Structural characterization, solution stability, and potential health and environmental effects of the Nano-TiO₂ bioencapsulation matrix and the model product of its biodegradation TiBALDH

Abstract

Dispersions of small TiO₂ nanoparticles in alcohol, stabilized by surface complexation with protonated triethanolamine ligands, are proposed as bio-encapsulation matrices. They were characterized by TEM and EXAFS spectroscopy, and DLS was used to investigate the nanoparticles stability over time in their pure form and after insertion into aqueous medium. This study reveals unusual structural features that explain the recently demonstrated facile formation of dense encapsulates on the surface of biological objects. In the view of the potentially broad application of this already industrially available material in agriculture as an encapsulation matrix for biocontrol organisms, its potential health and environmental effects were characterized by employing a number of model systems. The potential health effects of the produced stable aqueous dispersions were studied in vitro using A549 and U1810 lung carcinoma cell lines. The nanotitania in the environment is partly bio-digested with the formation of citrate and lactate complexes. The effects on the growth of tobacco pollen grains by the nanotitania and of the ammonium lactato-oxo-titanate (TiBALDH, a product already broadly used in biomineralization studies) were investigated to gain insight into the impact of these materials on the environment and specifically on plant reproduction. TiBALDH was used as a model product of the bio-digestion and its structure has been probed in this work by X-ray powder diffraction and EXAFS spectroscopy. No acute negative bio-effects could be observed for the studied materials at significantly high concentrations, such as 50 μg ml⁻¹ for the viability of human lung cancer cells and up to about 120 μg ml⁻¹ for the growth of pollen grains, corresponding to the conditions of proposed field applications. This observation was contrasted by the apparently high toxicity of the LaAlO₃ based nanophosphor which was applied as a positive control.

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1. Introduction

Recently, increased interest in versatile and cheap nanostructured matrices for bio-encapsulation and drug delivery has urged the development and investigation of biocompatible inorganic nanomaterials. Some examples of materials proposed for these applications are calcium carbonate¹ and calcium phosphate,² silica,^3,4 and, more recently, titanium dioxide.^5,6 These materials are highly attractive, particularly due to their high versatility and ability to display sensitivity to the chemical conditions in surrounding media, which is applicable for regulated release of the encapsulated content. Examples of such behaviour are the facile dissolution of CaCO₃ at decreased pH¹ and the solubilisation (bio-digestion) of nanosized TiO₂ when the concentration of chelating carboxylate ligands such as citrate or lactate is increased (as occurs in biological media subject to oxidative stress).^5a,6 It is important to note that this bio-digestion can apparently be reversed by the action of enzymes, producing a new titania shell via biomineralization. This was recently demonstrated using an ammonium titanium lactate complex (TiBALDH) as the starting material.^5b

A number of studies have demonstrated that titanium dioxide is non-toxic for many species of microorganisms⁵ with limited effect on the biochemical balance in soil associated with removal of phosphorus and even of nitrogen.^7a In fact, several types of microorganisms have skeletons that consist mostly of titania, while magnetic iron titanate, FeTiO₃ ilmenite, is used within the organs of orientation in a variety of species.^7b An attractive titania-based nanomaterial for environmental applications is CaptiGel, a solution of uniform TiO₂ nanoparticles (4 ± 1 nm) stabilized by electrostatic charges from surface capping with triethanol-ammonium cations.⁸ It was demonstrated to be able to preserve biomaterials and microorganisms through the formation of dense coatings via aggregation of nanoparticles. The titania particles coalesce, producing dense membranes on the surface of living cells. It was originally supposed that the driving force in this process was the electrostatic interaction between the positive charges on the surface of the modified titania particles and the effective negative charges of phosphate head groups in the membrane lipids.^5a It is important to get a better insight into its structure and trace the potential transformation of this material in aqueous medium, especially in view of the recent data that positively charged nanoparticles can damage cell membranes.⁹ We sought also to trace longer term effects that can be caused by this type of material on broader scale applications. We focus on two types of possible impact, the first on human health through exposure via the respiratory tract, and the second on plant reproduction by influencing viability and growth of the pollen grains. It also appeared important to identify the chemical nature of the bio-digestion products of nano-titania and evaluate their own potential environmental impact.

