Nanotoxicológia Fodor Bertalan Miskolci Egyetem, Egészségügyi Kar Nanobiotechnológiai és Regeneratív Medicina Tsz.

Key challenges in medicine Translating breakthroughs in understanding disease into preventive medicine How to increase productivity dramatically How to align reimbursement with better outcome and efficiency How to reap the benefits of healthcare while reducing the inefficiencies Paul Smit, Philips: ETP Nanomedicine 2007 The key challenges today in the conventional medicine are:

Conventional “Modern” The Progression of Medicine Conventional “Modern” Medicine “Personalized” or “Molecular” Medicine Nanomedicine Single-cell Medicine Today we see a changing paradigm from the conventional medicine to individualization, to personalized molecular medicine. The nanomedicine is a very usefull tool in this process.

Definition of nanomedicine Applying of nanotechnology in the theoretical and practical medical sciences: in diagnosis in treatment in prevention in disease control in medical researches Investigation and/or treating of life processes and diseases with nanoscale (10-9- 10-6 m) devices/drugs What is the nanomedicine?

Why does nanomedicine represent a huge promise for healthcare Why does nanomedicine represent a huge promise for healthcare? Earlier diagnosis increases chances of survival. By the time some symptoms are evident to either the doctor or the patient, it may be already too late. Conventional medicine is reactive to tissue-level problems that are happening at the symptomatic level. Nanomedicine will proactively diagnose and treat problems at the molecular level inside single-cells, prior to traditional symptoms, and hopefully prior to irreversible tissue and organ damage. Conventional medicine is not readily available to much of humanity because it is labor-intensive and that labor is sophisticated and expensive. Nanomedicine will be much more preventive, comparatively inexpensive because it will minimize use of expensive human experts, and can be more readily mass produced and distributed.

Trends in the nanomedical research Main targets: cardiovascular-, infectious diseases, cancer, diabetes, musculo-sceletal-, mental illness Early and accurate diagnosis – Nanodiagnostics -biosensors, nanoscale devices Targeted drug delivery – transfer the drug only to the diseased tissue site and monitor its impact Regenerative medicine – intelligent biomaterials, targeted cell implantation, polymers with programmable conformation, etc. Ethical, Legal, Social, Toxicological, etc.? The main targets of these researches are:……………… And the main goal is the early an accurate……………………………….. But in parallel these processes we have to answe a lot of new related questions.

In this slide you can see the number of registred nanomaterials and releated nanoclaims in the last five years.

USA nanotechnology patents Total number of patents And here you can see the increasing number of the nanotechnology patens in the USA Ray Bawa: Nanotechnology patent proliferation and the crisis at the U.S. patent office Albany Law Journal of Science and Technology 17 (3), 699-736, 2007

Supported EU FP7 topics 3.4 10 Topic Goals € billion % 1. Health DNA sequencing, tissue, cell and gene therapies, as well as biotech medicines 6.1 18 2. Food, agriculture, fisheries and biotechnology EuropeanKnowledge Based Bio- Economy (KBBE) (food, feed, forest, fisheries, agriculture, aquaculture, chemistry 1.9 6 3. Information and Communication Bioinformatics, personal healthcare, computer power to speed up DNA sequencing plus research into ‘Future and emerging technologies 9,1 27 4. Nanoscience get to the bottom of a disease, and develop and integrate new technologies and materials. 3.4 10 5. Energy A major opportunity for biotech. From the development of bio refineries to marine biomas 2.3 7 6. Environment emphasize the sustainable management of resources, climate change, pollution, and conservation. 1.8 5 7. Transport safer, 'greener' and 'smarter' pan European transport systems that will benefit all citizens, respect the environment, and increase the competitiveness of European industries in the global market. 4.1 12 8: Socio-economic Sciences and Humanities Every technological development has a societal consequence. Opportunities especially for National Association led projects like BioImpact, EuroBioJobs portal, BioLife TV, BioPicture Festival. 9: Space Biotech can support the EU’s long term needs, including space transportation (biofuels), bio-medicine, life and physical sciences in space 1.4 4 10: Security The biotech industry contributes to the safety of citizens not only by developing detection technologies and the knowledge needed to ensure security, but also by producing biomedical vaccines. Among the supportes EU FP7 frameprogramms, the nanomedicine has fourth priority with .4 billion Euros. It means approximetly 10 % of total budget. 10

