Article pubs.acs.org/crt
Cite This: Chem. Res. Toxicol. 2019, 32, 49−56
Engineering Protective Polymer Coatings for Liver Microtissues Xi Chen,†,‡,○ Wen Jiang,†,§,○ Ayman Ahmed,† Clare S. Mahon,∥ Markus Müllner,∥,⊥ Bocheng Cao,# and Tian Xia*,†,∇
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Center for Environmental Implications of Nanotechnology, California NanoSystems Institute, University of California, Los Angeles, California 90095, United States ‡ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States § Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States ∥ Key Centre for Polymers and Colloids, School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia ⊥ The University of Sydney Nano Institute (Sydney Nano), Sydney, New South Wales 2006, Australia # Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States ∇ Division of NanoMedicine, Department of Medicine, University of California, Los Angeles, California 90095, United States S Supporting Information *
ABSTRACT: Three-dimensional (3D) hepatocyte microtissues (MT), also known as spheroids, have proven to be advantageous in providing more accurate information and physiologically relevant and predictive data for liver-related in vivo tests; therefore, spheroids have increasingly been used to study hepatotoxicity, drug delivery to the liver, and tissue engineering. However, variabilities in the generation of 3D MT remain a major challenge. Methods that encapsulate and protect hepatocytes offer a promising pathway in prolonging cell survival, as well as maintaining its liver cell functions. Herein, we studied the encapsulation and resultant protective effects of hydrogen bonded, biocompatible polymer coatings for hepatocyte MT in 3D cell culture. We exposed the MT to hepatotoxic nanomaterials (NMs), such as graphene oxide (GO) and cobalt oxide (Co 3 O 4 ), to assess the protective effects of poly(vinylpyrrolidone) (PVPON) and tannic acid (TA) coatings. The polymer coating allowed the MT to maintain its morphology. More significantly, it increased the viability of hepatocyte-composed MT by hampering the cellular interaction between hostile NMs and hepatocytes. Based on alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, the liver cell function was maintained throughout the coating process, including after NM treatment. The study provides a straightforward and safe methodology for maintaining the morphology as well as cellular function of hepatocyte MT in vitro.
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patients with liver failure.5 Long-term and stable liver-specific functions of hepatocytes should be considered in the development of BAL support systems. Therefore, in the context of mimicking and predicting biological responses of liver hepatocytes, various approaches have emerged to advance in vitro hepatocyte cell culture; examples include cocultures with nonparenchymal cells and three-dimensional (3D) cell culture methods.6,7 Culturing hepatocytes in 3D typically yield spherical microtissue (MT) also known as hepatocyte spheroids, which is an effective method in the upregulation of liver-specific functions of hepatocytes due to the high similarity to real tissues.8,9 Moreover, spheroidal MT present high biological relevance due to the tighter intercellular interactions, establishing a more realistic “cell architecture” which also features active uptake and secretion pathways.10,11
INTRODUCTION The liver is the largest internal organ and gland in the human body and maintains many vital functions, including bile production to break down fats, detoxification of drugs and chemicals, the synthesis of blood plasma proteins, and the regulation of the mononuclear phagocyte system.1 Hepatocytes are the predominant cell type in the liver. These are epithelial cells that perform important roles in metabolic, endocrine, and secretory functions of the body.2 Many studies have used hepatocytes as an in vitro model to investigate liver functions; however, isolated hepatocytes generally dedifferentiate when cultured in traditional two-dimensional (2D) monolayers in vitro, resulting in the loss of phenotype and specific functions.3 For example, the detection of metabolism-mediated hepatotoxicity of drugs in vitro often fails, as hepatocytes in 2D monolayers rapidly lose their morphologies and functions.4 In addition, various bioartificial liver (BAL) systems have been used for clinical trials as a bridge for liver transplantations in © 2018 American Chemical Society
