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Recent progress in biomedical applications of

Cite this: DOI: 10.1039/c3cp43938k
titanium dioxide
Zi Fei Yin,wa Long Wu,wb Hua Gui Yang*b and Yong Hua Su*a
Published on 28 January 2013 on http://pubs.rsc.org | doi:10.1039/C3CP43938K

As one of the most common chemical materials, titanium dioxide (TiO2) has been prepared and widely
used for many years. Among all the applications, the biomedical applications of TiO2 have motivated strong
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Received 6th November 2012,
Accepted 25th January 2013
DOI: 10.1039/c3cp43938k
www.rsc.org/pccp
interest and intensive experimental and theoretical studies, owing to its unique photocatalytic properties,
excellent biocompatibility, high chemical stability, and low toxicity. Advances in nanoscale science suggest
that some of the current problems of life science could be resolved or greatly improved through applying
TiO2. This paper presents a critical review of recent advances in the biomedical applications of TiO2, which
includes the photodynamic therapy for cancer treatment, drug delivery systems, cell imaging, biosensors for
biological assay, and genetic engineering. The characterizations and applications of TiO2 nanoparticles, as
well as nanocomposites and nanosystems of TiO2, which have been prepared by different modifications to
improve the function of TiO2, are also offered in this review. Additionally, some perspectives on the
challenges and new directions for future research in this emerging frontier are discussed.

1. Introduction colouring, cosmetics, and toothpastes. Since the first report

As a well-known functional material, titanium dioxide (TiO2)
has been widely used as a white pigment in paints, food
a
Department of Traditional Chinese Medicine, Changhai Hospital, Second Military
Medical University, 168 Changhai Road, Shanghai, 200433 China.
E-mail: suyh@smmu.edu.cn; Fax: +86 21 8187 1559; Tel: +86 21 8187 1569
b
Key Laboratory for Ultrafine Materials of Ministry of Education, School of
Materials Science and Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, China.
E-mail: hgyang@ecust.edu.cn; Fax: +86 21 64252127; Tel: +86 21 64252127
† These authors contributed equally to this work.
of photocatalytic splitting of water on a TiO2 electrode under
ultraviolet (UV) light in 1972,1,2 TiO2 has been extensively
studied and remained as one of the most important candidates
used as photovoltaic cells, photocatalysis, photodegradation,
electrochromic devices etc,1,3–8 due to its nontoxicity, strong
optical absorption, low cost, and high chemical stability.
Among all the applications, the medical applications of TiO2
are undoubtedly promising, which may play an important role in
the improvement of health care, especially cancer treatment. For
instance, based on its excellent photocatalytic activity, photo-
excited TiO2 demonstrates the capability to kill cancer cells

Zi Fei Yin received her Bachelor Long Wu received his Bachelor

degree in Traditional Chinese
Medicine from Second Military
Medical University in 2009. She
is now a Master degree candi-
date, majoring in Integrated
Traditional and Western Medicine,
under the supervision of Prof.
Yong Hua Su. Her research
interests focus on the thera-
peutic effect and mechanism of
photodynamic therapy and
Zi Fei Yin Chinese medicine on cancer.
degree in School of Chemistry
and Chemical Engineering from
Yantai University in 2009. He is
now a PhD student in East China
University of Science and Techno-
logy under the supervision of
Prof. Hua Gui Yang. His current
research interests focus on the
design, synthesis and charac-
terization of novel functional
semiconductor materials for clean
Long Wu and renewable energy, especially
solar energy.

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effectively, and it can also be applied as a nucleic acid endo-

nuclease, which is of vital importance to genetic engineering.9–11
Furthermore, when combined, doped or tailored with different
methods,12–19 the nanocomposites and nanosystems of TiO2
may serve as candidate tools for cell imaging, biological analysis,
and drug delivery.13,20–23
To date, the synthesis, physicochemical properties and
applications (especially in clean energy and environmental
protection fields) of TiO2 have been reviewed extensively.24–32
However, the biomedical applications of TiO2 in life science have
been rarely summarized so far. Herein, we summarize the recent
progress of various biomedical applications of TiO2 including
photodynamic therapy for cancer, drug delivery system, cell
Published on 28 January 2013 on http://pubs.rsc.org | doi:10.1039/C3CP43938K

imaging, biosensors for biological assay, genetic engineering, Fig. 1 Bulk crystal structure of rutile (left) and anatase (right). Titanium atoms
and some perspectives are also provided finally. are gray, and oxygen atoms are red. (Obtained from ref. 36).
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2. Fundamental properties of TiO2