2. Experimental

The titania colloids described in the literature as nano-TiO₂ stabilized with surface-capping protonated tea-ligands⁵ were received from CaptiGel AB, Sweden, as an ethanol solution with a TiO₂ concentration of 120 mg ml⁻¹ and was diluted for further applications by either anhydrous ethanol or LiChrosolv water (Merck, pH = 7.00). The titanium(IV) lactate complex solution, with the commercial name TiBALDH, was at 50 wt% concentration in relation to the formula C₆H₁₈N₂O₈Ti and was diluted by water for further tests. LaAlO₃ and Tb-doped LaAlO₃ nanoparticles were produced from alkoxide precursors using the Bradley reaction with acetophenone as the solvent as described in ref. 10.

The solutions were measured on a Malvern Zetasizer DLS to obtain information about size distribution and surface charge of the particles. Polymer electrolyte titrations were carried out at the Polymer Chemistry Department of the Royal Institute of Technology (KTH) in Stockholm. Electrospray mass-spectrometry measurements were carried out with a Bruker Esquire-LC.

The solutions were dried in air for 2 weeks under a hood and the produced solids were investigated with an optical MOTIC digital microscope and a TM-1000-μ-DeX SEM-EDS. TEM images were obtained with a TOPCON EM002B operating at 200 kV (at the Laboratory of Multimaterials and Interfaces, CNRS UMR 5615, University Claude Bernard Lyon-1). The samples were analyzed by X-ray powder diffraction using a Bruker SMART Apex-II diffractometer (sealed-tube Mo-Kα radiation, λ = 0.71073 Å) as-obtained and after thermal treatment at 500 °C in 1 h. Rietveld refinement of powder data was carried out using an EXPO 2009 program package.¹¹ The thermal decomposition of the as-obtained dried samples was followed by TG-FTIR analysis using a Perkin-Elmer Pyris-1 Spectrum 100 combined installation, using a heating rate of 10 °C min⁻¹ up to a final temperature of 600 °C. The phase composition of the heat-treated samples was established using a Bruker EVA program package.

EXAFS-Data collection. Titanium K edge X-ray absorption spectra were recorded at the wiggler beam line I811 at the MAXLab, Lund University, Sweden. The EXAFS station was equipped with a Si [111] double crystal monochromator. The data collection was performed in luminescence mode. Higher order harmonics were reduced by detuning the second monochromator to 30% of the maximum intensity at the end of the scans. The energy scale of the X-ray absorption spectra were calibrated by assigning the first inflection point of the K edge of a titanium foil to 4966 eV.¹² The IFEFFIT program package¹³ was used for the data treatment.

Cell culture and treatments

Human non-small cell lung carcinoma (NSCLC) cell lines (A549 and U1810) were cultured in RPMI 1640 with 10% fetal calf serum (FCS), 2 mM glutamine, penicillin (100 μg ml⁻¹) and streptomycin (100 μg ml⁻¹). Cells were seeded in 6 well plates twenty four hours prior to treatment with titanium oxide (5–100 μg μl⁻¹), doxorubicin (0.5 μg μl⁻¹) or their combination. They were counted after 120 h using Beckman cell counter, according with manufacturers instruction.

Immunoblotting

Cells were lysed in RIPA buffer (25 mM Tris·HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with a protease inhibitor cocktail (Complete-M, Roche). The protein concentration was determined using a BCA protein assay (Pierce). The samples were mixed with Laemmli's buffer, boiled for 5 min, subjected to SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% non-fat milk in PBS and probed with primary antibodies diluted in PBS containing 2% BSA and 0.05% Tween-20. The following antibodies were used: mouse anti-cleaved PARP (Cell Signaling Technology), p53 (Santa Cruz), rabbit anti-actin (Sigma-Aldrich). The recognized proteins were detected using horseradish peroxidase-labelled secondary antibodies: anti-mouse IgG or anti-rabbit IgG (all from Pierce), and an enhanced chemiluminescence kit (Western blot detection reagent, GE Healthcare UK Limited).

