Effect of extracts of poly(ether imide) microparticles on cytotoxicity,ROS generation and proinflammatory effects on human monocytic (THP-1) cells

Abstract

Current haemodialysis techniques are not capable to remove efficiently low molecular weight hydrophobic uremic toxins from the blood of patients suffering from chronic renal failure. With respect to the hydrophobic characteristics and the high level of protein binding of these uremic toxins, hydrophobic adsorber materials might be an alternative to remove these substances from the plasma of the chronic kidney disease (CKD) patients. Here nanoporous microparticles prepared from poly(ether imide) (PEI) with an average diameter of 90 ± 30 μm and a porosity around 88 ± 2% prepared by a spraying/coagulation process are considered as candidate adsorber materials. A prerequisite for the clinical application of such particles is their biocompatibility, which can be examined i.e. indirectly in cell culture experiments with the particles’ extracts. In this work we studied the effects of aqueous extracts of PEI microparticles on the viability of THP-1 cells, a human leukemia monocytic cell line, as well as their macrophage differentiation, reactive oxygen species (ROS) generation and inflammation.

A high cell viability of around 99 ± 18% and 99 ± 5% was observed when THP-1 cells were cultured in the presence of aqueous extracts of the PEI microparticles in medium A and medium B respectively. The obtained microscopic data suggested that PEI particle extracts have no significant effect on cell death, oxidative stress or differentiation to macrophages. It was further found that the investigated proinflammatory markers in THP-1 cells were not up-regulated. These results are promising with regard to the biocompatibility of PEI microparticles and in a next step the hemocompatibility of the microparticles will be examined.

Keywords

Chronic kidney disease (CKD)cytotoxicity human monocytic (THP-1) cells poly(ether imide) microparticles reactive oxygen species (ROS)

1 Introduction

Chronic renal failure is considered to be a condition, in which kidneys malfunction to remove uremic toxins and fluids from the body. Chronic kidney disease (CKD) is associated with inflammation and as a consequence with oxidative stress. Both inflammation and oxidative stress cause adverse health effects in CKD patients especially cardiovascular diseases (CVD). It is anticipated that retained uremic metabolites are one potential cause of oxidative stress and inflammation [4]. Hemodialysis (HD), hemofiltration (HF), hemodiafilteration (HDF) and kidney transplantations are commonly used procedures for treatment of CKD patients, but current conventional, high flux haemodialysis techniques, which are operated under aqueous conditions, are less efficient in removing low molecular weight hydrophobic uremic solutes from the blood of CKD patients. In uremic erythrocytes certain protein kinases or endothelial NO synthase are activated, which play a significant role in maintaining the mechanical and morphological properties of the erythrocyte membrane [14].

With respect to the hydrophobic characteristics and the high level of protein binding of such uremic solutes, some scientific studies investigated therapeutic approaches like combined usage of hydrophobic adsorber materials, such as apheresis particles, along with dialysis as an artificial kidney [28]. Recently, nanofibrous polymeric membranes prepared from poly(ethylene-co-vinyl alcohol) zeolite composites by electrospinning have been reported to adsorb uremic solutes like creatinine in a blood purification system [15]. Another approach for the removal of hydrophobic substances from the blood of patients is the utilization of hydrophobic polymers such as poly(ether imide) (PEI), which are applied as highly porous microparticulate adsorber materials. These porous, spherically shaped PEI microparticles can be prepared by a spraying/coagulation process having a mean diameter in the range of 70–100 μm and a total porosity based on weight in the range of 75–80% depending on the polymer solution composition [1]. PEI microparticles are of high mechanical robustness and steam-sterilisable, while the pore sizes of these microparticles are well defined so that the binding of uremic toxins of various sizes should be very effective. For qualifying such microparticles in view of potential future clinical applications their biocompatibility needs to be demonstrated in vitro and in vivo. In a first step typically aqueous extracts of the adsorber materials are investigated regarding in vitro effects on human cell lines, which are caused by leachable toxic substances originating from the particle processing, before studying the application of the adsorber in vivo. It is further essential to ensure that adsorber materials are free of bacterial contaminations and do not release any toxic chemical substances, which otherwise evoke inflammation and lead to failure of the device in patients. Human monocytes are sensitive and get activated on exposure to various factors like toxins released by infectious agents and cause inflammation by secreting various proinflammatory markers including cytokines and chemokines. These secreted proinflammatory products lead to onset as well as progression of chronic inflammatory disorders. In this study, we investigated potential toxic effect of PEI microparticle extracts, on human monocytic (THP-1) cells by assaying cytotoxicity, intracellular ROS levels, gene expression of proinflammatory markers at transcripts and protein levels and differentiation of monocytes to macrophages.