MICRO- AND NANOSCALE COMPARISON CHART • ~ 0.5-0.8 mm (10-4 m): Coverslip for microscopic slides • ~ 50-200 μm: Human hair • ~ 20-50 μm: Many primary and cultured cells • ~ 7 μm: Human red blood cells • ~ 1 μm=1000 nm (10-6 m): Bacteria • ~ 100 nm: Viruses • ~ 25 nm: Microtubule width • ~ 15 nm: Antibodies (IgG) • ~ 1-20nm: Most proteins • ~ 10 nm (10-8 m): Intermediate Filaments (Vimentin) • ~ 5 nm: Microfilament width (Actin) • ~ 2-4 nm: Ribosome • ~ 2.4 nm: DNA width • ~ 1.2 nm: Amino acid (tryptophane) • ~ 1 nm: Aspirin molecule • ~ 1 nm -100 nm : Nanoparticles • ~ 0.2 nm: Individual atom 1 nm=10 Å Yuri Volkov, PhD, MD

Nano-periodic system Tomalia, 2009 J. Nanoparticle Research The engeneered nanomaterials we can categorize into Tomalia, 2009 J. Nanoparticle Research

Nano-periodic system Tomalia, 2009 J. Nanoparticle Research 13

Interactions Between Technologies for Development of Nanomedical Systems Nanoparticle fabrication and quality control labs Nanochemistry Dynamic Light scattering sizing Zeta Potential Atomic Force Microscopy Cell and intracellular targeting labs Flow cytometry Imaging (laser opto-injection and ablation) cytometry Confocal (one- and multi-photon analysis) Transient Gene Therapy (“gene drugs”) Construction of therapeutic genes for specific biomedical applications Animal testing/comparative medicine Human clinical trials Nanomaterials biocompatibility labs Microscopy/image analysis/LEAP Gene expression microarray analyses Biosensor Labs Biosensor molecular biology Results evaluated in targeting labs

Carbon nanotubes Allotropes of carbon A single-walled carbon nanotube (SWNT) is a one-atom thick sheet of graphite (called graphene) rolled up into a seamless cylinder with diameter on the order of a nanometer. Length-to-diameter ratio exceeds 1,000,000. Extraordinary strength

Intracellular targeting of nucleus Cell biology of nanomaterials can reveal previously unknown cellular mechanisms and responses. On the right, multiwalled carbon nanotubes (MWNT-NH3 – blue arrow) penetrating a human cell line (HeLa) imaged by TEM. On the right, confocal laser scanning microscopy of single-walled carbon nanotubes (SWNT-NH3) trafficking to the perinuclear region of epithelial lung carcinoma cells (adapted from Refs. Pantarotto, et al. Angew.Chem.Int.Ed. 2004, 43, 5242-5246; and Kostarelos, K. et al. Nature Nanotech. 2007, 2, 108-113 respectively).

Kidney function assay Chemically functionalised carbon nanotube body elimination through the renal route. The left image is a microSPECT image of an animal injected with radiolabelled f-CNT (red signal), indicating translocation to the kidneys within minutes. On the right handside, the two top images show single-walled carbon nanotubes (SWNT) and the rest of the images multi-walled carbon nanotubes as imaged by TEM from urine samples (Singh et al, PNAS, 2006).

CAGED ATOMS. A water-soluble contrast agent being developed for magnetic resonance imaging encapsulates two gadolinium metal atoms (purple) and one scandium metal atom (green) that are attached to a central nitrogen atom (blue). The molecule's tail (gray and red) makes the cage water-soluble. Water molecules (red and yellow Vs) surround the molecule.

Concept: Smart Nanomedicine Systems with Control of Gene/Drug Delivery within Single Cells Cell targeting and entry Y Y Intracellular targeting Therapeutic genes Magnetic or Qdot core (for MRI or optical imaging) Biomolecular sensors (for error-checking and/or gene switch) Here is a simple animation of how this appears from the point of view of the nanocapsule. Targeting molecules (e.g. an antibody, an DNA, RNA or peptide sequence, a ligand, a thioaptamer), in proper combinations for more precise nanoparticle delivery Leary and Prow, PCT (USA and Europe) Patent pending 2005

The Multi-Step Targeting Process in Nanomedical Systems

SPIO MRI Agents Why? Enhanced sensitivity (SNR) Super Paramagnetic Iron Oxide (SPIO) Nanoparticle Magnetic Core Biocompatible Shell Targeting Ligand Core Ligand Shell Why? Enhanced sensitivity (SNR) Contrast Control (T1 & T2) Targeting Capability Improved Biocompatibility Acceptable Toxicity How? Control Size Control Composition Control Shell Chemistry Next Gen Molecular Imaging Agents

