Abstract

Currently, nanoparticles are a hot topic in the medical field. They are being used and have

potential applications in medicine such as drug delivery and diagnostics. However, some

nanoparticles may be dangerous at certain doses and have fuelled studies which link them

to autoimmunity. Ongoing in vitro as well as in vivo experiments in this laboratory led by

Prof. Yuri Volkov have provided a link between various nanomaterials and the possible

development of autoimmune diseases by the induction of protein citrullination. However,

it is not clear whether nanomaterials induced protein citrullination is persistent. The

present study investigates the sustainability of protein citrullination induced by silica

nanoparticles. We utilized A549 lung cancer epithelium cell culture model to analyze

protein citrullination by High Content Screening and Western immunoblotting techniques.

Silica nanoparticles significantly increased the level of protein citrullination in A549 cells

in comparison to non treated negative controls. This increase in protein citrullination was

sustained in cells even after the removal of these particles for up to another 96 h, indicating

that citrullination is not reversible and is persistent. Nanomaterials induced irreversible

protein citrullination explain harmful effect on the possible development of autoimmune

diseases such as Rheumatoid Arthritis and Multiple Sclerosis and raise a serious concern to

human health.

TCD Faculty of Health Sciences

Assignment Submission

Student Name

Student Number

Course MSc. Molecular Medicine 2010

Project Title

Supervisor

Word Count

Project Due Date July 16th 2010

Declaration

I, the undersigned declare that the project material, which I now submit, is my own work. Any assistance received by way of borrowing from the work of others has been cited and acknowledged within the work. I make this declaration in the knowledge that a breach of the rules pertaining to project submission may carry serious consequences.

Signed: ____________________________________

Contents

1. Introduction 1.1 Nanotechnology.………………………………………………. 11.2 Nanoparticles ………………………………………….……… 11.3 Potential health risks associated with nanoparticles………….. 21.4 The effect of nanoparticles on Immune function………………31.5 Nanoparticles and protein citrullination ……………………… 51.6 Project aims…………………………………………….............6

2. Materials and methods…………………………………………………… 72.1 Reagents and Antibodies……………………………………… 7

2.2 SiNP…………………………………………………………… 72.3 Cell culture……………………………………………………. 72.4 High Content Screening and Analysis (HCA) of protein citrullination ………………………………………………………. 82.5 Western blot analysis of protein citrullination ………………. 8

3. Results3. 1 SiNP induced protein citrullination as analysed by HCA…….. 123. 2 SiNP induced protein citrullination as analysed by Western blotting ……………………………………………………………. 17

4. Discussion………………………………………………………………… 215. Acknowledgments………………………………………………………….256. References …………………………………………………………………. 26

1. Introduction

1.1 Nanotechnology

Nanotechnology, as defined by the United States (US) Nanotechnology Initiative, is ‘the

understanding and control of matter at dimensions of roughly 1-100 nanometres, where

unique phenomena enable novel applications (Medina et al., 2007; Lin et al., 2006). It is

among the fastest growing areas of scientific research widely perceived as one of the key

technologies of the 21st century. This technology is being increasingly used for

manufacturing diverse industrial items such as cosmetics or clothes and for infinite

applications in electronics, aerospace and computer industry and many products are

already on the market (Medina et al., 2007; www.nanotecproject.com). Nanotechnology,

in combination with biomedical developments, offers the promise of revolutionary tools

for biomedical analysis. One of the most promising applications of such a combination is

the application of highly luminescent nanomaterials as tags for identification and

quantitation of small amounts of biological targets (Jin et al., 2007). There has been

significant advances worldwide and increased global funding in nanotechnology research.

In the coming few years, nanotechnologies are expected to bring a fundamental change

with an enormous impact on life sciences, including drug delivery diagnostics,

nutriceuticals and the production of biomaterials. A huge breakthrough has been the use of

nanotechnology in cardiovascular medicine and cancer treatment, see figure 1 (Medina et

al., 2007).

