant concentrations of pristine NM To simulate clinically relevant concentrations of NM for exposure to cells, the dosages used for subsequent assays were derived based on the concentrations obtained via ICP-MS. On the assumption that each milligram of Ca, Si or Ti detected was derived directly from HA, SiO2 or TiO2 respectively, we divided the amount of Ca, Si or Ti detected, by its molecular weight to obtain the number of molecules of HA, SiO2 or TiO2 present in toothpastes. Amount of Ca / Si / Ti detected Mole of HA/ SiO2 / TiO2 = present in toothpastes Molecular weight of Ca / Si / Ti The amount of mole obtained was then multiplied to the molecular weight of HA, SiO2and TiO2 and normalized to size of pellet obtained in order to assess the amount of NM in each toothpaste pellet. (Mole of HA / SiO2 / TiO2 present in toothpastes) x Amount of HA/ SiO2/ TiO2 present in toothpastes = (Molecular weight of HA / SiO2 / TiO2) Weight of toothpaste pellets obtained 20 The amount of HA, SiO2 and TiO2 NM present in the toothpastes tested were calculated based on the above formula. A standardized range of concentrations for HA, TiO2 and SiO2 NM was used in order to compare the effects of the different NM. 2.2 Pristine NM HA NM and SiO2 NM were purchased from Sigma-Aldrich, USA while TiO2 NM (AEROXIDE® P25) was purchased from Evonik Degusa, USA. All NM suspensions were prepared by adding the NM in various solvents used in different experimental setup; namely: ultrapure water, and DMEM – F12 (1:1) complete media (Invitrogen, USA). One minute of probe sonication was used to help to disperse the NM in the solvents. 2.2.1 Tagging of Pristine NM with FITC Fluorescein isothiocyanate (FITC) tagged NM were prepared using 3- Aminopropyltriethoxysilane (APTES; Sigma-Aldrich, USA), which was used to attach aloxysilane groups to the surface hydroxyl groups. These amines readily formed thiourea bonds with FITC (Sigma-Aldrich, USA). Briefly, 100 mg of NM were dispersed in 100 mL of anhydrous ethanol (Fisher Scientific, USA); thereafter 5 mL of APTES was added drop wise to the suspension and stirred continuously at 80 o C for 5 hours. For HA NM, 50 mg of FITC (final concentration of 0.5 g/l) was added whereas 25 mg of FITC was added to SiO2 or TiO2 NM (final concentration of 0.25 g/l). The mixture was stirred continuously for another 16 hours at 80 oC and the FITC 21 tagged NM were then collected. To remove excess FITC, the NM pellets were washed with ethanol and subsequently with ultrapure water thrice by centrifugation at 750 g for 20 minutes. During each washing step, exposure of FITC-NM to light was minimized to prevent photobleaching of FITC. Finally, the FITC-NM were freezedried and stored as dry powders. 2.2.2 Characterization of pristine NM Transmission Electron Microscopy (TEM; JEOL JEM-2010, Japan) was used to view the NM morphology. HA and TiO2 NM were suspended in ethanol whereas SiO2 NM were pre-coated with 0.2 % (v/v) Triton-X100 to reduce aggregation and subsequently suspended in ethanol. Thereafter, a drop of NM suspension was placed onto carboncoated grids. The NM size was determined by scoring 50 randomly chosen NM using ImageJ software. The hydrodynamic size, polydispersity index (PDI), and surface charge of the NM in various solvents were analyzed using Dynamic Light Scattering (DLS) (Malvern Co., UK). Each sample was measured in triplicate, using Image J, and the mean values were reported. 2.3 TR146 Human buccal epithelial cells culture Human buccal epithelial cells (TR146; Sigma-Aldrich, USA) was cultured in 1:1 complete high glucose Dulbecco’s Modified Eagle’s Media : nutrient mixture F-12, (DMEM/F12) (Invitrogen, MA, USA) supplemented with 10 % fetal bovine serum (FBS), 1 % L-glutamine and 1 % Penicillin/Streptomycin at standard culture condition of 37 °C, 5 % CO2 and sub-cultured when 90 % confluent. 22 2.4 2.4.1 Internalization of NM into cells Confocal microscopy imaging TR146 cells were grown overnight on 8-well chamber slides with seeding density of 30,000 cells/cm2. The cells were treated by replacing the cell culture medium with medium containing different concentrations of FITC tagged NM (0, 125 and 1250 µM) in DMEM/F12 complete medium for 2, 4, 8 and 12 hours. Following treatment, the cells were washed thrice with cold phosphate buffered saline (PBS) and fixed with 4 % paraformaldehyde for 15 min at 4 oC. Subsequently, the cells were blocked for 1 hour with 2 % BSA, 0.1 % Triton-X100 in PBS to reduce non-specific antibody binding. Thereafter, the cells were washed thrice with PBS, and counterstained with CF568-phalloidin (Biotium, USA) for actin filaments. Finally, the labelled slides were mounted with ProLong® Gold anti-fade reagent with DAPI (Invitrogen, USA). The images were captured using Nikon Eclipse C1 confocal microscope. 2.4.2 Quantification of cellular uptake of NM The internalized NM were quantified by plating TR146 cells overnight on 24 well plates with seeding density of 30,000 cells/cm2. The following day, 250 µM of FITCNM in DMEM/F12 complete medium were introduced to the cells for 2, 4, 8, 12, 16, 20 and 24 hours. Cells were washed thrice with PBS, followed by lysing the cells with 0.1 % Triton-X100 in TE buffer. The lysates were collected and transferred into new 96 well plates where the fluorescence intensity was measured at an excitation/emission wavelength of 488/525 nm using Tecan Infinite 200 microplate reader (Tecan Inc., Switzerland). The amount of FITC-NM uptake was obtained by 23 normalizing the fluorescence values of the treated cells to untreated control. Data obtained represented the mean values of three independent experiments. 2.4.3 Membrane and cytosolic fractions To determine the fraction in which NM are localized, TR146 cells were plated overnight on 6 cm dish with seeding density of 30,000 cells/cm2. 125 and 1250 µM of FITC-NM in DMEM – F12 (1:1) complete medium were introduced to the cells for 12 hours. Cells were washed thrice with PBS and membrane proteins extracted as per manufacture’s protocol (Mem-PER Eukaryotic Extraction Kit; Thermo Scientific, USA). The extracted membrane and cytosol fractions were collected and transferred into new 96 well plates, where the fluorescence intensity was measured at an excitation/emission wavelength of 488/520 nm using Tecan Infinite 200 microplate reader (Tecan Inc., Switzerland). The amount of FITC-NM in each fraction was obtained by normalizing the fluorescence values of the treated cells to untreated control. Data obtained represented the mean values of three independent experiments. 2.5 Evaluation of cytotoxicity All lysis buffers were supplemented with 1 % protease inhibitor (Sigma- Aldrich, USA) and 1 % phosphatase inhibitors cocktail (Sigma-Aldrich, USA). Protein extracts were resolved using pre-cast gradient SDS-polyacrylamide gel electrophoresis (4 - 15 %, Mini-Protean, Biorad Laboratories Inc, USA), and electro-transferred (25 mM Tris, 192 mM glycine, 10 % methanol, 0.05 % SDS) onto a nitrocellulose membrane for immunoblot analysis. Antibodies probing were done as per manufacturers’ 24 recommendation. The appropriate Horseradish Peroxidase (HRP) conjugated goat antibody (Santa Cruz Biotechnologies, USA) was used for protein detection. Protein bands were detected using ImmobilonTM Western Chemiluminescent HRP substrate (Millipore, USA) in Chemiluminescence Imaging System (Syngene, UK). For apoptotic signalling pathway, TR146 cells were plated overnight on a 6 cm dish with seeding density of 30,000 cells/cm2. Thereafter, the cells were treated with various concentrations of HA, SiO2 and TiO2 NM (0, 62.5, 125, 250, 500 and 1250 µM) for 24 hours. After treatment, cells were washed thrice with cold PBS and lysed with standard Laemmli’s sample buffer (2 % SDS, 10 % glycerol, 5 % ßmercaptoethanol, 0.002 % bromophenol blue and 62.6 mM Tris-HCl, pH 6.8). The cell lysates were sonicated for 5 seconds and centrifuged at 15,000 g for 20 minutes at 4 ºC to remove cell debris, after which supernatant was collected. Protein extracts were boiled at 95 ºC for 5 minutes and resolved using a pre-cast gradient (4 – 15%) polyacrylamide gel. Apoptosis markers were detected by employing the following antibodies: anti-phospho p53, anti-total p53, anti-pMDM2, anti-PARP, anti-caspase 3, anti-caspase 8, anti-caspase 9, anti-Bax (Cell Signaling Technology, USA) and anti β-actin (Santa Cruz Biotechnology, USA). For cell cycle analysis, TR146 cells were plated overnight on a 6 cm dish with a seeding density of 30,000 cells/cm2 and synchronized at G1/S phase with 400 nM Lmimosine (Sigma-Aldrich, USA) for 24 hours. After which, the cells were released and treated with various concentrations of HA, SiO2 and TiO2 NM (0, 62.5, 125, 250, 500 and 1250 µM) for 6 hours. Thereafter, the proteins were extracted. Cell cycle 25 related markers were detected by employing the following antibodies: anti-phospho p53, anti-total p53, anti-pMDM2, anti-cyclin B1, anti-cyclin D, anti-cyclin E, antiCDK4, anti-CDK6, anti-phospho cdc2 (Cell Signaling Technology, USA) and anti – β-actin (Santa Cruz Biotechnology, USA). 