Article pubs.acs.org/molecularpharmaceutics
Mechanisms of Nanoparticle Internalization and Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge Azzah M. Bannunah, Driton Vllasaliu,† Jennie Lord, and Snjezana Stolnik* Division of Drug Delivery and Tissue Engineering, School of Pharmacy, Boots Science Building, University of Nottingham, Nottingham NG7 2RD, U.K.
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ABSTRACT: This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC50) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein) but was not significantly affected by the inhibition of clathrin pathway. KEYWORDS: Cell uptake, Caco-2, endocytosis, epithelial cells, nanoparticles, nanoparticle transport, nanotoxicity
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INTRODUCTION Understanding the interaction of nanoparticles with the epithelium as a portal of entry into the body is essential in the areas of nanotoxicology and drug delivery as this interaction dictates the systemic absorption and organ-specific toxicity of nanomaterials. In terms of biomedical uses, there has been a recent explosion of interest in nanosized systems with potential application across a range of biomedical fields, including drug delivery,1 device-based therapy, medical imaging,2 and tissue engineering.3 The unique properties of nanoparticles afforded by their small size and high surface area offer significant potential for exploitation in biomedicine. Considering drug delivery, nanoparticulate systems can potentially be used to break through biological barriers and achieve accumulation at target sites.4 However, safety issues regarding biomedical application of nanoparticles remain5,6 and need to be addressed extensively before their widespread use in the clinic.7 Furthermore, full understanding of how physicochemical characteristics of nanomaterials influence their interaction © 2014 American Chemical Society
with the biological systems is required before safe and effective clinical application. Physicochemical characteristics of nanoparticles that influence their interaction with the biological systems, including the uptake via the epithelium, comprise nanoparticle material,4 size,8 surface charge,9 and surface chemistry.10 An almost infinite number of different possible combinations of these nanoparticle parameters are possible and their effect on biological interactions needs to be studied in order to predict nanotoxicology or design ideal nanosized drug delivery systems. Yet, this area remains relatively unexplored. This is especially the case with respect to the present knowledge regarding nanoparticle interaction with the epithelium, with a relatively small number of publications addressing this topic.11,12 Received: Revised: Accepted: Published: 4363
June 20, 2014 October 15, 2014 October 19, 2014 October 19, 2014 dx.doi.org/10.1021/mp500439c | Mol. Pharmaceutics 2014, 11, 4363−4373
Molecular Pharmaceutics
Article
Instruments, Ltd., Malvern, U.K.). Aminated and carboxylated polystyrene lattices of 50 and 100 nm and were suspended in 1 mM L−1 NaCl (pH 5.5) or in the biological solution used in cell experiments (HBSS:HEPES; pH 7.4), and the measurements were taken under these conditions. The reported zeta potential values represent a mean of three measurements. Cell Culture. Caco-2 and Calu-3 cells were cultured using EMEM supplemented with 10% v/v FBS, 1% v/v nonessential amino acids, 1% v/v L-glutamine, and 1% v/v antibiotic− antimycotic solution. Cells were seeded on 96-well plates (for toxicity studies) at a density of 1 × 104 cells per well and on Transwell permeable supports (12 mm diameter; 0.4 μm pore size) at 1.25 × 105 cells per well. Caco-2 cells were cultured on Transwell supports for 21 days prior to their use in nanoparticle uptake and transport experiments, with medium replenishment every 2 days. Cell layer integrity and tight junction formation was assessed by periodic measurements of transepithelial electrical resistance (TEER), conducted using an EVOM epithelial voltohmmeter (World Precision Instruments, USA). Nanoparticle Toxicity. MTS Assay. The effect of nanoparticles on cell viability was determined using the CellTiter 96 AQueous (MTS) cell proliferation assay kit. Caco-2 and Calu-3 cells were cultured on 96-well plates for 2 days prior to the study. Culture medium was removed and replaced with 100 μL of nanoparticle suspensions (diluted in HBSS) at different concentrations. Cells incubated with HBSS were used as a negative control, whereas cells incubated with Triton X-100 (0.2% v/v in HBSS) were used as a positive control. Cells were incubated with the samples and controls at 37 °C/5%CO2 for 4 h. After this interval, nanoparticle samples were removed and cells washed with PBS. One hundred microliters of culture medium was then added into each well, followed by 20 μL of the MTS reagent; cells were then incubated for 2 h. MTS absorbance was thereafter measured at 490 nm using a Dynex absorbance microplate reader (Dynex Technologies, USA). Relative cell viability was calculated using the following equation:
We adopted a systematic approach in investigating the effect of nanoparticle size and surface charge on their toxicity, in addition to studying the impact of these parameters on cell uptake and translocation across an intestinal epithelial cell monolayer model. Specifically, we determined whether negatively and positively charged model nanoparticles of 50 and 100 nm, which were initially characterized, show different effects in epithelial cells in terms of toxicity, cell uptake, and transepithelial transport, including the mechanisms involved in epithelial cell uptake and translocation. Toxicity studies were initially conducted in two epithelial cell lines, intestinal Caco-2 and airway Calu-3, to determine whether nanoparticle size or charge influences their toxicity, in addition to identifying nanoparticle concentrations with no notable cytotoxicity, to be used in further studies. Cell uptake and transport experiments were conducted in an intestinal model based on polarized Caco-2 cell monolayers, with uptake mechanisms elucidated by pharmacological inhibitors.
