Bioavailability of Fullerene under Environmentally Relevant

Mar 4, 2016 - Carbon fullerene (C60) has emerged at the forefront of nanoscale research and application due to its unique properties. As the productio...
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Bioavailability of Fullerene under Environmentally Relevant Conditions: Effects of Humic Acid and Fetal Bovine Serum on Accumulation in Lipid Bilayers and Cellular Uptake Yeonjeong Ha,† Xianzhe Wang,‡ Howard M. Liljestrand,† Jennifer A. Maynard,§ and Lynn E. Katz*,† †

Department of Civil, Architectural and Environmental Engineering, The University of Texas at Austin, Austin, Texas 78712, United States ‡ Department of Biochemistry, The University of Texas at Austin, Austin, Texas 78712, United States § Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, United States S Supporting Information *

ABSTRACT: Carbon fullerene (C60) has emerged at the forefront of nanoscale research and application due to its unique properties. As the production of this nanoparticle rapidly increases, it can be released into natural aquatic environments and can accumulate in biological systems. This research examined the effects of humic acid and fetal bovine serum (FBS), which are ubiquitous in aquatic environments and representative of blood plasma in living organisms, respectively, on bioavailability of fullerene. Bioavailability was investigated using in vitro methods for lipid membrane accumulation and cellular uptake studies. Humic acid and FBS significantly changed the characteristics of fullerene including its particle size and surface charge. The effects of humic acid on lipid accumulation of fullerene depended on the lipid head charge. FBS also significantly decreased the lipid accumulation when positively charged and zwitterionic head groups were present on the lipids, possibly due to the higher steric repulsion of the protein coated nanoparticles. In addition, both humic acid and FBS protein effectively lowered the amounts of fullerene taken up by Caco-2 cells, which are derived from a human colorectal adenocarcinoma and have similar functions to the small intestinal epithelium. Results of this study suggest that surface modification of fullerene by environmentally relevant matrices can significantly affect the biological transport, as well as the possible toxicity of this nanomaterial.

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INTRODUCTION Carbon fullerene (C60) which was discovered by Kroto et al., (1985)1 is at the center of nanotechnology due to its unique properties such as its electron rich cage structure, high reactivity, and ability to accept and release electrons. Recently, fullerene use has expanded to a broad range of commercial2−4 (e.g., batteries, fuel cells, photovoltaics, and face cream) and biomedical applications5−8 (e.g., inhibitor of the HIV protease, drug carrier, and anticancer drug). As usage of this nanomaterial rapidly increases, the potential for its release into aquatic environments also increases. Thus, concerns related to the possible harmful effects of fullerene nanoparticles (NPs) within mammalian cells and aquatic organisms9−12 must be addressed. It is now well accepted that when nanoparticles are released into natural aquatic and biological environments, surface modification of nanoparticles occurs due to interactions with natural organic matter (NOM) present in aquatic environments13−15 and biological macromolecules16 in living organisms. Many previous studies have reported that this surface modification of nanomaterials affects nanoparticle interactions with living systems as well as possible toxicity toward them.17−21 Researchers have also suggested that NOM in © 2016 American Chemical Society

natural waters as well as biological macromolecules (e.g., protein) affect the stability of carbon based nanomaterials such as fullerene and carbon nanotubes.22−26 For example, dispersion of fullerene particles is typically enhanced in the presence of NOM,25 and NOM−fullerene interactions enhance the photochemical reactivity of fullerene.27 Previous studies have suggested that these changes in dispersion status and photochemical reactivity of fullerene have critical effects on transport and toxicity,10,25,27−29 but the mechanisms have not been fully elucidated. In addition, it was suggested that protein coated NPs can affect the immune system of living organisms.30 For example, when fullerene NPs interact with the amyloid protein, they can inhibit fibrillation of amyloid which causes Alzheimer’s disease, and therefore improving cognitive Special Issue: Jerry Schnoor's Lasting Influence on Global and Regional Environmental Research Received: Revised: Accepted: Published: 6717

