the nanoparticle industry has expanded substantially in recent years leading to exposure of various nanomaterials to human and environment .
particle size plays an important role in determining the particular biological behavior of nanomaterials . due to their extreme small size ,
nanoparticles possess specific large surface area , which makes the number of surface atoms or molecules increasing exponentially .
hence , particles at nanorange exhibit much higher chemical and biological reactivity than fine particles .
the risks associated with nanoparticles exposure require investigation due to evidence that these particles can be more inflammogenic and toxic than larger particles comprising of the same material . in recent years
, size - dependent toxicity between micro- and nanoscale particles has been demonstrated [ 35 ] .
carbon black ( cb ) has wide industrial applications , and used as a reinforcement agent in rubber products , black pigment in printing inks and lithography , electrode for batteries in electrical conductors and during the finishing process of leather goods production .
elemental carbon - based nanoparticles with a diameter of less than 100  nm are a major part of diesel exhaust and ambient pollution . after deposition in the lungs
, larger particles are phagocytized by alveolar and airway macrophages [ 6 , 7 ] , but the fine and ultrafine carbon particles remain in the lungs for a longer period of time .
ultrafine particles are phagocytized to a minor extend but they can still enter macrophages and epithelial cells and even penetrate into the circulation .
thus , ultrafine particles not only trigger local inflammatory reactions in the lungs but also cause systemic extrapulmonary effects .
in addition to performing phagocytosis , they can release inflammatory mediators such as cytokines and chemokines , crucially involved in destruction of microbes and particles using various enzymatic systems .
cb nanoparticles are reported to cause cytotoxic injury , increase levels of proinflammatory chemokines , and inhibit cell growth .
epidemiological as well as experimental studies have confirmed the role of cb nanoparticles in aggravating pulmonary disorders such as asthma , lung cancer , pulmonary fibrosis , and systemic cardiovascular disorders . in this study ,
cb particles were chosen considering their production in huge quantities posing high environmental risk compromising health of general population [ 13 , 14 ] . because of the sporadic information on in vitro size - dependent effect of cb particles , the present study was conducted to determine the effect of their nano- and micron - sized particles on viability , phagocytosis , and cytokine induction in human monocytes , thp-1 cells .
these undifferentiated cells express many of the properties of monocytes and represent a model of innate immune system .
these cells are an essential link between the adaptive and innate immune responses because they develop into various forms of antigen - presenting cells ( macrophages and dendritic cells ) .
they are often used as a model to study human inflammatory responses , which allow for the possibility of elucidating the interactions of nanoparticles with innate immune cells [ 16 , 17 ] .
carbon nanopowder < 50  nm and carbon powder ~500  nm were purchased from sigma - aldrich .
physicochemical properties of particles were analyzed using transmission electron microscopy ( tem ) , dynamic light scattering ( dls ) , and zeta potential analyzer .
dry powder of particles was suspended in cell culture medium at a concentration of 1  mg / ml and then sonicated at room temperature for 10 minutes to form a homogeneous suspension .
after sonication and stabilization , the tem samples were prepared by drop coating of the stock suspension on carbon - coated copper grids .
tem measurements were performed at an accelerating voltage of 120  kv ( model 1200ex , jeol ltd . , tokyo , japan ) .
zetapals ( brookhaven instruments corporation , holtsville , ny ) was used to determine the hydrodynamic size and zeta potential of particle suspension in cell culture medium . the human monocytic cell line , thp-1 was obtained from national centre for cell sciences , pune , india .
they were maintained in rpmi 1640 medium supplemented with 10% heat inactivated fetal bovine serum ( fbs ) , l - glutamine ( 2  mm ) , streptomycin ( 100  g / ml ) , and penicillin ( 100  u / ml ) .
thp-1 , cells were seeded into 24-well plates at 1  10 cells / ml and exposed to particles in the concentration range of 50 to 800  g / ml for 24  h. based on the results of screening study ( data not shown ) done with different concentrations of particles and previous reports [ 18 , 19 ] , a concentration range was selected .
the cells were exposed within 10  min of preparation of nano- and micro - cb suspensions .
cell - free controls were included in order to assess the interference of particles with each assay .
thp-1 cells were incubated with cb particles ( nano and micro ) in a concentration range of 50 , 100 , 200 , 400 , and 800  g / ml for 24  h. following this incubation period , cytotoxicity of particles was assessed using mtt assay . briefly , mtt ( 20  l per well of 5  mg / ml stock ) was added and incubated for 4  h. supernatants were removed by centrifugation and then 300  l of dmso was added .