TiO2 occurs as four major crystal structures: the stable rutile
(tetragonal, a = b = 4.584 Å, c = 2.953 Å), metastable anatase
(tetragonal, a = b = 3.782 Å, c = 9.502 Å), brookite (rhombohedral,
a = 5.436 Å, b = 9.166 Å, c = 5.135 Å) and TiO2 (B) (monoclinic,
a = 12.16 Å, b = 3.74 Å, c = 6.51 Å).1,5,33–37 The brookite and TiO2
(B) structures are less studied in experimental investigations.
Both the rutile and anatase crystals are formed by a basic building
block consisting of a titanium atom surrounded by six oxygen
atoms in a more or less distorted octahedral configuration. Fig. 1
depicts the unit cell structures of the rutile and anatase TiO2.
The differences in the two crystal structures are the distortion
of each octahedron and the assembly pattern of the octahedral
chains. In rutile, the distortion of the cubic lattice is slight so
that the unit cell is stretched beyond a cubic shape. In anatase,
the octahedron is significantly distorted, and thus its symmetry
is lower than orthorhombic. These polymorphs determine
electronic band structures, surface structure, and thus the bulk
diffusion, surface transfer capability and redox potentials of
photoinduced charge carriers. Among various polymorphs of TiO2,
rutile is the most stable phase for particle having a size above
35 nm,38 while anatase is considered to be the most thermodyna-
mically stable for the nanoparticles with a size below 10–20 nm.1,5,39
The unique physicochemical properties of TiO2 crystals are
affected not only by the intrinsic electronic structure and crystal
structures but also by their size, shape, and doping.24,40–42 In
particular, intensive research attention has been paid to the
design and synthesis of TiO2 with high-reactivity facets. For a
long period, tailored synthesis of faceted anatase TiO2 single
crystals has been a great challenge. Recently, on the basis of
theoretical predictions, Yang et al.43 successfully synthesized
uniform anatase TiO2 single crystals with 47% of the highly
reactive facets using hydrofluoric acid (HF) as a morphology
controlling agent for the first time. After that, many promising
applications such as photocatalytic degradation of organic dye
molecules, photocatalytic water splitting and dye-sensitized
solar cells (DSSCs) using facet dominated anatase TiO2 have
also been developed.44–48

Hua Gui Yang completed his PhD
in 2005 at the National Univer-
sity of Singapore. He then joined
the General Electric (GE)
Company as a Research Scientist
and moved to the University of
Queensland (UQ) in 2007 as a
Postdoctoral Research Fellow.
Currently he is a professor of
East China University of Science
and Technology. His work has
been published in top journals
Hua Gui Yang such as Nature, and featured in
the Nature Materials, RSC’s
Chemistry World and ACS Chemical & Engineering News.
Currently he has interests in design and synthesis of metallic and
semiconducting crystals for renewable clean energy and
environmental protection applications.
Yong Hua Su received her PhD
in Integrated Traditional and
Western Medicine from Second
Military Medical University in
2003, under the supervision of
Prof. Chang Quan Ling. She is
presently a Professor in the
Department of Traditional Chinese
Medicine in Changhai Hospital,
which is affiliated to the Second
Military Medical University. Her
research interests focuses on
Yong Hua Su cancer treatment and the bio-
medical applications of
nanomaterials.