MTS assay

Cells were cultured at a density of 2 × 10³ cells per well in 96-well plates and some CellTiter 96® AQueos One Solution Reagent (Promega, Madison, WI) was added to each well according to the manufacturer's instructions. After 48 of treatment with titanium nanoparticles the cell viability was determined by measuring the absorbance at 490 nm using a Labsystems Multiskan MCC/340 plate reader.

Plant culture and treatments

For the preparation of the growth medium 3.2 ml of 0.5 M H₃BO₃, 1 ml of 1 M CaCl₂·2H₂O, 2 ml of 0.5 M Ca(NO₃)₂·4H₂O, 2 ml of 0.5 M MgSO₄·7H₂O and 100 g sucrose were added to a flask with 500 ml water. The pH value was subsequently adjusted to 6.5 with 2 M potassium hydroxide. Afterwards the growth medium was filtered to make it sterile and stored at 4 °C.

Mature stamens covered with pollen were collected from flowering N. tabacum plants, which were kept in growth chambers under standard conditions (16 h illumination per day at 23 °C; night temperature: 20 °C). The stamens were transferred into a growth medium and stirred to release pollen. The pollen suspension was filtered through a grid to remove anthers and mixed with different concentrations of CaptiGel or TiBALDH to test the toxicity. 400 μl of the pollen suspension with different particles concentrations were added to a plate with 24 wells. The cells were kept at room temperature in the dark for 2.5 h before analysis. Then the pollen tubes were imaged using an epifluorescence microscope. Microscope pictures were taken with 2.5× magnification and afterwards the length of the pollen tubes was measured. To look more carefully at the pollen some pictures were also taken at 10× magnification. For the TiBALDH-containing samples it was also possible to count the number of germinated pollen after 1.5 h. Addition of CaptiGel produced in the growth medium a non-transparent gel, so the following treatment was applied to produce samples permitting observation: 1 ml of TiO₂ solution was added to 1 ml CaCl₂ (1M) and 8 ml water in a tube and mixed. A TiO₂ gel was formed, giving a white suspension. The evolved clumps were centrifuged for 15 min (1800 rpm) and the supernatant, which was a transparent solution, was poured out. The precipitate was washed three times with 10 ml water and ground mechanically with a spatula. After pouring out the supernatant the precipitate was mixed with 10 ml water and subjected to ultrasound treatment to get a suspension of TiO₂ particles for observation under the optical microscope.

The introduction of LaAlO₃ nanoparticles was made by shooting a portion of the suspension onto a filter with collected pollen grains with subsequent desorption and dilution until the desired concentration was achieved (see ESI†).

3. Results and discussion

Characterization of applied inorganic materials

According to DLS measurements, the initial ethanol solutions of nano-TiO₂ stabilized by surface complexation with protonated triethanolamine (CaptiGel) contain uniform 4 ± 1 nm particles. The colour of the solution changes on storage from light yellow to orange, but no change in the particle size was observed even after 24 months of storage. Immersion of CaptiGel into a 10-fold excess of water produced clear solutions that were stable in a closed vessel for at least 2 weeks without the appearance of either opalescence or precipitation. DLS measurements for these aqueous solutions were carried out in triplicate with high reproducibility and showed the average particle size was about 60 nm (complex particle size distribution, see Table TS1 in the ESI† with major peaks at 140, 15 and 5 nm). The size decreased to about 24 nm after 15 min of ultrasound treatment through redistribution between the fractions in favour of the 5 nm sized fraction, which corresponded to single non-aggregated particles. Only a marginal decrease in size was observed after 48 h of storage. The particles in the aqueous medium are negatively charged with an average ζ potential of −11.1 mV, indicating complete loss of the tri-ethanol-ammonium ligand (clearly detectable by ESI-MS in the produced medium) and re-charging of the originally positively charged particles, see ref. 5. Titration with poly-dimethylammonium chloride gives a charge value of 0.006 equivalents per gram in relation to TiO₂. This confirms the negative surface charge of the particles, which is important for decreasing the threat of membrane damage for encapsulated biomaterials commonly caused by positively charged particles.⁹

The stability of the suspensions produced by immersion of CaptiGel into a 10-fold excess of isotonic NaCl solution is much lower: considerable opalescence appears after 2 h and complete gelation of the whole solution occurs within 24 h. Gelation in the presence of a CaCl₂ solution occurs within 15 min. The reason for this behaviour is apparently charge compensation through adsorption of cations.

