Effect of TiO₂ nanoparticle surface functionalization on protein adsorption, cellular uptake and cytotoxicity: the attachment of PEG comb polymers using catalytic chain transfer and thiol–ene chemistry

Abstract

A successful modification of titanium dioxide (TiO₂) nanoparticles surfaces by a grafting-to polymer technique combining catalytic chain transfer and thiol–ene click chemistry is reported. Vinylic end functional polymers were first prepared by catalytic chain transfer polymerization (CCTP) using oligo(ethylene glycol) methacrylate as a monomer. The presence of vinylic end groups was then exploited to attach the polymers to thiol functionalized TiO₂ nanoparticles via thiol–ene Michael nucleophilic reactions. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-infrared (ATR-IR), dynamic light scattering (DLS), and thermogravimetric analyses (TGA) were used to verify the successful modification of the TiO₂ surface. The modified TiO₂ nanoparticles were stable in cell culture media and formed smaller aggregates when compared to non-surface modified nanoparticles. Cellular toxicity of the hybrid TiO₂–polymer particles towards human lung cell lines A549 and H1299 in vitro was evaluated. Results from one-dimensional gel electrophoresis show the presence of polymer layers around the particles affects the adsorption of protein onto the TiO₂ surface. The reduction in particle aggregate size and changes to the particle surface chemistry, following polymer grafting, was found to reduce cellular uptake and diminish cytotoxicity for both human lung cell lines tested.

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Introduction

Hybrid organic–inorganic nanoparticles are of interest for use in various applications due to high surface functionality, afforded by their small size, yielding properties that were substantially different to equivalent bulk materials.^1–6 The controlled production of nanoparticles is a growing research area with potential applications in biomedical, optical, and electronic fields.^2,5–10 The novel properties of nanomaterials are imbued by large surface effects influencing surface energies, presenting opportunities for functional materials but also in some cases raising concerns over cytotoxicity.^11–15 A large number of studies have shown that nanoparticles are able to pass through cell membranes in organisms (leading to their use as drug delivery vehicles),^1,6,16 but presently nanoparticle interactions with complex biological systems are still not fully understood.^16–18 TiO₂ nanoparticles have extended applications in the areas of environmental technologies,^19,20 clean energy production,²¹ self-cleaning surfaces,²² textiles,²³ sensors,²⁴ antimicrobial agents, pharmaceuticals, as well as cosmetics, and sunscreens.^25,26 The wide-spread use of these particular particles has attracted much research aimed at understanding the biological impact of TiO₂ nanoparticles and reducing any toxicity issues.^{10,13–16,27–29}

Recently, Tedja et al. showed that the aggregate size of TiO₂ nanoparticles in biological media had a significant influence on biological impact of TiO₂ on human lung cell lines in vitro (a reduction in TiO₂ toxicity was observed when a sonication method was used to reduce the aggregate size³⁰). Micrometer-sized TiO₂ aggregates have been found to be more readily taken-up by human lung cell lines when compared to sub-micrometer aggregates; with a corresponding impact on cytotoxicity in the human lung cell lines A549 and H1299. In addition to the particle aggregate size, the surface chemistry of nanoparticles has been shown to affect their biological interactions. Our recent study has shown that adsorption of serum proteins on to the surface of TiO₂ nanoparticles resulted in a lower biological impact on both human lung cell lines A549 and H1299.³¹ Interestingly, the reduction of biological impact due to serum adsorption has been noted to be accompanied by a higher particle uptake compared to the non-serum-treated TiO₂ after a 24 h exposure period. Therefore, the adsorption of serum proteins was identified as a potential protection strategy to reduce cytotoxic effects of endocytosed TiO₂ nanoparticles.³¹ However, the adsorption of protein(s) on to nanoparticles results in systems difficult to fully characterize (and therefore control), following the possibility of conformational changes in protein structure and folding. For instance, gold³² and supermagnetic iron oxide³³ nanoparticles have been reported to capture serum proteins with significant induced conformational changes. Denatured protein layers may have significant effects on cellular immune responses.³⁴ As a result, strategies to mediate TiO₂ nanoparticle surfaces to reduce cytotoxicity whilst minimizing protein adsorption are required.