2.6 Evaluation of inflammatory response 2.6.1 Intracellular Reactive Oxygen Species (ROS) level measurement TR146 cells were grown on 96-well plates at a density of 30,000 cells/cm2 overnight. The media was replaced with media containing different concentrations of NM (0, 62.5, 125, 250, 500 and 1250 µM) and incubated for 24 hours. Intracellular ROS level was detected using 10 µM of 2',7'-Dichlorofluorescin diacetate (DCFH-DA; Sigma Aldrich, USA). Additionally, the intracellular ROS level was normalized against the amount of cells which were detected with 1 µg/mL of Hoechst 33342 (Invitrogen, USA). The DCF and Hoechst dye intensity were detected at excitation/ emission wavelengths of 488/525 nm and 350/461 nm respectively. The fluorescence intensity of each fluorophore was measured using microplate reader (Tecan Infinite 200, Tecan Inc., Switzerland). The amount of ROS generated was obtained by normalizing the fluorescence values of the treated cells to untreated control. Data obtained represented the mean values of three independent experiments. 26 2.6.2 Inflammatory genes expression profile using Quantitative Real Time PCR (qPCR) TR146 cells were exposed to various concentrations (0, 62.5, 125, 250, 500 and 1250 µM) of HA, SiO2 and TiO2 NM for 24 hours after which total RNA was isolated using RNeasy Mini kit (Qiagen, Germany) as per manufacturer’s protocol. The concentration and integrity of RNA were measured using Nanodrop2000 Spectrophotometer (Thermo Scientific). RNA was then converted to cDNA using RevertAid™ H Minus First Strand cDNA synthesis (Fermentas, USA) kit as per manufacturer’s recommendation. KAPA SYBR® FAST qPCR kit was used to perform the quantitative PCR reactions using ABI 7300 real-time PCR system (Applied Biosystem, USA). Human TATA-box binding protein (hTBP) was used as an internal control. No template controls (NTC) were included to ensure that the samples were not contaminated with DNA. Fold induction was calculated using 2∆∆C(T) [178]. The sequences of the forward and reverse primers for various genes assessed are as listed: TNF-α forward 5' CCT CTC TCT AAT CAG CCC TCTG 3' TNF-α reverse 5' GAG GAC CTG GGA GTA GAT GAG 3' IL-6 forward 5’ GAA AGC AGC AAA GAG GCA CT 3’ IL-6 reverse 5’ TTT CAC CAG GCA AGT CTC CT 3’ hTBP forward 5’ TGC CCG AAA CGC CGA ATA TAA TC 3’ hTBP reverse 5’ GTC TGG ACT GTT CTT CAC TCT TGG 3’ 27 2.6.3 Secreted embryonic alkaline phosphatase (SEAP) assay RAW 264.7 macrophage cells were stably transfected with NFκB-SEAP (secreted embryonic alkaline phosphatase) reporter gene (a gift from Dr Tan; Faculty of Dentistry, National University of Singapore). The cells were maintained in complete DMEM medium supplemented with 10 % fetal bovine serum (FBS), 1 % L-glutamine and 1 % Penicillin/Streptomycin, at standard culture condition of 37 °C, 5 % CO2 and sub-cultured when 90 % confluent. RAW 264.7 - NFκB-SEAP cells were treated with 0, 62.5, 125, 250, 500 and 1250 µM of HA, SiO2 and TiO2 NM for 6 hours. As a positive control, RAW 264.7 NFκB-SEAP cells were treated with heat killed E. coli. Phospha-Light™ SEAP reporter gene assay (Applied Biosystem, USA), which measures amount of NFκB production was performed according to manufacturer’s protocol. Chemiluminescence signal was detected using a luminometer (Glomax 96 well microplate luminometer, Promega). The amount of NFκB-SEAP secreted was obtained by normalizing the obtained values of the treated cells to untreated control. Data obtained represented the mean values of three independent experiments. 2.7 Wound healing assay Wound healing assay was performed by plating TR146 cells overnight on 6 well plates with seeding density of 90,000 cells/cm2. Upon reaching confluence, a scratch was made on the cell monolayer using a p200 pipette tip. Thereafter the cells were washed thrice with PBS to remove cell debris. Complete media containing different 28 concentration of NM (0, 62.5, 125, 250, 500 and 1250 µM) were added to the wells immediately after scratching and incubated for 12 hours. The cells were imaged using Olympus DP21 (Olympus, USA) at t = 0 hour and t = 12 hours. Width of the scratch was quantified using ImageJ (n = 50). 2.7.1 Blocking of clathrin-mediated endocytosis Monodansylcadaverine (MDC) was used to block clathrin-mediated endocytosis. The effective concentration of MDC to block NM uptake was tested to be 50 µM. TR146 cells were plated overnight on 6 well plates with seeding density of 90,000 cells/cm2. Pre-treatment of the cells with 50 µM of MDC was performed for 1 hour. Subsequently, 125 and 1250 µM of NM was added for 12 hours to examine the effectiveness of inhibitors in blocking the uptake of NM into cells and wound healing assay was performed as described above. 2.8 Statistical analysis Statistical significance was ascertained using Student’s t-test with data analysis tool of MS EXCEL 2007. p value of < 0.05 was considered significant. 29 Chapter 3: Results 30 3.1 Determination of NM found in commercially available toothpastes 3.1.1 Characterization of NM found in commercially available toothpastes Inorganic materials present in four commercially available toothpastes: DLE, COL, SEN, COLPS (actual brand and make are concealed because of copyright issues) were isolated. Using XPS, we analysed the surface chemistry of the toothpaste extracts and detected calcium (Ca) in DLE, COL and COLPS and titanium (Ti) in SEN (Figure 1) and the amount of each element (Table 1). The presence and concentration of Ca, Ti and silicon (Si) (Table 2) in all four toothpastes was further verified by ICP-MS. Figure 1 | Identification of elements present in inorganic extracts of commercially available toothpastes. XPS spectrum reflected presence of calcium in (A) DLE (B) COL (D) COLPS and titanium in (C) SEN 31 Samples Element detected Percentage of element in sample (Normalized to carbon) DLE Calcium 15.41% COL Calcium 16.21% SEN Titanium 6.24% COLPS Calcium 19.15% Table 1 | Elemental composition detected in the surface chemistry of inorganic extracts from commercially available toothpastes. Samples Calcium Silicon Titanium (mg of metal per 1 gram of pellet) DLE 130.76 29.28 0.29 COL 107.02 28.95 0.41 SEN 10.24 157.32 0.006 COLPS 147.72 35.73 0.003 Table 2 | Identification and concentration of metal oxide-based nanomaterials present in inorganic extracts of commercially available toothpastes via ICP-M/S 32 The hydrodynamic sizes of particles present in the DLE, COL, SEN and COLPS toothpaste extracts were 126.74 ± 5.12 nm, 145.99 ± 2.15 nm, 116.26 ± 1.29 nm and 53.70 ± 5.93 nm respectively (Figure 2). Figure 2 | Distribution of particle size present in toothpaste extracts. 3.1.2 Effect of toothpaste extracts on cell proliferation rate Exposure of cells to COL, DLE, SEN and COLPS toothpaste extracts of two different concentrations (50 and 500 µg/ml) for 24 hours had no effect on the cell proliferation rate. When the cells were exposed to 50 µg/ml of COL, DLE, SEN and COLPS for 48 hours, the cell proliferation rate was reduced by 9 %, 10 %, 18 % and 23 % respectively. At the higher concentration of 500 µg/ml, DLE, COL, COLPS and SEN reduced cell proliferation rate by 11 %, 13 %, 21 % and 22 % respectively (Figure 3). 