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However, for practical applications, the large band gap of
TiO2 (B3.2 eV for anatase and brookite, B3.0 eV for rutile) is so
large that it can be only activated under ultraviolet (UV)
irradiation. To overcome the serious weaknesses of low separa-
tion probability or the photoinduced electron–hole pairs and
narrow light-response range in pristine TiO2, many modification
strategies have been proposed to achieve this target, including
doping, co-doping with two or more foreign ions, surface sensitiza-
tion by organic dyes or metal complexes, and decoration with noble
metal deposition, etc.36,39,42,49–51 In particular, doping is a powerful
strategy for modifying electronic structure and the construction of
heteroatomic surface structures that allow high-efficiency photo-
catalysis under solar light irradiation. In photocatalysis, a break-
Published on 28 January 2013 on http://pubs.rsc.org | doi:10.1039/C3CP43938K
through work by Asahi et al. in 2001 reported nitrogen-doped TiO2
can improve its photocatalytic activity for the photodegradation of
methylene blue and gaseous acetaldehyde under visible light
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irradiation.52 After that, nearly all metal and non-metal ions have
been explored as dopants in modifying the electronic structure of
TiO2 to improve its photocatalytic activity.
Photocatalysis of TiO2 involves three processes: the excita-
tion, bulk diffusion and surface transfer of photoinduced
charge carriers. Fig. 2 shows the three processes in a photo-
catalytic reaction.
Firstly, the semiconductor photocatalyst particles absorb a
photon with energy larger than the band gap. The electrons in the
valence band of the photocatalyst are excited to the conduction
band. Simultaneously, the holes are left in the valence band.
Secondly, the excited electrons and holes separate and
migrate to the surface of photocatalyst which is drastically
affected by crystal structure, crystallinity, and by the particle
size of the photocatalyst. However, recombination of photo-
generated electron and hole pairs results in a decrease in the
photocatalytic activity.
Lastly, the electrons and holes can lead to produce surface
chemical reactions. The holes can react with surface adsorbed
Fig. 2 Schematic illustration of main processes in a photocatalytic reaction.
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H2O to produce hydroxyl radicals. Meanwhile, the electrons are
usually scavenged by O2 to yield superoxide radical anions.
These species in solution can react to give other cytotoxic
reactive oxygen species (ROS) such as hydrogen peroxide and
peroxy radicals, which are harmful to cancer cells. The major
reactions that result in the formation of ROS are shown as
simplified eqn (1)–(6).
TiO2 + hu - h+ + e (1)
H2O + h+ -  OH + H+ (2)
O2 + e - O2 (3)
O2 
+ H - HO2
+ 
(4)
2 HO2 - H2O2 + O2 (5)
H2O2 + O2 -  OH + OH + O2
(6)
3. Biomedical applications of TiO2
So far, plentiful research works focusing on the biomedical
applications of TiO2 have been reported. Based on different
functional activities concerned with different research fields,
these biomedical applications can be classified as follows:
(i) Photodynamic therapy for cancer
(ii) Drug delivery system
(iii) Cell imaging
(iv) Biosensors for biological assay
(v) Genetic engineering
3.1 Photodynamic therapy for cancer
Photodynamic therapy (PDT) for cancer involves administration
of a tumor localizing photosensitizing agent, which may require
metabolic synthesis, followed by activation of the agent by light
of a specific wavelength.53 Compared with surgical, radiological,
chemotherapeutic treatments and other conventional therapies,
PDT is increasingly being recognized as an alternative and
promising non-invasive treatment for cancer.54–56
When TiO2 nanoparticles were illuminated by UV light
with wavelength less than 385 nm, photo-induced electrons
and holes could be created.34 Moreover, these photo-induced
electrons and holes could further react with hydroxyl ions or
water to form powerful oxidative radicals (e.g.,  OH, HO2 ),57
which are capable of destroying the structure of bacteria, fungi,
tumor cells, and so on. In view of the rising incidence of cancer
and the unparalleled advantages of PDT, many researchers
have focused on the application of TiO2 as a photosensitizer
to treat cancer due to its high photocatalytic efficiency, low
toxicity and excellent photostability.
The photokilling activity of TiO2 to malignant cells was first
reported on a TiO2 film electrode by A. Fujishima et al.58,59 In
the system, Hela cells were cultured on the surface of TiO2