after thorough mixing , optical density at 570  nm was detected by microplate reader ( biotek , usa ) .
control values ( without stimuli ) were set at 100% viable and all values were expressed as a percentage of the control and respective tc-50 ( particle concentration inducing 50% cell mortality ) concentrations were calculated using graphpad prism software .
the phagocytic ability of thp-1 cells after 4  h exposure to varying concentrations of cb particles ( namely , 1/2tc,50 , tc50 and 2tc50 ) was assessed by measuring their ability to phagocytose 1  m latex beads ( 1  m latex beads , carboxylate modified polystyrene , and fluorescent yellow - green ) .
culture medium containing latex beads at a bead - to - cell ratio of 10  :  1 was transferred to the culture wells .
beads not phagocytosed were removed by centrifugation at 225  g for 5  min ; the cell pellet was then resuspended in phosphate buffer saline .
the process was repeated three times and finally the cells were vortexed for 10  s and fluorescence of the cells was determined at an excitation and emission wavelengths set at 440 and 485  nm , respectively .
microscopic images of phagocytosed latex beads , only at one concentration , that is , tc-50 concentration of test particles , were taken .
dic images or paired dic and fluorescence images of phagocytosed beads by monocytes were acquired using fluorescent microscope ( zeiss , germany ) with 40x dry and 100x ( oil immersion ) objectives . to investigate the effect of cb particles on cytokine production , an enzyme linked immunosorbent assay ( elisa ) was performed . for determination of il-1 , il-6 , tnf- , and il-8 , monocytes were cultured at 1  10 cells / ml and were exposed to tc-50 concentration of cb particles ( nano carbon : 591.4  g / ml and micro carbon 687.1  g / ml ) for 6 , 18 , 24 , and 48  h. after particle exposure , cell - free supernatants were harvested via successive 10  min centrifugations ( 2,000  rpm , 7,000  rpm , and 13,000  rpm ) and stored at 80c until analysis .
elisa was performed according to the manufacturer 's protocol ( abcam elisa kit ) and absorbance values were measured using microplate reader ( biotek instruments , usa ) .
a minimum of three independent experiments were performed and concentrations calculated from the linear regression equation were derived from a set of standard absorbance values .
gene expression analysis of cox-2 and mcp-1 was evaluated by exposing cells to tc-50 concentration of cb particles ( nano carbon : 591.4  g / ml and micro carbon 687.1  g / ml ) for 24  h. total rna were isolated using rneasy mini kit ( qiagen , usa ) .
the concentration and integrity of rna was measured using multimode microplate reader ( biotek , usa ) prior to the experiment .
the enhanced avian hs rt - pcr kit ( sigma , usa ) was used for the amplification of cox-2 , mcp-1 , and 18 srna gene , according to the manufacturer 's instructions .
thp-1 cells exposed to nano- and microparticles of cb ( 0 , 50 , 100 , 200 , 400 , and 800  g / ml ) for 24  h were collected into tubes and washed with pbs .
the cells were incubated for 3  h in lysis buffer ( 20  mm tris - hcl , ph 8.0 , 5  mm edta , 0.1  m nacl , 0.5% sds , and 100  g / ml rnase ) at 37c .
after incubation , phenol  :  chloroform ( 1  :  1 ) mixture was used to extract dna . by adding an equal volume of ice - cold absolute isopropanol , dna was precipitated .
dna was dissolved in 50  l of 1x te ( 10  mm tris , 1  mm edta , and ph 8.0 ) buffer .
20  g of dna was loaded onto 1.2% agarose gel and electrophoresis was carried out at 60  v for 2  h with tbe as the running buffer .
statistical analysis was carried out with graphpad prism 4 statistical software ( graphpad software inc . ,
one - way analysis of variance ( anova ) with tukey 's method for multiple comparisons was used to evaluate the various responses induced by different concentrations of particles and statistical comparisons between particle sizes were performed with two - way anova , followed by a bonferroni posttest .
tem analysis was performed to determine the morphology and size of the particle and micrographs are shown in figure 1 .
the primary sizes of the particles estimated from tem images ( figure 1 ) were presented in table 2 .