1.2 Nanoparticles

Nanoparticles have unique properties not shared by non-nanoscale particles with the same

chemical composition (Auffan et al., 2009). They represent a transition between bulk

materials and atomic or molecular structures and, at this level, quantum effects lead to the

occurrence of specific physicochemical properties including conductivity, strength,

durability and reactivity (Lison et al, 2009). These properties make nanoparticles useful for

many applications. Several nanomaterials are made of metals or metal oxides, including

silica, zinc, cerium, zirconium, gold, silver, copper, lead, cadmium, geranium and

selenium. Production of nanomaterials is currently estimated to be thousands of tonnes per

year worldwide, and is expected to increase substantially in the next decade.

1

Much effort is being put into the technological research and development on

nanomaterials for their potential application in biology and medicine. For example,

inorganic ceramic nanoparticles, such as silica, with porous characteristics emerged as

drug vehicles in the 90’s (Cherian et al., 2000). As a non-metal oxide, silica (SiO2)

nanoparticles (SiNP) have found extensive applications in chemical mechanical polishing

and as additives to drugs, cosmetics, printer toners, varnishes, and food (Lin et al., 2006).

The use of SiNP has been extended to biomedical and biotechnological fields in recent

years such as biomarkers for leukaemia cell identification using optical microscopy

imaging, cancer therapy, DNA delivery, drug delivery, and enzyme immobilization (Lin et

al., 2006). Silica occurs in crystalline or amorphous forms. Crystalline silica is abundant in

most rock types such as granites, sandstones, quartz and sands, as well as soils. Based on

several toxicity studies, the International Agency for Research on Cancer (IARC) has

classified crystalline silica as a Group 1 carcinogen (Cho et al., 2007).

Exposure to silica at micro-scale size is associated with the development of several

autoimmune diseases, including systemic sclerosis, rheumatoid arthritis, lupus, and chronic

renal disease, while certain crystalline silica polymorphs may cause silicosis and lung

cancer (Lin et al., 2006). Although the pulmonary toxicity of crystalline silica is well

known, there is relatively little information about the toxicity of amorphous silica

(Napierska et al., 2009). Therefore, there is an urgent need to investigate the possible toxic

effect of SiNP on the human health.

1.3 Potential health risks associated with nanoparticles

Increased industrial and biomedical applications of nanoparticles in recent years has raised

safety concern (Myllynen, 2009) and attracted attention of researchers to investigate

potential toxic effect they may have on human health. Humans have been exposed to

nanoparticles throughout their evolutionary phases (Medina et al., 2007). As a result of

their small size and thus more surface area nanoparticles may easily be taken up by

humans and other mammals through the the respiratory system, blood, central nervous

system (CNS), gastrointestinal (GI) tract and skin have been shown to be targeted by

nanoparticles (Medina et al., 2007). Inhalation of nanoparticle has been associated with

neurological disease: Parkinson’s and Alzheimer’s disease, lung disease: Asthma,

Bronchitis and emphysema and lymphatic system: Kaposi’s sarcoma. Figure 1 shows the

2

possible health effects associated with atmospheric NP’s after inhalation. Nanoparticles

ingested have been associated with gastro-intestinal disease: Crohn’s disease (Buzea et al.,

2007). However, a direct link between exposure and disease manifestations is extremely

difficult to establish because of the inherent limitations of epidemiological studies to draw

causal conclusions (Cho et al., 2007).

Figure 1. Potential effects of inhaled ultrafine particles. After inhalation of NP’s the above

diagram shows three possible routes which all ultimately damage the heart. (Source: Ostiguy et al.,

2006).

1.4 The effect of nanoparticles on Immune function

Nanoparticles have also been associated with the development of autoimmunity. Smoking

has been linked in the pathogenesis of certain autoimmune diseases such as RA, systemic

sclerosis, multiple sclerosis and Crohn’s disease (Lee et al., 2007). More recently, the

association of silica and development of systemic autoimmune diseases such as Systemic

lupus erythematosus (SLE) and scleroderma stems from exposure during occupations such

as mining, construction, and glass and pottery production has been reported (Pollard et al.,

2010).

3

Because the immune system is dispersed throughout most tissues and organs,

exposure of humans to chemicals and drugs is certain to involve some immune cell contact

regardless of the route of exposure (Dietert et el., 2008). Cytotoxicity or cell death induced

by nanomaterials has been reported to be associated with immune response (Figure 2).