Received: May 6, 2018 Published: November 30, 2018 49
DOI: 10.1021/acs.chemrestox.8b00120 Chem. Res. Toxicol. 2019, 32, 49−56
Chemical Research in Toxicology
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A persistent problem of multicellular spheroids is their variability of morphologies or lack of uniformity, often linked to medium composition, cell density, and duration in cell culture. For example, liver spheroids from commercial sources may show a loss in uniformity, morphology, and integrity after transportation (see Figure S1). These hurdles affecting reproducibility and proliferation rates in 3D cell culture need to be overcome to warrant comparable data evaluation.12 In this context, the protective encapsulation of the hepatocytes provides a feasible avenue to prolong cell survival in vitro, while maintaining the protein secretion and avoiding hepatotoxicity.13 Encapsulation using biocompatible polymers provides a semipermeable protective barrier for the environmental pathogens (e.g., engineered nanomaterials), while permitting the diffusion of nutrients, oxygen, and metabolic products which maintain cell survival and function, which could be useful for toxicity screening of small molecule drugs but not nanomaterials.14 Mitry et al. have recently shown that human hepatocytes can be embedded into alginate to protect the cells during transplantation into a patient with acute liver failure−who then benefited from improved cell viability and liver function compared to nonencapsulated hepatocytes.15 Specifically, the encapsulation and cell surface modification to preserve cell function can be achieved using layer-by-layer (LbL) assembly methods.16,17 The LbL technique is a robust methodology allowing for alternating deposition of polymer layers on surfaces. This in turn permits the wrapping of substrates (e.g., cells) with ultrathin coatings of controllable thickness and composition.18 Hydrogen-bonding systems based on tannic acid (TA), a natural organic polyphenol, have received increasing interest as effective protective coating due to their intrinsic antioxidant and antibacterial properties.19 Kozlovskaya et al. have used TA and its hydrogen-bonding interaction with poly(N-vinylpyrrolidone) (PVPON) to perform multilayer coating of pancreatic islet cells.6 The TA/ PVPON layers deposited on the islets surface could maintain the cell viability and β-cell functionality for at least 96 h in vitro, which has a great potential use in the pancreatic islets transplantation. In this context, to enhance cell viability and maintain the cell morphologies and functionalities of MT, we hypothesized that a protective encapsulation of the MT may provide a feasible avenue to prolong cell survival in vitro, while maintaining the protein secretion and avoiding hepatotoxicity induced by environmental pathogens exposure including nanomaterials.13 Herein, we created hepatocyte MT using hanging-drop 3D cell culture technique and fabricated MT encapsulation with multilayer PVPON/TA coating through hydrogen bonding via LbL assembly. To test the protective effects of PVPON/TA coating, we used two types of nanomaterials (NMs), graphene oxide (GO) nanosheet and cobalt oxide (Co3O4) nanoparticle, as model environmental toxicants to study the cytotoxicity, cellular uptake, and hepatocyte function, as well as the interaction of NMs with coated and uncoated spheroids. These two nanomaterials have been demonstrated by us to be able to induce cytotoxicity in vitro. We found that the polymer coating significantly decreased the toxicity induced by NMs but maintained the hepatocyte functions upon exposure to NMs. Our study demonstrates an avenue to preserve hepatocyte MT in vitro functions in the presence of toxic nanomaterials.