One practical way to maintain a small aggregate size of nanoparticles is to use a polymer stabilizing outer corona, to increase the steric repulsion between individual nanoparticles. There are several techniques available to coat nanoparticles with polymer, including “grafting to”, “grafting from”, and “layer-by-layer” (LbL) approaches.^1,6,35–43 The different synthetic techniques create hybrid organic–inorganic particles with different surface structures. One disadvantage of the LbL approach is that it requires several steps, which can be time consuming and the resulting material can be less stable and sensitive to the ionic concentration, since the surface layers are not covalently bonded.³⁵ Stable polymer coatings may be formed by covalent bonding between the nanoparticle surface and preformed polymers; with either of the grafting approaches.^37,38,40 Comparing the two different grafting approaches, the “grafting from” technique usually gives increased grafting density, however subsequent characterization of the polymer layer can be problematic. In contrast, the “grafting to” approach allows for a more facile synthesis and characterization of the polymer prior to nanoparticle surface attachment. In our previous work, we used a variety of functional anchoring groups for the covalent attachment of molecules to the surface of nanoparticles bearing hydroxyl groups, including (for example) dopamine, cysteine, amine, phosphonic acid, carboxylic acid, and trimethoxy silane.^40,41,44 A recent study by Tucker-Schwartz et al.⁴⁵ demonstrated the attachment of a silane compound (that also contained a free thiol) to iron oxide nanoparticles, for subsequent modification via thiol–ene Michael addition.⁴⁵ In our current work, we used thiol–ene chemistry to modify TiO₂ nanoparticles with polymeric chains. The polymer chains were first synthesized by catalytic chain transfer – a polymerization technique ideally suited to making short chains with a very high terminal vinyl functionality for subsequent reaction with thiols.^46,47 Thiol–ene addition can be induced easily using two different approaches: thiol–ene Michael nucleophilic addition or photo-initiated radical addition.^48–50 After modification and characterization, the TiO₂ nanoparticles were assessed in a number of in vitro experiments with the human cell lines A549 and H122.

Materials and methods

Materials

All reagents were used without further purification. The monomer oligo(ethylene glycol) methyl ether methacrylate (Aldrich, 99%) was used as received and stored at −18 °C. The initiator 2,2′ azobisisobutyronitrile (AIBN) was re-crystallized twice from methanol. The catalytic chain transfer polymerization agent bis-(difluoroboryl)dimethylglyoximato cobalt(II) (CoBF) was synthesized according to the method of Bakac et al.⁵¹ The solvents acetonitrile (Aldrich, 99%), hexyl amine (Aldrich, 99%), and (3-mercaptopropyl) trimethoxy silane (Aldrich, 98%) were used as received. TiO₂ used was Aeroxide-P25 (Evonik, USA) which is a 80 : 20 mixture of anatase : rutile with a surface area reported to be approximately 50 m² g⁻¹. Roswell Park Memorial Institute-1640 (RPMI1640), Dulbecco's modified Eagle medium: nutrient mixture F-12 (DMEM/F-12), defined keratinocyte serum free media (KSFM) cell culture media, fetal bovine serum (FBS), phosphate buffered saline (PBS), Hank's buffered salt solution (HBSS) and Trypsin/EDTA were all purchased from GIBCO Invitrogen. L-glutamine was purchased from Sigma Chemical Co., St. Louis, MO, USA. Plastic culture microplates and flasks used in the experiment were supplied by Sarstedt AG & Co., Germany. CellTiter-Blue® (AlamarBlue®), CellTiter 96® MTS and CellTiter-Glo® Luminescent cell viability assays were purchased from Promega Co., Madison, WI, USA. Hydrochloric acid (HCl) and hydrofluoric acid (HF) were provided from Ajax Finechem Pty Ltd, Sydney, Australia.

Cell lines

Human lung epithelial cell lines, A549 (ATCC, CCL-185™) and NCI-H1299 (ATCC, CRL-5803™), kindly provided by Dr Louise Lutze-Mann, were separately cultured in RPMI1640 media supplemented with 10% FBS and 1% L-glutamine. The cells were cultured in treated T75 flasks and incubated at 37 °C in a humidified incubator with 5% CO₂ atmosphere (Heraeus). Cell culture experiments were performed in 96-well treated-culture plates for the biological assays (Alamar blue, MTS and ATP luminescent assays) and 6-well treated-culture plates for the cell count and quantification of particle uptake.

Synthesis of POEGMA polymer

The CCTP was performed in a similar method to that published elsewhere.⁵² A typical CCTP procedure is described as follows; the monomer oligo(ethylene glycol) methyl ether methacrylate (OEGMA₄₇₅) (10 g, 2.1 × 10⁻² mol) was mixed with acetonitrile as solvent (10 mL). The reaction mixture was then sparged with N₂ for at least an hour. In a different flask, CCTP catalyst CoBF, at a concentration determined by the target molecular weight, was mixed with AIBN (1 mg, 2 × 10⁻⁶ mol) and also degassed with N₂ to ensure the absence of O₂. The reaction solution was then transferred to the flask containing CoBF via a cannula. Polymerization was then run for 14 h at 70 °C and the resulting polymer was purified by precipitation in diethyl ether. The product polymers were characterized using GPC and NMR before use in grafting experiments.

