Gene expression in nanotoxicology: A search for biomarkers of exposure to cobalt particles and ions

Despite the wide use of nanoscale materials in several industrial applications as well as in biology and medicine, very little research has been carried out on the potential toxicity of nanoparticles. We had previously obtained 10 differentially expressed mRNAs in BALB3T3 fibroblasts exposed to different forms of cobalt, i.e., microparticles, nanoparticles, and ions. Those genes represented candidate biomarkers for indicating specific cellular effects after cobalt nanoparticle exposure. In the present paper, we have further evaluated the expression of those genes by real-time RT-PCR after exposure to different forms of cobalt. Moreover, we also tested some genes associated with cobalt toxicity, such as VEGF, HIF-1α, and Bnip3. We identified biomarkers that are sensitive to cobalt ions that we think to be the reactive form. Our data, in fact, are consistent with the possibility that Co-nano, due to their large surface area, once inside the cell dissolve and act as ions.     

Preparation and improvement of release behavior of chitosan microspheres containing insulin

Sophisticated delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (Np) were prepared using a solvent emulsion evaporation method (SEEM) to load zinc(II) phthalocyanine (ZnPc). Np were obtained using poly (d,l latic-co-glycolic acid) (PLGA). ZnPc is a second generation of photoactive agents used in photodynamic therapy.

ZnPc loaded PLGA nanoparticles were prepared by SEEM, characterized and available in cellular culture. The process yield and encapsulation efficiency were 80 and 70%, respectively. The nanoparticles have a mean diameter of 285 nm, a narrow size distribution with polydispersive index of 0.12, smooth surface and spherical shape. ZnPc loaded nanoparticles maintains its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained with a moderate and burst effect of 15% for 3 days. The photocytotoxicity of ZnPc loaded PLGA Np was evaluated on P388-D1 cells what were incubated with ZnPc loaded Np (5 μM) by 6 h and exposed to red light (675 nm) for 120 s, and light dose of 30 J/cm². After 24 h of incubation, the cellular viability was determined, obtaining 61% of cellular death. All the physical–chemical, photophysical and photobiological measurements performed allow us conclude that ZnPc loaded PLGA nanoparticles is a promising drug delivery system for photodynamic therapy.     

Chronic morphine-mediated adenylyl cyclase superactivation is attenuated by the Raf-1 inhibitor, GW5074

The utility of morphine for the treatment of chronic pain is limited by the development of analgesic tolerance. Adenylyl cyclase (AC) superactivation, induced by chronic opioid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present work, we tested the role of Raf-1 in morphine-mediated AC superactivation in CHO cells stably expressing the human μ-opioid receptor. We found that pretreatment of CHO cells stably expressing the human μ-opioid receptor with the selective Raf-1 inhibitor, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one (GW5074, 10 μM, 60 min) completely abolished chronic morphine-mediated AC superactivation (P < 0.01). This finding indicates that Raf-1 may have a crucial role in compensatory feedback regulation of cellular cAMP levels by clinically important opioid analgesics.     

Cationic nanoparticles induce caspase 3-, 7- and 9-mediated cytotoxicity in a human astrocytoma cell line

Abstract

On a daily basis we are exposed to cationic nanoparticulates in many different ways. They are known to distribute to many organs of the body, and while some evidence suggests that these nanoparticles are toxic to cells, the mechanism of their toxicity is not clear. Here we apply a combination of biochemical and imaging techniques to study the mechanism by which amine-modified polystyrene nanoparticles induce cell death in a human brain astrocytoma cell line. Flow cytometry analysis of cells exposed to cationic nanoparticles revealed an increase in cell membrane permeability of the dyes YoPro-1 and propidium iodide, indicating onset of an apoptotic followed by a secondary necrotic response. Activation of caspases 3/7 and 9 and cleavage of poly(ADP-ribose) polymerase (PARP)-1 was also detected, providing clear molecular evidence of the apoptotic pathway induced by the nanoparticles. Transmission electron microscopy also revealed that these nanoparticles induce morphological changes in lysosomes and mitochondria, consistent with our observation of a rapid increase in the formation of reactive oxygen species in these cells. Together these results suggest that amine-modified polystyrene nanoparticles can mediate cell death through an apoptotic mechanism mediated by damage to the mitochondria.     