Article
EXPERIMENTAL SECTION
Chemicals. Reagents were purchased from Aldrich and used as received unless otherwise indicated. Tannic acid (TA), polyvinylpyrrolidone (PVPON, MW 10K), fluorescein isothiocyanate-modified bovine serum albumin (BSA-FITC), N-hydroxysuccinimide (NHS), and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich. Vinylpyrrolidone (VPon, 97%) and hydroxyethyl acrylate (HEA, 96%) were destabilized using a silica gel column. High purity methanol was received from Sigma-Aldrich, and diethyl ether was received from Merck. Deuterium oxide (D2O) and deuterated chloroform (CDCl3) were purchased from Cambridge Isotopes Laboratories. CH2Cl2 was dried over 4 Å molecular sieves prior to use. Azobis(isobutyronitrile) (AIBN) and 2-[(ethoxythioxomethyl)thio]propanoic acid (Xanthate) were recrystallized and provided by the Key Centre for Polymers & Colloids, The University of Sydney, Australia. The Co3O4 nanoparticles were from Lutz Madler’s lab at the University of Bremen, and GO were from Mark Hersam’s lab at Northwestern University. Preparation of Fluorescently Labeled PVPON. (1) 7(Diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (compound 1), 4-diethylsalicylaldehyde (3.86 g, 20.0 mmol), diethyl malonate (6.38 g, 40.0 mmol), and piperidine (2 mL) were combined in EtOH (60 mL), and the mixture was heated under reflux for 16 h. 10 % v/v NaOH(aq) (60 mL) was added, yielding immediately a yellow precipitate that redissolved to produce a brown solution, which was heated under reflux for a further 15 min. The solution was then cooled to room temperature before acidification to pH 2 using 37% HCl(aq). The orange solid produced was isolated by filtration and recrystallized from EtOH, to obtain the final product as orange needle-like crystals (0.389 g, 8%).20 1H NMR (200 MHz, CDCl3) δ 1.26 (6H, t J = 7.1 Hz, CH2CH3), 3.50 (4H, q, J = 6.4 Hz, CH2CH3), 6.53 (1H, d, J = 2.9 Hz) 6.70 (1H, dd, J = 3.0, 9.0 Hz), 7.45 (1H, d, J = 8.8 Hz) 8.65 (1H, s), 12.32 (1H,s, OH) 13C NMR (75 MHz, CDCl3) δ 12.4, 45.3, 96.8, 105.4, 108.6, 110.9, 132.0, 150.2, 153.8, 158.0, 164.4, 165.5. Melting point is 223.5−225 °C. (2) Synthesis of poly(vinylpyrrolidone-co-hydroxyethyl acrylate) copolymer−poly(VPon-co-HEA). Xanthate (9.60 mg, 0.05 mmol), VPon (1.00 g, 9.01 mmol), HEA (0.06 g, 0.51 mmol), and 2.0 mL of MeOH were mixed in a Schlenk flask and degassed via three freeze−pump−thaw cycles. The polymerization was carried out at 70 °C and stopped after 6 h by cooling and exposing the mixture to air. The polymer was purified via precipitation into cold diethyl ether, subsequently dialyzed into MeOH, and finally freeze-dried from water. (3) Diethylaminocoumarin-labeled poly(VPon-co-HEA). Poly(VPon-co-HEA) (20 mg, 2.1 × 10−6 mol, 1.0 equiv) was dissolved in CH2Cl2. Compound 1 (0.5 mg, 2.0 × 10−6 mol, 1.0 equiv) and dimethylaminopyridine (0.02 mg, 2.0 × 10−7 mol, 0.1 equiv) were added before addition of N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (0.4 mg, 2.2 × 10−6 mol, 1.1 equiv). The solution was left to stir at room temperature for 8 h and then dialyzed against CH2Cl2 (MWCO 2 kDa). The solution was evaporated to dryness, yielding a yellow film which was submerged in H2O and dialyzed again against H2O. The solution was lyophilized to afford the title product (15 mg, 71%). Fluorescent Labeling of GO with BSA-FITC. BSA-FITC labeled GO samples were prepared via a diimide-activated amidation as described before.21 5 mg of N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were dissolved in 2 mL of GO sample suspensions (100 μg mL−1) in DI water, and the mixture solutions were then stirred at room temperature for 2 h. Subsequently, the GO pellets were washed twice with DI water and collected by centrifugation at 15,000 rpm for 10 min. The obtained GO samples were then reacted with 1 mL of BSA-FITC solution (0.1 mg mL−1 in DI water). After a 12 h reaction with stirring, the resultant BSA-FITC labeled GO samples were washed three times with DI water and then resuspended in cell culture water for future use. Characterizations of Nanomaterials and Polymers. The sizes and morphologies of GO nanosheets and Co3O4 nanoparticles samples were determined using transmission electron microscopy 50
DOI: 10.1021/acs.chemrestox.8b00120 Chem. Res. Toxicol. 2019, 32, 49−56