Synthesis of TiO₂–POEGMA nanoparticles

The synthesis of the nanoparticle–polymer hybrids was done using a two-step process as follows:

(1) TiO₂ nanoparticles (140 mg, 1.8 × 10⁻³ mol) were mixed in water (50 mL) with (3-mercaptopropyl) trimethoxysilane (300 mg, 1.5 × 10⁻³ mol) followed by serial sonication at a frequency of 20 Hz for 10 min intervals (three times with a 5 min break between each). The suspensions were then incubated at 60 °C, with stirring, for 6 h followed by 18 h incubation at room temperature without stirring. The thiolated nanoparticles were purified by a series of washing and centrifugation steps (1 h at 6000 rpm) to ensure the removal of (3-mercaptopropyl) trimethoxysilane.

(2) The thiolated particles were then mixed with a series of POEGMAs with different molecular weights (in acetonitrile and hexylamine). The deoxygenated samples were then placed in a 40 °C oil bath overnight and subsequently purified by centrifugation and freeze-drying.

Polymer and particle characterizations

Gel permeation chromatography. DMAc GPC analyses of the polymers were performed in N,N-dimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2,6-di-butyl-4-methylphenol (BHT)] at 50 °C (flow rate = 1 mL min⁻¹) using a Shimadzu modular system comprised of an SIL-10AD auto-injector, a PL 5.0 mm bead-size guard column (50 × 7.8 mm) followed by four linear PL (Styragel) columns (10⁵, 10⁴, 10³, and 500 Å) and an RID-10A differential refractive-index detector. GPC was calibrated using polystyrene standards (Polysciences) with a molecular range from 104 to 30 000 000 g mol⁻¹.

Nuclear magnetic resonance (NMR). Structures of the synthesized compounds were analyzed by ¹H NMR spectroscopy using a Brucker DPX 300 spectrometer at 300 MHz.

X-ray photoelectron spectrometer (XPS). A Kratos Axis ULTRA XPS incorporating a 165 mm hemispherical electron energy was used. The incident radiation was monochromatic A1 X-rays (1486.6 eV) at 225 W (15 kV, 15 ma). Survey (wide) scans were taken at an analyzer pass energy of 160 eV and multiplex (narrow) higher resolution scans at 20 eV. Survey scans were carried out over 1200 eV binding energy with 1.0 eV steps and a dwell time of 100 ms.

Dynamic light scattering (DLS). The sizes of the unmodified and modified TiO₂ aggregates were characterised using dynamic light scattering (DLS, Nano-DLS, Brookhaven) at 37 °C (temperature at which cell culture was conducted). The unmodified and modified particles were dispersed in either water, PBS, RPMI1640 media supplemented with 1% L-glutamine, DMEM/F12 with Glutamax or KSFM (with and without 10% FBS for all media) and the d₉₀ values were calculated from the cumulative frequency intensity.

Particle uptake measurement

After 24 h incubation with unmodified and modified TiO₂ particles at a concentration of 150 μg mL⁻¹, the cell media containing particles was removed and the cells were washed three times with HBSS to remove the non-internalized particles. The cells were then incubated with 1 mL of 6 M HCl overnight. The entire contents of the well were retrieved and each well was washed three times with 1 mL of Milli-Q water. The combined HCl extract together with the washes were treated with 1 mL of 40% (w/w) HF and incubated overnight at room temperature. The samples were then diluted to a final volume of 10 mL with Milli-Q water and the Ti contents were quantified using ICP-OES (PerkinElmer Optima, 3000DV).

Biological impact evaluation

The biological impact of nanoparticles was evaluated using three different assays to assess different biological end points: (1) CellTiter-Blue® (AlamarBlue) assay to determine the cell metabolic activity; (2) MTS assay to determine the cell mitochondrial activity; and (3) CellTiter-Glo® luminescent cell viability assay to determine the level of cellular energy (ATP). For each assay, cell cultures were established at an initial cell density of 16 700 cells per cm² and the cells were exposed to a concentration of 150 μg mL⁻¹ of TiO₂ for 24 h prior to evaluation.

Cell metabolic activity. After 24 h incubation with TiO₂ particles, the cells were washed three times with HBSS, and 20 μL of the CellTiter Blue® reagent was added to each well with 100 μL of RPMI1640 media supplemented with 1% L-glutamine only. The fluorescence signal was then quantified by measuring absorbance at excitation 544 nm and emission 590 nm using an f_max fluorescence plate reader (Molecular Devices) after 4 h incubation with the assay reagent.

Cell mitochondrial activity. After 24 h incubation with TiO₂ particles, the cells were washed three times with HBSS, and 20 μL of MTS/PMS reagent mixture was added to each well with 100 μL of RPMI1640 media supplemented with 1% L-glutamine only. The plate was then incubated at 37 °C for 4 h before it was quantified by measuring the absorbance at 490 nm.