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MATERIALS AND METHODS Materials. Caco-2 cells (human intestinal adenocarcinoma) were obtained from the European Collection of Cell Cultures (ECACC) and were used between passages 63−67. Calu-3 cells (human airway epithelial cells) were obtained from the American Type Culture Collection (ATCC, USA) and were used at passage 36 and 37. Eagle’s minimum essential medium (EMEM), Trypsin/EDTA, antibiotic/antimycotic solution, fetal bovine serum (FBS), L-glutamine, and nonessential amino acids (NEAA) were purchased from Sigma-Aldrich (Poole, U.K.). Hank’s balanced salt solution (HBSS), Triton X100, 4-(2-hydroxyethyI)-1-piperazineethanesulfonic acid (HEPES), paraformaldehyde, Hoechst (33342) nucleic acid stain, and 1,4-diazabicyclo-octane (DABCO, UV mountant) were also obtained from Sigma-Aldrich. Phosphate buffered saline (PBS) was purchased from Oxoid Ltd. (U.K.). Lactic dehydrogenase assay (LDH) was obtained from Sigma-Aldrich, and CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) was purchased from Promega (USA). Nocodazole, 5-(N-ethyl-N-isopropyi)-amiloride (EIPA), methyl-βcyclodextrin, and chlorpromazine were obtained from SigmaAldrich (U.K.). Genistein and dynasore were purchased from Tocris Bioscience and Calbiochem (U.K.). Orange aminated latex polystyrene nanoparticles (100 nm) and blue aminated latex particles (50 nm) were obtained from Sigma-Aldrich. Fluoresbrite carboxy-modified microspheres with mean diameters of 50 and 100 nm were purchased from Polysciences Ltd. (Germany). Transwell permeable supports (12 mm diameter, 0.4 μm pore size) were obtained from Corning Life Sciences (USA). Nanoparticle Characterization. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were performed at room temperature (25 °C). Nanoparticles of different surface charges, sizes, and concentrations were measured by a Viscotek DLS instrument, using OmniSIZE software (Malvern, U.K.). Stock suspensions of nanoparticles were diluted to varying concentrations using HEPES-buffered HBSS as the suspension medium. Nanoparticle suspensions were left for 1 h at room temperature (25 °C) and particle size measured to determine the stability of submicron particles in the biological medium. The reported results represent the mean of 10 measurements in three independent experiments. Zeta Potential. Zeta potential measurements were performed at 25 °C using a Malvern Zetasizer 2000 (Malvern
cell viability(%) =
S−T × 100 H−T
where S is the absorbance obtained with the tested samples, T is the absorbance observed with Triton X-100, and H is the absorbance observed with HBSS. LDH Assay. The LDH assay was employed as a measure of membrane integrity as a function of the amount of cytoplasmic LDH released into the medium (LDH is a soluble enzyme located in the cytosol and is released into the culture medium upon cell membrane damage). Caco-2 and Calu-3 cells were cultured on 96-well plates for 2 days before the assay. Culture medium was removed and cells washed with PBS before nanoparticle application (100 μL; diluted in HBSS). Triton X100 (0.2% v/v in HBSS) was used to induce LDH release (positive control), whereas HBSS was used as the negative control. Cells were incubated with the samples (and controls) for 4 h. After this period, 50 μL of the sample solution was removed from each well and placed on another multiwell plate. One hundred microliters of the LDH reagent was applied to the samples, and an incubation time of 20−30 min at room temperature was allowed. After this period, absorbance at 490 nm was measured using a Dynex absorbance microplate reader. LDH release(%) = 4364
S−H × 100 T−H
dx.doi.org/10.1021/mp500439c | Mol. Pharmaceutics 2014, 11, 4363−4373
Molecular Pharmaceutics
Article
Figure 1. Relative cell viability (%), as indicated by the MTS assay, after incubation of Caco-2 (i) and Calu-3 cells (ii) with positively (NPs+) or negatively (NPs−) charged (50 and 100 nm) nanoparticles in a 4 h experiment. Nanoparticles were applied in HBSS:HEPES at different concentrations (a). Data also presented as nanoparticle numbers (b) and surface area (c) versus cell viability. Data represents the mean ± standard deviation (n = 7).