2 Materials and methods

2.1 Particle preparation

Poly(ether imide) (trade name Ultem^® 1000) was purchased from SABIC Deutschland GmbH (Düsseldorf, Germany) and used without any further purification. PEI microparticles were prepared by a spraying/coagulation process with a 130 μm spinneret utilizing a water coagulation bath according to the method previously reported [1]. Afterwards, the obtained PEI particles were sterilized via steam sterilization (121°C, 2.0 bar, 20 minutes) using a Systec Autoclave D-65 (Systec GmbH, Wettenberg, Germany).

2.2 Particle characterization

Shape and size of the prepared microparticles were characterized by scanning electron microscopy (SEM) with a Phenom G2 pro (L.O.T. - Oriel, Darmstadt, Germany) after coating with a conductive layer. For analysis of the average particle diameters the image processing software, Image J (Version 1.48v, Wayne Rasband, USA) [23] was applied and the standard deviation (error) was determined by measuring 350 particles. In addition, the particles‘ surface and cross section was visualized by scanning electron microscope (SEM) at higher magnification with a Gemini Supra 40 VP (Zeiss AG, Oberkochem, Germany). The cross sectional samples were prepared via cryo-ultramicrotomy. The frozen samples were cut into sections of 500 nm thickness and coated with a 5 nm thin conductive layer before SEM investigation.

Hg-porosimetry was applied to determine the overall porosity of the microparticles as well as the cumulative pore volume, the average pore diameter and the pore size distribution utilizing a PASCAL 140–440 porosimeter (POROTEC, Germany). A powder dilatometer type CD3P was filled with ca. 0.1 g of the dried microparticles and examined in the low pressure range of 0.013 to 0.4 MPa with the 140 PASCAL system and in the pressure range from 0.1 to 400 MPa with the PASCAL 440 system. Data analysis was conducted with the SOL.I.D Software Version 1.4.1., while applying the “sphere” surface area model.

2.3 Cell culture experiments

THP-1 cells procured from NCCS (Pune, India) were cultured in RPMI-1640 (Invitrogen, New York, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Biowest, Kansas, USA) and 1% (v/v) Penstrep (Invitrogen, New York, USA). Cells were maintained in an incubator at 37 ° C and 5% CO₂. THP-1 cells were incubated with the prepared PEI particle extracts (as described below) for 5 h and subjected to the assays described in the following.

2.4 Preparation of PEI microparticle extracts

200 mg of steam sterilized PEI microparticles were suspended in 12 mL of THP-1 cell growth medium without (medium-A) and with FBS of 10% (v/v) (medium-B) and agitated for 5 h on a rotatory shaker for proper mixing. Then the media were centrifuged at 3000 rpm for 5 min at room temperature and the collected supernatants. FBS (10% v/v) was added to medium-A. Medium A and B were prepared to distinguish the toxic or inflammatory effects, if any, of PEI particles on THP-1 cells, due to the depletion of essential components of the medium by binding to serum albumin or release of toxic substances, which may cause toxicity to the cells.

