Synthesis and characterisation of biologically compatible TiO2 nanoparticles
© Cheyne et al; licensee Springer. 2011
Received: 31 August 2010
Accepted: 14 June 2011
Published: 14 June 2011
We describe for the first time the synthesis of biocompatible TiO2 nanoparticles containing a functional NH2 group which are easily dispersible in water. The synthesis of water dispersible TiO2 nanoparticles coated with mercaptosuccinic acid is also reported. We show that it is possible to exchange the stearic acid from pre-synthesised fatty acid-coated anatase 5-nm nanoparticles with a range of organic ligands with no change in the size or morphology. With further organic functionalisation, these nanoparticles could be used for medical imaging or to carry cytotoxic radionuclides for radioimmunotherapy where ultrasmall nanoparticles will be essential for rapid renal clearance.
Organically functionalised inorganic nanoparticles are being increasingly studied as a result of their many technological applications. In particular, the synthesis of inorganic nanoparticles for biomedical applications is being widely researched. Biomedical applications of inorganic nanoparticles include biosensing , targeted drug delivery agents  and contrast agents in magnetic resonance imaging (MRI) [3, 4]. Surface-coated superparamagnetic iron oxide nanoparticles have been extensively employed as magnetic resonance signal enhancers that can resolve the weakness of current MRI techniques. Most recently, it has been shown that by conjugating surface-coated Au-Fe3O4 nanoparticles to both herceptin and cis-platin, the nanoparticles can act as target-specific nanocarriers to deliver platin into Her2-positive breast cancer cells with strong therapeutic results . Furthermore, these nanoparticles can act as both a magnetic and optical probe for tracking the platin complex in cells and biological systems. However, the iron oxide nanoparticles commonly used as MRI contrast agents have a radius of over 50 nm so that they have a limited extravasation ability and are subject to easy uptake by the reticuloendothelial system [6, 7]. In order to enhance biological targeting efficiency, ultrasmall nanoparticles with greatly reduced hydrodynamic sizes are desired. Recently, ultrasmall (core size of 4.5 nm) c(RGDyK)-coated Fe3O4 nanoparticles have been synthesised , and results show a dramatic increase in cellular uptake. These nanoparticles were synthesised via thermal decomposition of Fe(CO)5 in the presence of the ligand 4-methycatechol (4-MC). The 4-MC-coated nanoparticles were then conjugated with a peptide c(RGDyK) via the Mannich reaction. There has been much research into the synthesis and properties of TiO2 nanoparticles since surface-modified TiO2 nanoparticles have many applications including photocatalysis  and photoelectric conversion [10, 11]. Such research has shown that it is facile to make surface-coated TiO2 nanoparticles with an ultrasmall core size of 3 to 5 nm [12, 13]. However, the study of TiO2 nanoparticles for biological applications, which have been shown to be non-toxic at low doses  (5 mg/kg body weight), has thus far been limited as such TiO2 nanoparticles are generally synthesised via a nonhydrolytic method and hence are non-dispersible in water. There are a couple of examples of functionalised TiO2 nanoparticles which are dispersible in water [15, 16]; however, in these reports, a broad size distribution is evidenced (3 to 8 nm).
In this paper, we show that it is possible to synthesise ultrasmall TiO2 nanoparticles with a core size of 5 nm with a range of coated short-chain organic functional groups which are comparable in size to diabodies which exhibit rapid renal excretion . The organically functionalised nanoparticles are highly dispersible in a range of solvents, and results show that when coated with aspartic acid or mercaptosuccinic acid, the nanoparticles are easily dispersible in water. Hence, for the first time, ultrasmall biocompatible TiO2 nanoparticles containing a functional NH2 or SH group have been synthesised. With further organic functionalisation and conjugation to a targeting moiety such as a single-chain antibody fragment or to biotin, these nanoparticles could be used to carry multiple short-lived radionuclides including 99mTc and 67Ga for medical imaging or to cytotoxic radionuclides for radioimmunotherapy where ultrasmall nanoparticles will be essential for rapid renal clearance.