October 9, 2015 February 8, 2016 March 4, 2016 March 4, 2016 DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Environmental Science & Technology functions.31 Thus, it is imperative to investigate the influence of NOM and biomolecules on the bioavailability of nanomaterials. The objective of this study is to explore the effect of environmentally and biologically relevant solutions on fullerene bioavailability. To understand bioavailability, we developed two in vitro methods: (i) lipid accumulation by solid supported lipid membranes (SSLMs) with synthetic lipid bilayers of varying composition and (ii) cellular uptake experiments using Caco-2 cell monolayers. Transport of fullerene NPs from aquatic and biological environments to an organism occurs via lipid membranes which serve as cell barriers. Thus, bioaccumulation and biouptake of fullerene NPs through these lipid membranes are of critical importance to assess bioavailability of this nanoparticle. To investigate the effects of lipid membrane composition on the lipid accumulation of fullerene, three unsaturated lipids (DOTAP, DOPC, and 18:1 PG) which have the same acyl chain length (C18) but different head charges were chosen. The biological relevance of theses lipids was summarized in our previous studies.32,33 In brief, cell membranes of living creatures mainly consist of unsaturated lipids which have acyl chain lengths from C14−C22 with zwitterionic and negative head charges. Lipid membranes with cationic head charges can be applied to gene delivery due to the electrostatic interactions between cationic head groups and anionic phosphate groups of the DNA/RNA. Thus, three unsaturated lipids in this study are representative of lipid membranes in real environments and biological applications. The characteristics of lipid membranes selected in this study are summarized in Table 1. As representative NOM and

changes in the particle characteristics of fullerene due to the presence of humic acid and FBS and evaluate the effect of these substances on fullerene cellular uptake.

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MATERIALS AND METHODS Materials. Carbon fullerene (100% solid, 99.5+ %) was obtained from SES Research (Houston, TX). Suwannee river humic acid standard II and FBS were purchased from the International Humic Substances Society (IHSS, St.Paul, MN) and Life Technologies (Grand Island, NY), respectively. Nonfunctionalized silica microspheres (Bangs Laboratories, Inc., Fisher, IN) were used as the solid support for the solid supported lipid membranes (SSLMs). The mean diameter of the microspheres was 5.2 μm. Three lipids, cationic 1,2dioleoyl-3-trimethylammonium-propane (DOTAP), zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (18:1 PG) were purchased from Avanti Polar Lipids (Alabaster, AL). To perform the cellular uptake tests, Caco-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). All solvents used in this study were of analytical grade. Preparation of Aqueous Fullerene Suspensions. Fullerene suspensions in water (nC60) were prepared by a liquid exchange method by ultrasonication (SON/nC60) and details are described elsewhere.32,36 After dissolving 3 mg of fullerene in 5 mL toluene, 30 mL of deionized water was added to the solution. Under ultrasonic treatment with air purging, the toluene layer was removed, leaving yellowish fullerene aggregations in water. This fullerene suspension was filtered through a 0.8 μm membrane filter (Millipore, Billerica, MA) to remove particles that were not dispersed in water. The size and zeta potential of the resultant fullerene suspension were measured using Dynamic Light Scattering (DLS, Malvern Zetasizer Nano ZS), and fullerene aggregates were used within 2 days to prevent any changes in particle characteristics. To investigate the effect of humic acid and FBS on the bioavailability of fullerene, fullerene dispersions were incubated in the dark for 24 h with humic acid (5−50 mg/L, concentration ratio of humic acid to nanoparticle ranged from 1.1−11) or 10% FBS for 24 h. The humic acid or FBS coated fullerene was also characterized by DLS and used for lipid accumulation as well as cellular uptake experiments. Determination of Accumulation of Fullerene in Lipid Membranes. SSLMs with three different lipid membranes (DOTAP, DOPC, and PG) were synthesized. The detailed procedure was reported in our previous study.32 In brief, the synthetic membrane vesicles for each lipid were mixed with silica microspheres by vigorous vortexing prior to 2 h of gentle mixing on a shaker. Then, the mixtures were centrifuged and supernatants that contained excess lipids not adsorbed onto silica beads were transferred to other vials. The mass of lipids that adsorbed onto the SSLMs was determined by measuring differences between initial lipid concentration and supernatant concentration. The synthesized solid supported lipid membranes and fullerene dispersions were placed into 40 mL vials with PTFE/silicon septa. The vials were incubated at room temperature using a custom-made tumbling device. After the desired incubation time, each vial sat for 1 h to allow settling of the solid supported lipid membranes (SSLMs). Then, 0.5 mL of supernatant containing the fullerene particles that remained in solution were transferred to a new vial for liquid extraction of