electrode. When the electrode was anodically polarized under
UV-irradiation, Hela cells were damaged from the membrane.
However, cancer cells were not killed when the electrode was
located 10 mm away from the cell surface, so they prepared a
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polarized, illuminated TiO2 microelectrode in 1995,60 which
showed selective antitumor activity of a single cancerous
T24 cell.
Considering the electrode system was not feasible and
available, TiO2 particles were applied, even nanoparticles were
also prepared, which could yield an extremely large surface area
and be incorporated by the living cells, resulting in higher reaction
rates. Under these experimental conditions, with the photoinduc-
tion of UVA, more and more researchers showed the phototoxic
effect in vitro of TiO2 nanoparticles on a series of human cancer
cells, such as cervical cancer cells (HeLa),9 bladder cancer cells
(T24),10 monocytic leukemia cells (U937)61 and adenocarcinoma
cells (SPC-A1),62 colon carcinoma cells (Ls-174-t),63 breast epithelial
Published on 28 January 2013 on http://pubs.rsc.org | doi:10.1039/C3CP43938K
cancer cells (MCF-7, MDA-MB-468),64 glioma cells(U87),65 and
human hepatoma cells (Bel 7402).66
TiO2 nanoparticles are typically insoluble in aqueous medium,
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hampering the utilization of TiO2 in biological applications, so
how to enhance the water-soluble activity of TiO2 needs to be
investigated. J. Seo et al.67 successfully fabricated water-soluble
TiO2 nanoparticles through a high-temperature non-hydrolytic
method. These nanoparticles are short and rod shaped and are
3.5 nm in diameter and 10.4 nm in length (Fig. 3A), and showed
higher toxicity on human melanoma cells (A375) with the
presence of UV irradiation than commercially available
Degussa P-25 nanoparticles (Fig. 3B).
Importantly, the anticancer effect of TiO2 has been also
demonstrated in vivo,9,32,68 indicating that photoexcited TiO2
nanoparticles may be a potential way to treat cancer in the
future. R. Cai et al.9 injected Hela cells under the skin of nude
mice to cause tumors to form, when the size of the tumors grew
Fig. 3 (A) TEM image of synthesized water-soluble TiO2 nanoparticles; inset:
HRTEM lattice-fringe image. (B) The surviving fraction of melanoma A375 cells
when treated with water-soluble TiO2 nanoparticles (K), Degussa P-25 nano-
particles (m) after UV irradiation for 30 min, and water-soluble TiO2 nanoparticles
without UV irradiation (’). (Obtained from ref. 67).
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Fig. 4 The tumors of nude mouse before treatment (A) and 4 weeks after
treatment (B). TiO2 powder (0.4 mg) was injected into Tumor 1. Tumor 2, which
was not injected with TiO2 particles, was also opened surgically. Both tumors
were irradiated by a Hg lamp for 1 h. (Obtained from ref. 68).
to about 0.5 cm, they injected a solution containing fine particles
of TiO2 to the tumor. After 2 or 3 days, the skin covering
the tumor was cut open to be exposed and irradiated by UVA.
This treatment clearly inhibited the tumor growth. After 13
more days, the treatment was repeated and a further marked
antineoplastic effect was observed (Fig. 4). Furthermore, TiO2
particles can significantly suppress the growth of bladder and
glioma cancer cells implanted into nude mice as well, even
prolong the survival time.10,65
The photo-generated holes are easy to recombine with
the photo-induced electrons, which greatly reduced the photo-
catalytic inactivation efficiency of TiO2,69 so how to enhance the
photocatalytic efficiency of TiO2 has been deeply investigated.
Surface modification with other elements is a plausible and
extensively used method. J Xu et al.70 synthesized gold-doped
TiO2 nanocomposites through the deposition–precipitation (DP)
method and confirmed that the loading amount of Au on the
surface of TiO2 nanoparticles can strongly affect the photocatalytic
inactivation efficiency of TiO2. The most efficient nanocomposites
were TiO2 nanoparticles doped with 2%. When 50 mg mL 1 2 wt%
Au/TiO2 nanocomposites were used, all of the Human Colon
Carcinoma LoVo cells were killed under the irradiation of UV
light within 100 minutes, while only 40% cancer cells were killed
on the same condition of TiO2 nanoparticles (Fig. 5).
TiO2 can only be activated by UV light due to its energy gap
(3.0–3.2 eV). However, UV light is harmful to the human body,
and UV only accounts for almost 5% of the sun’s energy
compared to visible light (45%), the shift in the optical
response of TiO2 from the UV to the visible spectral range will
have a profound positive effect on the practical applications. In