In addition to concentration dependency, toxicity is also expressed as changes in cell viability and LDH release versus nanoparticle numbers and surface area. Nanoparticle number was calculated by converting nanoparticle concentration into numbers using an equation supplied by nanoparticle manufacturers. It must be noted that conversion assumes monodisperse particle size distribution, and DLS data illustrate that particle size distribution is narrow. Total nanoparticle surface area was then calculated by calculating the area of a single sphere (nanoparticle) and multiplying it by the number of applied nanoparticles. Uptake and Transport of Nanoparticles Across Cell Monolayers. Cell uptake and transport studies were conducted on confluent Caco-2 and Calu-3 cell layers. To
ensure cell layer intactness, TEER was measured prior to the studies, and only Caco-2 cell layers displaying TEER values >1000 Ω/cm2 and Calu-3 cell layers with TEER of >500 Ω/ cm2 were included in the experiments. The culture medium was removed and cells washed (×3) with warm PBS. HBSS (HEPES-buffered at pH 7.4) was applied to both compartments of the Transwell, and cells equilibrated in this solution for 1 h at 37 °C. Apically present HBSS was then replaced with 0.5 mL of nanoparticle suspensions, which were applied at 100, 50, and 25 μg mL−1. Nanoparticle transport across the cell layers was determined by sampling the basolateral solution at regular time points (every 30 min for 4 h), with the replacement of sampled solution with fresh HBSS. Nano4365
dx.doi.org/10.1021/mp500439c | Mol. Pharmaceutics 2014, 11, 4363−4373
Molecular Pharmaceutics
Article
Figure 2. LDH release in the cell culture medium after Caco-2 (i) and Calu-3 (ii) exposure to positively (NPs+) and negatively (NPs−) charged nanoparticles (100 and 50 nm). Nanoparticles were applied in HBSS:HEPES at different concentration for 4 h (a). Data also presented as nanoparticle numbers (b) and surface area (c). Triton X-100 (TX; 0.2% v/v) was employed as a positive control to induce LDH release, and HBSS:HEPES was used as a negative control. Data represents the mean ± standard deviation (n = 7).
particles were quantified by florescence using an Infinite M200 plate-reader (Tecan, Switzerland). Cell internalization of nanoparticles was determined by confocal microscopy (using an Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope) and quantified by fluorescence measurement following the last sampling point in the transport experiment. The nanoparticle samples were removed from the apical surface of the cell layers, and cells were washed extensively with PBS and Trypan blue to quench the fluorescence13 of membrane-associated nanoparticles. Cells were then permeabilized with Triton X-100 (0.2% v/v in PBS), as reported previously,14−17 and removed from the permeable supports. Permeabilized cells were thereafter
pelleted by centrifugation and nanoparticles quantified in the supernatant by fluorescence measurements (excitation/emission a 481/644 nm for 100 nm particles, 358/410 nm for 50 nm particles, and 529/590 nm for 100 and 50 nm carboxylated particles). Nanoparticle Uptake Pathway Determination. Pharmacological inhibitors were employed to determine the endocytic pathways responsible for cellular uptake and transport of positively and negatively charged nanoparticles. Caco-2 cell monolayers were incubated with previously optimized doses (in terms of toxicity) of dynasore (20 μg mL−1), genistein (50 μg mL−1), 5-(N-ethyl-N-isopropyi)amiloride (EIPA) (10 μg mL−1), nocodazole (12.5 μg mL−1), 4366
dx.doi.org/10.1021/mp500439c | Mol. Pharmaceutics 2014, 11, 4363−4373
Molecular Pharmaceutics
Article
Figure 3. Internalization of positively (NPs+) and negatively (NPs−) charged nanoparticles by Caco-2 monolayers: (a) 50 nm nanoparticles and (b) 100 nm nanoparticles. Nanoparticles were applied to cell monolayers at concentrations of 25, 50, and 100 μg mL−1 suspended in HBSS:HEPES for 240 min. Data represents the mean ± standard deviation (n = 4).
chlorpromazine (20 μg mL−1), and methyl-β-cyclodextrin (6650 μg mL−1) in HBSS; these were applied individually to the apical chamber and cell monolayers incubated for 30 min at 37 °C. Inhibitors were then removed, cells washed with PBS, and 100 nm sized nanoparticles added, suspended in HBSS (50 μg mL−1), containing one of the inhibitors at concentrations described above. In a control experiment, nanoparticles (50 μg mL−1) were applied to the cell monolayers without the inhibitors (and cells were not treated with the inhibitors prior to nanoparticle application). Internalization and translocation of nanoparticles were measured using fluorescence quantitation as described previously. Data Analysis. All data are displayed as mean ± standard deviation (n = total number of cell monolayers), with three repeats. Student’s t-tests were performed for comparisons of two group means, while one way analysis of variance (ANOVA) followed by Bonferroni posthoc test was applied for comparison of three or more group means. P value of