33 Figure 3 | Toothpaste extracts reduced TR146 human buccal epithelial cells proliferation rate. SYBR green quantification data for cells exposed to 50 µg/ml and 500 µg/ml of toothpaste extracts for (A) 24 hours and (B) 48 hours. Data represent mean ± SD. *p < 0.05, compared against control group. 3.1.3 Calculation of physiologically relevant concentrations of NM From our preliminary studies, HA, TiO2 and SiO2 NM were found in commercially available toothpastes. The amount of HA present in 0.25 gms of toothpaste extracts 34 ranged from 3591.87 µg to 246,141.93 µg, SiO2 ranged from 9,376.72 µg to 9,423.79 µg and TiO2 ranged from 0.28 µg to 104.11 µg (Table 3). For subsequent assays in this study, we chose to determine the dose response at which the NM will produce a potential toxic effect on the cells by exposing the cells to HA, TiO2 and SiO2 at concentrations of 0 µM, 62.5 µM, 125 µM, 250 µM, 500 µM and 1250 µM, which is equivalent to 0 µg to 300 µg (based on the formula stated in section 2.1.6). Samples HA (µg) SiO2 (µg) TiO2 (µg) DLE 246,141.93 9,408.81 73.17 COL 203,060.82 9,376.72 104.11 SEN 3,591.87 9,423.79 0.28 COLPS 225,807.65 9,387.42 0.66 Table 3 | Amount of HA, SiO2 and TiO2 present in 0.25 gms of commercially available toothpastes. 3.2 Characterization of pristine NM All pristine NM employed in this study were spherical in shape (Figure 4A-C), with primary particle sizes of 48.63 ± 14.17 nm, 14.01 ± 2.00 nm, and 21.12 ± 8.21 nm for HA, SiO2 and TiO2 NM, respectively (Table 4). The hydrodynamic sizes of HA, SiO2 and TiO2 NM in ultrapure water were 958.07 nm, 665.53 nm and 392.17 nm. Whereas, in complete cell culture medium, the 35 hydrodynamic sizes of HA, SiO2 and TiO2 were 236.17 nm, 244.90 nm and 271.53 nm respectively (Table 4). Pristine HA NM registered a zeta potential of -16.10 mV, pristine SiO2 NM were found to be -20.10 mV and pristine TiO2 NM to be +21.10 mV in ultrapure water. In complete cell culture medium, HA NM registered smaller zeta potential value of -6.50 mV, SiO2 NM were found to be -2.00 mV and TiO2 NM to be -7.10 mV (Table 4). Figure 4 | Physical characterization of pristine NM. TEM micrographs of (A) HA, (B) SiO2 and (C) TiO2 NM. Sample Particle Size (nm) HA 48.63 ± 14.17 SiO2 14.01 ± 2.00 TiO2 21.12 ± 8.21 Hydrodynamic size (nm) Zeta Potential (mV) DMEM/F12 MilliQ water / PDI / PDI MilliQ water DMEM/F12 -16.10 -6.50 -20.10 -2.00 +21.10 -7.10 958.07 / 236.17 / 0.38 0.47 665.53 / 244.90 / 0.29 0.32 392.17 / 271.53 / 0.16 0.20 Table 4 | Size characterization of pristine NM and their surface charges. 36 3.3 Effects of pristine NM on human buccal epithelium 3.3.1 Internalization and bio-distribution of NM into TR146 human buccal epithelial cells All three NM were internalized into the cells regardless of their size and surface charge. The amount of NM internalization was directly dependent on the concentration and exposure time; i.e. increased internalization was observed with increased concentration and exposure time for all three NM. Throughout the experimental time-point (up to 12 hours), internalized HA, SiO2 and TiO2 NM were observed to accumulate in the cells (Figures 5 - 7). A few small aggregates of HA NM were observed in the cytoplasm of the cells (Figure 5). SiO2 NM were localized in both the cytoplasm and the nuclei (Figure 6). TiO2 NM were internalized into the cytoplasm and localized around the nuclei (Figure 7). 37 Figure 5 | Internalization of FITC-HA NM by TR146 human buccal epithelial cells. The cells were exposed to two different concentrations (125 µM and 1250 µM) of FITC-tagged HA NM for four different time-points of t = 2, 4, 8 and 12 hours. Few FITCtagged HA NM were observed in the cytoplasm of the cells. Scale bar = 50 µm 38 Figure 6 | Internalization of FITC-SiO2 NM by TR146 human buccal epithelial cells. The cells were exposed to two different concentrations (125 µM and 1250 µM) of FITC-tagged SiO2 NM for four different time-points of t = 2, 4, 8 and 12 hours. With increasing concentrations and time-points, more SiO2 NM were observed to be internalized into the cells. SiO2 NM can be clearly observed to accumulate in the cell nuclei at higher concentrations and time-points. Scale bar = 50 µm 39 Figure 7 | Internalization of FITC-TiO2 NM by TR146 human buccal epithelial cells. The cells were exposed to two different concentrations (125 µM and 1250 µM) of FITC-tagged TiO2 NM for four different time-points of t = 2, 4, 8 and 12 hours. TiO2 NM were internalized and accumulated in the cytoplasm, near the nucleus. More TiO2 NM were observed in the cells with increasing concentrations and time-points. Scale bar = 50 µm 40 3.3.2 Uptake of NM by human buccal epithelial cells and interaction with membrane and cytosolic proteins Our data showed a significant uptake (> 1.5 fold increase, p < 0.001) of all three NM by the cells; even after 1 hour of exposure (Figure 8). All three NM followed a similar trend of increased cellular uptake with increased exposure time (Figure 8). With extended exposure time of 16 hours, cells exposed to HA NM reflected up to 10-fold increase in HA NM uptake (Figure 8A). Whereas, cells treated with SiO2 (Figure 8B) and TiO2 NM (Figure 8C) displayed a maximum of a 2-fold increase in uptake which was observed at 4 hours and 2 hours after exposure, respectively. For all tested NM, the amount of NM internalized peaked at a particular exposure time and subsequently declined. HA NM were retained in the cell for the longest period of time compared to SiO2 and TiO2 NM (Figure 8). HA NM were retained in the cells for 16 hours, thereafter the amount of HA NM detected decreased (Figure 8A). The amount of SiO2 NM detected in the cells peaked at 4 hours and subsequently declined (Figure 8B). The amount of TiO2 NM detected in the cells peaked after 2 hours of exposure and was sustained until 8 hours of treatment, thereafter the quantity of TiO2 NM declined (Figure 8C). Although few HA NM aggregates were observed in the cells in the imaging study (Figure 5), they displayed the most internalization (Figure 8A) of the three NM studied. 41 Figure 8 | Tracking of NM uptake by TR146 human buccal epithelial cells. The cells were subjected to treatment of 250 µM of (A) HA, (B) SiO2 and (C) TiO2 NM for various time-points. It was observed that all three NM followed a similar trend of increased cellular uptake of NM with increasing time-point. With extended exposure time, the amount of NM internalized by the cells declined. Controls are non-treated sample. Data are expressed as mean ± SD. *p < 0.05, compared against control group. The bio-distribution of HA, SiO2 and TiO2 NM was further determined and it was found that more HA NM were located on the cell membrane (Figure 9A) compared to being localized in the cytoplasm (Figure 9D). There was a dose-dependent interaction between NM and the cellular proteins. When cells were exposed to 125 µM of HA NM, an 8-fold increase (p = 0.003) was observed in the membrane fraction (Figure 9A) and 5-fold increase (p < 0.0001) in the cytosolic fraction (Figure 9D) compared to the control group. The exposure of the cells to 1250 µM HA NM led to an 80-fold increase (p = 0.0001) of HA NM detected in the membrane fraction (Figure 9A) and a 25-fold increase (p = 0.0002) in the cytosolic fraction (Figure 9D). At 125 µM of SiO2 NM, there were no significant differences in the amount of SiO2 NM detected in the membrane (Figure 9B) and cytosolic fractions (Figure 9E) compared to the control group (p > 0.05). Cells exposed to 1250 µM of SiO2 NM gave rise to a 1.5-fold (p < 0.0001) and a 1.2-fold (p < 0.0001) increase in the amount of SiO2 NM localized in 42 membrane (Figure 9B) and cytosolic fractions (Figure 9E) respectively. When the cells were exposed to 125 µM of TiO2 NM, a 1.2-fold increase was detected in the amount of TiO2 NM localized in membrane (p = 0.015) (Figure 9C) and cytosolic fractions (p = 0.003) (Figure 9F). At 1250 µM of TiO2 NM, an approximately 2-fold increase (p = 0.0004) was observed for TiO2 NM localized in the membrane fraction (Figure 9C) and a 1.7 fold increase (p < 0.0001) was detected in the cytosolic fraction (Figure 9F). 