Table 1. Characteristics of selected unsaturated phospholipid components

phospholipids DOTAP DOPC 18:1 PG

carbon chain: double bonds (C18:1, 18:1) (C18:1, 18:1) (C18:1, 18:1)

main transition temperature (°C) ∼−0 −22 −18

physical state at room temperature liquid crystalline liquid crystalline liquid crystalline

charge of lipid head positive zwitterionic negative

biomacromolecules, we chose a commercially available humic acid and fetal bovine serum (FBS), respectively. Humic acid is ubiquitous in aquatic environments, which consists of a hydrophobic backbone and various functional groups such as phenolic and carboxylic groups which facilitate nonspecific adsorption onto NPs.24 The presence of these hydrophilic funtional groups alters the polarity of NPs coated with humic acid as compared to bare NPs.34 FBS contains a variety of proteins, including albumin and immunoglobulins, which are present in the blood plasma of living organisms, and is used as a cell culture supplement. Frequently, when NPs are incubated with a protein solution, the proteins adsorb onto the particle surface, forming a protective layer which alters the nanoparticle surface chemistry.16,22 This modification may influence interactions between NPs and biological systems as well as potential risks associated with occupational and environmental nanoparticle exposure. To date, only one study has suggested that humic acid reduced the uptake of fullerene by aquatic living organisms,35 and no study has reported the effects of humic acid and FBS on lipid accumulation and cellular uptake of fullerene. Thus, the focus of this research was to investigate 6718

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Figure 1. Effects of humic acid on (a) particle size and (b) zeta potential of fullerene NPs (C0 = 4.7 mg/L). Insertions in Figure 1(a) and Figure 1(b) are size distribution and zeta potential distribution of fullerene nanoparticle, respectively. The average sizes were calculated based on intensity, and average ± standard deviation of polydispersity index (PDI) values of all measurement in this Figure is 0.228 ± 0.0370.

minimize emulsion creation. GAA was successfully used in previous studies for extracting fullerene suspensions from solutions containing environmentally relevant materials and biomolecules.23,37,38 Cell Culture. Caco-2 cells were maintained in flasks in Dulbecco’s modified Eagle medium (D-MEM) containing 10% FBS, 0.2 mM nonessential amino acids, penicillin/streptomycin (100 U/mL), and 4 mM L-glutamine in an atmosphere of 5% CO2 at 37 °C. Cell passages were performed every two to three days, and were passaged at least twice before being seeded for cellular uptake experiments. Cellular Uptake Studies. To investigate the effect of humic acid coating on cellular uptake of fullerenes, fullerene stock solutions incubated with different concentrations of humic acid (5, and 10 mg/L, concentration ratios of humic acid to nanoparticle were 0.625 and 1.25) were prepared. Caco-2 cells in 150 cm2 cell culture flasks were washed with phosphatebuffered saline (PBS) three times before being detached by a

fullerene. For bare fullerene suspensions in water, liquid extraction was performed by adding 0.1 M Mg(ClO4)2 (0.4 mL) and toluene (0.5 mL) followed by vigorous mixing. After liquid extraction, the initial fullerene concentration (C0) and supernatant concentration (Cw) were measured. Fullerene accumulation in the lipids was calculated using eq 1 fullerene accumulated in the lipid(mg/kg‐lipid) =

C lip m

=

C 0 − Cw m

(1)

where Clip (mg/L) is the fullerene concentration in the lipid, which can be determined from the difference between the initial fullerene concentration (C0) and the fullerene concentration in the supernatant (Cw). m is the concentration of lipid (kg-lipid/L) in the system. For liquid extraction of fullerene dispersions incubated with humic acid and protein, we additionally added 2.5 mL of glacial acetic acid (GAA) to 0.1 M Mg(ClO4)2 and toluene to 6719

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Figure 2. Effect of fetal bovine serum on the fullerene particle distribution. The average size was calculated based on intensity, and the average ± standard deviation of PDI values without and with FBS are 0.188 ± 0.00950, and 0.813 ± 0.0.108, respectively.