order to extend the optical absorption of TiO2 to the visible light
region, different methods have been developed. Dye adsorbed
and doping methods are the extensively applied approaches.
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Fig. 5 (A) TEM images of TiO2 nanoparticles (a), and 2 wt% Au/TiO2 (b and c). The
magnifications of (a), (b), and (c) are 150 000, 400 000, and 1 500 000, respectively.
(B) Surviving fraction of LoVo cells under the irradiation of UV light after being
incubated in culture mediums: (a) with no TiO2, (b) containing 50 mg mL 1 TiO2, and
(c) containing 50 mg mL 1 2 wt% Au/TiO2. (Obtained from ref. 70).
The dyes such as hypcrellin B,71 chlorine e6,72 and zinc phthalo-
cyanine73 which are well-known PDT sensitizers, were applied in
the dye-adsorbed method. As regards to the doping method,
several metal elements (Pt, Fe)74–76 and nonmetal elements (C,
N)77–80 have been used. Z. Li et al.80 synthesized nitrogen-doped
TiO2 (N-TiO2) nanoparticles by calcining the anatase TiO2 nano-
particles under an ammonia atmosphere. The prepared N-TiO2
nanoparticles showed a higher absorbance in the visible region
than the pure TiO2 nanoparticles (Fig. 6A). Higher antitumor
activity of N-TiO2 nanoparticles under the irradiation of visible
light was also reported (Fig. 6B). Recently, a co-doped method was
also utilized, which means that TiO2 was modified with two
different elements (Fe@N,Fe@Ag).81,82 For example, according
to the research of Md. Abdulla-Al-Mamun et al.,82 Ag@Fe-doped
TiO2 nanopowders (Fig. 7A) could produce higher amount of  OH
under the presence of visible light irradiation (Fig. 7B), and
exhibited more efficient in cancer cell killing than Fe-doped TiO2.
Though the anticancer effect of TiO2 is effective, the killing was
devoid of specificity. Hence, it is necessary to prepare improved
TiO2 nanoparticles, which can specifically identify and bind with
the receptors of cancer cells, in order to increase the selective
antitumor activity and reduce the non-selective cell death.83
Recently, monoclonal antibody proteins (CEA,83 pre-S1/S2,84
IL13a2R85 and EGFR86) with high affinity and specificity have been
immobilized on the surface of TiO2 nanoparticles. These proteins
are overexpressed on the surface of certain cancer cells, therefore
the modified TiO2 nanoparticles with a specific antibody are useful
for directing the nanoparticles towards the specific cell population.
For example, E. A. Rozhkova et al.85 utilized a platform of 5 nm
TiO2 nanoparticles tethered through a DOPAC linker to the
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Fig. 6 (A) Diffuse reflectance absorption spectra of pure TiO2 and N-doped TiO2
(Sample N-550-1 and N-550-2). Sample N-550-2 exhibited the highest absor-
bance in the visible region. (B) The cytotoxicities and the photokilling effects of
pure TiO2 and N-doped TiO2 (N-550-2) with the concentration of 200 mg mL 1 on
HeLa, hepatocellular carcinoma cells(QGY), and human nasopharyngeal carci-
noma cells (KB) cells. (Obtained from ref. 80).
antihuman-IL13 2R. These bioconjugated nanoparticles were
demonstrated to target brain cancer cells and avoid normal brain
cells (Fig. 8). Moreover, with the new method, TiO2 nanoparticles
can be modified with various exclusive targeting proteins, so that
TiO2 nanoparticles may be used as a target therapy of specific
cancer cells more effectively.83 At present, the cancer treatments
can shrink the size of the tumour, but these effects are transient.
Viewing that cancer may be considered as a cancer stem cell
disorder rather than that of rapidly growing cells, eliminating
cancer stem cells may be the possible cure of cancer.87,88 Therefore,
how to recognize the cancer stem cells is a great challenge. The
monoclonal antibody (mAb) Nilo1, which can recognize the surface
antigen in neural stem cells, has been successfully coupled to
TiO2 nanoparticles by G. Elvira et al.89 The Nilo1-TiO2 complexes
were verified to specifically deplete in vitro cancer stem cells upon
UV-irradiation, which provided the basis for further applications of
TiO2 in cancer stem cells therapy.
3.2 Drug delivery system
For the ultimate goal of maximizing the therapeutic activity
while minimizing the side effects, an ideal drug distribution
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Fig. 7 (A) TEM micrographs of (a) Fe-doped TiO2 and (b) Ag@Fe-doped TiO2.
a1 and b1 show the corresponding electron diffraction patterns of (a) and (b),
respectively. (B) The photoluminescence (PL) spectral changes observed during
irradiation of (a) Fe-doped (5%) TiO2 and (b) Ag@Fe-doped (5%) TiO2 samples
under visible light irradiation. The intensity of the PL is in proportion to the