Results and discussion
The two-phase thermal synthesis of titanium dioxide nanoparticles was adapted from a previously described procedure . Typically, a solution of tert- butylamine dissolved in water was added to a Teflon-lined steel autoclave. Separately, titanium(IV) n-propoxide and stearic acid (SA) were dissolved in toluene and added to the autoclave. The autoclave was sealed and heated to 180°C for 16 h and allowed to cool to room temperature. TiO2 nanoparticles were recovered by precipitation with acetonitrile and isolated by filtration. The "SA-coated" nanoparticles are dispersible in chloroform and methanol but are not dispersible in water or acetonitrile. The approximate number of SA molecules bound to each nanoparticle core was calculated to be 500 by following an established procedure .
Characterisation of surface-functionalised nanoparticles
Mean hydronamic radius for the different carboxylic acid-coated TiO2 nanoparticles determined from DLS measurements.
Carboxylic acid (ligand)
Mean hydrodynamic radius (nm)
In summary, we have created a facile route to synthesise ultrasmall surface-coated TiO2 nanoparticles with a range of organic coatings. Furthermore, the surface-coated nanoparticles are incredibly robust so that it is possible to perform ligand exchange reactions on the outer capping groups without disturbing the overall size or structure morphology of the nanoparticles. Results suggest that ligand exchange is most successful with bidentate ligands as a result of the availability of two carboxylic acid groups which bind to the TiO2 core.
This two-step approach toward the synthesis of surface-modified TiO2 nanoparticles allows for fine tuning of the nanoparticle core size in the first step before surface modification with suitable ligands in the second. By separating the surface modification step from that of the nanoparticle formation, this method allows for the production of identical nanoparticle cores before differentiation by surface modifications. Additionally, the use of bifunctional ligands to form the nanoparticle coating allows for the possibility of post-synthesis modifications to further functionalise the nanoparticle. This may be beneficial for use in biological applications as the initial surface functionalisation can convey improved water solubility before addition of more biologically relevant moieties. With further organic functionalisation and conjugation to a targeting moiety, the biological applications of the nanoparticles described here include the transport of multiple short-lived radionuclides including 99Tc and 67Ga for medical imaging or to cytotoxic radionuclides for radioimmunotherapy. The biological potential of these new nanostructures is currently being investigated.
All ligand exchange reactions were performed under an argon atmosphere. All reagents were purchased from Sigma-Aldrich (Sigma-Aldrich Company Ltd, Dorset, England) and used without further purification. Cleavage of Boc protecting groups was achieved by stirring in 4 M HCl/dioxane for 3 h under argon.
Routine 1H NMR and COSY data for TiO2 nanoparticles were obtained at 400 MHz on a VarianUnity INOVA instrument (Agilent Technologies Ltd, UKInfrared spectra were obtained from 400 scans at 4 cm-1 resolution using a Nicolet 380 spectrometer (Thermo Electron Corporation, Franklin, MA, USA) fitted with a diamond attenuated total reflectance (ATR) platform. IR and NMR data reported were obtained at room temperature. Room temperature X-ray diffraction patterns were collected for the organically coated TiO2 nanoparticles on a Bruker D8 Advance diffractometer (Bruker AXS Ltd, Coventry, UK) with twin Gobel mirrors using Cu Kα1 radiation. Data were collected over the range 20° < 2θ < 80°, with a step size of 0.02°. Transmission electron microscopy images were obtained for the organically coated TiO2 nanoparticles on a Philips CM10TEM (FEI Ltd, Netherlands). Dynamic light scattering (DLS) was performed using a Malvern mastersizer (Malvern Instruments Ltd, Malvern, UK).