Cationic nanoparticles induce caspase 3-, 7- and 9-mediated cytotoxicity in a human astrocytoma cell line

On a daily basis we are exposed to cationic nanoparticulates in many different ways. They are known to distribute to many organs of the body, and while some evidence suggests that these nanoparticles are toxic to cells, the mechanism of their toxicity is not clear. Here we apply a combination of biochemical and imaging techniques to study the mechanism by which amine-modified polystyrene nanoparticles induce cell death in a human brain astrocytoma cell line. Flow cytometry analysis of cells exposed to cationic nanoparticles revealed an increase in cell membrane permeability of the dyes YoPro-1 and propidium iodide, indicating onset of an apoptotic followed by a secondary necrotic response. Activation of caspases 3/7 and 9 and cleavage of poly(ADP-ribose) polymerase (PARP)-1 was also detected, providing clear molecular evidence of the apoptotic pathway induced by the nanoparticles. Transmission electron microscopy also revealed that these nanoparticles induce morphological changes in lysosomes and mitochondria, consistent with our observation of a rapid increase in the formation of reactive oxygen species in these cells. Together these results suggest that amine-modified polystyrene nanoparticles can mediate cell death through an apoptotic mechanism mediated by damage to the mitochondria.     

Nanotoxicology and in vitro studies: the need of the hour

Nanotechnology is considered as one of the key technologies of the 21st century and promises revolution in our world. Objects at nano scale, take on novel properties and functions that differ markedly from those seen in the corresponding bulk counterpart primarily because of their small size and large surface area. Studies have revealed that the same properties that make nanoparticles so unique could also be responsible for their potential toxicity. Nanotechnology is rapidly advancing, with more than 1000 nanoproducts already on the market. Considering the fact that intended as well as unintended exposure to nanomaterials is increasing and presently no clear regulatory guideline(s) on the testing/evaluation of nanoparticulate materials are available, the in vitro toxicological studies become extremely relevant and important. This review presents a summary of nanotoxicology and a concise account of the in vitro toxicity data on nanomaterials. For nanomaterials to move into the applications arena, it is important that nanotoxicology research uncovers and understands how these multiple factors influence their toxicity so that the ensuing undesirable effects can be avoided.     

In vitro toxicity of nanoparticles in BRL 3A rat liver cells.

This study was undertaken to address the current deficient knowledge of cellular response to nanosized particle exposure. The study evaluated the acute toxic effects of metal/metal oxide nanoparticles proposed for future use in industrial production methods using the in vitro rat liver derived cell line (BRL 3A). Different sizes of nanoparticles such as silver (Ag; 15, 100 nm), molybdenum (MoO(3); 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe(3)O(4); 30, 47 nm), and titanium dioxide (TiO(2); 40 nm) were evaluated for their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; 1 microm), manganese oxide (MnO(2); 1-2 microm), and tungsten (W; 27 microm), to compare the cellular toxic responses with respect to the different sizes of nanoparticles with different core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24h of exposure). Results showed that mitochondrial function decreased significantly in cells exposed to Ag nanoparticles at 5-50 microg/ml. However, Fe(3)O(4), Al, MoO(3) and TiO(2) had no measurable effect at lower doses (10-50 microg/ml), while there was a significant effect at higher levels (100-250 microg/ml). LDH leakage significantly increased in cells exposed to Ag nanoparticles (10-50 microg/ml), while the other nanoparticles tested displayed LDH leakage only at higher doses (100-250 microg/ml). In summary the Ag was highly toxic whereas, MoO(3) moderately toxic and Fe(3)O(4), Al, MnO(2) and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag (15, 100 nm) in liver cells is likely to be mediated through oxidative stress.     