2.5 Cell viability- trypan blue method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

THP-1 cells at a density of 5×10⁵/ mL were incubated with PEI extracts for 5 h at 37°C and 5% CO₂. The treated cells were tested for their viability by trypan vital staining exclusion method and MTT assay. For the trypan vital assay 20 μL of the PEI treated cell suspension were mixed with 10 μL of trypan blue solution and the morphology of cells observed under an inverted microscope using a hemocytometer [10]. For the MTT assay 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) were added to the PEI treated cell suspension and incubated for 4 h. The cells were washed twice with growth medium after the incubation period to remove excess dye. Purple coloured insoluble formazans were dissolved in 100 μL of dimethyl sulfoxide (DMSO) and the absorbance was quantified at 570 nm with the reference wavelength at 630 nm.

2.6 Macrophage differentiation

The effect of PEI microparticle extracts on THP-1 cells differentiation into macrophages was investigated by microscopic analysis. Differentiation of cultured THP-1 cells into macrophages can be achieved by stimulating THP-1 cells with phorbol-myristate acetate (PMA) for 48 h [18]. Phenotype of PEI treated THP-1 cells after 5 h was observed microscopically using an inverted microscope (Olympus) at a magnification of 20X. THP-1 cells induced with PMA for 48 h were used as positive control [18, 26].

2.7 Intracellular reactive oxygen species (ROS) production

PEI extracts pretreated cells were loaded with 5 μM 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA) fluorescent probe for 15 min and the fluorescence intensity observed under a confocal microscope (Zeiss NLM 710, Carl Zeiss MicroImaging GmbH, Germany) [11]. Cells stimulated with 100 μM arachidonic acid (AA) for 10 min were used as positive control [13].

2.8 Quantification of cytokines by ELISA

Cells pretreated with PEI extracts for 5 h were centrifuged at 3000 rpm for 3 min and the supernatants collected. Secretory porinflammatory cytokines like tumor necrosis factor (TNF-α), monocyte chemotactic protein (MCP-1) and interleukins (IL-6, IL-8) in supernatants were quantified by using BD-Bioscience (San Diego, USA) ELISA kits according to the manufacturer’s protocol. THP-1 cells induced with lipopolysaccharide (LPS) were applied as positive control for comparison [31].

2.9 Gene expression studies of proinflammatory and cell differentiated markers by quantitative real-time PCR

RNA was isolated from the cells using Trizol reagent and c-DNA was synthesized by Iscript c-DNA synthesis kit (Bio-Rad laboratories, Hercules, USA). The gene expression levels of TNF-α, MCP-1, IL-6, IL-8, and toll like receptor (TLR-4) were analysed by using qRT-PCR, which was performed by using SYBER Green PCR kit. The primer sequences for the tested genes were purchased from Eurofins genomics (Bangalore, India) and are given in the Table 1 [6]. The 10 μl reaction mixture consisted of 2 μl of cDNA template and 8 μl of cocktail mix (5 μl of 1x SYBR Green, 0.5 μl of 1x forward and reverse primers, 2 μl of water). The mRNA levels of all genes were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Negative controls contained 8 μl of cocktail mix without cDNA and 2 μl of water.

2.10 Statistics

All data were expressed as mean value of minimum of 3 independent experiments ± standard deviation.

3 Results

In this study we explored the viability, the monocytic differentiation, the proinflammatory behaviour and the intracellular ROS levels of human monocytic cells, which were treated with extracts prepared from porous PEI microparticles.

3.1 Microparticle characterization

Morphological characterization by SEM and Hg-porosimetrie revealed that spherical, highly porous particles with a bulk porosity of 88 ± 2% and an average diameter of 90 ± 30 μm were obtained by the spraying/coagulation process. In Figs. 1A, C and D representative SEM images of PEI microparticles are shown, visualizing their spherical shape as well as the porous morphology of the particles. Here a nanoporous structure becomes obvious from SEM images taken at high magnification at the particles’ cross section. The particle diameter distribution obtained from the analysis of 350 particles is shown in Fig. 1B.