43 Figure 9 | Internalization of NM and interaction of NM with membrane and cytosolic proteins. The cells were subjected to treatment of 125 µM and 1250 µM of HA, SiO2 and TiO2 NM for 12 hours. It was observed that there was a dose-dependent interaction between NM and the cellular proteins. More HA NM were found in the membrane fraction than cytosolic fraction, whereas similar amount of SiO2 and TiO2 NM were found in the membrane and cytosolic fractions. Data represent mean ± SD. *p < 0.05, compared against control group. 44 3.3.3 HA, TiO2 and SiO2 NM are non-cytotoxic Cell exposure to all concentrations (62.5 to 1250 µM) of HA, SiO2 and TiO2 NM up to a maximum of 24 hours did not result in an increase in phosphorylation of MDM2. Furthermore, all tested NM did not give rise to changes in the expression level of phosphorylated p53 or activated PARP (Figure 10). Cells synchronized at G1/S phase of the cell cycle and subsequently exposed to the various concentrations (62.5 to 1250 µM) of HA, SiO2 and TiO2 NM for 6 hours did not induce any significant changes in protein expression level of cell cycle related genes: cyclin D, CDK4, CDK6, cyclin E1, phospho-cdc2 and cyclin B1 (Figure 10A). After the cells were treated with all concentrations (0 to 1250 µM) of HA, SiO2 and TiO2 NM for 24 hours, active caspase 9, caspase 8 and caspase 3 expressions were not detected. The protein expression level of Bax also remained constant (Figure 10B). 45 Figure 10 | Exposure to NM did not affect cell cycle progression or activated apoptotic pathway. TR146 human buccal epithelial cells were treated with HA, SiO2 and TiO2 NM. No significant changes in cell cycle related genes were detected after 6 hours of NM exposure. Similarly, apoptotic pathway genes were not activated after 24 hours of exposure. This signifies that exposure of cells to NM did not induce genotoxicity to the cells. 3.3.4 Nanomaterials induced mild inflammatory response 3.3.4.1. ROS production There was an increase in ROS production when cells were exposed to increasing concentrations of HA, SiO2 and TiO2 NM. All concentrations (62.5 to 1250 µM) of HA NM gave rise to significant increase (p < 0.05) in ROS expression levels compared to the control group; the highest concentration (1250 µM) caused an approximate 40 % increase (p = 0.027) in ROS production (Figure 11A). In 46 comparison, only the higher concentrations of SiO2 (500 and 1250 µM) and TiO2 NM (250 to 1250 µM) resulted in a significant increase (p < 0.05) in ROS production. For cells exposed to the highest concentration (1250 µM) of SiO2 NM, a 15 % increase (p = 0.018) in ROS expression level was detected (Figure 11B) compared to the control group. Of the three NM studied, 1250 µM of TiO2 reflected the highest increase in ROS expression levels; approximately 60 % higher (p = 0.003) compared to the control group (Figure 11C). Figure 11 | NM induced elevated ROS expression level in TR146 human buccal epithelial cells. After being exposed to various concentrations of (A) HA, (B) SiO2 and (C) TiO2 NM for 24 hours, increasing Reactive Oxygen Species (ROS) levels were detected in TR146 cells with increasing concentrations of NM. Data represent mean ± SD. *p < 0.05, compared against control group. 3.3.4.2. Interleukin-6 (IL-6) expression We observed a dose dependent elevation in IL-6 expression when the cells were treated with increasing concentrations of HA NM (Figure 12A). All concentrations (62.5 to 1250 µM) of HA NM resulted in a significant increase (p < 0.05) in IL-6 expression level (Figure 12A). Cells exposed to 1250 µM of HA NM triggered almost a 4-fold increase (p < 0.0001) of IL-6 expression when compared to the control group. 47 Only cells exposed to concentrations of 500 and 1250 µM of SiO2 NM gave rise to a significant increase (p < 0.05) in IL-6 expression, with a 1.5-fold increase (p = 0.007) observed for the highest concentration (1250 µM) of SiO2 NM (Figure 12B). All concentrations of TiO2 NM gave rise to a significant increase (p < 0.05) of IL-6 expression, with the highest concentration (1250 µM) of TiO2 giving rise to the greatest change; approximately 3.5-fold increase (p < 0.0001) of IL-6 (Figure 12C). Figure 12 | NM induced dose dependent elevation of IL-6 expression. (A) Cells treated with HA NM triggered a significant expression level of IL-6. (B) In comparison, only cells exposed to higher concentrations of SiO2 NM reflected a significant increase in expression level of IL-6. (C) All concentrations of TiO2 NM exposed cells gave rise to a significant increase in IL-6 expression. Data represent mean ± SD. *p < 0.05, compared against control group. 3.3.4.3. Tumor necrosis factor-alpha (TNF-α α) expression Cells exposed to all concentrations of HA NM (62.5 to 1250 µM) resulted in a significant (p < 0.05) increase in the expression level of TNF-α (Figure 13A). However, significant up-regulation (p < 0.05) of TNF-α was only observed for concentrations of 500 and 1250 µM of SiO2 (Figure 13B) and TiO2 NM (Figure 13C). A 5-fold increase (p < 0.0001) was observed following 1250 µM of HA and TiO2 NM 48 treatment, while 1250 µM of SiO2 NM only induced an approximate 3-fold increase (p = 0.0008) in TNF-α expression. Figure 13 | NM induced dose dependent elevation of TNF-α expression. (A) The cells exposed to all concentrations of HA NM resulted in a significant increase in expression level of TNF-α. In comparison, only cells exposed to higher concentrations (≥ 500 µM) of (B) SiO2 NM and (C) TiO2 NM resulted in a significant increase in TNF-α expression. Data represent mean ± SD. *p < 0.05, compared against control group. 3.3.4.4. Nuclear factor kappa B (NFκ κB) expression We observed a trend of elevated expression of NFκB when cells were exposed to increasing concentrations of all three NM (Figure 14). Lower concentrations (62.5 and 125 µM) of HA NM did not significantly affect the amount of NFκB secreted. However, concentrations of ≥ 250 µM of HA NM gave rise to a significant increase (p < 0.05) in amount of NFκB secreted. Cells treated with 1250 µM of HA NM induced an approximate 1.2-fold increase (p < 0.001) in the amount of NFκB secreted (Figure 14A). SiO2 NM of concentrations higher than 62.5 µM reflected significant increase in amount of NFκB secreted. The highest concentration of SiO2 (1250 µM) gave rise 49 to a 1.5-fold increase (p < 0.0001) in NFκB secreted compared to the control group (Figure 14B). TiO2 NM also induced a significant increase (p < 0.05) in NFκB secreted. Up to 1.2-fold increase (p = 0.0002) in NFκB secreted by the cells was observed for 1250 µM of TiO2 NM (Figure 14C). For comparison, RAW-NFκB cells that were exposed to heat-killed bacteria (E. coli) showed a drastic 30-fold increase (p < 0.0001) in secreted NFκB (Figure 13D). Figure 14 | NM induced dose dependent elevation of NFκB. (A) The cells exposed to HA NM of concentrations higher than 125 µM resulted in significant increase in amount of NFκB secreted. (B) Whereas, cells treated with SiO2 NM of concentrations higher than 62.5 gave rise to significant increase in amount of NFκB secreted. (C) Cell exposure to TiO2 NM also resulted in a general trend of significant increase in level of NFκB secreted. (D) Cells treated with heat-killed E. coli served as a positive control with a significant amount of NFκB secreted. Data represent mean ± SD. *p < 0.05, compared against control group. 