The level of cellular energy (ATP). After 24 h incubation with TiO₂ particles, the cells were washed three times with HBSS, and 100 μL of CellTiter-Glo® luminescent cell viability assay reagent was added to each well and the plate was then mixed using an orbital shaker for 2 min, followed by 10 min incubation to stabilize the luminescence signals. Luminescence was read using a luminometer (Turner Biosystems, Model 9100-102).

Statistical analysis

Statistical comparison of multiple groups of data was analysed using one way ANOVA followed by a Dunnett test which was used to compare means from the control group and each of the group exposed to particles. The statistical test was performed using Minitab v13 statistical program (Minitab Inc.). The values are expressed as the mean ± standard error of the mean (SEM). The statistical significance versus control group was established as p < 0.05.

Results and discussion

Biocompatible polymer synthesis

The biocompatible polymer chosen for this work was a polyethylene glycol macromonomer analogue made from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) units. OEGMA₄₇₅ was polymerized using the catalyst chain transfer polymerization (CCTP) technique using bis-(difluoroboryl)dimethylglyoximato cobalt(II) (CoBF), an established and widely used CCT catalyst.⁵¹ COBF was shown previously to be highly reactive towards various OEGMA macromonomer radicals with a chain transfer constant (C_S) = 1800 for the macromonomer OEGMA₄₇₅.⁵²

In this study, three different polymers with different molecular weights were synthesized via CCTP (Scheme 1) and subsequently characterized by ¹H NMR spectroscopy and GPC chromatography (Fig. S1†) – the summary of the polymers obtained is tabulated in Table 1. PDIs of the polymers were obtained via GPC showing PDIs < 1.5 (the actual PDIs from a transfer dominated polymerization should be close or equal to 2, the results obtained herein reflect the inaccurate GPC calibration and losses on purification). Molecular weight values were obtained from both GPC and from ¹H NMR data using eqn (1) (below).

where MW^OEGMEMA, I^CH₂OCO, and I^CH₂=C correspond to the molar masses of OEGMA₄₇₅, integral of signal at 4.1 ppm (attributed to CH₂ in adjacent position of ester group), and of the vinyl group at 5.6–6.0 ppm respectively. Polymer characterization by NMR and GPC were in accord. It is noteworthy that data from the proton NMR (Fig. S1†) can be used to confirm the progress of polymerization and the build up of polymer vinylic end-groups. The polymer vinyl end-groups are distinct from the signals emanating from the monomer vinyl groups (i.e. 5.5 and 6.1 ppm for monomer and 5.6 and 6.0 ppm for polymer respectively).

Scheme 1 Catalytic chain transfer polymerization of OEGMA yielding vinylic terminated POEGMA.

Table 1 Summary of polymer products used to modify titanium dioxide nanoparticles

Code	M n, GPC (g mol^−1)	M n, NMR (g mol^−1)	PDI
POEGMA2.5	2200	2600	1.15
POEGMA16	15 000	16 000	1.36
POEGMA20	20 000	20 000	1.40

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Synthesis of polymer-coated TiO₂ nanoparticles

In this work, surface modification of TiO₂ nanoparticles was targeted to; (i) minimize nanoparticle aggregation (on dispersion), and (ii) introduce surface functionality for subsequent attachment to biocompatible polymers. The synthesis of the nanoparticle–polymer hybrids was achieved using a two-step sonochemical and thiol–ene Michael addition process. Firstly, TiO₂ nanoparticles were mixed with (3-mercaptopropyl) trimethoxysilane, as a linker with the silyl group attached to TiO₂ surfaces leaving exposed free thiol groups (–SH) on the nanoparticle outer surface. The free thiols were then reacted with pre-synthesized polymers using thiol–ene Michael addition reactions as shown in Scheme 2.

Scheme 2 Overall synthetic approach for the surface modification of TiO₂ nanoparticles with POEGMA.

In order to ascertain reaction conditions for successful thiol–ene Michael addition reactions, a model reaction was first attempted using the monomer OEGMA₄₇₅ and the dimer of OEGMA₄₇₅ (to represent the polymer with vinylic end group) in a reaction with (3-mercaptopropyl) trimethoxysilane catalyzed by hexylamine in acetonitrile. The reaction was followed by ESI-MS mass spectral analysis (as shown in Fig. S2†). It is evident from the mass spectrometry data that on reaction, a shift to higher molecular weights is observed with the m/z peaks increased by 196.4 daltons consistent with the molecular weight of the organo-silane compound. This model reaction confirmed that the reaction of POEGMA (synthesized via CCTP) with 3-mercaptopropyl trimethoxysilane proceeded in a quantitative fashion and (hopefully) could be translated to surface modification (Scheme 2). It is important to note that an attempt was made to attach the resulting polymer bearing silane groups to the surface of nano-TiO₂. However, there were difficulties with attachment, as the resulting polymer readily cross-linked forming an insoluble gel network following self-condensation of the terminal silane groups under basic conditions. Hence, the approach shown in Scheme 2, where 3-mercaptopropyl trimethoxysilane was firstly attached to the TiO₂ surface was chosen as the preferred synthetic approach. The polymer components (synthesized via CCTP) could be made with a range of different functional monomers (potentially increasing the applicability of the CCT approach). While there has been some work reported in the literature, showing modification of polymers made via CCTP with small thiol bearing molecules,^47,52 this is the first time (to our knowledge) that CCT/thiol–ene has been applied to the surface modification of nanoparticles. This synthetic route to PEGylated nanoparticle surfaces provides an alternative to linear PEG with the advantage of significant control over the nature of the PEG surface (possible expansion to copolymerization with control over molecular weight and architecture).^53,54