since nanoparticles often form agglomerates in a solution , the hydrodynamic sizes of the dispersed particles and their agglomerates in cell culture medium were estimated using zetapals .
these values were found larger than the per se particle size measured by tem ( table 2 ) .
after 24  h exposure of thp-1 cells to varying doses ( 50 , 100 , 200 , 400 , and 800  g / ml ) of nano- and micro - cb particles , cellular metabolic activity was detected by mtt assay .
the cell viability decreased in a concentration- and size - dependent manner following exposure to cb particle ( figure 2 ) and a significant decrease was observed at concentration from 100 to 800  g / ml for nano carbon particles and at 400 and 800  g / ml for micro carbon particles .
the nano carbon particles decreased the percentage of cell viability from 82% to 41% while micro carbon particles decreased it from 69% to 56% . the significant size selective difference ( p < 0.01 ) between nano- and micro carbon
was found only at highest concentration of 800  g / ml . the tc-50 value ( particle concentration causing 50% cell mortality ) calculated for nano- and micro carbon was found to be 591.4  g / ml and 687.1  g / ml , respectively ( table 3 ) .
the assay was carried out by taking 3 different concentrations of test particle , namely , 1/2 tc-50 , tc-50 , and 2tc-50 for each particle . a significant reduction in the phagocytosis of indicator latex beads occurred after exposure to all concentrations of nano - cb particles ( p < 0.01 ) , while no significant reduction was observed with their microsize ( figure 3 ) .
qualitatively control cells were found to phagocytose more beads as compared to nano - cb particle exposed monocytes whereas the phagocytic capacity of monocytes was not affected by the presence of micro - cb particles ( figure 5 ) .
the cellular release of the proinflammatory cytokine ( il-1 , tnf- , and il-6 ) and chemokine ( il-8 ) into the culture medium was measured when monocytes were exposed to tc-50 concentration of cb particle at different time points ( 6 , 18 , 24 , and 48  h ) ( figure 6 ) .
carbon particles showed significant size- and time - dependent release of il-1 , il-6 , tnf- , and il-8 from thp-1 cells ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) . in case of nano - cb particles , there was time - dependent release of cytokine up to 24  h which declined thereafter while it remained increasing up to 48  h in case of micro - cb particles .
the nanosized particles were more potent than the larger particles in inducing the release of all the cytokines ( p < 0.001 ) .
a significant upregulation of mcp-1 gene expression was observed with both sizes of cb particles ( figure 7(a ) ) .
micron size of cb particles exhibited more expression of mcp-1 gene than nanosize ( figure 7(c ) ) .
however , no significant change was observed in cox-2 gene expression by both sizes of cb particles ( figures 7(a ) and 7(b ) ) as compared to control .
exposure of thp-1 cells to both sizes of cb particles for 24  h did not exhibit any smearing pattern on agarose gel ( figure 8) .
the inconsistencies of the effects of different cb samples in relation to carcinogenicity and toxicity were reported [ 23 , 24 ] .
these inconsistencies are likely to be related to variation in the particle sizes between the different samples .
therefore , the present in vitro study compared the potential differences in the ability of nano- and micro - cb particles to produce toxicity on exposure to human monocytes , thp-1 .
the average size of the particles was in agreement with the size provided by the supplier .
dls analysis revealed a hydrodynamic size of dispersed particles in cultured medium indicating agglomeration in aqueous media which was further corroborated by zeta potential measurement .
needless to surprise , the mean particle sizes and size distribution of particles ( measured by dls ) were found enhanced when measured in aqueous media compared to measurements in the dry phase ( measured by tem ) . to minimize the effects of particles
aggregation and sedimentation , suspension of particles was always freshly prepared and sonicated before each experiment . in this study
, the cytotoxicity results demonstrated that nanoparticles of cb were more potent than its micron size in causing toxicity to thp-1 cells as revealed by mtt assay .
it was found that only nano - cb particle impaired the phagocytic capacity of monocytes .
our findings of impaired phagocytosis in nano - cb particle exposed cells support the findings of renwick et al . who demonstrated that ultrafine ( cb and tio2 ) particles impaired macrophage phagocytosis to a greater extent than fine particles compared on a mass basis .
the study of inflammatory responses of thp-1 cells on exposure to nano- and micro particles of cb revealed the induction of proinflammatory cytokines il-1 , il-6 , and tnf-. many cytokines including il-1 , il-6 , and tnf- activate functions of inflammatory cells during acute inflammatory responses .