Magnetic Nanoparticles for MRI Monodisperse Core Biocompatible Shell Magnetic Core Biocompatible Shell Targeting Ligands Targeting Capability

Quantum dot Quantum dot is a semiconductor whose excitons are confined in all three spatial dimensons. As a result, they have properties that are between those of bulk semiconductors and those of discrete molecules. Colloidal quantum dots irradiated with a UV light, different sized quantum dots emit different color light due to quantum confinement

Quantum Dot Applications in Cancer Management Quantum dots Protein binding & internalization Laboratory diagnostics Quantum dot labelling of mouse colon cancer Sentinel node visualization for breast cancer through 1 cm of tissue

Nanocell drug delivery Targeting molecules on nanocell attach to or enter cancer cells Nanocell releases chemotherapy drugs and imaging particles into the cells from its core. The drugs attack the tumour while the imaging particles help monitor tumour death

Drug delivery systems, nanocarriers 1 (nm) 100 nm 0.1 nm 10-6 10-7 10-8 10-9 10-10 Visible Spectrum liposome Szén nanocső micell dendrimers carbon nanotubes fullerens quantum dot 30

Use of liposomes in pharmacotherapy Localized and rate controlled delivery Improved therapeutic response Achieve appropriate tissue or blood levels Reduced adverse reactions Less drug administered Targeted drug release Lower dosing frequency Improved patient compliance Simpler dosing regimens Lower cost per dose Utilization of otherwise un-useable compounds

Liposomal drugs in the market and under development Trade Name Encapsulated agent Application Approval date Doxil, Caelyx Doxorubicin Ovarian cancer, breast cancer, Kaposi’s sarcoma 1995 Abelcet Amphotericin B Systemic fungal infections DaunoXome Daunorubicin Solid tumors 1996 Ambisome Fungal infections 1997 Epaxal-Berna Hepatitis A Hepatitis vaccine DepoCyt Cytarabin tumors 1999 Amphotec 2000 Myocet Visudyne Verteporfin Macular degeneration, ocular histoplasmosis Name Encapsulated agent Application status ATRA ATRA-IV Hematiological tumors Phase I-II Aroplatin oxeliplatin Colorectal tumors Onco-ts vincristine Phase III Topotecan TC topotecan Various tumors preclinical Vinorelbine TC Various solid tumors LED doxorubicin Breast cancer LEP-ETU paclitaxel LEM-ETU mitoxantrone Prostate cancer LEraFAON Pancreatic cancer Camptosar cancer

Liposomal Targeting Passive a process by which the physical properties of the liposomes combined with the microanatomy of the vasculature at the target tissue determine drug selective localization (EPR effect). Active requires a homing device (antibody, receptor ligand, etc.) as part of the liposome surface so that the liposomes can recognize the ‘‘sick’’ cells, bind to them selectively, and either be internalized by these cells or be broken down by either enzymatic hydrolysis or processes such as ultrasonic irradiation to release the drug near the cell surface so it will be taken up by the target cells

Types of hypersensitvity reactions to nanomedicines Acute Anaphylactic –toid reactions idiosyncratic pseudoallergic infusion reactions C activation-related pseudoallergy (CARPA) Late (chronic) late pseudoallergic reactions

APT070 39

2004 Donaldson és mtsai: Nanométeres részecskék viselkedése alapvetően eltér a nagyobbaktól Nanotoxicológia

CNT, egyéb C alapú nanorészecskék 1991 óta 21.236 cikk CNT (2007) 100 cikk/hét (2006) Gyártott CNT mennyisége 100 tonna 2004-ben 294 tonna 2005-ben 2400 tonna 2010-ben (becsült adatok) Keleti és Ázsiai piacok Korea vezető

MWCNT falak között kb. 0.34 nm távolság DWCNT – külső funkcionalizálható, belső intakt (mint az SWCNT) A CNT vége lehet zárt, vagy nyitott. Ha zárt, különböző rácsszerkezetű lehet – toxicitás A CNT erősen hajlamos az aggregációra Erősen insolubilis Kémiai anyagokkal szemben ellenálló A levegőben 500 C-on ég

Szintézisének 3 lehetséges módja Leggyakoribb: chemical vapor deposition (CVD) Elv: C tartalmú fragmentek képzése, feloldás – tube képződés - általában valamilyen fém katalízissel, magas hőmérsékleten (500–1200 ° C). Lehet katalízis nélkül is, de a hatékonysága alacsony, SWCNT kevés képződik így 3 kulcs összetevő: szénforrás (metán, metanol, acetilén, benzén, CO) katalizátor (felszíni: SiO2, por: MgO, zeolit, aluminium származékok, szilikát, vagy levegőben nincs katalizátor: HiPCO – nagy nyomású CO Minden tényező hatással lehet a CNT toxicitására, nemcsak maga a CNT szerkezet!!