The attachment of linker and polymer were monitored by XPS (Fig. 1). A wide scan survey of the nanoparticle for all elements showed clearly that the bare nano-TiO₂ has only Ti and O as the main signals, with slight contamination of carbon from the atmosphere evident (Fig. S3†). After modification with 3-mercaptopropyl trimethoxysilane (the “linker”) and POEGMA there are new elemental signals corresponding to Si, S, O, and C (Fig. S3†), showing successful modification. However, differences reflecting the successive attachment steps involving silane and thiol–ene Michael addition could not be detected by the XPS wide scan. Therefore, high-resolution XPS analysis was carried out to investigate the chemical binding state of each element. XPS evaluation of the different stages of particle functionalization is shown in Fig. 1. XPS of bare TiO₂ indicates carbon traces (with a low intensity) attributed to surface contamination by organic compounds during the synthesis or storage. From XPS measurements, after modification with the linker, the particle surface displays signals for C–C or C–H bonds and after attachment of the POEGMA, the XPS spectra confirms the presence of C–O and O–C=O bonds in addition to C–C and C–H peaks – clear evidence for the attachment of polymer to the nanoparticle surface. The ratio of C/Ti was also calculated for the nano-TiO₂ during different stages of the nanoparticle functionalization. The C/Ti ratio for bare TiO₂ is only 0.4 compared to 10.1 after attachment of the linker and 13.5 after attachment of the POEGMA consistent with the successful attachment of linker and subsequent modification with POEGMA.

Fig. 1 XPS spectra focused on the C1s binding energy of the TiO₂–polymer hybrid during different steps of the synthetic process high-resolution spectrum for C1s signal. The C/Ti ratio is indicated in this figure.

The XPS data were further supported by ATR-FTIR analysis (Fig. S4†). It can be seen that the bare TiO₂ does not have any signals consistent with organic species except for the broad absorption in the range 3000–3500 cm⁻¹ corresponding to O–H peaks, while analysis of the purified POEGMA modified TiO₂ gave characteristic signals at around 1000–1200 cm⁻¹, at 1730 cm⁻¹ and at 2700–2800 cm⁻¹ corresponding to ether bonds (C–O), carbonyl esters (C=O) and C–H (aliphatic). The ATR-FTIR results are in good accord with the XPS data.

The synthesized hybrid particles were also characterized by TGA (Fig. S5†) to assess the amount of organic material, viz., OEGMA₄₇₅ and POEGMA, attached to the TiO₂. As expected, TiO₂ was stable up to 800 °C. In contrast, those nanoparticles modified with polymer underwent a partial degradation at approximately 330 °C, characteristic of the thermal degradation of POEGMA. The synthesized hybrid nanoparticles underwent thermal weight losses of 12–15%, with the loss slightly higher as the molecular weight of the polymer increased. The grafting density was calculated using the following equation: grafted per nm² = (ΔM^Polymer* × N_A)/(M^Polymer_n × m^TiO₂ × S), with ΔM^Polymer*, M^Polymer_n, N_A and S corresponding to the mass of polymer grafted (without silane), molecular weight of polymer, Avogadro's number, mass of TiO₂ nanoparticles and surface area of TiO₂, respectively (Table 2). The number of grafted chains decreased as the molecular weight of the polymer increased. This may be attributed to steric hindrance effects on the thiol–ene Michael additions as the polymer molecular weight increased. As the grafting density is lower than 0.10 chain per nm² for POEGMA16.0 and 20.0, this indicates that the polymer attached on the surface has a mushroom type conformation. For short OEGMA chain, i.e. 475 g mol⁻¹, the conformation of the polymer attached on TiO₂ corresponds to a brush polymer. In summary, the XPS, ATR-IR and TGA analyses were consistent in showing that the polymer modified particles were successfully synthesized.