Hg-porosimetry was employed for quantification of the particles’ bulk density, cumulative pore volume, average pore size and pore size distribution as well as the pore accessibility. The microparticles exhibited a low bulk density of 0.12 ± 0.02 g×cm³, a mean pore diameter of 320 ± 20 nm and a cumulative volume of 1.33 ± 0.07 g×cm³, while a relative low pore accessibility of 43 ± 2% was found. In Fig. 1E the pore size distribution and the cumulative pore volume are displayed.

3.2 Effect of microparticle extracts on cell viability

Trypan blue exclusion method and MTT assays were performed to assess cell viability of PEI extract treated THP-1 cells. Integrity of the cell membrane can be detected by the Trypan blue assay. Dead cells take up the dye and as a result the cytosol appears blue in colour, while the cytosol of viable cells remains unstained. A high viability around 99% was observed when THP-1 cells were cultured in both medium extracts A (99 ± 18% ) and B (99 ± 5% ) of the PEI microparticles. This suggests that extracts of PEI particles did not cause a significant cell death rate (data not shown). The MTT assay further confirmed no toxic effects of PEI particle extract treated cells since 100 ± 5% cell viability was seen similar to untreated cells (Fig. 2).

3.3 Influence of microparticle extracts on ROS production in THP-1 cells

Pretreatment of THP-1with PEI microparticle extracts (medium A and medium B) for 5 h did not increase intracellular ROS production as indicated by confocal microscopy images (Fig. 3C, D) similar to control cells (Fig. 3A) whereas AA treated cells showed high amount of ROS production (Fig. 3B) within 10 min of stimulation.

3.4 Effect of microparticle extracts on proinflammatory markers and differentiation to macrophages

Transcripts of proinflammatory genes like TNF-α, MCP-1, IL-6 and IL-8 were quantified using real time PCR. Bacterial endotoxin or LPS upregulated the gene transcripts of proinflammatory markers while PEI mircoparticle extracts treated cells did not show such upregulation (Figs. 4A–D). Secretion of the protein markers TNF-α, MCP-1, IL-6 and IL-8 were quantified using ELISA and observed at basal level in control cells as well as in microparticle extracts treated cells, whereas LPS treatment enhanced the secretion of above proinflammatory markers by several folds (Figs. 5A–D).

Differentiation of monocytes to macrophages is a well-established biomarker indicating inflammatory stress. Macrophages are key immune cells generating ROS and secreting various proinflammatory cytokines. Phenotype of THP-1 suspension cells (Fig. 6A) treated with PMA for 48 h changed to adherent macrophage like structures (Fig. 6B). In contrast, no macrophage differentiation was observed for THP-1 cells treated with PEI extracts for 5 h (Figs. 6C and D). At the molecular level PMA enhanced the gene expression of TLR-4, which is a marker for differentiated macrophages, whereas PEI microparticle extract treated cells had a low level of TLR4 similar to the undifferentiated THP-1 cells (Fig. 6E).

4 Discussion

Chronic kidney disease (CKD) is described as a serious condition associated with ageing, diseased or failure of kidneys in removing the waste from the body. CKD is preferentially recognized in aged persons, who were affected with diabetes and hypertension [16]. Patients suffering from CKD have a high risk of experiencing cardiovascular diseases like stroke, heart diseases or peripheral arterial diseases, which account for approximately 30% mortality worldwide [21]. Dialysis is a common procedure employed in reducing the progression of the disease, but existing dialysis procedures can only help the patients partially. A major limitation of conventional hemodialysis therapies is that such treatments can only remove hydrophilic substances efficiently, while hydrophobic or protein bound substances such as uremic toxins remain in the patients’ blood. Recently, a good in vitro absorption capacity for protein bound middle and high molecular weight uremic toxins could be demonstrated for a nanoporous activated carbon monolith [20]. Another approach for the removal of hydrophobic substances from the blood of patients is the utilization of highly porous, microparticulate adsorber materials prepared from hydrophobic polymers such as poly(ether imide) (PEI). Such steam-sterilisable and mechanical robust PEI microparticles possess well defined pore sizes so that the binding of uremic toxins with various molecular weights should be possible. For qualifying such microparticles in view of potential future clinical applications, the absorber particles have to be tested extensively for their side effects on toxicity or activation of human monocytic cells, since human plasma subjected to hemodialysis will get in contact with blood cells. It is established that activation of monocytic cells participates in onset and progression of inflammation processes and in turn is responsible for atherosclerosis [24].