acid. As shown in Figure 1(a), the presence of humic acid significantly decreased the particle size, indicating that humic acid can adsorb onto fullerene and prevent fullerene particle aggregation. This result is in agreement with Xie et al.24 who concluded that the particle size distribution of fullerene shifted to smaller diameters when incubated with 20 mg/L humic acid. In addition, many previous studies13,34,39 have reported that the presence of humic acid effectively prevents aggregation of other NPs (e.g., ZnO, TiO2, SiO2, and Al2O3) over time, which implies increasing stability in natural aquatic environments. Also, the addition of humic acid decreased the surface charge of the fullerene NPs (Figure 1(b)). This result is also consistent with other studies13,34,39 which suggested that NPs incubated with humic acid became more negatively charged due to the deprotonated carboxylic acid functional groups on the humic acid at circumneutral pH. It was suggested that the presence of multiple functional groups on the relatively large humic acid molecules can lead to steric hindrance effects which can increase the stability of NPs coated with humic acid. As the humic acid concentration increased (Figure 1), differences in both size and zeta potential of fullerene particles became negligible, indicating that 10 mg/L of humic acid was sufficient to saturate the fullerene surface (for a fullerene concentration of 4.7 mg/L). Figure 2 shows the effect of FBS on the particle size distributions. After 24 h of incubation with 10% FBS, the size distribution becomes narrower, and there is a shift in the distribution to slightly higher mean particle size. This shift can be explained by nonspecific adsorption of FBS on the fullerene surface. Deguchi et al., (2007) also have reported that protein coated fullerene exhibits a slightly higher particle size compared to bare fullerene. When fullerene NPs are exposed to protein containing solutions, the formation of a protein coating can prevent the formation of large particle aggregates, which increases the stability of the NPs. Indeed, it has been reported that stability of fullerene increases due to protein adsorption.40 In addition, previous studies have indicated that the presence of protein makes carbon nanotubes22,41 and polystyrene particles20 more stable in water. Studies20,21 have also demonstrated that the surface charge of negative polystyrene NPs became less negative after adsorption of serum proteins. However, it was not possible to obtain zeta potential data for fullerene incubated with 10% FBS solution in cell culture media

trypsin-EDTA solution. Then, cells were resuspended in complete growth medium (with 10% FBS), seeded in 6-well polycarbonate plates (Corning Inc., Corning, NY) at a density of 105 cells/cm2 and incubated in an atmosphere of 5% CO2 at 37 °C. The next day, the cell culture media was changed to FBS-free medium. After another day of incubation, the cell culture media was replaced with 2 mL of fresh FBS-free medium with 200 μL of fullerene stock solutions that had been incubated with different concentrations of humic acid. After 24 h of incubation, cells were washed three times with PBS and detached from the plates with 400 μL of trypsin-EDTA solution. The cells were lysed with 30 min of ultrasonication. Then, fullerene was destabilized with 400 μL of 0.1 M Mg(ClO4)2 and 2.5 mL glacial acetic acid (GAA) and extracted with toluene. To evaluate the effect of FBS on cellular uptake of fullerene, fullerene suspensions were incubated in cell culture media with and without FBS for 12−24 h. As above, 2 mL of fullerene suspensions were added to Caco-2 cells in 6-well polycarbonate plates and incubated for 2 days. Other procedures (e.g., cell lysis, liquid extraction of fullerene, and fullerene concentration analysis) for performing the uptake experiments are as described above. Instrumental Information. Fullerene concentrations in the toluene phase were measured using a Waters 2690 high performance liquid chromatography system equipped with a Waters 996 photodiode array detector (Milford, MA) after liquid extraction. Lipid concentrations in water were determined using a TOC analyzer (Tekmar Dohrmann Apollo 9000, Cincinnati, OH). Statistical Analysis. To compare results of cellular uptake studies, statistical analyses were performed using a student’s ttest.

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RESULTS AND DISCUSSION Impact of Humic Acid and Fetal Bovine Serum on the Fullerene Particle Characteristics. To investigate the effects of humic acid and FBS on the fullerene particle characteristics, particle size and zeta potential were measured after 24 h of incubation of fullerene with humic acid or FBS. Figure 1 highlights the significant changes in particle size and zeta potential of fullerene due to the addition of 10−50 mg/L humic 6720

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Figure 3. Rates of fullerene NPs (C0 = 4.7 mg/L) accumulation in three different lipid membranes with differently charged head groups. Solid lines represent model fits using eq 2.