Recently, a number of investigators have found nanoparticles responsible for toxicity in

different organs (Medina et al, 2007). Developmental immunotoxicology (DIT) is a

subarea of immunotoxicology focusing on the effects of exposure to biological materials,

chemicals, drugs, medical devices, physical factors (e.g., ionizing and ultraviolet

radiation), and, in certain instances, physiological factors on the developing immune

system (Dietert et el, 2008).

Figure 2. Putative mechanism of drug/chemical-induced autoimmunity. The diagram above illustrates the subsequent path of cells when exposed to a toxic particle. Immune receptors [Nod-like receptor (NLR) and toll-like receptors (TLR)] are activated by toxicant such as nanomaterials induced cell death and leading to an immune response (Source: Pollard et al., 2010).

One of the significant challenges facing autoimmune disease research is in identifying

the specific events that trigger loss of tolerance and autoimmunity (Pollard et al., 2010).

Pathological protein citrullination is associated with inflammation, occurring in a range of

autoimmune diseases such as Rheumatoid arthritis, multiple sclerosis, glaucoma, myositis,

and Alzheimer’s disease (Wegner et al, 2009).

4

1.5 Nanomaterials and protein Citrullination

Protein citrullination is a post-translational modification of proteins and involves the

enzymatic conversion (deimination) of protein-contained arginine residues. This enzymatic

conversion is caused by activation of a family of calcium dependent enzymes

peptidylarginine deiminase (PAD) (Vossenaar and Venrooij 2004). In mammals, five

isotypes of PAD have been described (Vossenaar and Venrooij 2004). This conversion

causes a small change in molecular mass (less than 1 Da) and the loss of one positive

charge. The consequence of losing this positive charge might change (loss or gain) its

ability to interact with neighbouring proteins (Venrooij and Pruijn). It can be presume that

the modification alters the protein structure and results in a somewhat looser, less

organised, more open configuration (Gyorgy et al, 2006). Since citrullinated residues are

not natural amino acid in proteins, they induce an immune response (Gyorgy et al, 2006).

The immune system should be kept tolerized continuously against all of these

modifications on self-proteins. Permanent failure of tolerization to such modifications of

proteins might cause the escape of auto-reactive immune cells that can give rise to severe

autoimmune-mediated chronic inflammation (Uysal et al., 2010).

Increased peptidylarginine deimination correlates with the severity of MS

(Moscarello et al., 2002b). In MS, the myelin protein undergoes deimination of arginine to

citrulline by PAD (G. Harauz et al, 2004). Deimination limits the ability of myelin basic

protein (MBP) to maintain a compact myelin sheath by perturbing the protein’s structure

and its interactions with itself and with lipids. (Harauz et al, 2004; Moscarello et al, 2007;

Vossenaar and Robinson, 2005). Antibodies to autoantigens modified by citrullination

through deimination of arginine to citrulline were present in about two-thirds of all RA

patients but are rare (2%) in healthy individuals and relatively rare in other inflammatory

conditions. (Klareskog et al, 2006).

Ongoing studies carried out in Professor Yuri Volkov’s laboratory showed

nanomaterial induced protein citrullination in vivo and in vitro in various cell lines

including A549 human lung epithelium cells (unpublished data). However, it is not clear

whether protein citrullination induced by these nanomaterials is a reversible or irreversible

phenomenon.

5

1.6 Project aim

Based on the above-mentioned facts and ongoing experiments in Prof. Volkov’s

laboratory, the aim of the present study was to investigate whether protein citrullination

induced by SiNP is a reversible or irreversible phenomenon.

6

2. Materials and Methods

2.1 Reagents and Antibodies

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine,

and streptomycin were from Gibco BRL (Grand Island, NY, USA). Mouse monoclonal

anti-α-tubulin antibody was purchased from Sigma (St Louis, MO, USA). Alexa fluor 488

conjugated anti-mouse was from Molecular Probes (Invitrogen Corporation, California,

USA). Polyvinylidene fluoride (PVDF) membrane was obtained from Pall Gelman

Laboratories (Ann arbor, MI, USA). Acrylamide-bisacrylamide solution, Acrylogel (30%)

was purchased from BDH (VWR International Ltd., England). Enhanced

Chemiluminescence (ECL) plus reagent was purchased from Amersham (Arlington

Heights, IL, USA). All the reagents unless attributed specifically, were from Sigma (St