50 3.3.5 NM impaired wound healing Cells exposed to all concentrations (0, 62.5, 125, 250, 500 and 1250 µM) of HA, SiO2 and TiO2 NM significantly (p < 0.05) impaired wound healing. Wound healing ability was increasingly affected when cells were exposed to increasing concentrations of NM. Of the three NM studied in this study, HA NM had the greatest impact on epithelial cell migration. Cells treated with 1250 µM of HA NM displayed approximately 50 % reduction (p = 0.0005) in wound healing was observed (Figure 15); even at the lowest concentration of HA NM (62.5 µM), wound healing was hindered by 30 % (p = 0.016) (Figure 15). Of the three NM tested, SiO2 NM had the least impairment on the cells’ wound healing capability. The lowest concentration (62.5 µM) of SiO2 NM impaired the cells' wound healing ability by 14 % (p = 0.0003). The highest concentration (1250 µM) of SiO2 NM hindered wound healing by 30 % (p = 0.0003) (Figure 16). Cells exposed to TiO2 NM showed a similar trend. When cells were exposed to 62.5 µM of TiO2 NM, a 10 % reduction (p = 0.037) in wound healing was observed. Cells exposed to the highest concentration (1250 µM) of TiO2 NM displayed an approximate 40 % (p < 0.0001) impairment of wound healing (Figure 17). 51 Figure 15 | HA NM impaired wound healing. Images captured for different concentration of HA NM treated cells at t = 0 and 12 hours. Exposure to HA NM resulted in the greatest impact in epithelial cell migration, with the highest concentration (1250 µM) reducing wound healing by approximately 50 %. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 52 Figure 16 | SiO2 NM hindered wound healing. Images captured for different concentration of SiO2 NM treated cells at t = 0 and 12 hours. Exposure to SiO2 NM resulted in the least impact in epithelial cell migration, with the highest concentration (1250 µM) reducing wound healing by approximately 30 %. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 53 Figure 17 | TiO2 NM impaired wound healing. Images captured for different concentration of TiO2 NM treated cells at t = 0 and 12 hours. Exposure of human buccal epithelial cells to the highest concentration (1250 µM) of TiO2 NM resulted in approximately 40 % reduction in wound healing ability. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 54 3.3.6 Uptake of NM into human buccal epithelial cells via clathrin mediated endocytosis A specific inhibitor of clathrin-mediated endocytosis, MDC, was used to block the internalization of NM. This resulted in a significant recovery (p < 0.05) of the cells' wound healing capability (Figures 18 - 20). When cells were pre-treated with MDC and subsequently exposed to 125 µM of HA NM, only a 3 % reduction (p = 0.302) in wound healing was observed. Treatment of the cells with 1250 µM of HA NM resulted in a 10 % impairment of wound healing, however, this was not statistically significant (p = 0.055) compared to the control group (Figure 18). The pre-treatment of the cells with MDC and subsequent exposure to SiO2 NM also led to recovery in cell migration (Figure 19). Cells treated with 125 µM of SiO2 NM resulted in a 10 % reduction in wound healing (p = 0.051). When the cells were pre-treated with MDC and subsequently exposed to 1250 µM of SiO2 NM, a significant 25 % impairment in wound healing was observed (p < 0.0001) (Figure 19). Cells pre-treated with MDC and followed with exposure to 125 µM of TiO2 NM, resulted in a 5 % reduction (p = 0.251) in wound healing; while 1250 µM of TiO2 NM resulted in an 8 % reduction (p = 0.103) in wound healing (Figure 20), both were not significantly different from the control group. 55 Figure 18 | HA NM internalized into cells via clathrin-mediated pathway. Images captured for different concentration of HA NM treated cells at t = 0 and 12 hours. Cells pre-treated with MDC and subsequently exposed to HA NM significantly improved the cells' wound healing ability. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 56 Figure 19 | SiO2 NM internalized into cells via clathrin-mediated pathway. Images captured for different concentration of SiO2 NM treated cells at t = 0 and 12 hours. Although cells pre-treated with MDC and subsequently exposed to SiO2 NM still impaired wound healing significantly, an improvement in the cells migration rate was still reflected. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 57 Figure 20 | TiO2 NM internalized into cells via clathrin-mediated pathway. Images captured for different concentration of TiO2 NM treated cells at t = 0 and 12 hours. Cells pre-treated with MDC and subsequently exposed to TiO2 NM resulted in a significant improvement in the cells' wound healing ability. Data are expressed as means ± SD. *p < 0.05, compared against control group. Scale bar = 50 µm 58 Chapter 4: Discussion 59 4.1 Characterization of NM in commercially available toothpastes In recent years, potential toxicity of NM have sparked numerous studies. In order to begin to investigate the effects of NM on an in vitro model of oral mucosa, there is a need to first determine the types of NM present in oral care products. Thus far, there are no studies that have characterized the NM found in commercially available oral care products. Toothpastes contain a variety of organic and inorganic materials such as sorbitol, hydroxyethyl cellulose, peppermint and triclosan [179]. In this study, we focused on inorganic NM present in commercially available toothpastes. Even though we detected Ca and Ti in the toothpaste extracts, there exist limitations for detection of elements using XPS as only the surface elemental composition of the extracts can be analyzed. Consequently, this may not be an accurate representation of the elements within the toothpaste extracts. Thus, ICP-MS was utilized to verify the type and concentration of inorganic elements present. Using ICP-MS, Si, in addition to Ca and Ti, was detected in the toothpaste extracts. The NM detected had average sizes of less than 200 nm and it was assumed that they were added in toothpastes to aid in polishing of teeth surfaces or repairing of damaged enamel. However, the other unknown inorganic materials present in the toothpaste extracts could have affected the size measurement which is a limitation of this study. Based on the ingredients in the toothpastes, we assumed that the presence of Ca, Si and Ti were derived from HA, TiO2 and SiO2 NM the toothpastes [86, 89, 90, 104, 123, 124, 180]. 60 4.1.1 Effect of inorganic toothpaste extracts on cell proliferation rate A reduction in cell proliferation rate was observed only when the cells were exposed to the toothpaste extracts for 48 hours, DLE and COL reduced cell proliferation rate by approximately 10 % whereas SEN and COLPS reduced cell proliferation rate by 20 %. This suggests that particle size may play an important role in material toxicity as SEN and COLPS have smaller hydrodynamic size compared to DLE and COL. However, we cannot exclude the possibility that unidentified inorganic materials in the toothpaste extracts could have resulted in the reduced cell proliferation. Additionally, the cells were exposed to the toothpaste extracts for an arbitrarily determined period of time that may not be clinically relevant. As such, our preliminary findings warranted further studies to explore the toxicity effects of the individual NM. 4.2 Characterization of pristine NM Many studies investigating similar parameters as this study may be clinically irrelevant because of the use of high NM doses [181]. In an effort to design a clinically relevant study, physiologically relevant dosages of pristine NM were chosen for the experiments. The dosages were determined (range: 0 to 1250 µM) based on the concentrations of detected elements in the toothpaste extracts. All pristine NM employed in this study had the same shape - spherical. Interestingly, we observed that the pristine NM formed aggregates 2 to 5 fold smaller in complete cell culture medium compared to ultrapure water. As the cell culture medium was 61 constituted of various proteins, this suggested that NM disaggregated in complex protein environments [182, 183]. This observation is likely due to the proteins forming corona and breaking down the natural aggregates formed by the NM in an aqueous environment [182]. In the complete cell culture medium, HA, TiO2 and SiO2 NM registered smaller zeta potential value compared to ultrapure water. The tested NM had smaller hydrodynamic sizes in complete cell culture medium than in water, which suggests that the high surface area of NM could facilitate protein and ions adsorption, and mask the surface charge of the NM [184, 185]. Furthermore, the increase in surface area in NM could influence and change the NM’s surface properties, which in turn may also affect dispersion stability. Collectively, these factors may explain the smaller zeta potential value when the NM were in complete cell culture medium. 