Karakterizálás szükségessége Karakterizáció – „as-produced” Karakerizálás a formulázás során (tárolás, szállítás, etc.) – ahogy „beadásra” kerül Karakterizálás az alkalmazás után (in vivo hatás)

Méret A méret a leginkább vizsgált paraméter. A biológiai aktivitás a méret csökkenésével arányosan nő A nanométeres mérettartományú részecskék biológiai aktivitása (toxicitása) jóval nagyobb, mint a nagyobb részecskéké (akár mikro méretű) Azonban a méretnél nagyon gyakran csak az individuális méretet vesszük figyelembe és nem a tényleges méretet (aggregáció – gyártás, szállítás, tárolás)

Inhaled NP: membrán receptorok számára kezelhető méret (könnyebb transzlokáció), alacsonyabb alveoláris makrophág uptake (redukált clearence, redukált inflammatorikus válasz) Ugyanakkor!! Hosszabb ideig tartózkodik a szabad NP az alveoláris régióban – elhúzódó cytopathiás, etc. hatás

Felszín Alacsonyabb méret – növekvő felszín/volumen arány Növekvő felszínen több hozzáférhet atom, atomcsoport, etc. – növekvő toxicitás

A szervezetbe jutó NP tömeg vagy felszín a lényeg a toxicitásnál (dózis?) Ugyanazon tömegű nano és micro anyag közül nano toxicusabb Felszínre normalizálva egyenlő Nagyobb felszín – nagyobb abszorpciós kapacitás LDH (életképesség vizsgálat), cytokin mennyiségi vizsgálat – CB felszínén ezek a fehérjék absorbeálódhatnak – a vizsgálatok téves alulértékelése

Alak NP különböző alakú lehet (gömb, tű, cső, pálca, „korong”, etc.) Két fő hatás: 1. hidrodinamikus radius oldatokban és aerosolban (szférikus vs. hosszúkás) 2. depozíciós és absorpciós kinetika a biológiai rendszerekben, blocking mechanizmusok (ion csatornák, aktív transzport, etc. CNT – stimulálja a thr aggregációt a fullerén nem

Kémiai összetétel NP lehet inorganikus, fémoxid, organikus, core-shell, stb. Alapvetően befolyásolja a toxicitást (biokompatibilitás) QD – metal core – inorganic shell Fizikai (termál) vagy kémiai (fotokémia, oxidáció) coat degradáció – QD toxicus Fém core – biokompatibilis shell – degradáció - toxicitás A fém tisztasága??

Rácsszerkezet Rutil – anatáz (TiO2) stabil – metastabil <20 nm a tulajdonság változik Fényvédő krémek – rutil – anatáz alapvetően másként viselkedik a fotokatalizált oxidációs reakciókban

Felszín kémiai összetétele NP – erős hajlam az aggregációra (nagy surface/volume – erős interpartikuláris kölcsönhatások – van der Waals) Stabilizálás specifikus bevonatokkal, ami az aggregációt gátolja (hidrofil terminális csoportokat –SH, –CN, –OH, –COOH, –NH2 tartalmazó organikus anyagok) a felszínre kovalens kötéssel kötődnek Ez az insolubilis NP-át solubilissá teszi – nagyobb biológiai hozzáférhetőség – nagyobb biológiai aktivitás – nagyobb toxicitás (pl. funkcionalizált CNT)

Méret, alak meghatározási módok Transmission Electron Microscopy (TEM) resolution is in the 0.5–3 nanometers, and it is the most useful and appropriate technique for the direct investigation of NM (63). In particular, TEM resolution is nominally below 1 nm for high-resolution transmission electron microscopy (HRTEM) (64). SEM and TEM observations must be performed in a vacuum environment, and can be applied only to solid samples. Other microscopy techniques are also available for the determination size and shape, as well as the aggregation state, such as scanning probe microscopy (SPM). SPM includes both atomic force microscopy (AFM) and Photon correlation spectroscopy (PCS), in its subtechniques dynamic scattering (DLS) and quasielastic light scattering (QELS), can measure article size of NM in liquid dispersions, with the great advantage of g a nondestructive technique (73–77). The obtained dimension is actually