Louis, MO, USA). All the plastic wares were from Nunc (©2010 Thermo Fisher

Scientific,)

Rabbit polyclonal anti-citrullinated protein antibody (AbCam, #ab6464, UK) and rabbit

polyclonal anti-citrullinated protein antibody (Upstate #07-377, Placid Lake, NY) were

used for immunofluorescence staining and Western blot analysis respectively. FITC-linked

goat anti-rabbit antibody was from Sigma. Horseradish peroxidase conjugated anti-rabbit

IgG and anti-mouse IgG antibodies were from Dako A/S (Denmark).

2.2 SiNP

The non-metal oxide SiNP (30 and 400 nm; Glantero Let., Cork, Ireland), positively

charged alumina coated chloride-ion stabilized SiNP (40 nm; Sigma-Aldrich, LUDOX CL

420891). SiNP concentrations were as follows: 30 nm- 3.44± 0.27 mg/ml, 40 nm- 2.5±

0.06 mg/ml, 80 nm- 1.25± 0.07 mg/ml, 400 nm- 5.43± 0.04 mg/ml.

2.3 Cell culture

A human lung epithelial cell line A549 originated from a alveolar cell carcinoma was used

and cultured in DMEM medium containing 10% (v/v) FBS and 100 µg/ml streptomycin in

a humidified chamber at 37ºC containing 5% CO2.

7

2.4 High Content Screening and Analysis (HCA) of protein citrullination

Five 96 well plates were seeded with 4× 102 cells in 100µl of DMEM. After 24 h, cells

were treated with indicated concentrations if SiNP (sizes 30nm, 40nm, 400nm and 80nm)

in triplicate. Triplicate wells were also left untreated for a negative control. Cells were

incubated with SiNP overnight at 37˚C. After 24 h, the SiNP were removed from plates

and further incubated for additional 1 to 4 days. The culture medium was changed daily in

the remaining plates. After completion of treatments, plates were washed with PBS and

fixed with 3% PFA for 10 min. Goat serum (10% in phosphate buffer solution (PBS)) was

added to each well as a blocking agent and left overnight at 37ºC. The wells were then

washed in PBS and 50 µl of the primary antibody (rabbit anti-citrulline protein, dilution

1/1000) was added and incubated at 37ºC for 2 h. Wells were washed in PBS and the

secondary antibody (goat anti-rabbit antibody) was added and incubated at 37ºC for 2 h.

Cells were further stained with phallodin for actin and hoechst for nuclei. Wells were

washed three times in PBS. Plates were scanned (three randomly selected fields per well

containing at least 300 cells) using IN Cell Analyzer 1000 automated microscope (GE

Healthcare). Images were acquired using 20X objective. Protein citrullination was

analyzed and quantified by IN Cell Investigator software (GE Healthcare) measuring

cytoplasmic fluorescence intensity.

2.5 Western blot analysis of protein citrullination

Cells were grown in 6-well tissue culture plates (0.2×106 cells per well). Three non treated

wells and three wells with cells treated with 30 nm, 40 nm and 400 nm. Each time point 0

h, 24 h, 48 h, 72 h and 96 h used one 6-well plate.

2.5.1 Cell lysis

Cells were washed with ice-cold PBS and lysed in lysis buffer [HEPES 50 mM (pH 7.4),

NaCl 150 mM, MgCl2 1.5 mM, EGTA 1 mM, sodium pyrophosphate 10 mM, sodium

fluoride 50 mM, -glycerophosphate 50 mM, Na3VO4 1 mM, 1% Triton X-100,

phenylmethylsulphonyl fluoride 2 mM, leupeptin 10 g/ml and aprotinin 10 g/ml]. Lysis

was carried out at 4ºC for 30 min. Lysates were centrifuged at 16,000 g for 15 min at

4ºC. The protein content of the supernatant was determined by Bradford assay and stored

8

at -20ºC until needed.