4.3 Effects of pristine NM 4.3.1 Internalization and bio-distribution of pristine NM All three NM were internalized into the cells regardless of their size and surface charge. However, no disruption in the actin network was observed. In general, higher concentrations of NM led to increase internalization and accumulation of the NM in the cells. NM were observed in the cells even after 12 hours of exposure, indicating their stability within the cells. The low fluorescence signal detected for HA NM could be due to the large size of the HA NM rendering most of them unable to penetrate through the protective barrier of the cells. Furthermore, more HA NM were located in 62 the membrane fractions compared to the cytosolic fractions, reinforcing the earlier observation that internalization of NM is size-dependent. SiO2 NM were found in both the cytoplasm and the nuclei, and the small size is likely the reason for their ability to diffuse through the nuclear pore complex [47]. Another possible explanation could be the adsorption of proteins that signalled nuclear localization on the surface of SiO2 NM, allowing them to enter and accumulate in the nuclei [186-188]. The particle size of TiO2 NM is larger than SiO2 and they were observed to be localized only in the cytoplasm. Our observations are in agreement with other studies [71, 189-191] in that particle size is an important factor in determining the extent of NM internalization into the cell cytoplasm or nucleus. The accumulation of internalized NM in the cells increases the probability of interaction with biological molecules which in turn increases the risk for long term effects [42]. The small sizes of the NM allowed ease of uptake into the cells even after 1 hour of exposure. Interestingly, the amount of NM internalization peaked at a specific exposure time and subsequently declined. An explanation for the decline may be due to the cell-recognition of the NM resulting in the degradation and/or exocytosis of the NM after prolonged exposure. It is noteworthy that even though few HA NM aggregates were observed in the cells in the imaging study, HA NM displayed the most internalization of the three NM studied. This could possibly be due to the binding of HA NM to the surface membrane rather than truly being internalised into the cells. In addition, HA NM were possibly either adsorbed to the cell membrane and/or retained in the cell for the longest period of time compared to SiO2 and TiO2 NM, suggesting the higher stability of HA NM. 63 4.3.2 Interaction of HA, SiO2 and TiO2 NM with membrane and cytosolic proteins Cell membranes consist of cell receptors that are essential for cell-cell signaling, cell adhesion and interactions as well as immune system recognition of foreign substances [192]. In all NM (HA, SiO2 and TiO2) studied, a dose-dependent interaction between NM and cellular proteins was observed. We proposed that the uptake of NM into the cells was triggered by the binding of the NM to the surface receptors of the cell membrane. Factors such as shape and size can affect the binding of NM to surface receptors and the subsequent uptake via active transport [190]. All three NM are spherical in shape, indicating that their internalization into cells was size dependent. For HA NM, more NM were located in the membrane fractions compared to the cytosolic fractions. Whereas, similar amounts of SiO2 and TiO2 NM were located in the membrane and cytosolic fractions, suggesting that the smaller sizes of SiO2 and TiO2 NM allowed them to be more easily internalized by the cells. Collectively, the observed adsorption of NM to the cell membrane and accumulation in the cells for up to 24 hours results suggest the stability of NM in the cellular environment. Although, it is unlikely that the time exposure to toothpaste in daily living is of the same magnitude as in our study (up to 24 hours), it is plausible that the daily use of oral care products with NM additives could lead to accumulative detrimental effects. As such, more stringent profiling of the hazard risks of NM is needed before addition in consumer products. 64 4.3.3 Cell cycle progression and apoptosis DNA damage response is regulated by the tumor suppressor protein, p53 and consists of several steps - DNA repair mechanism, cell cycle arrest and apoptosis. MDM2 plays a vital role in stabilizing p53 [193], and phosphorylation of MDM2 leads to the degradation of p53 [194]. Although, NM are known to trigger DNA damage [195, 196], we did not observe an increase in the phosphorylation of MDM2, phosphorylated p53 and total p53 when the cells were treated with all concentrations of HA, SiO2 and TiO2 NM. This implied the stability of the p53 protein and that there was no significant activation of DNA damage response above the basal levels of the p53 protein. PARP, which is involved in DNA repair when the cells are under environmental stress [197], was also not activated. Both experiments indicated that the NM did not induce DNA damage nor increased the amount of stress in the cells [198]. Even though no DNA damage was detected in the cells treated with HA, SiO2 and TiO2 NM, a hindered cell cycle progression would provide evidence of early DNA damage. When cell cycle arrest induced by DNA damage is activated, the cells accumulate in the gap1 (G1), DNA synthesis (S) or in gap2/mitosis (G2/M) phase. Cell cycle progression is controlled by a family of cyclins and cyclin dependent kinases (CDK). In order to progress through the restriction point in G1 phase, cyclin D will form a complex with cyclin dependent kinases (CDK) 4 or 6 [199]. The absence of detectable changes in protein expression of cyclin D, CDK4 and CDK 6 indicated that cell cycle progression past the restriction phase was not affected. To facilitate cell progression into S phase, cyclin E1 interacts with and activates CDK2 65 [200-202]. Similarly, the insignificant change in expression level of cyclin E1 indicated absence of cell cycle arrest. Cyclin B1 controls the activation of cdc2, which in turn regulates the entry of cells into mitosis [203]. In accordance to the other cell cycle related proteins, there were no changes in expression level of phospho-cdc2 and cyclin B1. Collectively, the results signified that cell cycle progression was not affected when the cells were exposed to HA, SiO2 and TiO2 NM. This is in line with reported studies whereby internalized NM did not disrupt cell cycle progression [204, 205]. Apoptosis is triggered via two pathways: the extrinsic and intrinsic pathways [206]. The extrinsic pathway, also known as the death receptor pathway, is activated through ligand binding to death receptors on the cell surface. No activation of caspase 8 was observed after cell exposure to NM, indicating that the tested NM did not mimic ligands and did not bind to the death receptors on cell membrane. The intrinsic pathway involves permeabilization of mitochondria and release of cytochrome c from mitochondria into the cytoplasm, leading to the activation of caspase 9 and mitochondrial stress related proteins [207]. Expression levels of activated caspase 9 and Bax were not detected. This suggests that even though the NM were internalized into the cells, they did not permeabilize the mitochondria membrane nor led to release of cytochrome c. 66 As expected, activated caspase 3 was not detected, which confirmed that exposure of cells to NM did not trigger apoptotic events via death receptors ligands mimicry or mitochondria damage. 