Placental transport of brevetoxin-3 in CD-1 mice

The purpose of this study was to examine the distribution of brevetoxin-3 administered to pregnant dams and to determine the extent of placental transport to fetuses. Twenty-nine pregnant CD-1 mice were administered ³H-brevetoxin-3 (1.3 μCi/animal; 2.8 μg compound/kg) by intratracheal instillation on one of gestational days 15–18. Groups of four or five dams were killed at selected times through 48 h post-dosing. Four pregnant dams were administered ³H-brevetoxin-3 on gestational day 15 or 16 via osmotic minipump to provide continuous delivery of compound (0.13 μCi, 7.5 ng compound/day) over a 72-h period. Then the dams and fetuses were killed. Brevetoxin-associated radioactivity was detected in placentas and fetuses within 0.5 h of intratracheal administration. Concentrations of brevetoxin equivalents in fetuses were approximately 0.3 ng/g throughout the 48-h post-dosing, resulting in a calculated dose to fetuses of 19 ng/g h. Following brevetoxin infusion, concentration of brevetoxin equivalents in fetuses was 0.1 ng/g, lower than that present in most maternal tissues. Results demonstrated placental transport of brevetoxin or its metabolites following maternal acute exposure and repeated low-dose exposure. The consequences of these findings for pregnant women exposed to brevetoxins by inhalation or ingestion remain to be determined.     

High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay

Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2–3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 μg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 μg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 μg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring <1 mg test material, giving a substantial increase in productivity. Any material selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity.     

Alterations in human red blood cell properties induced by 3-(dimethylamino)phenol (in vitro)

3-(Dimethylamino)phenol (3-DMAP) exists in the environment as a transformation product of ureic herbicides and may also be considered as a derivative of phenoxyherbicides.

In this study, the activity of glutathione peroxidase, catalase and superoxide dismutase, as well as the level of free radicals and changes in cell morphology were measured in human erythrocytes exposed (in vitro) to 3-(dimethylamino)phenol. Human erythrocytes were incubated for 1 h in 3-DMAP at concentrations of 10–500 μg per 1 ml erythrocytes of 5% haematocrit.

The results show that 3-(dimethylamino)phenol increased the level of free radicals and changed the activity of glutathione peroxidase, catalase, superoxide dismutase and acetylcholinesterase. It also changed cell morphology.

All these results corroborated the thesis that 3-DMAP induces oxidative stress in cells. 3-DMAP changed the properties of the cell membrane, caused strong oxidation of haemoglobin, inhibited the levels of enzymatic and non-enzymatic antioxidants, which, in result, lead to generation of free radicals (ROS and semiquinones) that occurred in the exposed cells, predisposing them to oxidative damage.     

Validation of metallothionein,interleukin-8, and heme oxygenase-1 as markers for the evaluation of cytotoxicity caused by metal oxide nanoparticles

Metal oxide nanoparticles have an industrial value, although their harmful effects are also known. Induction of respiratory inflammation through their inhalation is a serious indicator of their toxicity. Although the phenomenon of metal ion release is involved in the induction of inflammation, all metal ions are not necessarily toxic. However, currently, no particular index to evaluate cytotoxicity caused by nanoparticles exists. An index based on biological response is critical. In the present study, we examined the gene expression-based index for nanoparticle-derived cytotoxicity. The cellular effects of six kinds of metal oxide nanoparticles, ZnO, NiO, CuO, MgO, Bi₂O₃, and MoO₃ on A549 cells were examined. It was seen that lactate dehydrogenase (LDH) assay, which is one of the most important assays for assessing cell membrane damage, is inhibited by metal ions released from the metal oxide nanoparticles. In some cases, enzyme activity-based assay was not suitable for the evaluation of cytotoxicity of nanoparticles. ZnO and CuO nanoparticles displayed severe cytotoxicity and enhanced gene expression of heme oxygenase-1 (HO-1) and interleukin-8 (IL-8). The IL-8 gene expression was also increased from Bi₂O₃ exposure. Additionally, the gene expression of metallothionein 2A (MT2A) was enhanced in the ZnO, CuO, and Bi₂O₃ exposed cells. These results suggest that these nanoparticles released metal ions in the cells. The enhancement of HO-1, IL-8, and MT2A gene expressions was related to the cytotoxic activity of metal oxide nanoparticles. Thus, the expression level of these genes is a good indicator of nanotoxicology of metal oxide nanoparticles.     