2.5.2 Sample preparation

Cell lysates were normalized for equal protein content as indicated for particular immuno-

blotting experiment and final volumes were adjusted with lysis buffer. Protein samples

were then boiled at 100C for 5 min with Laemmli sample buffer [final concentration:

Tris-HCl 62.5 mM (pH 6.7), Glycerol 10% (v/v), sodium dodecyl sulphate 2% (w/v),

bromophenol blue 0.002% (w/v) containing - mercaptoethanol 143 mM] for 5 min and

centrifuged briefly (1 min) to remove any insoluble solids.

2.5.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein samples were resolved by electrophoresis on SDS-PAGE gels to separate the

proteins according to their size and molecular weight. The gel apparatus (Atto, Japan) was

assembled according to the manufacturer’s instructions. The resolving gel (10 %

acrylamide gels) was made by adding the components in the following order (final volume

20 ml).

2.5.4 Components of resolving gel solution (10 %)

Distilled water (8.03 ml), 30 % polyacrylamide (6.67 ml), 1.5 M Tris-HCl pH 8.8 (5 ml),

10 % w/v SDS (200 µl), 10 % w/v Ammonium persulphate (APS) (200 µl),

Tetramethylethylenediamine (TEMED) (10 µl). The gel was quickly poured in between

the two glass plates until the polyacrylamide solution reached 1 cm below the plastic

combs. The gel was overlaid with distilled water to prevent oxidation of the gel. The gel

was allowed to set for approximately 30 minutes at room temperature.  The distilled water

was drained off. The stacking gel was made by adding the components in the following

order as indicated.

2.5.5 Components of the stacking gel solution (5%)

Distilled water (3.05 ml), 30 % polyacrylamide (0.65 ml), 1.5 M Tris-HCl pH 6.8 (1.25

ml), 10 % w/v SDS (200 µl), 10 % w/v APS (200 µl), TEMED (10 µl). The stacking gel

solution was poured on top of the resolving gel. The plastic combs were inserted into the

9

stacking gel to make protein sample loading wells and the gel was allowed to set for

approximately 30 minutes at room temperature. The combs were removed from the gel and

any gel lanes that were not straight were straightened using a pin. The gels were placed

into the gel electrophoresis box and the box was filled with 1X SDS-PAGE running buffer

(prepared as given below), making sure bubbles are removed.  Each loading well was

washed extensively by pipetting up and down with running buffer to remove any

unpolymerised acrylamide solution. Protein samples (20 µl each) and the protein

molecular weight ladder were loaded into the wells and electrophoresed at 100 V/20

mAmp per gel until the sample buffer migrated to the bottom of the gel. The gel was then

ready to be transferred to PVDF membrane by Western blotting.

2.5.6 Western blotting

Western blotting was carried out for the transfer of electrophoresed proteins to PVDF

membrane using the semi-dry transfer technique according to the manufacturer’s

instructions (ATTO Corporation, Japan). The PVDF membrane (0.45 m) was activated

by soaking it in methanol for 1 min. The membrane was then immersed in transfer buffer

for 10 min at room temperature. A gel sandwich was made by placing 4 sheets of

Whatmann 3 mm filter paper (pre-soaked in transfer buffer) on the anode of the Western

blotting apparatus.  The PVDF membrane was then placed on top of the filter papers and

kept moist by flooding it with transfer buffer (prepared as given below). The gel was then

placed on top of the PVDF membrane and any air bubbles between the gel and membrane

were carefully removed. Another 4 sheets of Whatmann 3 mm filter (pre-soaked in transfer

buffer) were placed on top of the gel. The cathode was lowered carefully onto the gel

sandwich and any excess transfer buffer remaining on the anode was removed with paper

towels to prevent the apparatus from short-circuiting. Western blotting was performed for

1 h at 100 mAmp / 200 V per gel at room temperature.

2.5.7 Immuno-detection and development of blots

Following Western blotting, non-protein bound sites on the PVDF membrane were

blocked by incubating the membrane in freshly prepared 5% non-fat milk in PBST

(Blocking solution, prepared as given below) for 1 hour at room temperature with constant

gentle agitation. Blots were then washed three times with PBST and incubated with

10

appropriate primary antibodies (diluted according to the manufacturer’s instructions in

blocking buffer) overnight at 4°C with constant agitation. Following incubation with

primary antibody, blots were washed five times with 0.1% PBST to remove any unbound

antibody. Blots were then incubated with the relevant horseradish peroxidase (HRP)

conjugated secondary antibody (diluted according to the manufacturers instructions in

blocking buffer) for 1 h at room temperature with constant agitation. Unbound secondary

antibody was then removed by washing the membrane five times with PBST for 10

minutes. 