Table 2. Kinetic Parameters Obtained by Fitting the Data Using eq 2 fullerene coating materials

Clip,0a (mg/kg-lipid)

DOTAP

lipid membrane

ka (L/mg/h) ‑4

kd (1/h)

Kb (L/mg) ‑5

τc (h)

r2

bare fullerene humic acid FBS

8

3.93 × 10 3.14 × 108 3.93 × 108

1.80 × 10 9.38 × 10−7 1.50 × 10−4

8.57 0.028 9.36

2.10 × 10 3.35 × 10−5 1.61 × 10−5

0.12 35.71 0.11

99.8 90.3 84.5

DOPC

bare fullerene humic acid FBS

3.93 × 108 3.14 × 108 3.93 × 108

1.24 × 10‑6 3.83 × 10−7 4.04 × 10−7

0.066 0.022 0.043

1.86 × 10‑5 1.72 × 10−5 9.38 × 10−6

15.03 45.05 23.20

96.3 95.6 89.2

PG

bare fullerene humic acid FBS

3.93 × 108

7.33 × 10‑7

0.13

5.59 × 10‑6

7.63

82.6

3.93 × 108

5.16 × 10−7

0.058

8.95 × 10−6

17.33

61.8

a

Clip,0 for bare and FBS coating fullerene was estimated with the assumption of a single layer adsorption of 150 nm NPs onto lipid membranes. Because humic acid decreased the mean size of fullerene NPs, we used 120 nm instead of 150 nm for estimating Clip,0 for humic acid coated fullerene. b K (=ka/kd) is the affinity constant and high value of K indicates a strong affinity between fullerene and lipid membranes. cCharacteristic time (τ) is defined as 1/(kaC + kd) and smaller K indicates faster interaction.

(i.e., results did not meet quality criteria due to the increased number of subruns per measurement), possibly due to the fluid characteristics of the 10% FBS containing solution (e.g., color and viscosity). Instead, we measured zeta potential of fullerene NPs incubated with 2.5% FBS solution in PBS overnight (Figure S1). As shown in Figure S1, fullerene incubated with the FBS solution has significantly less negative charge compared with bare fullerene. This result demonstrates that protein adsorbed onto the fullerene surface after 24 h of incubation. We also characterized the particle size of fullerene nanoparticles with and without NOM/FBS in cell culture media after 2 days of incubation (Figure S2, Figure S3); aquatic Suwannee River NOM was used. As shown in Figure S2, bare fullerene in cell culture media aggregates relatively quickly due to the high ionic strength of cell culture media. On the other hand, the size of NOM coated fullerene in cell culture media is significantly smaller compared with that of bare fullerene after 2 days of incubation, which indicates that NOM effectively increased colloidal stability. Figure S3 shows that FBS also increases the stability of fullerene in cell culture media, and the

stability of FBS coated fullerene in cell culture media is even higher than the fullerene suspension in water. Kinetics of Lipid Accumulation of Fullerene. Time dependent lipid accumulation studies for bare fullerene NPs were performed to investigate the effects of lipid surface charge on the rates of distribution of fullerene between lipid and water. Results are shown in Figure 3. We chose three unsaturated lipids with different head charges but similar acyl chain length (C18). They are all present in a liquid crystalline phase at room temperature. Lipid accumulation of fullerene with the cationic DOTAP lipid reached steady state very quickly (within 30 min), and accumulation values with both the zwitterionic DOPC and anionic PG increased continuously, reaching a plateau after approximately 48 h. At all times, fullerene accumulation values were highest for DOTAP, followed by DOPC, and then PG lipid. Due to the negative charge associated with the fullerene nanoparticle surface in water, a strong electrostatic interaction between fullerene NPs and the positive DOTAP lipid would be expected compared to DOPC (zwitterionic) and PG (negative) lipids. Thus, it can be concluded that adsorption of charged NPs onto oppositely 6721

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Environmental Science & Technology charged lipid membranes or oppositely charged sites on the lipid bilayer could play a critical role for bioaccumulation of NPs. Although cationic lipid membranes are rare in natural systems, membrane proteins might have cationic patches42 which can facilitate fullerene adsorption. To better understand the effects of lipid composition on the accumulation rates, we fit the Langmuir adsorption model (eq 2) to our experimental data and obtained rate parameters, assuming that the first step of the distribution mechanism of fullerene nanoparticles is the physical adsorption on the lipid membranes. Fullerene adsorption on the fixed portions of the lipid membrane surfaces can occur by electrostatic interaction.43,44 This empirical model has been used in previous studies to describe cellular uptake kinetics of gold NPs45 and oxide iron NPs.44 This model was also employed by Hou et al.43 for describing the effect of gold nanoparticle size on the lipid membrane accumulation. The model equation is C lip(mg/kg‐lipid) =