Polysaccharides isolated from Ganoderma lucidum occurring in Southern parts of India, protects radiation induced damages both in vitro and in vivo

The in vivo and in vitro radioprotective property of the polysaccharides isolated from Ganoderma lucidum were determined by survival studies, induction of micronucleus in reticulocytes of mice, strand breaks in plasmid pBR322 DNA and inhibition of lipid peroxidation (TBARS assay). Polysaccharides were administered as a single dose after whole body exposure to 10 Gy ⁶⁰Co γ-radiation to Swiss albino mice. At a dose of 500 μg/kg body wt, the polysaccharides were most effective in protecting animals from radiation induced loss of lethality. Administration of 500 μg/kg body wt to animal exposed to 10 Gy gamma radiation resulted in more than 60% survival on the 30th day compared to the dose of 300 mg/kg/body wt administration of amifostine, a clinically used radioprotective drug. The induction of micronuclei was reduced by the administration of polysaccharides. The decrease in micronuclei induction was dose dependent. Thus following 4 Gy exposure the micronuclei in polychromatic erythrocytes (MNCE) was reduced from 28.16 ± 3.049 to 16.0243 ± 2.074 and 6.30 ± 2.422 by polysaccharides at doses of 250 μg/kg body wt and 500 μg/kg body wt, respectively, and to 10.4 ± 2.581 by amifostine at a dose of 300 mg/kg body wt. The results indicate the significant protective effect of Ganoderma polysaccharides against radiation induced damages. The findings thus suggest the potential use of Ganoderma polysaccharides as novel radioprotective agent.     

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1–7 cells, it clearly enhances Pb²⁺-induced cytotoxicity. It is likely that Pb²⁺ must enter cells to exert most of its toxic effects. Pb²⁺ is known to substitute for Ca²⁺ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb²⁺ into GT1–7 neuronal cells with a special focus on the role of extracellular calcium (Ca²⁺), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb²⁺ (1 μM or 10 μM), i.e. without any external stimulus, clearly increased in nominally Ca²⁺-free buffer and was partially abolished by 13 mM Ca²⁺ when compared to uptake in the presence of a physiological concentration of extracellular Ca²⁺ (1.3 mM). Depolarization by 25 mM K⁺, or antagonists of VSCCs, verapamil (10 μM) or flunarizine (10 μM) had no clear effect on basal Pb²⁺ uptake. Glutamate (1 mM) increased Pb²⁺ uptake, but only when cells were treated with 1 μM Pb²⁺ in the presence of 1.3 mM Ca²⁺. Our data suggest that Pb²⁺ competes for the same cellular uptake pathways with Ca²⁺, although not via VSCCs. In addition, enhancement of Pb²⁺-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb²⁺.     

纳米毒理研究进展

随着纳米技术的迅速发展,人们对纳米材料安全性及其生物效应信息的需求不断增加。纳米毒理学,作为一门“关于纳米设备和纳米结构的相关生物效应及其问题的科学”已逐渐引起了人们的关注。纳米微粒的小尺寸效应、表面效应、量子尺寸效应和宏观量子隧道效应使其与宏观材料相比具有特殊的理化性质、生物活性和生物动力学过程,从而对人体产生各种潜在危害。本文就目前纳米毒理的研究进展作一综述。     

In vitro induction of cytotoxicity and DNA strand breaks in CHO cells exposed to cypermethrin, pendimethalin and dichlorvos