The immuno-blots were visualized using the ECL method according to manufacturer’s

instruction (Amersham, Arlington Heights, IL, USA). ECL reagents were placed on the

membrane and incubated for 1 min. Membranes were then exposed to Kodak X-OMAT S

film for the appropriate time period (range 15 sec to 10 min). Exposed films were

developed using an automatic developer (CURIX 60, AGFA, Type 9462/100/140, Agfa-

Gevaert AG, Munich, Germany).

10X SDS-PAGE Running Buffer: Prepared by adding 30g Trizma base, 142g glycine and

10g SDS and made up to 1000ml with dH20.  This solution was diluted to 1X final

concentration with distilled water just prior to use for running the SDS-PAGE gels.

Western Blot Transfer Buffer: Prepared by adding 5.8g of Trizma base, 29g glycine, 1g

SDS and made up to 800ml with dH2O and 200 ml methanol was added to final volume

1000ml.

Western Blot Blocking Solution: This was prepared by 5 % w/v marvel powder in

phosphate buffered saline (PBS) with 0.1 % v/v Tween-20.

11

3. Results

3. 1 SiNP induced protein citrullination is irreversible as analysed by HCA

To investigate whether SiNP induced protein citrullination is reversible or irreversible

phenomenon, A549 human lung epithelial cells were exposed to various size SiNP (30 nm,

40 nm, 80 nm, or 400 nm) for 24 h. After 24 h, the SiNP were removed by aspiration (0 h)

and cells were washed with PBS. Cells were further incubated for up to additional 4 days

and medium were changed daily. At the end of all the treatments, cells were fixed and

immuno-stained for citrullinated proteins. HCA was performed using an automated

microscope (the IN Cell Analyzer) and protein citrullination was quantified by IN Cell

Investigator software (GE Healthcare) measuring cytoplasmic fluorescence intensity.

There was an evident increase in citrullination by SiNP at 0 h in comparison to non treated

cells as expected (Figure 3-5). However, significant amount of citrullinated proteins

induced by all the SiNP tested were present in cells even after removing the particles for

up to additional 96 h (Figure 3-5).

12

Figure 3. High Content Screening and Analysis of protein citrullination by 30 nm SiNP. A549 cells (N/T) were treated with 30 nm SiNP for 24 h. Cells were then washed (0 h) and incubated with culture medium for additional 24 h, 48 h, 72 h and 96 h. Cells were fixed with 3% PFA and immunostained for citrullinated proteins. Protein citrullination was analyzed by high content screening tool and quantified as described in the ‘Materials and Methods’ and presented. Results are mean values of three independent experiments performed in triplicates. Three fields per well were scanned with at least 300 cells. 30 nm SiNP show a capability of maintaining protein citrullination due to the RFU reading at 96 h.

13

Figure 4. High Content Screening and Analysis of protein citrullination by 40 nm SiNP. 24 h time point shows a remarkable decrease of protein citrullination, it is lower than N/T cells showing protein citrullination is not sustained. However, it increases again at 48 h and follows the trend of protein citrullination in figure 3 until 96 h.

14

Figure 5. High Content Screening and Analysis of protein citrullination by 400 nm SiNP. At 0 h there is a decrease in protein citrullination in comparison to N/T cells. 400nm have not induced citrullination in protein as 30 nm and 40nm. However, at 24 h protein citrullination increases and is maintained up until 96 h. At 72 h there is a significantly higher protein citrullination reading which is also seen in the previous graphs.

15

Figure 6. High Content Screening and Analysis of protein citrullination by 80 nm SiNP. The level of protein citrullination induced by 80 nm SiNP is similar to figure 3. Protein citrullination is not sustained at 48 h. Disregarding the prominent reading at 72 h, 96 h shows a fractional decrease in protein citrullination in comparison to N/T cells. Therefore after removal of 80 nm SiNP (0 h), citrullination is barely maintained at 96 h.