kaCC lip,0 kaC + kd

(1 − exp(− (kaC + kd))t ) (2)

where Clip (mg/kg-lipid) is the mass of fullerene that accumulated per unit mass of lipid as a function of time, t is time (hr), ka (L/mg/h) is association rate constant, kd (1/h) is a dissociation rate constant, C (mg/L) is the initial fullerene concentration, and Clip,0 (mg/kg) is the maximal concentration that can be adsorbed onto a unit mass of lipid membrane. It is difficult to calculate the exact Clip,0 value because the size of the fullerene aggregates is not uniform. Here, we estimated Clip,0 using an assumption that there is a single adsorbed layer of nanoparticle aggregates which have a uniform size of 150 nm on lipid membranes based on the calculations in Hou et al.,.43 Although Clip,0 used in this study might not be accurate, that does not affect the lipid membrane composition dependence of the rate parameters summarized in Table 2. In Table 2, K (=ka/ kd) is the affinity constant and a high K value indicates a strong affinity between fullerene and the lipid membrane. Characteristic time (τ) is defined as 1/(kaC + kd) and a smaller τ value means faster rates. As shown in Table 2, the affinity constant is highest with DOTAP lipid and lowest with PG lipid. It is noteworthy that the affinity constant with the zwitterionic lipid (DOPC) is slightly lower than with the positive lipid (DOTAP) but still much higher than with the negative lipid (PG), probably due to the electrostatic interaction between fullerene and the positive portion of the zwitterionic lipid membrane (N+ terminus). Effects of Humic Acid and Protein on Lipid Accumulation. Figure 4 shows the impact of humic acid on fullerene accumulation in the three lipid membranes. Fullerene incubated with humic acid and FBS was extracted to a toluene phase, and the recovery from the extracted solution was 110 (±0.29) % for 20 mg/L humic acid and 96 (±23) % for 10% FBS. With DOTAP lipid (Figure 4(a)), initial lipid accumulation values in the presence of 20 mg/L of humic acid were much lower than without humic acid, but eventually reached a plateau at 80 h that was equivalent to the accumulation without humic acid. The possible reason for the slower adsorption process in the presence of humic acid may be due to competitive adsorption between fullerene and humic acid present in the background solution. Because humic acid is negatively charged, adsorption between the negatively charged humic acid and positively charged lipid membranes is favorable.

Figure 4. Rates of fullerene NPs (C0 = 4.7 mg/L) accumulated in (a) DOTAP (positive head), (b) DOPC (zwitterionic head), and (c) PG (negative head) with and without humic acid (20 mg/L). Solid lines represent model fits using eq 2.

Indeed, as shown in Figure S4, more than 20% of the humic acid rapidly adsorbed onto the DOTAP lipids, however, humic acid did not adsorb onto DOPC or PG lipids. In Figure 4(a), lipid accumulation values with 20 mg/L humic acid decreased slightly after 6 h of incubation. To better understand the lipid accumulation of fullerene in the presence of humic acid, one additional test was performed. Two vials were prepared: a control vial containing only solid supported lipid membranes (SSLM) with DOTAP lipid in 6722

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Figure 5. Rates of fullerene fullerene NPs (C0 = 4.7 mg/L) accumulated in (a) DOTAP (positive head), (b) DOPC (zwitterionic head), and (c) PG (negative head) with and without 10% fetal bovine serum. Solid lines represent model fits using eq 2.

water, and another vial containing DOTAP lipid with 20 mg/L humic acid. After 80 h of incubation, humic acid was added to

the control vials to provide the same humic acid concentration, that is, 20 mg/L of humic acid in the sample vial and the 6723

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Figure 6. Effect of humic acid on cellular uptake of fullerene. The injected fullerene concentration was 8 mg/L. * denotes the value is lower than the value without HA at P < 0.05, ** denotes the value is lower than the value without HA at P < 0.01 based on the student’s t test.