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Chemical Research in Toxicology
Figure 1. Preparation and characterization of polymer coated 3D hepatocyte spheroids. (A) Scheme of 3D hepatocyte spheroid fabrication. (B) The spheroid was imaged using a DIC microscope at day 2, 5 and 7, respectively. Scale bars are 100 μm. (C) Schematic figure of polymer coating (PVPON/TA) on the spheroid via LbL assembly. (D) FITC-labeled PVPON was used to visualize the polymer coating (4 bilayers, termed as 4L) by fluorescence microscopy. Scale bars are 100 μm. (TEM JEOL 1200 EX microscope) operating at 120 kV. The average size of all samples was obtained by analyzing at least 30 individual nanomaterials. Hydrodynamic size and ζ-potential measurements of all GO and Co3O4 suspensions with a concentration of 50 μg mL−1 in DI water or cell culture medium were evaluated at 25 °C using a ZetaPALS instrument (Brookhaven Instrument, Holtsville, US). NMR spectra were recorded on a Bruker Avance 300 spectrometer, with 1H at 300 MHz and 13C at 75 MHz, or on a Bruker Avance 200 spectrometer, with 1H at 200 MHz, with the residual solvent as an internal standard. Gel permeation chromatography (GPC) was performed on an UFLC Shimadzu Prominence system at 50 °C using DMAc/LiBr as eluent and a flow rate of 1.0 mL·min−1. The GPC sample was taken from the reaction mixture, diluted with DMAc, and pressed through a 220 nm Nylon filter prior to injection. The GPC system was calibrated using PMMA standards. Maintenance of the Hepatocytes and Preparation of Hepatocyte Microtissues. Mouse liver hepatocytes (Hepa 1−6, obtained from ATCC, US) were maintained in completed Dulbecco’s Modified Eagle’s Medium (DMEM containing 10% fetal bovine serum, 100 units mL−1 penicillin and 100 g mL−1 streptomycin) at 37 °C in a 5% CO2 humidified atmosphere and subcultured prior to confluence using trypsin−ethylenediaminetetraacetic acid (EDTA) solution with a concentration of 0.25% (w/v) trypsin−0.53 mM EDTA. To prepare the 3D hepatocyte tissues, 500 Hepa 1−6 cells in 40 μL of cell culture medium were seeded into each well in a 96-well hanging drop plate (HDP1096 Perfecta3D) on the first day. To facilitate the tissue growth, 5 μL of fresh medium was added to each well every day from the second day to the sixth day. The tissues were then harvested by transferring into a 96-well U-shape plate once the
tissue spheroids formed on the seventh day. Specifically, the hanging drop plate was lined up on top of the U-shape plate, and then 40 μL of fresh medium was added to each well to flush the tissue spheroids from the hanging drop plate down to the U-shape plate (Ultra low attachment, Corning Costar, US). Fabrication of LbL Polymer (PVPON/TA) Coated Hepatocyte Microtissues. To fabricate the LbL polymer coated hepatocyte tissues, a group of 8 tissues was plated into each well of a 12-well plate (Falcon polystyrene microplates. Corning, US). The tissues were then resuspended in a 0.5 μL PVPON solution (1 mg mL−1 in cell culture medium), followed by mixing and gently shaking (50 rpm) at 37 °C (performed on Thermo Scientific MaxQ 4450 shaker) for 3 min to allow for polymer adsorption. After incubation, the polymer-coated tissues were washed by two cycles of removal and redispersion in fresh cell medium. 0.5 μL of TA solution (0.3 mg mL−1 in cell culture medium) was then added to the PVPON-coated tissues and incubated for 3 min. The tissues were washed as described above. The sequential adsorption of PVPON and TA was repeated until 2/ 4/6 bilayers were deposited on the tissues. Characterization of 3D Hepatocyte Microtissues. The growth of 3D tissues from the first day to the seventh day was monitored by a differential interference contrast (DIC) microscope (Observer D1, Zeiss, US) with a 2.5× objective. Fluorescently labeled tissues were imaged on a fluorescence microscope (Observer D1, Zeiss, US) equipped with a standard FITC filter cube. A transmission electron microscope (TEM) was also used to observe the morphology of the 3D hepatocyte tissue. Harvested tissues were fixed with 2% glutaraldehyde in 0.1 M phosphate-buffered saline (1× PBS) for 2 h at room temperature. After postfixation in 1% osmium tetroxide 51
DOI: 10.1021/acs.chemrestox.8b00120 Chem. Res. Toxicol. 2019, 32, 49−56