these cytokines increase the vascular permeability and thus cause swelling and redness associated with inflammation .
il-1 and il-6 are responsible for fever reactions while tnf- stimulates endothelial cells and is responsible for hypotension .
the nanoform of cb particle showed increase in release of il-1 and il-6 with increasing time of exposure up to 24  h and then declined thereafter distinctly from its micron form .
tnf- secretion was induced at early time point , that is , at 6  h , which later on decreased with time .
it was observed that the secretion of il-1 , il-6 , and tnf- cytokines was more profound with nanosize than with micron size .
chemokines are secondary proinflammatory mediators ; that is , they are typically induced by primary proinflammatory mediators such as il-1 or tnf . in the present study , gene expression of mcp-1 , a cc chemokine , and release of il-8 , a member of the cxc chemokine subfamily ,
the result of the present study demonstrated the stimulation of chemotaxis as revealed by upregulation of mcp-1 gene and increased release of il-8 on exposure to cb particles .
have also shown the upregulation of il-6 and mcp-1 in rats after inhalation exposure of cb and substantiate the findings .
il-8 is a chemokine which plays a key role in the activation of neutrophils and their recruitment to the site of inflammation .
results demonstrated increase in the release of il-8 with time after exposure to both sizes of cb particles .
kim et al .   reported increased expression of il-8 mrna and protein on exposure of ultrafine carbon particles in normal human bronchial epithelial cells and supported the findings .
noticeably , the mcp-1 gene expression was more upregulated by micron size of cb than nanosize .
similar results were observed with another chemokine , il-8 , where micron size of cb was able to induce more secretion of il-8 at later time of exposure .
cox-2 , an inducible isoform of cyclooxygenase , is induced by several mitogenic and proinflammatory stimuli including lps , interleukin-1 ( il-1 and il-1 ) , and tnf- [ 30 , 31 ] . in the present study , cb particles did not show any change in the cox-2 expression .
overall , in accordance with the results of ferin et al .   and li et al . , it was found that nano - cb induced a greater toxicity and inflammatory response than micro - cb in human monocytes . in the present study ,
the effect of cb particles on dna damage of human monocytes was also studied . in assessing toxicity ,
dna damage to macrophages is an important outcome since ( i ) these cells remove inhaled nps   and ( ii ) dna damage is considered to be an important initial event in various diseases including carcinogenesis .
the results showed that there was no smearing pattern observed on agarose gel indicating that both sizes of cb particles did not cause any dna damage . in conclusion , a comparative toxicity study between nano- and micro - cb particle resulted in size - dependent cytotoxicity and increased inflammatory responses in human monocytes , thp-1 cells .
nano - cb particles altered the phagocytic capacity of monocytes although micro - cb had no significant effect on phagocytic capacity of monocytes .
however , both sizes of cb particle did not have any effect on dna of thp-1 cells .
further studies are required to elucidate the exact pathway of inflammatory response induced by cb particles in immune cells .
 increased levels of particulate air pollution are associated with increased respiratory and cardiovascular mortality and morbidity . 
 some epidemiologic and toxicological researches suggest ultrafine particles ( < 100  nm ) to be more harmful per unit mass than larger particles . in the present study , 
 the effect of particle size ( nano and micro ) of carbon black ( cb ) particle on viability , phagocytosis , cytokine induction , and dna damage in human monocytes , thp-1 cells , was analysed . 
 the cells were incubated with nanosize ( ~50  nm ) and micron ( ~500  nm ) size of cb particles in a concentration range of 50800  g / ml . the parameters like mtt assay , phagocytosis assay , elisa , gene expression , and dna analysis were studied . 
 exposure to nano- and micron - sized cb particles showed size- and concentration dependent decrease in cell viability and significant increase in proinflammatory cytokines il-1 , tnf- and il-6 as well as chemokine il-8 release . 
 gene expression study showed upregulation of monocyte chemoattractant protein-1 gene while cyclooxygenase-2 gene remained unaffected . 
 nano cb particles altered the phagocytic capacity of monocytes although micron cb had no significant effect . 
 cb particles did not show any significant effect on dna of monocytes . 
 the investigations indicate that cb particles in nanosize exhibit higher propensity of inducing cytotoxicity , inflammation , and altered phagocytosis in human monocytes than their micron size . 
1. Introduction
2. Materials and Methods 
3. Results
4. Discussion