4.3.4 Inflammatory response Increased ROS production was observed when cells were exposed to increasing concentrations of HA, SiO2 and TiO2 NM. Interestingly, cell exposure to all concentrations (62.5 to 1250 µM) of HA NM gave rise to a significant increase in ROS expression levels despite its claim for being biocompatible [208, 209]. This could be attributed to the Ca2+ ions in HA, giving rise to high surface energy and formation of free radicals [210]. SiO2 NM which is another NM widely used in drug delivery also resulted in elevation of ROS production. This is possibly due to SiO2 NM’s ability to generate free radicals in aqueous medium following reduction of dissolved molecular oxygen [211]. Of the three NM evaluated, TiO2 NM caused the highest increase in ROS expression levels. TiO2 NM could have generated ROS due to absorption of photons from UV light. The absorbed energy promotes electron excitation across the TiO2 NM’s band gap, creating a hole in the valence band. When both the electrons and holes migrate to the surface of the excited NM, it increases the possibility for the electrons to react with oxygen to produce superoxide anion O2●-, or with water molecules to produce the hydroxyl radicals [212, 213]. Our observations are aligned with other studies whereby HA, SiO2 and TiO2 NM were reported to exert their toxic effects through oxidative stress [91, 214, 215]. Additionally, the high surface area: volume ratio has been reported to potentiate the oxidative stress effect of NM [216]. The increase in ROS production after cell exposure also suggests that NM 67 could induce inflammation [72, 217]. ROS production could have inflicted damage to the cell membrane by oxidizing lipids and proteins [218, 219] which could contribute to NM internalization into the cells. IL-6 and TNF-α are vital mediators of inflammatory processes mediated by NFκB [220] and their expressions can be induced in multiple cell types such as human oral tissue after exposure to compounds of dental materials [221]. In addition, IL-6 expression is induced in response to TNF-α [222]. As such, expression levels of these inflammatory markers would provide valuable insight as to how HA, SiO2 and TiO2 NM could affect NFκB mediated inflammatory response. There was a correlation between ROS production and IL-6 expression level in cells exposed to all concentrations of HA and SiO2 NM. This could be due to the positive feedback relationship between IL-6 and ROS production [223-225]. The presence of IL-6 could also evoke ROS production through the enzyme complex of the NADPH oxidase pathway [226]. Since IL-6 functions to protect the cells from oxidative injury [227, 228] and regulates acute phase inflammatory [220, 227, 229], the observed IL-6 expression in conjunction with increased ROS production suggested that the cell expression of IL-6 was an attempt to counteract oxidative stress. However, the correlation between ROS and IL-6 expressions was not observed in cells exposed to TiO2 NM. Intriguingly, only TiO2 NM with concentrations ≥ 250 µM led to a notable increase in ROS production, while a significant increase in IL-6 expression level was observed for all TiO2 NM concentrations. It is probable that IL-6 68 was expressed to stimulate release of anti-oxidant enzymes [230] to counteract and nullify the ROS expressed after NM exposure. However, since IL-6 is also involved in carcinogenesis regulation [231, 232] and cell proliferation [233-235], it is possible that the observed IL-6 expression was involved in a cellular response other than inflammation. A 2-fold increase in TNF-α production was only observed when the cells were exposed to higher concentrations (≥ 500 µM) of HA, SiO2 and TiO2 NM. A correlation between expression level of TNF-α and ROS was also observed, which is in accordance to the role of TNF-α generating ROS via the pro-apoptotic TNF-α signalling complex II [236, 237]. We observed a trend of elevated expression of NFκB with increasing concentrations of NM. Cells that were treated with higher concentrations (≥ 250 µM) of NM resulted in a statistically significant (p < 0.05) increase in amount of NFκB secreted. NFκB plays a central role in inflammatory response because it induces transcription of proinflammatory genes [238]. Concomitantly, TNF-α activates expression of NFκB to provide a defence mechanism for the cells by preventing cell death induced by TNF-α [239, 240]. RAW-NFκB cells exposed to heat-killed bacteria showed a drastic 30-fold increase in level of NFκB secreted. This implied that although NM exposed cells led to a statistically significant (p < 0.05) up-regulation of approximately 1.5 fold of NFκB secreted, this change was negligible compared to that in a conventional inflammatory response model. 69 As NFκB activation aids in the prevention of cell death induced by TNF-α, this could explain an absence of apoptotic activity after cell exposure to NM. Thus, even though NM exposure elicited an increase in oxidative stress and expression levels of inflammatory response genes, it is unlikely that the tested NM elicited a clinically significant response. The absence of cell cycle arrest and apoptotic events suggests the presence of an adaptive mechanism to counteract NM induced oxidative stress and inflammatory responses. 4.3.5 NM impaired wound healing and their route of uptake into cells Since all three NM were internalized into the cells, it would be interesting to examine if this would hinder wound healing in the trauma-prone oral cavity [164]. All three tested NM affected the cells’ migration mechanism. However, NM did not affect cell cycle progression which suggests that the NM likely impair wound healing by hindering cell migration and not by halting cell proliferation. As such, it is probable that the wounds will still heal given sufficient time, albeit slower. However, the delayed wound healing may be a concern in individuals with systemic conditions such as diabetes mellitus where their healing ability is already slow [173, 241]. Though NM were observed in the cells, a direct causation between NM internalization and wound healing cannot be drawn as it is possible that the NM can inhibit cell migration simply via physical blockage – adsorbed to the cell membrane or lining the cell culture vessel and preventing the cells from migrating. However, NM have increased affinity with proteins to form a protein corona [242] and they might mimic biological molecules to facilitate receptor mediated uptake. This in turn, can lead to 70 interference of biological processes such as expression of cell migration genes, impair wound healing or even disrupt the integrity of cytoskeleton. Clathrin-mediated pathway was posited as the most probable route of entry based on the size of the NM (≈ 200 nm) used in this study and our preliminary findings that NM endocytosis were highly influenced by particle size [243]. As such, MDC, a specific inhibitor of clathrin-mediated endocytosis in mammalian cells [244, 245] was used. Pre-treatment with MDC followed by cell exposure to all studied NM resulted in a significant recovery of wound healing capacity which provided evidence that clathrin-mediated endocytosis was likely the main route of entry for the cell internalization of HA, SiO2 and TiO2 NM. However, the MDC treated cells still displayed a lower wound healing capacity than the control cells, which highlights the possibility that other mechanisms of uptake, via either endocytosis pathway or passive diffusion exist. 