Trypan blue staining and MTT assay have shown that the cell viability of THP-1 were not affected when treated with PEI microparticle extratcs for 5 h. Differentiation of monocytes to macrophages initiates the progression and pathogenesis of various chronic diseases [29] as macrophages are major sources of ROS generation [3] and cytokine secretions upon activation [25]. Macrophages are capable to form foam cells, which are considered to be a crucial step in leading to atherosclerosis [17]. However PEI microparticles extracts did not affect the phenotype of THP-1 cells on incubation for 5 h which is equivalent to the time of a single hemodialysis treatment.

Activation of key immune cells like monocytes can be triggered by various factors when they come in contact with endotoxins, cytokines or dietary assimilates of glucose and polysaccharides. These factors can induce an oxidative stress by generating intracellular ROS, which attack macromolecules of the cells like nucleic acids, proteins and lipids. Lipids present in the membrane form peroxide adducts upon oxidation [12], which activate various stress signaling pathways by modifying enzymes like caspases leading to apoptosis [27]. Oxidative stress can lead to many metabolic disorders including atherosclerosis [19]. Oxidative stress generated by ROS has been implicated in the pathological systems of both CKD and CVD, most importantly chronic inflammation through activation of proinflammatory cytokines via NF-κb signaling pathway. In this study THP-1 cells pretreated with PEI microparticle extracts for 5 h did not show enhanced ROS, the cells were physiologically similar to untreated control cells, whereas THP-1 cells treated with arachidonic acid had higher levels of intracellular ROS.

Inflammatory response is characterized by the secretion of various proinflammatory cytokines like TNF-α, MCP-1, IL-1 IL-6, IL-8, and IL-18 etc. through the activation of TLR-2 and TLR-4 [22]. TLRs act as sensors for bacterial contaminants and activate the cells through NF-κb signaling pathway [9]. These secreted products lead to various chronic inflammatory diseases like CKD and CVD. It is essential to ensure that PEI microparticle extracts are free of bacterial contamination and do not release any toxic chemical substances, which otherwise evoke inflammation and lead to failure of the device applied in patients.

Endotoxins of gram negative bacteria or LPS bind to TLRs and evoke inflammatory response by activating translocation of NF-κb into the nucleus [2, 8]. NF-κb binds to the promoters of various proinflammatory genes and initiates inflammation. Proinflammatory cytokines upregulate adhesion molecules like vascular cell adhesion molecules (VCAM-1), intracellular adhesion molecules (ICAM-1) and endothelial selectin (E-selectin) and can lead to atherosclerosis [5 , 30]. Therefore, we analysed the inflammatory response of PEI microparticle extracts in THP-1 cells and we observed that the particle extracts did not activate any of the inflammatory markers both at protein and transcript levels.

5 Conclusion

The obtained results clearly demonstrate that the extracts of PEI microparticles did not exhibit negative side effects on the viability, the differentiation and the proinflammatory behaviour of human monocytic cells. In a next step the hemocompatibility of the presented PEI microparticle will be examined. These results support that PEI microparticles are interesting absorber material candidates in the context of CKD.

Footnotes

Acknowledgments

Indo-German Science and Technology Centre (Grant No. IGSTC/NPORE/SDT/2012) and German Federal Ministry for Education and Research (BMBF), (Grant No.s 01DQ13006A, 01DQ13006B and 01DQ13006C) are acknowledged for financial support. Reddi Kiran Kumar, first author of this manuscript, is a recipient of fellowship as an SRF (senior research fellowship) from IGSTC funded project (Grant No. IGSTC/NPORE/SDT/2012).

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