According to TEM observations (see Fig. 1) the non-heat-treated gels consist of strongly aggregated TiO₂ particles with a size of about 5 nm. The material is amorphous under X-rays, but the distance between fringes in the high-resolution TEM and the selected area diffraction patterns (see ref. 5 for the latter) indicate that the particles have an anatase structure in the core, but are covered with an amorphous shell. The small size of the structurally coherent region precludes obtainment of the X-ray diffraction pattern. The air-dried gel contains high amounts of organic stabilizer, tri-ethanolamine and tri-ethanol ammonium salt, and residual water (weight loss of 55% upon heat treatment, see Fig. FS1†). Heat treatment at 500 °C provides a mixture of anatase and rutile phases (Fig. FS2†) indicating typical behavior for strongly aggregated small anatase particles that facilitates phase transition into rutile.¹⁴ When the gels were prepared using isotonic NaCl instead of water, the resulting xerogels contained relatively large, 30–50 nm, single crystals of NaCl. Hindered nucleation of salts in inorganic gels, resulting in the formation of single crystals was reported earlier for the drying of silica gel droplets.¹⁵

Fig. 1 Air-dried gel produced from CaptiGel diluted by water (a, b) and via gelation in isotonic NaCl solution (c, d). The cubic NaCl crystals are visible in C as phase-separated inclusions.

The EXAFS study of the CaptiGel xerogel reveals a striking difference between this material, bulk, and well-crystallized nano-sized anatase¹⁶ (see Fig. FS3†). The spectrum of anatase can be distinguished as a weaker contributing component. The strongest signals, however, indicate a shortened Ti–O distance (testifying decreased average coordination number) and also drastically weakened signals in the area corresponding to Ti–Ti interactions, which are apparently quenched by the truly amorphous structure. It is probably the latter that is behind the ability of CaptiGel particles to coalesce and form dense TiO₂-shells on the surface of biological objects.⁵ The ability to adhere to the surface of biomembranes may also be explained by the enhanced strength of surface complexation of phosphate entities in phospholipids with the amorphous titania.^6,17

It was previously reported that nano-titania is bio-digestible and dissolves in the presence of chelating carboxylate ligands such as citrate or lactate. The reaction in the presence of an excess of such ligands should lead to tris-carboxylato-titanate species that are reported and well structurally characterized in literature for environmentally relevant conditions.¹⁸ However, application of the CaptiGel material is proposed for field conditions with relatively low concentrations of lactic acid, e.g. in soil, and should lead to a product with lower Ti : lactate ratio. Such a compound is commercially available under the trivial name TiBALDH, which stands for titanium bis-ammonium lactato-dihydroxide. It appeared logical to use this product as a model for investigating the potential environmental effects of bio-digested nano-titania. We felt that it needed further chemical characterization since a tentative chemical formula from following from its common name was apparently erroneous. In spite of the relatively broad application of this product in the synthesis of titanium dioxide¹⁹ and even drawings of it's “structure” in quite serious journals²⁰ the latter has never before been investigated. Moreover, it is well-known that titanium cations do not form stable hydroxide species in aqueous media and not a single reliably defined structure of titanium(IV) species with two hydroxide groups attached to one and the same Ti atom can be found in the Cambridge Structural Database.