71 Chapter 5: Conclusion and Future Work 72 5.1 Conclusion An in vitro model of human buccal epithelium was used to assess the impact of NM found in oral care products. The characterization of the inorganic toothpaste extracts identified the presence of Ca, Si and Ti, possibly derived from HA, SiO2 and TiO2 NM. This study showed that there was reduced cell proliferation rate upon prolonged exposure (48 hours) to toothpaste extracts. In order to conduct controlled experiments to identify which NM gave rise to the detrimental effects, pristine NM were utilized. We demonstrated that pristine HA, SiO2 and TiO2 NM were able to cross the cell membrane of human buccal epithelial cells and be internalized by the cells, regardless of their surface chemistry or size. HA and TiO2 NM were mainly deposited in the cytoplasm, while the small size of SiO2 NM allowed for diffusion through the nuclear pore and accumulation in the nuclei. We provided further evidence that the amount of NM uptake into the various compartments of the cell was size-dependent as more HA NM were found in the membrane fractions rather than in the cytosolic fractions. On the other hand, similar proportions of smaller sized SiO2 and TiO2 NM were found in both the membrane and cytosolic fractions. The amount of internalized NM in the cells declined over time, raising the possibility that these NM were recognized as foreign particles and exocytosed by the cells. Cell exposure to all tested NM resulted in elevation of ROS production and approximately 1.5 fold increase in inflammatory response. However, NM did not severely affect protein expression involved in cell cycle progression and apoptotic 73 events, suggesting the existence of an adaptive mechanism to counteract NM induced oxidative stress and inflammatory responses. Of the three NM, HA NM had the greatest impact on wound healing. A causative link between internalization of NM and impaired wound healing was shown by blocking endocytosis. We identified clathrin-mediated endocytosis as the main route of entry for the NM. Preventing internalization of NM via clathrin-mediated endocytosis resulted in a significant recovery of wound healing. This study demonstrated that commercially available toothpastes with NM additives can potentially impair wound healing in an in vitro model of human buccal epithelium. However, the cell cycle was not arrested and apoptosis was not detected. This suggests that the observed impaired wound healing would still heal, given sufficient time. 5.2 Recommendations for Future work We have shown that NM elevated the mRNA expression level of inflammatory response genes. However, the mRNA expression levels may not always correlate to the protein levels [246]. Further studies need to be conducted to assess the cytokine levels of inflammatory mediators using enzyme-linked immunosorbent assay (ELISA). More inflammatory cytokines such as IL-1, IL-8 and prostaglandin E2 can also be used to provide a more complete assessment of NM in toothpastes on inflammation. Although, we showed that NM can be found in the membrane and cytosolic fractions, the specificity binding of NM to different proteins is still 74 unknown. A causative link between internalization of NM and wound healing capability was identified, but the molecular mechanism of how NM hindered wound healing is not yet understood. Future studies can be conducted to determine which migration genes are affected after treatment with NM. It is well documented that monolayer cultured cells behave differently from cells maintained in three dimensions. Hence, experiments on a multilayer model of human buccal epithelial cells or tissue engineered oral mucosa may be carried out to more accurately define the impact of NM on the oral mucosa. 75 Chapter 6: References 76 [1] Pidgeon N, Harthorn B, Satterfield T. Nanotechnology risk perceptions and communication: emerging technologies, emerging challenges. Risk Anal. 2011;31:1694-700. [2] Morris VJ. 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Genome Biol. 2003;4:117. 94 [...]... cell proliferation, induce cytokines production and apoptosis [91, 92, 95] Currently, most of the research studies conducted in the field of dentistry are focused on the ability of HA NM to manage biofilm growth and re-mineralize the tooth surface [87, 88, 9698] There remains limited information on the cellular effects of HA NM on the human buccal mucosa despite the widespread use of HA NM in oral... studies have focused on the use of NM for oral drug delivery [165167] and the absorption and bio-distribution of NM following oral administration [65, 66, 168] The high permeability of the oral mucosa amplifies the importance of understanding the impact of NM on the oral mucosa Yet there are no studies that have evaluated the toxic effects of NM on oral epithelium With the prevalence of NM found in oral... were being internalized into human buccal epithelial cells and their bio-distribution in the cell if internalized Thereafter, we sought to evaluate the cellular responses (i.e presence of cell cycle arrest, evidence of apoptosis and inflammatory response) of human buccal epithelium to the NM - a potential toxicant Next, we determined the route of uptake of NM and their effects on wound healing This would... an excitation and emission wavelength of 485 nm and 535 nm, respectively The relative percentage of viable cells was obtained by normalizing the fluorescence values of the treated cells to untreated control Data represented the mean of three independent experiments 2.1.6 Determination of physiologically relevant concentrations of pristine NM To simulate clinically relevant concentrations of NM for exposure... USA) Briefly, 100 mg of NM were dispersed in 100 mL of anhydrous ethanol (Fisher Scientific, USA); thereafter 5 mL of APTES was added drop wise to the suspension and stirred continuously at 80 o C for 5 hours For HA NM, 50 mg of FITC (final concentration of 0.5 g/l) was added whereas 25 mg of FITC was added to SiO2 or TiO2 NM (final concentration of 0.25 g/l) The mixture was stirred continuously for another... to be considered when evaluating the toxicity of the NM [44] In addition to the size and surface chemistry, the structure of NM also plays an important role in determining the toxicity of materials The properties are further affected by the aggregation of NM that could change the reactivity of NM and result in detrimental effects 1.2.2.1 Route of entry for nanomaterials The main routes of entry of NM... NM on fundamental biological functions of the human buccal epithelium The hypotheses of this study were firstly; NM found in commercially available toothpastes would not have a toxic impact on the human buccal epithelial cell line in vitro Secondly, the NM found in commercially available toothpastes would not hinder wound healing To evaluate the toxicity profile of NM, we first investigated whether... Project on Emerging Nanotechnology (PEN) listed at least 1317 globally sold consumer products containing NM Some of the consumer products which frequently contain engineered NM include personal care products, textiles, and sports equipments [8-10] The number of consumer products with NM additives is projected to exceed 3000 products by 2020 [11] Silver (Ag), carbon (C), titanium oxide (TiO2), silicon dioxide... [51] Animal studies have demonstrated that Ag NM deposited in the liver can inhibit mitochondria activity and reduce the amount of available energy for the cells [52, 53] The constant low level of energy available for the cells could result in impaired antioxidant defence and/or weak immunity response [54] Furthermore, the presence of Ag NM reduces the concentration of glutathione which is necessary to... by ROS [51, 55] The association of low levels of glutathione [56] and auto-immune and degenerative diseases (i.e Alzheimer's disease, Parkinson's disease) has been reported which suggests that repeated exposure to Ag NM may put individuals at risk for development of these conditions The skin is the largest organ in the human body that is constantly exposed to the environment and therefore is potentially ... Calculation of physiologically relevant concentrations of NM 34 3.2 Characterization of pristine NM 35 3.3 Effects of pristine NM on human buccal epithelium 37 V 3.3.1 Internalization... characterization of pristine NM and their surface charges 36 3.3 Effects of pristine NM on human buccal epithelium 3.3.1 Internalization and bio-distribution of NM into TR146 human buccal epithelial... potential toxic effects The internalization and bio-distribution of pristine NM in human buccal epithelium were determined with confocal microscopy The mechanism of NM uptake into human buccal epithelium