Table 2 Grafting density of POEGMA chains on TiO₂ using grafting ‘onto' approach with thiol–ene chemistry^a

Samples	M n (g mol^−1)	Weight loss (wt%)	Grafting density (chains per nm^2)
a Note: grafting density = (ΔM^Polymer × NA)/(M^Polymern × m^TiO2 × S^TiO2), with ΔM^Polymer, M^Polymern, NA and S correspond to mass of polymer grafted determined by TGA, molecular weight of polymer, Avogadro's number and surface area of TiO2, respectively. The grafting density was calculated using 50 m^2 g^−1 as surface specific).
TiO2–silane	196	6.0	—
TiO2–OEGMA	480	12	1.25
TiO2–POEGMA2.5	2600	13.5	0.39
TiO2–POEGMA16.0	16 000	14	0.07
TiO2–POEGMA20.0	20 000	15	0.064

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Finally the polymer-coated nanoparticles were characterized by DLS to investigate particle aggregate sizes in aqueous and cell culture media RPMI1640 supplemented with 10% FBS and 1% L-glutamine. The cell culture media we used has been commonly used for in vitro cell culture systems to support cell growth and proliferation for evaluating material toxicity. In this study, the particle exposure to the cell lines was conducted in this cell culture media; and therefore, it is important to carefully characterize the particles once suspended in this media. Fig. 2 shows that the surface modification of TiO₂ nanoparticles by POEGMA-grafting altered the subsequent particle aggregation significantly. The bare TiO₂ nanoparticles exhibit aggregation sizes of above 1 μm in all biological media tested in this study. After modification with the linker (silane), the particle aggregate sizes were similar to the bare TiO₂. This minimal effect of silane on aggregation may be partly explained by the potential reactivity of the free surface thiols towards disulphide formation. Subsequent attachment of the PEG monomer had no effect on nanoparticle aggregation in the cell culture medium; however, in contrast, attachment of PEG monomer significantly reduced aggregation in water (Fig. 2). Once the TiO₂–monomer particles were diluted using the cell culture medium, the particle size aggregate increased to almost 1 μm. This result suggests that the attachment of PEG monomer alone does not confer enough steric stabilization to avoid the aggregation of the nanoparticles in cell culture media. After the attachment of POEGMA, the hybrid particles were successfully stabilized with a particle size of approximately 300 nm (Fig. 2) in the cell culture media (with and without 10% FBS). The data suggests that changing the polymer chain length (between 2.5; 16.0; and 20.000 g mol⁻¹) does not significantly affect the aggregation state of POEGMA–TiO₂ particles. A similar observation has been reported with PEG-coated nanospheres designed for drug delivery.⁵⁵ DLS measurements were also done in various other cell culture media (Fig. S6†), including Dulbecco's modified Eagle medium (DMEM) and keratinocyte serum-free medium (KSFM). The data reported herein is consistent with stabilization yielding a similar aggregate size of 300 nm in all cell culture media tested. The particle stability was monitored over a 24 h period revealing that the POEGMA–TiO₂ particles maintained stability with a steady aggregate size of 300 nm (Fig. S7†).

Fig. 2 Aggregate sizes of naked and modified TiO₂ nanoparticles in different diluents (data taken from at least 5 independent experiments).

Following the modification process, the particles were sterilized by an autoclaving process (121 °C, 1 atm, 20 minutes) and stored at 4 °C for 16 h prior to subsequent biological testing. The DLS measurement shown in Fig. 2 indicates that the aggregate sizes were unaffected by the autoclaving process. Interestingly, the autoclaved bare TiO₂ nanoparticles suspended in PBS buffer, (involving a high temperature (121 °C) and storage at 4 °C for 15–16 h), resulted in aggregate sizes significantly smaller than aggregates that were not subjected to an autoclaving process. This suggests that the autoclaving process might have altered the surface of bare TiO₂ yielding different interactions with the proteins when suspended in the RPMI1640 cell culture medium supplemented with 10% FBS and 1% L-glutamine. This autoclaving-induced stability effect was not observed for other modified particles.

Cellular uptake of polymer-modified TiO₂ nanoparticles by human lung cell lines in vitro

The extent of particle uptake by the cells is shown in Fig. 3 clearly showing that the surface chemistry modifications by silane, as a linker, followed by attachment of POEGMA monomer or polymer significantly affected the amount of particle uptake by both the human lung cell lines A549 and H1299. Fig. 3 shows the amount of particle uptake per cell after a 24 h period of exposure to the naked, silane-, monomer- or polymer-modified TiO₂ particles after the sterilization process (autoclaving) at an exposure concentration of 150 μg mL⁻¹. There is a significant decrease of up to 4-fold and 2-fold, of the uptake, after the particles have been modified with silane, by human lung cell lines A549 and H1299, respectively. In our previous investigation, TiO₂ nanoparticles with the same surface chemistry comprised of larger particle aggregates were reported to be more readily taken up by cells, compared to smaller particle aggregates.³⁰ In the present study, an increase in particle aggregate size (following attachment of silane, which theoretically made the surface negatively charged), was found to result in a lower cellular uptake, indicating that the surface chemistry plays an important role alongside aggregate size in determining interactions (uptake) with human lung cell lines.