Synthesis of titanium dioxide nanoparticles
Titanium dioxide nanoparticles were synthesised by a two-phase thermal approach adapted from a previously described procedure . Typically, a solution of 0.15 mL of tert- butylamine (1.43 mmol) dissolved in 14.5 mL of water was added to a 45-mL Teflon-lined steel autoclave. Separately, 0.225 g of titanium(IV) n-propoxide (0.792 mmol) and 0.75 g of stearic acid (2.64 mmol) were dissolved in 14.5 mL of toluene and added to the autoclave without additional stirring. The autoclave was sealed and heated to 180°C for 16 h and allowed to cool to room temperature. The TiO2 nanoparticles were recovered by precipitation with 90 mL of acetonitrile and isolated by filtration. Off-white solid; 1H NMR (CDCl3); δ 0.88 (t, 3H), 1.25 (s, 30H) and 2.03 (s, 2H); IR ν max 2,960, 2,915, 2,848, 1,620, 1,521, 1,455, 1,400, 1,300, 1,258, 1,220 and 1, 066 cm-1.
Procedure for surface modification of nanoparticles
A solution of carboxylic acid (150 mg) in 5 mL chloroform was added to a reaction vessel containing a dispersion of "SA-coated" TiO2 nanoparticles (100 mg) in 10 mL chloroform. The reaction was stirred for 18 h under reflux. The resultant surface-modified nanoparticles were recovered by evaporation of the solvent in vacuo, re-suspension in acetonitrile and filtration. Unbound starting material was removed by repeated washings of the nanoparticles with acetonitrile.
Benzoic acid exchanged TiO2
Off-white solid; 86% yield; 1H NMR indicates an incomplete exchange (37%) of stearic acid with benzoic acid; 1H NMR (CDCl3); δ 0.88 (t, 3H), 1.28 (s, 28H), 1.65 (t, 2H), 2.34 (t, 2H), 7.42 (t, 1.2H), 7.53 (t, 0.6H) and 8.06 (d, 1.2H); IR ν max 2,956, 2,919, 2,849, 1,630, 1,599, 1,513, 1,448 and 1,411 cm-1.
Glycine exchanged TiO2
Synthesis was performed from Boc-glycine. Cleavage of the protecting group was achieved by stirring the resulting nanoparticles under argon in 4 M HCl/dioxane for 3 h. Off-white solid; 91% yield; 1H NMR indicates an incomplete exchange (30%) of stearic acid with glycine; 1H NMR (CDCl3); δ 0.88 (t, 3H), 1.25 (s, 30H), 2.02 (d, 2H), 2.33 (s, 1H), 3.75 (s, 1.4H); IR ν max 3,319, 3,115, 2,991, 2,928, 1,742, 1,613, 1,495, 1,435, 1,406, 1,337, 1,305, 1,248, 1,118, 1,066 and 901 cm-1.
Aspartic acid exchanged TiO2
Synthesis was performed from Boc-aspartic acid. Cleavage of the protecting group was achieved by stirring the resulting nanoparticles under argon in 4 M HCl/dioxane for 3 h. Off-white solid; >95% yield; 1H NMR (D2O); δ 1.40 (s, 0.4H), 2.03 (s, 0.4H), 2.13 (s, 0.3H), 3.09 (d, 2H, J = 5.2 Hz), 4.25 (t, 1H, J = 5.6 Hz); COSY clearly shows coupling between the protons of the doublet (δ 3.09) and triplet (δ 4.25); IR ν max 3,316, 3,166, 2,970, 2,910, 1,721, 1,615, 1,506, 1,410, 1,346, 1,296, 1,253, 1,220, 1,151 and 1,066 cm-1.
Phthalic acid exchanged TiO2
Off-white solid; purification not possible; resulting nanoparticles not dispersible.
Mercaptosuccinic acid exchanged TiO2
Synthesis was performed using mercaptosuccinic acid. To reduce the possibility of oxidation occurring between mercaptosuccinic acid moieties, the reaction was performed under anhydrous conditions but in an otherwise identical manner to previous exchange reactions. Pale-yellow solid; >95% yield; 1H NMR (D2O); δ 2.62 (m, 1H) and 2.91 (m, 1H); IR ν max 2,915, 2,848, 1,685, 1,535, 1,515, 1,442 and 1,384 cm-1.
We thank Mr Kevin Mackenzie for making TEM measurements. This work was supported by the Breast Cancer Campaign.
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