16

3.2 SiNP induced protein citrullination is irreversible as analysed by Western blotting

We next analyzed by western blotting whether SiNP induced protein citrullination was

irreversible phenomenon. For this purpose, A549 cells were exposed to various size SiNP

(30 nm, 40 nm, or 400 nm) for 24 h. After 24 h, the SiNP were removed by aspiration (0 h)

and cells were washed with PBS. Cells were further incubated for up to additional 4 days

and medium were changed daily. At the end of all the treatments, cells were lysed and

Western immunoblotted for citrullinated proteins. There was an evident increase in

citrullination of proteins of sizes 25 KDa, 250 KDa and between 50-100 KDa by SiNP at 0

h in comparison to non treated cells as expected (Figure 7-9). However, significant amount

of citrullinated proteins induced by all the SiNP tested were present in cells even after

removing the particles for up to additional 96 h (Figure 7-9).

Taken together both sets of results, HSA and western blot, it can be concluded that

SiNP induced protein citrullination is an irreversible process. The irreversible protein

citrullination by SiNP in cultured human cells provides an explanation for the possibility

of development of autoimmune diseases and therefore raise a serious concern to human

health.

17

4. Discussion

Using high content screening analysis and western blotting techniques we investigated the

reversibility/irreversibility of protein citrullination in A549 cells after 24 h incubation with

silica particles. The human bronchoalveolar carcinoma- derived cell line, A549 has been

used in many studies investigating the toxicity of nanomaterial (Jin et al., 2007: Lin et al.,

2006). The Respiratory airways make first contact with inhaled nanoparticles therefore

A549 cells were ideal. In this study nanoparticle induced protein citrullination in cells was

confirmed to be sustained and irreversible.

In recent years nanoparticles have been investigated as drug delieverants for treating

diseases. Various classes of nanoparticles exist, including polymers, liposomes, ceramic

nanomaterials, metallic particles, carbon nanomaterials and quantum dots (Medina et al,

2007). Carbon nanotubes and quantum dots have been extensively studied and proven to

be useful and relatively safe in relation to most of the other classes of nanoparticles used in

the medical industry. Using nanotechnology in the health industry is currently very limited

as toxicology experiments are still being performed on many nanomaterials, in vitro and in

vivo. What makes nanoparticles so damaging is there extensive surface area which enables

them to be absorbed very quickly and easily. To date, there are very few studies

investigating the toxic effects of nanomaterials, and no guidelines are presently available

to quantify these effects (Lin et al, 2006). While the number of publications dealing with

nanotoxicology and referenced in PubMed (keywords, ‘‘nanostructures/*toxicity’’) was

limited to 12 in 2004, we counted 236 publications in January 2008. (Lison et al, 2009).

Silica particles do not degrade in the body and can cause serious side effects at certain

doses. Silica is an important particle to evaluate, being the major constituent in sand, rock,

and rubber and naturally atmospheric particles of silica in East Asia continue to increase

along with the desertification of China. (Fujiwara et al., 2008).

As our results have shown silica induces protein citrullination. Many studies have shown

that protein citrullination is one of the first steps in attracting and triggering autoantibodies

and ultimately ending in the onset of an autoimmune disease e.g. MS and RA (Vossenaar

and Robinson 2005, Kim et al 2003 and Moscarello et al 2007).

21

The HSA results demonstrated the variance in protein citrullination levels at specific time

points of fixation and western blot results further demonstrated that there was a

considerably more amount of protein which was citrullinated in treated cells than non

treated cells and that citrulline did not revert back to arginine once nanomaterials have

been removed.

The results of HSA, showed that non-treated cells in figures 3-6 had a similar reading of

approx 8 rfu. All graphs except 400nm (figure 5) show citrullination was reduced after

particles had been removed (24 h). It appears that in the presence of SiNP’s citrullination

is much higher (0 h), however once the particles have been removed from the environment

the level of citrullination drops but protein citrullination is still sustained. The decrease in

citrullination may be due to the silica particles in the media being removed while some

silica particles were endocytosed which preserve the level of protein citrullination. A

constant state of citrullination is observed. Noticeably, in all four graphs after 72 h there is

a massive increase in the protein citrullination level and a plunge again after the next 24 h.