Fig. 3 Cellular uptake of naked and modified particles by human lung cell lines (a) A549 and (b) H1299 after a 24 h exposure at a concentration of 150 μg mL⁻¹ TiO₂. The * shows the cellular uptake of modified TiO₂ that was significantly different from the amount of cellular uptake of the naked TiO₂ (by one-way ANOVA followed by Dunnett's test) with p < 0.05, from at least 3 independent experiments.

Modification with OEGMA (monomer) and POEGMA with various chain lengths, resulted in a further reduction in nanoparticle cellular uptake (Fig. 3). This difference in uptake may result from the stability of the particle aggregates; requiring, a longer time period to access the adhered cells on the bottom of the culture dish. The gravitational settling rate of nanoparticles has been reported to significantly affect the amount of internalized particles in the cell culture system.⁵⁶ Our data shows that particle uptake was reduced by all of the surface modifications studied here. Similar to this finding, PEG-modified polyaspartamide nanoparticles have been shown to escape from being phagocytozed by the mouse monocyte macrophage cell line J774A1,⁵⁷ thereby minimizing the particle internalization. Conversely, an earlier investigation has reported that the attachment of PEG molecules onto iron oxide nanoparticles significantly increased the particle uptake by the human breast cancer cell line BT20.⁵⁸

The data in Fig. 3 indicates a reduced particle uptake by A549 cells following silane modification of the nanoparticles and a similar decrease in the particle uptake on further addition with monomer or polymers, while a different extent of cellular uptake reduction is shown for H1299 with the various surface modifications. This result might be attributed to different TiO₂ nanoparticle uptake pathways between the two cell lines. A549 and H1299 cell lines have been reported to internalize TiO₂ nanoparticles via different active uptake endocytosis pathways; clathrin-dependent and clathrin-/caveolae-independent endocytosis, respectively.³¹ Another possibility is that a very low uptake for silane-modified particles by A549 cells was minimally affected by further modifications.

Protein adsorption profiles of naked and modified nanoparticles

Fig. 4 shows the SDS-PAGE of proteins attached to the naked and modified TiO₂ nanoparticles following a 24 h period of incubation in RPMI1640 cell culture media supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine at 37 °C. Our previous study has shown that the amount of particle cellular uptake was directly affected by the quantity and identity of protein(s) adsorbed onto the surface of nanoparticles.³¹ From the SDS-PAGE gel, the bare TiO₂ particles are shown to adsorb at least 15 different proteins, as shown by the number of bands in lane 2 (Fig. 4).

Fig. 4 SDS-PAGE electrophoresis gel of proteins adsorbed onto the surface of non- and modified-TiO₂ nanoparticles after suspension in RPMI1640 supplemented with 10% FBS and 1% L-glutamine for an overnight period at 37 °C, line M: see Blue 2 marker, line 1: FBS, line 2: TiO₂, line 3: TiO₂–silane, line 4: TiO₂–silane–monomer, line 5: TiO₂–silane–POEGMA 2500 g mol⁻¹, line 6: TiO₂–silane–POEGMA 20 000 g mol⁻¹.

Fig. 4 shows the profile of adsorbed protein onto the surface of TiO₂ nanoparticles analyzed by protein (SDS-PAGE) gel electrophoresis. TiO₂–silane particles adsorbed fewer proteins, as some of the proteins (in particular with high molecular weights) that adsorb on the naked nanoparticle sample were no longer adsorbed onto the surface of TiO₂–silane particles. Furthermore, the results show that the low molecular weight proteins (approximately 14 kDa) appeared to be preferentially adsorbed onto TiO₂–silane particles, while the higher molecular weight proteins were not observed. Further modification with either monomer OEGMA₄₇₅ or polymer POEGMA with different chain lengths significantly changed the adsorbed protein profile and a reduction in the quantity of protein adsorbed (visualized by further reduction of the number of bands). The band associated with the adsorption of proteins in the low molecular weight range is shown to be more intense as the chain length of OEGMA increases, reflecting increased protein adsorption capacity of proteins in the low molecular weight range.