parameters obtained are summarized in Table 2. For estimating Clip,0 in eq 2 for fullerene coated with humic acid, we used a 120 nm particle size instead of 150 nm because humic acid significantly decreased the particle size of fullerene (Figure 1(a)); the Clip,0 value does not affect the dependence of humic acid and FBS on the rate parameters. With DOTAP lipids, humic acid coated fullerene has higher affinity coefficients compared to that of bare fullerene due to the strong electrostatic interactions. With DOPC lipids, the presence of humic acid and FBS significantly decreased fullerene affinity to the lipid membranes, and the affinity of FBS coated fullerene for lipid membranes was much lower than that of humic acid coated fullerene. This result suggests that FBS coating creates remarkably higher steric repulsion compared with the repulsion created by humic acid coating on NPs. In addition, humic acid and FBS coated fullerene interacts with all three lipid membranes more slowly, and except for the PG lipid, adsorption rates are slower with humic acid than FBS. Because both fullerene dispersions and humic acid are hydrophobic and exhibit a similarly negative surface charge, competitive adsorption of fullerene and humic acid can occur on the lipid, which results in slower lipid accumulation of fullerene in the presence of humic acid. Effects of Humic Acid and Protein on Cellular Uptake of Fullerene. To investigate the effect of humic acid coating on the cellular uptake of fullerene, fullerene was incubated with humic acid (5, 10, 20 mg/L) for 12 h prior to performing cellular uptake experiments. Results shown in Figure 6 demonstrate that humic acid coatings with 5 and 10 mg/L humic acid significantly decreased the overall mass of fullerene taken up by the cells for all humic acid concentrations. It is generally acknowledged that the net cell surface charge is negative and fullerene coated with humic acid has a lower surface charge compared to bare fullerene, as was evident in Figure 1. Thus, electrostatic repulsion between humic acid coated fullerene and the cell surface can be higher than that of bare fullerene, resulting in less fullerene uptake by the cells in the presence of humic acid. This result is in agreement with a study of Chen et al.35 which showed that humic acid addition decreased the uptake of fullerene by two aquatic organisms:

control vial. Then fullerene dispersions were added in the presence of these identical concentrations of humic acid (20 mg/L) in both vials and incubated for an additional 80 h. As demonstrated in Figure S6, fullerene NPs rapidly adsorbed onto the lipid membranes in the control vial. On the other hand, after 80 h of preincubation of the DOTAP lipid with humic acid, fullerene accumulation values were much lower than observed in the control vial. This result suggests that humic acid can strongly adsorb on the DOTAP lipid heads due to electrostatic interaction, and prevent fullerene adsorption. With DOPC lipid (Figure 4(b)), humic acid effectively decreased lipid accumulation of fullerene at all incubation times. The main driving force of fullerene interaction with zwitterionic lipid membranes is the electrostatic interaction between a positive portion of the lipid membrane and fullerene, and steric effects associated with humic acid coated fullerene can prevent the approach of fullerene to the lipid membranes. Moreover, negatively charged deprotonated functional groups on the humic acid decrease the surface charge of the particles. With PG lipid (Figure 4(c)), in the presence of humic acid, fullerene accumulation within the lipid was lower than without humic acid which can be explained by the stronger electrostatic repulsion between humic acid coated fullerene and a negatively charged lipid membrane (see Figure 1(b)). Initial experiments confirmed that adsorption of FBS to any of the three lipid membranes is negligible (Figure S5). However, the presence of 10% FBS significantly decreased the lipid accumulation of fullerene with both DOTAP (Figure 5(a)) and DOPC lipids (Figure 5(b)). When fullerene is exposed to the protein containing solution, fullerene was immediately coated with protein (Figure 2) reducing its surface energy.40 This decrease in surface energy contributes to the decrease in accumulation of fullerene with FBS. With PG lipid (Figure 5(c)), there is not a significant difference between lipid accumulation with and without FBS, because both bare fullerene and FBS coated fullerene have strong electrostatic repulsion with negative lipid membranes. To better understand the influence of humic acid and FBS on the lipid accumulation rates, the experimental data were fit using the kinetic model presented in eq 2, and the rate 6724

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Environmental Science & Technology

Figure 7. Effect of fetal bovine serum on the cellular uptake of fullerene. The fullerene concentration was added at 12 mg/L. *** denotes the value is higher than the value with FBS at P < 0.001 based on the student’s t test.