Chemical Research in Toxicology
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(OsO4) in PBS for 1 h, the tissues were dehydrated in a graded ethanol series (30%, 50%, 70%, 95%, and 100%) and then treated with propylene oxide and embedded in Epon for 48 h. Approximately 50−70 nm thick sections were cut on a Reichert-Jung Ultracut E ultramicrotome and picked up on Formvar-coated copper grids. The sections were stained with uranyl acetate (UA) and Reynolds lead citrate and imaged on a TEM (JEOL 100 CX, US) at 80 kV in the UCLA BRI Electron Microscopy Core. Further, to analyze the histology of 3D tissues, tissues were fixed in 4% paraformaldehyde (PFA) and embedded in histogel (Thermo Scientific Richard-Allan Scientific Specimen Processing Gel). The samples were further processed for resin embedding, sagittal sectioning, and hematoxylin/ eosin (H&E) staining in the Pathology and Laboratory Medicine Department at UCLA. Histological images of tissues were obtained using a light microscope (Digital micromaster premier microscopy, Fisher Scientific, US). In addition, to quantify the number of cells in each 3D hepatocyte tissue, a calibration curve of cell ATP value versus cell number was first set up. Specifically, a serial number of cells, 0, 250, 500, 750, 1000, 2000, 3000, and 4000, were seeded into a 96-well flat-bottom white plate, respectively. After incubation overnight to allow the cell attachment, an ATP assay was performed according to manufacturer’s protocol (ATPlite Luminescence Assay, PerkinElmer, US), and the ATP values were determined by a microplate reader (SpectraMax M5, Molecular Devices, US). A calibration curve of cell ATP values versus cell numbers was then obtained. Meanwhile, the ATP value of 3D tissues was evaluated by using a 3D cell viability ATP assay (CellTiter-Glo 3D Cell Viability Assay, Promega, US). The obtained ATP value was then interpreted into the above calibration curve, and the number of cells in each tissue was then determined. Assessment of Toxicity of Microtissues Using 3D Cell ATP Viability Assay. A single microtissue in 80 μL cell medium was seeded in each well of a 96-well clear bottom white plate. Tissues were then treated with 20 μL of GO nanosheets or Co3O4 nanoparticle suspensions (final concentration from 12.5, 25, 50, 100 μg mL−1). After 24 h treatment, the tissues were washed with PBS three times to remove free materials. Cell viability determined by cellular ATP content was accessed by the CellTiter-Glo 3D Cell Viability Assay (Promega, USA) according to the manufacturer’s instructions. The luminescence intensity was read on a SpectraMax M5 microplate spectrophotometer (Molecular Devices, US). Assessment of Cellular Uptake of Nanomaterials by Microtissue. Freshly prepared Co3O4 and GO nanomaterials at the concentration of 12.5 to 100 μg mL−1 were incubated with liver microtissues for 24 h in Corning Costar Ultra-Low attachment multiwell plates. Microtissues were then transferred to a new U bottom 96-well plate with 100 μL of PBS in each well and then centrifuged at 1500 rpm for 5 min to settle down into the bottom of the plate. The absorbance of the nanomaterials in the microtissues was read at 460 nm, and particle standards for both Co3O4 and GO at the dose ranged from 0 to 50 ppm were tested at the same time. The cellular uptake amount of the nanomaterials in each microtissues was calculated based on the standards. At least 5 microtissues were used at each dose. Assessment of ALT and AST Content in the Microtissues after Treatment with Nanomaterials. Five microtissues in 80 μL cell medium were seeded in each well of a 96-well clear bottom white plate (for CYP study) or a 96-well clear bottom plate (for ALT and AST study). Tissues were then treated with 20 μL of GO nanosheet or Co3O4 nanoparticle suspensions (final concentration was 50 μg mL−1). After 24 h treatment, the tissues were washed with PBS three times to remove free materials. ALT (Alanine Transaminase Activity Assay Kit, Abcam, Canada) and AST (Aspartate Aminotransferase Activity Assay Kit, Abcam, Canada) contents were determined according to the manufacturer’s instructions. The absorption intensity was read on a SpectraMax M5 microplate spectrophotometer (Molecular Devices, US).