A DLS study of TiBALDH indicated the presence of well-defined nanoparticles with a size of about 10 nm. Slow drying the 50 wt% solution in air provided a material that looked like a combination of a polycrystalline material and an opalescent gel/glassy solid under the microscope (Fig. 2a). The TGA study indicated weight loss of 71 wt% (Fig. FS4†), which reasonably correlated with the proposed C₆H₁₈N₂O₈Ti formula. The Rietveld refinement of the obtained X-ray powder data (Fig. 2b) is in agreement with a monoclinic unit cell a = 11.8685(2), b = 13.2396(2), c = 19.9578(2) Å, β = 107.718(3)°, Space group P2₁/c. These data closely resemble the unit cell parameters and symmetry settings for the ammonium titanyl oxalate, (NH₄)₈[Ti₄O₄(C₂O₄)₈](H₂O)₄ (a = 13.473(2), b = 11.329(1), c = 17.646(2) Å, β = 126.66(1)°, Space group P2₁/n), which also has an analogous chemical composition.²¹ The phase-corrected Fourier-transformed EXAFS spectrum of dried crystalline TiBALDH reveals relatively strong peaks in the range corresponding to the Ti–Ti distance (about 3.5 Å), confirming the oligo-nuclear nature of this species (Fig. 2c). With the view that in principle all other known titanium carboxylates with a Ti : L ratio lower than 1 : 3 are oxo-species with bridging oxygen atoms,²² this provides a very strong indication that TiBALDH has to be formulated as ammonium titanyl lactate, i.e. ammonium oxo-lactato-titanate, (NH₄)₈[Ti₄O₄(C₃H₅O₃)₈](H₂O)₄ (Fig. 2d). The commercial samples apparently contain a minor admixture of titania nanoparticle solutions stabilized by adsorbed lactate ligands. Thermal treatment of TiBALDH results in a pure anatase phase of TiO₂ (see Fig. FS5†), which is consistent with random nucleation in decomposition of a molecular precursor.

Fig. 2 Microscopic image of the air-dried TiBALDH sample (a), the fit of the Rietveld refinement of the powder X-ray pattern for this material (b), its phase-corrected Fourier transformed EXAFS spectrum (c), and the model of the centrosymmetric tetranuclear oxo-lactato-titanate anion (d).

Health effects on lung cells

In our study we intended to get insight into the possible effects of exposure to titania bio-encapsulation matrices on different types of lung cells and analyse the toxic effect of this compound . Two model cell lines were chosen for in vitro studies: adenocarcinomic human alveolar basal epithelial cells A549, which have become a common model today for screening the health effects of components in air pollution,²³ and also non-small lung cell carcinoma cells U1810, which have been broadly investigated as a model for potential effects of medicines and chemicals on lung cancer cells.²⁴ The cells were treated as described above using different concentrations of nanoparticles based on titanium oxide. Treatment of U1810 cells that express mutant p53 protein with nanoparticles did not induce cleavage of caspase-related substrate PARP-1, indicating that these nanoparticles do not activate apoptotic cell death, identified to be the typical mechanism in cytotoxic effect of small nanoparticles on the living cells.²⁵ The nanoparticles did not induce expression of p53 proteins in A549 wild type cells that express p53 and, importantly, we were also unable to detect apoptosis in these cells, as measured by the level of cleaved form of PARP-1 (Fig. 3).

Fig. 3 Western blot analysis of PARP-1 cleavage and p53 expression in A549 and U1810 cells treated with different concentrations of titanium oxide-based nanoparticles

To further explore the effect of nanoparticles on the studied cells, their effect on cell viability was measured. For this purpose, the cells were treated with titanium oxide for 48 h at concentrations of 1, 5, 10 and 50 μg μl⁻¹. The results for the highest concentration, 50 μg μl⁻¹, presented in Fig. 4 shows the absence of cytotoxicity of titanium oxide nanoparticles.

Fig. 4 Viability of A549 cells treated with titanium oxide nanoparticles

In addition, cell growth was measured in cells treated for 120 h with titanium oxide nanoparticles and the DNA damaging drug, doxorubicin. The results in Fig. 5 show a 16-fold increase in cell growth in untreated cells and the same fold increase in the number of cells observed after the treatment of cancer cells with the nanoparticles. We also examined if the nanoparticles had an additive effect in combination with doxorubicin (0.5 μg μl⁻¹). As shown in Fig. 5, doxorubicin inhibited cell growth, but its combination with the nanoparticles had no additive inhibitory effect on cell growth. Therefore, based on these experiments we can conclude that titanium oxide-derived nanoparticles are nontoxic to the studied cells.

Fig. 5 Growth of the cells treated with titanium oxide nanoparticles (50 μg μl⁻¹), doxorubicin (0.5 μg μl⁻¹) or their combination.