Felszín (terület) meghatározása Brunauer–Emmett–Teller (BET) method Szilárd, száraz NP felszín meghatározása Inert gáz (pl. N2) absorpciójának a meghatározása

Felszíni töltés Fontos tényező a biológiai válaszban (fagocytosis, genotoxicitás). Csökkenő felszíni töltés csökkent toxicitás (amine-terminated poly(amidoamine) dendrimers for drug delivery applications) Complement-aktiváció

Kémiai stabilitás és felszíni töltés oldatokban Vízben is és organikus oldószerekben is erős az aggregációs hajlama a NP Az aggregációs állapot és az aggregáció kinetikája sok faktortól függ. (NP type, concentration, coating, solvent, temperature, pH, salt concentration, ionic strength, presence of surfactant and/or dispersants, etc.) Zéta potenciál (i.e.,<30mV) or positive (i.e.,>30mV) kevésbé aggregálódik, a kettő között igen

Kémiai összetétel, tisztaság, rácsszerkezet meghatározás inductively coupled plasma (ICP), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), UV-Vis and fluorescence, A very recently commercially available technique, the aerosol time of flight–mass spectrometry (ATOF-MS), allows to get a detailed chemical analysis of NP under aerosol form, also subdividing the analyzed particles into with TOF-MS HPLC-FL Fourier transform infrared spectroscopy (FTIR) and Raman spectro- scopy (RS)

Az alkalmazásra került NP karakterizálása Talán a legfontosabb toxicológiai feladat Fluorescence microscopy (FLM), TEM, SEM, differential interference contrast microscopy (DIC), and confocal laser scanning microscopy (CLSM) Cryogenic SEM and TEM, as well as scanning transmission ion microscopy (STIM), are microscopy techniques specifically modified to be applied to unfixed, unstained vitrified biological samples as diagnostic tools in structural cell biology.

Néhány fiziko-kémiai jellemző the form of the particle (e.g., fibers more potent toxicant than isometric particles, spiky fractured crystals more potent than smooth roundish particles); the chemical composition and related surface reactivity (free radical generating surface sites, poorly coordinated and easily removable metal ions, strong adsorption and modification of endogenous antioxidants or of proteins); the time of residence in a given body compartment generally defined as biopersistence (a property related both to chemical factors, such as solubility, adsorption potential, and to the cellular and tissue response to it).

Biodistribution

PGA polyglycerol adipát Species függés!!

Farmakokinetika Kevés adat Növekvő vízoldékonyság – csökkent clearance a vérből, szöveti disztribúció NP akkumuláció májban, RES-ben Felszíni karakterisztikától függően akkumuláció pl. vesében Összességében nagy szöveti retenció – toxicitás potenciális növekedése Szöveti retenció – Vd (volume/kg) növekedése – elhúzódó C (volume/time-kg), elhúzódó T1/2 NP farmakokinetikai modellek species függése (kis állat – magasabb basal metabolic rate vs. nagy állat) NP – sejt interakció, aktív transzport, etc eltér a konvencionális kémiai farmakokinetikától

Légúti és cardiovascularis hatások Deposition models predict that deposition of coarse (>2.5 µm) and fine (0.1–2.5 µm) particles is governed by the processes of sedimentation and impaction. In the case of nanoparticles (<100 nm), particle mass and momentum are extremely small; thus, sedimentation and impaction are not significant factors in pulmonary deposition. Rather, nanoparticles behave like gas molecules, moving randomly by Brownian motion. Such movement would result in the random contact of nanoparticles with the epithelial and/or fluid lining the lung. In addition, as nanoparticles become smaller than 10 nm, nasal deposition by diffusional mechanisms becomes very high.

Fine particles are more effectively phagocytized and cleared by alveolar macrophages than nanosized particles. Oberdorster et al. (11). In this study, rats were exposed to nanosized (20 nm) or fine (250 nm) particles by inhalation for 12 weeks at respective concentrations that resulted in a similar mass deposition of the two particle types. At 1-year postexposure, 44% of deposited nanosized TiO2 had migrated to the interstitium compared to 13% for fine TiO2. In conclusion, not only are nanoparticles highly deposited in the alveoli, but particle number/mass is so great that some nanoparticles escape phagocytosis by alveolar macrophages and can enter the alveolar interstitium. The recognition of nanoparticles by alveolar macrophages is dependent on particle type, i.e., quantum dots and nanogold particles are rapidly phagocytized by alveolar macrophages while SWCNT are not. Therefore, a large fraction of deposited SWCNT rapidly migrates to the alveolar interstium. This high interstitial deposition of SWCNT after aspir ation has been associated with diffuse interstitial fibrosis of rapid onset, which progresses over 60 days postexposure (13).