The indiscriminate use of pesticides and herbicides to increase crop productivity has aroused a great concern among the environmental and health scientists due to their adverse effects in both target as well as non-target species. Although substantial information is available regarding their environmental and ecological impact, not much is known in regard to its toxicity in the mammalian system. Therefore a study was conducted for the assessment of cytotoxic and genotoxic effects of cypermethrin (Type II pyrethroid) dichlorvos (organophosphate) and pendimethalin (dinitroaniline herbicide) in Chinese hamster ovary (CHO) cells. CHO cells were exposed to 1 μM, 10 μM, 100 μM, 1000 μM, and 10,000 μM, cypermethrin, pendimethalin and dichlorvos for 3 h and cytotoxicity was assessed by MTT assay. Their genotoxic potential was also evaluated by Comet assay. The results demonstrate that dichlorvos and pendimethalin exhibited higher extent of cytotoxicity as compared to cypermethrin. A significant (p < 0.05) concentration dependent increase in DNA damage was observed with dichlorvos (0.01 μM and above) and pendimethalin (0.1 μM and above) as evident by Comet assay parameters viz., Olive tail moment (arbitrary units), tail DNA (%) and tail length (μM). Cypermethrin induced a significant (p < 0.05) DNA damage only at higher concentrations (1000 and 5000 μM). Our data indicates that these chemicals produce cytotoxicity and DNA damage in mammalian cells and should be used with caution.     

Artemia salina as a model organism in toxicity assessment of nanoparticles

Background

Because of expanding presence of nanomaterials, there has been an increase in the exposure of humans to nanoparticles that is why nanotoxicology studies are important. A number of studies on the effects of nanomatrials in in vitro and in vivo systems have been published. Currently cytotoxicity of different nanoparticles is assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on different cell lines to determine cell viability, a tedious and expensive method. The aim of this study was to evaluate the Artemia salina test in comparison with the MTT assay in the assessment of cytotoxicity of nanostructures because the former method is more rapid and convenient and less expensive.

Methods

At the first stage, toxicity of different nanoparticles with different concentrations (1.56–400 μg/mL) was measured by means of the brine shrimp lethality test. At the second stage, the effect of nanoparticles on the viability of the L929 cell line was assessed using the MTT assay. Experiments were conducted with each concentration in triplicate.

Results

The results obtained from both tests (A. salina test and MTT assay) did not have statistically significant differences (P > 0.05).

Conclusions

These findings suggest that the A. salina test may expedite toxicity experiments and decrease costs, and therefore, may be considered an alternative to the in vitro cell culture assay.     

Chelerythrine and dihydrochelerythrine induce G1 phase arrest and bimodal cell death in human leukemia HL-60 cells

A quaternary benzo[c]phenanthridine alkaloid chelerythrine displays a wide range of biological activities including cytotoxicity to normal and cancer cells. In contrast, less is known about the biological activity of dihydrochelerythrine, a product of chelerythrine reduction. We examined the cytotoxicity of chelerythrine and dihydrochelerythrine in human promyelocytic leukemia HL-60 cells. After 4 h of treatment, chelerythrine induced a dose-dependent decrease in the cell viability with IC₅₀ of 2.6 μM as shown by MTT reduction assay. Dihydrochelerythrine appeared to be less cytotoxic since the viability of cells exposed to 20 μM dihydrochelerythrine for 24 h was reduced only to 53%. Decrease in the viability induced by both alkaloids was accompanied by apoptotic events including the dissipation of mitochondrial membrane potential, activation of caspase-9 and -3, and appearance of cells with sub-G1 DNA. Moreover, chelerythrine, but not dihydrochelerythrine, elevated the activity of caspase-8. A dose-dependent induction of apoptosis and necrosis by chelerythrine and dihydrochelerythrine was confirmed by annexin V/propidium iodide dual staining flow cytometry. Besides, both alkaloids were found to induce accumulation of HL-60 cells in G1 phase of the cell cycle. We conclude that both chelerythrine and dihydrochelerythrine affect cell cycle distribution, activate mitochondrial apoptotic pathway, and induce apoptosis and necrosis in HL-60 cells.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007 ± 300 and 348 ± 62 Ω cm² were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 μm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 μm pores, a TEER value of 241 ± 90 Ω cm² was reached on day 5; on 3 μm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 μm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 μm pores no translocation was observed, on 3 μm pores 6% translocation was observed both for carboxyl- and amine-modified particles.