Effects on pollen grain survival and growth

The environmental impact and potential effects on plant reproduction were followed by visual observation of the growth of pollen tubes. Either washed titanium dioxide gel obtained from CaptiGel or TiBALDH diluted by growth medium were added to freshly collected grains. In view of the different form of application, suspension vs. solution of an individual water soluble complex species, the concentrations of the produced mixtures are expressed as mM in relation to the Ti(IV) content. Converted into weight/volume content of TiO₂ this gives 120 μg ml⁻¹ for 1.5 mM Ti(IV) content. As can be seen from Fig. 6b, no measurable effect on pollen tube growth could be observed for this relatively high concentration of titania gel produced from CaptiGel. At higher concentrations the growth of pollen tubes was sterically hindered as the solution was “crowded” by clumps of the gel. Observations at much higher concentrations were possible for TiBALDH, up to about 30 mM. No measurable effects could be observed at concentrations as high as 15 mM (1.2 mg ml⁻¹ TiO₂ equivalent—a truly huge concentration that is hardly achievable even in a direct field application of the formulated biomaterials). This permits us to conclude that neither titanium formulation matrices nor model products of their bio-digestion display any measurable toxicity.

Fig. 6 Growth of pollen tubes in a 100 μg ml⁻¹ suspension of titania gel produced from CaptiGel after 3 h of exposure (a). Length of pollen tubes measured in solutions with different concentrations of titania gel (b). Growth of pollen tubes in a 600 μg ml⁻¹ solution of TiBALDH (c). Length of pollen tubes measured in solutions with different concentrations of TiBALDH (d).

The reliability of the applied test model was evaluated using a LaAlO₃ nanophosphor as a positive control. It was surface-modified by adenosine tri-phosphate (ATP) according to a procedure described in literature¹⁷ for improved solution stability (to permit it to be applied in controlled concentrations). The epifluorescence microscope images (Fig. 7a and 7b) show enhanced autofluorescence of pollen grains, supposedly caused by interaction of membrane proteins with metal oxide nanoparticles (Fig. 7a and 7b) and thus indicates the interaction of particles with the grains. The increased autoluminescence was recently observed to be a general feature in the interaction of plant cells with oxide particles smaller than 15 nm.²⁶ It is important to note that the solution stability of the LaAlO₃ suspension was rather limited and aggregation (glowing clumps) can be seen even in the images of fresh samples. Precipitation of aggregates is apparent after 1 h and the negative effect of this material is clearly visible in the areas where the precipitate is concentrated. Even at rather low total concentrations of about 10 μg ml⁻¹ the pollen grains were dead within 4 h in the proximity of the precipitated nanomaterials (see Fig. 7c). This was probably caused by the evolution of Al³⁺ cations, which are known to be highly toxic for plant cells, upon partial dissolution of the nanoparticles.

Fig. 7 Pollen grains incubated with LaAlO₃ nanophosphor: as-produced (a), after 2 h (b) and after 4 h (c).

Acknowledgements

This research was supported by the Swedish Research Council grant “Molecular Precursors and Molecular Models of Nanoporous Materials”, and the Swedish Research Council FORMAS grant “Particle Impurities in Air: Express-analysis and Health Effects”, by the Swedish and Stockholm Cancer Societies. Vitaliy Kaminskyy was supported by a fellowship from the Swedish Institute and Karolinska Institutet. Julia Netrval (now at the Bruker-AXS Sweden) is gratefully acknowledged for assistance with the ζ potential measurements and polyelectrolyte titrations. We express our gratitude to Suresh Gohil for the help with electrospray mass-spectrometry studies and to Prof. Ingmar Persson for the help with EXAFS experiments. We thank also Prof. Stephane Parola and Dr Fernand Chassagneux at the University Claude Bernard Lyon-1 for help with TEM experiments and continuous fruitful discussions.

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Footnote

† Electronic supplementary information (ESI) available: TG curves for thermal decomposition of air-dried materials, XPD patterns for heat-treated materials, tables summarising results of DLS measurements, EXAFS spectra of CaptiGel in comparison with literature data on anatase, and details of shooting nanoparticles suspension using Bio-rad instrument. See DOI: 10.1039/c2ra20388j

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