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RESULTS AND DISCUSSION
Preparation and Characterization of Polymer-Coated 3D Hepatocyte Spheroids. Individual 3D hepatocyte spheroids were prepared using the hanging-drop approach as illustrated in Figure 1A. 500 hepatocytes were seeded into each well of a hanging-drop plate at day 1, and cells continued to aggregate and assemble from day 2 to 5. The spheroids started to form at day 5 and were harvested into a U-shaped plate at day 7. The hepatocytes and obtained spheroids were imaged by a differential interference contrast (DIC) microscope (Figure 1B). The average diameter of the spheroids harvested at day 7 was 230 ± 70 μm (analyzing at least 25 spheroids). The inner-structure of the spheroid was observed using a transmission election microscope (TEM), revealing tight cell− cell contact at the MT edges and a hollow center (Figure S2). The cell number of each spheroid was quantified based on an ATP-cell number calibration curve (Figure S3), and each spheroid was composed of 1500 ± 180 hepatocytes after 7 days incubation. The spheroid was subsequently coated with a protective polymer shell using the LbL approach as illustrated in Figure 1C. Briefly, PVPON and TA were sequentially deposited on the surface of the spheroid to yield a multilayered protective coating. FITC-labeled PVPON (the synthesis procedure of fluorescent-labeled PVPON is shown in Figure S4) was used to visualize the polymer coating (Figure 1D). Assessment of Cellular Toxicity of NMs to Hepatocyte Spheroid. To investigate whether the polymer coating affects cell viability and to assess the protective effect for spheroids upon exposure to NMs, we treated spheroids with NMs (GO and Co3O4) known to induce hepatotoxicity.21,22 The physicochemical properties of the NMs were characterized before exposure to spheroids (Figure S5). To determine the toxicity of NMs on the uncoated and coated spheroids, cell viability was quantified by a CellTiter-Glo 3D ATP assay. We first examined the effect of polymer layers on the protection of spheroids by tailoring the number of polymer layers (Figure 2). With the outer polymer layer number increased from 0L (uncoated) to 6L (6 bilayers), a negligible cell viability
Figure 2. Cytotoxicity of polymer coated (0−6 L) hepatocyte spheroids after 24 h incubation with GO and Co3O4 with the concentration of 50 μg mL−1, respectively. Cell viability was quantified by a CellTiter-Glo 3D ATP assay (Promega, US). Data represent the mean ± standard error from three independent experiments, and at least five spheroids were analyzed in each experiment. * P < 0.05 compared to the control based on Student’s t test. # stands for P < 0.05 compared polymer coated spheroids (2−6 L) with uncoated ones based on Student’s t test. 52
DOI: 10.1021/acs.chemrestox.8b00120 Chem. Res. Toxicol. 2019, 32, 49−56
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Chemical Research in Toxicology
polymer coating when exposed to a higher dose of GO. For Co3O4 treatment, uncoated spheroids showed a dramatic decrease in cell viability and only remained at 35% cell viability at the NM dose of 100 μg mL−1. In contrast, with the same dose of particles, the coated spheroids showed a cell viability of up to 65%. Collectively, the polymer coating remarkably increased the cell viability of spheroids under the exposure to NMs, with ∼18% and ∼30% increased viability for GO and Co3O4 treatment at the dose of 100 μg mL−1, respectively. In addition, the cell viability of spheroids was also expressed in heatmap format (Figure 3B), which clearly showed the differential cytotoxicity induced by GO and Co3O4 NMs. Assessment of Cellular Association of NMs to Hepatocyte Spheroids. NMs induced differential toxicity is likely due to differences in particle-spheroid interactions, including cellular association and subcellular processing.23,24 To determine the degree of cellular association, particle concentration within the spheroids after 24 h incubation was evaluated through the detection of particle absorbance intensity. The calibration curve of particle concentration versus particle absorbance intensity was determined using a wavelength of 450 nm (Figure S6). Uncoated spheroids showed a significant higher association of GO (≥50 μg mL−1) compared to the coated ones, indicating that polymer coating efficiently prevents GO uptake by spheroids, which suggests that the lower cellular association of