Daphnia magna and zebraf ish, presumably due to the more negatively charged surface of humic acid coated fullerene. The influence of FBS on cellular uptake of fullerene was also investigated (Figure 7). When higher concentrations of fullerene (>7 mg/L) were added to the cells, the mass of fullerene taken up by the cells in the presence of FBS was much lower than that under serum free conditions (Figure 7). This result supports our previous results which indicated that serum proteins effectively decreased lipid accumulation of fullerene due to steric hindrance and a possible decrease in surface energy. The proposed reduction in surface energy resulting from adsorption of proteins onto the fullerene surface likely decreased the interaction between particles and cell membranes, which consequently contributed to reduced cellular uptake. Many previous studies also have reported that serum proteins drastically reduced cellular uptake of NPs including polystyrene nanoparticles,21 silica NPs,19 and carbon nanotubes.22 In this study, we demonstrate that both humic acid and protein-coated fullerene have different surface properties. This change in surface properties leads to reductions in cellular uptake of fullerene by Caco-2 cells which have morphological and functional similarities to the small intestinal epithelial cells. In our previous study,46 we proposed that fullerene NPs can be transported through cell membranes via a combination of active transport (e.g., endocytosis) and passive diffusion. In the endocytosis process, NPs first bind to the cell surface, followed by internalization inside the cells. Thus, adsorption of NPs on the cell surface is a critical step for cellular uptake. Humic acid coated fullerene has a more negatively charged surface and FBS coated fullerene has less surface energy compared to bare fullerene, indicating that both humic acid and FBS can reduce the accessibility of fullerene to the cell surface. Thus, results of this study imply that humic acid and FBS can significantly affect active transport of fullerene NPs.

surface properties of fullerene NPs and affect the interaction of this nanoparticle with model cell membranes and cultured cells. In this study, fullerene accumulation in lipids was significantly influenced by the presence of humic acid and FBS, but the impact of each depended on the lipid head charges. With the positively charged lipid, DOTAP, fullerene coated with humic acid interacted more slowly with the lipid membrane but eventually reached comparable accumulation levels relative to bare fullerene. For the zwitterionic lipid (DOPC), both the rates and extent of distribution were reduced in the presence of humic acid. In contrast, for the negatively charged lipid, PG, humic acid had no significant impact on distribution and minimal effect on the extent of distribution into the lipid. In contrast to the humic acid coated fullerene, FBS coated fullerene did not yield the same extent of distribution to the positive lipid, DOTAP, as bare fullerene. However, the accumulation level of bare fullerene was higher than the accumulation of FBS coated fullerene with positive lipid membranes. Recent studies have shown that positive lipids can be used for gene therapy.47 Thus, accumulation of fullerene in positive lipid membranes under environmentally and biologically relevant conditions has implications for biological applications of fullerene. With the zwitterionic lipid (DOPC), both humic acid and FBS significantly reduced the amount of fullerene accumulated in the lipid and both coatings affected the rates; however, the impact of FBS was greater. Considering the most prevalent lipid composition in living organisms is zwitterionic lipids, the results of this study can provide fundamental information to evaluate toxicity toward living organisms in real environments. Using only lipid membrane components, we can envision passive adsorption and diffusion through lipid membranes. However, the cellular uptake mechanism of NPs into living cells is more complicated, including not only passive diffusion but also active transport (e.g., energy dependent endocytosis). Therefore, the effects of humic acid and FBS on cellular uptake of fullerene were also investigated. Results suggested that both humic acid and FBS decreased the quantitative mass of fullerene taken up by cells. To our knowledge, this is the first study to demonstrate surface modification of fullerene by humic acid and FBS and

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ENVIRONMENTAL IMPLICATIONS Natural aquatic systems and biological systems are the most likely environments where the presence of fullerene NPs will impart human and ecological effects. Humic acid and FBS, representative background organic species for environmental and biological systems, respectively, were shown to alter the 6725

DOI: 10.1021/acs.est.5b04964 Environ. Sci. Technol. 2016, 50, 6717−6727

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Environmental Science & Technology

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the resulting effects on nanoparticle lipid accumulation and cellular uptake. In particular, the use of lipids with varying surface charges provides insight into the impact of natural organic matter (e.g., fulvic acid and polysaccharide) and biomacromolecules (e.g., lipids, other type of proteins) on bioavailability of fullerene NPs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b04964. Details about zeta potential of FBS coated fullerene nanoparticles, the effect of NOM/FBS on fullerene stability in cell culture media, adsorption of humic acid/ FBS on three different lipid membranes, and effects of humic acid on the lipid accumulation of fullerene are available (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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