There are a number of possible low molecular weight serum-derived proteins that may be adsorbed onto the surface of TiO₂ nanoparticles, such as (but not limited to): transthyretin, haptoglobin α1 chain, lysozyme C, and apolipoproteins (ApoC-III, ApoC-II, and ApoA-II). Transthyretin is a thyroxin transport protein that is known to adsorb to toxic components present in the blood stream.⁵⁹ Transthyretin specifically interacts with receptor-associated proteins in the liver (it's major site of degradation) and has been reported to be strongly associated with silica nanoparticles.⁶⁰ Haptoglobin α1 chain is a protein known to interact with free hemoglobin (Hb) to prevent oxidative damage by Hb, and is also known to promote receptor recognition by monocytes/macrophages. Another protein that may be attached to our modified particles is lysozyme C, known to interact with TiO₂ nanoparticles.⁶¹ The adsorption of one or more of these proteins may have a significant impact how the cell interacts with the nanoparticles. Interestingly, the modification of TiO₂ nanoparticles with monomer OEGMA₄₇₅ or POEGMA is shown to significantly reduce the adsorption of protein(s) with molecular weights in the range of approximately 58 and 98 kDa. These protein bands include vitronectin (shown in our previous study), associated with an increase in the amount of particle taken up by human lung cell line A549.³¹ Vitronectin has also been demonstrated to increase crocidolite asbestos uptake by rabbit pleural mesothelial cells via integrin receptors.⁶² Therefore, it is possible that the reduction of particle cellular uptake of modified particles can be attributed to a reduction in adsorbed vitronectin.

Biological impact of the hybrid nanoparticles on human lung cell lines in vitro

Fig. 5 shows the biological impact of non- and modified-TiO₂ nanoparticles based on cellular metabolic and mitochondrial activities, and also cellular ATP level following 24 h exposure at a concentration of 150 μg mL⁻¹ TiO₂ nanoparticles. This particle concentration was particularly chosen as it was the concentration at which naked TiO₂ started showing biological impact on human lung cell lines in vitro.³⁰

Fig. 5 Biological impact of TiO₂ nanoparticles before and after modification in human lung cell lines (a–c) A549 and (d–f) H1299 after a 24 h exposure at a concentration of 150 μg mL⁻¹ TiO₂. The * shows the biological impact of modified TiO₂ that was significantly different from the amount of cellular uptake of the naked TiO₂ (by one-way ANOVA followed by Dunnett's test) with p < 0.05, from at least 4 independent experiments.

The data in Fig. 5, shows that modification of the nanoparticles with POEGMA polymer reduces the biological impact of TiO₂ particles especially in the case of H1299, which is more sensitive to particle exposure compared to A549. The sensitivity of H1299 has also been noted in previous investigations.^30,31 Modification with silane alone also reduced the biological impact of TiO₂ nanoparticles. This could be due to the reduction in protein adsorbed onto the silane-modified particles compared to the naked (unmodified) particles, resulting in significantly less particle cellular uptake.

Regardless of the different chain lengths of POEGMA (Fig. 5), the functionalization of the nanoparticles improved biocompatibility. This improvement in the biocompatibility of TiO₂ nanoparticles correlates with a lower particle uptake by both human lung cell lines A549 and H1299, which may be directly linked with a reduction in the quantity and different type of proteins adsorbed onto the particle surface. In our previous investigation, the adsorption of proteins (by pre-exposure to FBS) was shown to increase the particle uptake, while interestingly resulting in a lower biological impact after a 24 h exposure.³¹ The data from this study shows that modifying nanoparticle surface chemistry also influences the spectrum of proteins binding, suggesting that the type and amount of proteins binding to the particle surface, mediated by altering particle surface chemistry, is very important in determining how the particles interact with cells with a consequential impact on cell health.

Conclusion

In this work, we report a facile method to modify TiO₂ nanoparticles using thiol–ene click chemistry and CCT polymerization. The synthetic method described herein could be broadened to attach polymers onto a range of oxide type nanoparticles. The attachment of POEGMA to nano-TiO₂ aided in maintaining stable aggregates in various aqueous and biological media. The surface modifications were also shown to change the type and amount of serum proteins adsorbed onto the nano-TiO₂. The surface chemistry was shown to play an important role in determining the amount of particle uptake by cells (in addition to the aggregate sizes). It is noteworthy that the adsorption of vitronectin, a protein that has been highlighted to trigger TiO₂ cellular uptake, was reduced following polymer modification and this may therefore contribute to a lower cellular uptake of particles, especially for the A549 cell line. A reduced nanoparticle cell uptake leads to a demonstrated decrease in the biological impact of the TiO₂ on the two different human lung cell lines A549 and H1299. The results presented in this work have important implications for potentially addressing/investigating concerns regarding the nanotoxicity of nano-TiO₂ materials.

Acknowledgements

The authors acknowledge the Australian Research Council (ARC) for funding. CB is thankful for his APD fellowship from ARC (DP 1092640).

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Footnote

† Electronic supplementary information (ESI) available: Additional experimental information, Fig. S1–S7 and Table S1 are available. See DOI: 10.1039/c2py20450a

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