NMs leads to a decrease in cell toxicity (Figure 4A). Similarly, for the treatment with Co3O4, uncoated spheroids showed a significant higher association of Co3O4 (≥50 μg mL−1) compared to the coated ones, suggesting that the polymer coating protected the spheroids from a particle uptake-induced toxicity. The cellular association was also visualized using fluorescent microscopy (for GO, GO was fluorescently labeled as described in a previous study 21 ) and DIC microscopy (for Co 3 O 4 ) confirming that a higher extent of particles, both GO and Co3O4, was taken up into uncoated spheroids compared to coated spheroids (Figure S7). In addition, we further visualized the histology of spheroids after NMs (50 μg mL−1) uptake using hematoxylin and eosin (H&E) staining (Figure 4B). GO nanosheets were observed largely spreading inside the uncoated spheroids and interfering with tissue histology but were rarely seen in the coated ones. In contrast, Co3O4 nanoparticles mainly penetrated the uncoated spheroids with the damage of spheroids, however, internalized to a small extent inside the coated spheroids. The uptake of Co3O4 by coated spheroids may link to their toxicity examined in coated spheroids (as shown in Figure 3A). We think this is likely due to the small size of Co3O4 (D ≈ 10 nm), which could penetrate the semipermeable polymer that has a higher permeability under the cell culture condition as reported by previous studies.22,25 Collectively, the polymer coating effectively reduced the cellular association of NMs by spheroids, leading to a higher cell viability compared to uncoated spheroids. Determination of Polymer Coating Effect on the Hepatocyte Function of Spheroids through Assessment of Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST) Activity. ALT and AST are considered to be two of the most important enzymes to detect liver damage or injury from different types of diseases or conditions.26 ALT and AST levels are generally low but may spike during disease states or in the event of tissue injury.27 Thus, ALT and AST levels are routinely used as indicators of liver functions. In particular, marked elevation may signal acute liver damage.28
decrease was observed in the spheroids, confirming the polymer itself did not induce cytotoxicity in the spheroids. However, upon the exposure to GO (50 μg mL−1), spheroids showed a significant increase in cell viability with the increase of polymer layer number. In contrast, this layer effect (between 2L and 6L) was not observed in spheroids after treatment with Co3O4 (50 μg mL−1), even though multilayer coated spheroids showed a significant increase in cell viability compared to uncoated spheroids. It is notable that there were negligible differences in the cell viability (for both GO and Co3O4 treatment) between the spheroids coating with 4L and 6L polymers, indicating 4L polymer coating exhibited a nearly ultimate protective effect on the spheroids. Hence, we continued to use 4L-coated spheroids to examine the protective effect of polymer coating under exposure to NMs with a dose range from 12.5 to 100 μg mL−1. As shown in Figure 3A, the coated (4L) and uncoated spheroids showed significant differences in viability when
Figure 3. (A) Cytotoxicity of polymer coated (4L) hepatocyte spheroids after 24 h incubation with GO and Co3O4 with the dose range from 12.5 to 100 μg mL−1, respectively. Cell viability was quantified by a CellTiter-Glo 3D ATP assay (Promega, US). Data represent the mean ± standard error from three independent experiments, and at least five spheroids were analyzed in each experiment. *P < 0.05 compared to uncoated control; # p < 0.05 compared to coated control; ** p < 0.05 compare coated group to uncoated group based on Student’s t test. (B) Heat map summary of NM induced cytotoxicity in hepatocyte spheroids based on the proportion of cell viability obtained from ATP assay.
exposed to NMs (both GO and Co3O4) for 24 h. The polymer coating itself did not induce a significant decrease in cell viability of the spheroids. However, uncoated spheroids showed a significant decrease in cell viability after GO treatment and remained at 68% cell viability at the NM dose of 100 μg mL−1. In contrast, coated spheroids improved the cell viability up to 86%, compared to uncoated spheroids after treatment with the same dose of GO. There was no significant decrease in cell viability of spheroids after treatment with a lower dose of GO (