UFP DEP

Nanoparticles as the Most Toxic Component of PM10 PM is a complex mixture of particle types that vary widely depending on season, time of day, or the sampling site. CDNP originates principally from automobile tailpipes although there are other sources (4).

The oxidative stress then causes activation of signaling pathways for proinflammatory gene expression, including MAPK (30,35–37) and NF-kB activation (30,38) and histone acetylation that favors proinflammatory gene expression (39). Activation of these pathways culminates in transcription of a number of proinflammatory genes such as IL-8 in epithelial cells treated in vitro (40) and in human lungs exposed by inhalation (41).

Randomizált kettős vak study AMI (>6 hónap) DEP (300 µg/m3) or filtered air for 1 h 15 perc ergométer 12 csatornás Holter we documented painless myocardial ischemia thatwas increased up to threefold by diesel exhaust inhalation

Oxidative modification of low-density lipoprotein (LDL) particles is a dominant hypothesis of atherogenesis (17–19). Oxidized LDL particles are readily taken by macrophage scavenger receptors, leading to “foam cell” formation (macrophage loading with lipids), an obligatory step in atheroma development. Bioactive lipids derived from LDL oxidation can also modulate intracellular signal transduction and expression of genes coding inflammatory mediators and adhesion molecules (20–22)

Metodikai aspektusok Many studies have measured release of the cytoplasmic protein lactate dehydrogenase (LDH). This protein leaks into the extracellular media either as a consequence of direct membrane insult leading to death, or due to cell death resulting in membrane breakdown. Therefore the enzyme activity of LDH measurable within the cell culture medium of exposed cells should be directly proportional to the level of cell death. However, we (12) and others (29) have observed that micro and nanoparticles have a large capacity to either bind proteins or to induce their degradation, which could lead to an underestimation of that protein content in the cell culture supernatant. Our own recent data clearly shows that LDH mixed with nanoparticles results in an underestimation of the LDH content of media (15). It is interesting to note that activated carbons are used as medical absorbents and filters. They are commonly used to remove poisons and toxins and have been proposed to remove inflammatory markers in blood (31).

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is also widely published as a useful tool to measure cell viability (32). The MTT molecule is converted to a blue formazan product by the mitochondrial enzyme succinate dehyrogenase, and so a decrease in competence of the cells is used as an indicator of viability. However the colored formazan product is actually a combined measure of the amount of functional succinate dehydrogenase per cell and the number of cells per culture well. While a decrease in MTT absorbance can clearly be used as an indicator of toxicity, it is not clear whether this is due to decreased cell function or decreased cell number, although in fact for many purposes it may not be necessary to distinguish between the two. Interpretation of an increase in MTT absorbance, or in fact of no change, is also confused by these issues and may need to be considered carefully. The production of the colored formazan involves an oxidative reaction. In phagocytic cells generating an oxidative burst on exposure to nanoparticles, this additional oxidative effect could lead to an underestimation of cytotoxicity and again requires careful control. Since measurement of the formazan product is via absorbance, the light absorbing properties of any particles also needs to considered, monitored and controlled for. For example, black carbon particles will generate a significant background absorbance over many wavelengths.

Annexin V combined with propidium iodide staining is proving to be a reliable and relatively easy technique to quantify viability, but also to distinguish between cell death via apoptosis and necrosis (33). The technique involves staining the cells using immunofluorescence techniques followed by subsequent quantification by flow cytometery or flourimetry. It is also possible to image the cells by fluorescence or confocal microscopy to observe changes in individual cells within a culture. The Live/Dead assay has also been used in some studies (34). This assay involves treating the cells with calcein acetoxymethylester (AM) which diffuses into cells due to the lipophillic nature of AM. Once inside viable cells, cytoplasmic esterase enzymes will cleave the dye to release fluorescent calcein. In addition, the cells are treated with ethidium homodimer that can only enter cells if the cell membrane is compromised and hence the cell is dead. Once inside the cell the ethidium binds to DNA to generate a fluorescent signal which can be quantified or imaged alongside the calcein signal. With any of the assays using fluorescence as an endpoint, the ability of the particles to interfere with fluorescence quantification must be taken into consideration, for instance, TiO2 can reflect UV light, while carbon black absorbs light and can alter the background fluorescence