Results show that up until 72 h protein citrullination fluctuated but at 96 h, cell count

would have been much higher than at 0 h due to cell proliferation and new cells may not

contain silica particles which induce protein citrullination.

Our research suggests out of all four graphs 30nm induced the highest level of

citrullination whilst 40nm, 80nm and 400nm showed a similar amount of protein

citrullination in cells. All four graphs don’t follow a consistent trend apart from the

unusual citrullination increase at 72 h. This reading may be due to experimental error;

however it is unlikely as HSA experiments were done in triplicate and it is not a once of

phenomenon. This trend might be evidence of a unique event in the protein citrullination

of A549 cells by SiNP. In figures 4-5 the level of citrullination at 48 h is approx the same

as non-treated cells, which suggests citrullination, is not being sustained over a long period

and because of new dividing cells which do not have endocytosed particles, the levels of

citrullination drop.

The western blot results confirm and correlate with the HSA results. Unfortunately western

blot analysis was not carried out on 80nm silica particles. The non-treated cells show

protein citrullination is present without the influence of silica particle but once the

22

nanoparticles were added protein citrullination increased according to the band width and

intensity seen in figures 7-9. However the bands did not vary much between days. Unlike

the varying citrullination levels in the HSA results. Western blot shows no fluctuation in

band intensity between treated cells. From 0 h to 96 h it is evident that protein

citrullination is continuous. Western blot results of 400nm differ to 30nm and 40nm. The

bands are less intense and thinner suggesting 400nm may not have as much impact on

inducing protein citrullination in cells as it is that much bigger in size and less of a threat,

health wise. Exposure times in the dark room were all the same for each silica size

therefore it is not a reason for the difference.

At 0 h silica particles were removed and media was replaced, failure to remove all silica

particles may have influenced the results. Endocytosed NP’s could be the only reason

protein citrullination was sustained. Theoretically, if nanoparticles endocytosed by the cell

were exocytosed as well, citrullination may in fact be reversible. However, this is only a

speculation.

Auffan et al found that nanoparticles below 20–30 nm in size are characterized by an

excess of energy at the surface and are thermodynamically unstable. After analysing both

sets of data, HSA and western blot, the 30nm silica size nanoparticle was associated with

the highest level of protein citrullination at all time points. As the silica particle size

increases, protein citrullination appears to be less sustainable and consistent. Therefore

these results suggested the smaller the silica nanoparticle the better chance citrullination

has of occurring and being maintained. Yang et al, 200 also found the toxic effects were

closely related to the particle size and Lin et al 2006, demonstrated the cytotoxic effects of

Si02 but used different sized particles, 15nm and 46nm to our study.

In conclusion our study supported the evidence indicating that silica nanoparticles induce

protein citrullination which is a target of autoantibodies and considered a danger when it

occurs. Furthermore our study proposes that because protein citrullination is linked to the

onset of immune disorders, persistent protein citrullination could have a greater and more

harmful impact on health. The protein base change triggers autoantibodies such as anti-

CCP, which recruits inflammatory cells and causes chronic inflammation which is then

classified as an autoimmune disease e.g. RA.

23

Future studies may include the same protocol as used in this study but different cell lines

or nanoparticles. Yang et al, 200 results showed that nano-SiO2 exposure exerted toxic

effects and altered protein expression in HaCaT cells. Protein citrullination sustainability

was not a priority in this study but the data indicated the alterations of the proteins, such as

the proteins associated with oxidative stress and apoptosis, could be involved in the toxic

mechanisms of nano-SiO2 exposure. Many studies published to date of nanoparticle

toxicity are done in vitro, therefore more in vivo studies should be carried out. The most

interesting study continuing from this experiment will be the HSA repeat of SiNP induced

protein citrullination at 72 h.

24

5. Acknowledgments

I would like to thank Professor Yuri Volkov for giving me the opportunity to join his team

and be part of such a great project. I would also like to thank Dr Bashir Mustafa Mohamed

and Dr Navin Kumar Verma, my supervisors, who taught me new skills and techniques

that will benefit me tremendously in the future. I thoroughly enjoyed my 3months in the

lab and hopefully I will work with them again. I would finally like to thank my master’s

class who contributed to a great year for me.

25

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