amount of OH radicals produced in water. (Obtained from ref. 82).
system should deliver the drug when and where it is required.90,91
In recent years, controlled and targeted drug delivery systems have
gained increasing attention and the rapid expansion in advanced
materials and technology has resulted in a remarkable progress in
their development. Sustained and controlled drug release from
nanostructured reservoirs, which were implanted or injected into
human body at the site, targeted where the medication is needed,
is an attractive weapon to the regular intake of medication in the
case of chronic diseases, such as cancer and epilepsy.
Up to now, TiO2 has received considerable attention in
efficient drug delivery systems, and TiO2 nanoparticles have
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Fig. 8 Nanobiocomposites consisting of 5 nm TiO2 and IL13R-recognizing anti-
body linked via DOPAC linker to recognize and bind exclusively to surface IL13R.
(Obtained from ref. 85).
been fabricated into various shapes of drug delivery system, such
as whiskers, capsules, and porous shapes. Therefore, the drug can
be combined on the surface or contained in the reservoir of TiO2,
performing sustained controlled release activity. Additionally,
when the drug delivery system is modified with specific molecules
directing it to the targeted cells, it can be localized in the specific
cells to perform higher efficiency.
TiO2 has been studied as a carrier material for various drugs,
such as sodium phenytoin92 valproic acid,90 temozolomide,93
and daunorubicin.12–14 After an initial higher release rate, a
nearly constant release rate can be obtained over a long period
of time. These new drug delivery systems of TiO2 permit high
delivery efficiency, precise control of the dose, prolonged time
periods (i.e., days, weeks, or months) exposure to drugs and
significant reduction in toxicity. For example, Q. Li et al.12
confirmed that one-dimensional titanium dioxide whiskers
(TiO2 Ws) can obviously enhance the potential anti-tumor
intracellular efficiency of daunorubicin (DNR) by increasing
the concentration of DNR inside SMMC-7721, while alleviating
the toxic side-effects by efficiently making full use of DNR in the
environment (Fig. 9). Additionally, UV irradiation could further
enhance the growth inhibition of cancer cells by photocatalysis.
Furthermore, it has been found that the loading mode
between the drug and TiO2 can significantly affect therapeutic
efficacy. Y. Qin et al.13 loaded chemotherapeutic doxorubicin
(DOX) loaded on the highly water-dispersible TiO2 nano-
particles by non-covalent complexation (TiO2/DOX) or covalent
conjugation (TiO2-DOX) (Fig. 10A). In the case of TiO2/DOX, the
main fraction of DOX was found inside the nuclei, which was
probably due to the pH-responsive release of DOX from TiO2/
DOX in C6 glioma cells, while most TiO2-DOX was distributed
in the cytoplasm, and little TiO2-DOX penetrated the nuclei
(Fig. 10B). Consequently, TiO2/DOX exhibited a significantly
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Fig. 9 (A) Illustration of the possible mechanism account for the enhancing
uptake of DNR into SMMC-7721 cells via TiO2 whiskers (Ws) drug delivery. (B)
Microscopy images of SMMC-7721 cells (a), treated with TiO2 NPs (12.5 mg mL 1)
(b), TiO2 Ws (12.5 mg mL 1) (c), DNR (1.5  10 5 mg mL 1) alone (d), TiO2 NPs–
DNR (e), and TiO2 Ws–DNR (f). (Obtained from ref. 12).
higher cytotoxicity toward C6 glioma cells than free DOX
because of enhanced cellular uptake.
The site of the released drug delivery system plays key roles
in determining the therapeutic outcome in terms of selectivity and
specificity. Therefore, how to direct the drug carrier to targeted cells
is essential. Interestingly, C. Kim et al.94 has fabricated silica/titania
hollow nanoparticles (HNPs) with an average diameter of 50 nm
by using the dissolution and redeposition method, and mod-
ified HNPs with anti-[human epidermal growth factor receptor
2] monoclonal antibody (herceptin) (HER-HNP), and loaded
HER-HNP with large amount of hydrophobic anticancer drug
camptothecin (CPT) (HER-HNP-CPT) (Fig. 11A). The HER-
HNP-CPT efficiently internalized into the human breast cancer
SK-BR-3 cells, owing to their herceptin conjugation, and
released its entire load of CPT in 24 h, thus simultaneously
showed highly efficient anticancer activity and low toxicity to
normal cells (Fig. 11B). The new drug delivery system, with high
loading capacity for hydrophobic anticancer drug and high
selectivity for cancer cells, offers a new research direction for
anticancer drug carriers.
3.3 Cell imaging
Cell imaging and observation provides insight into the cell fate.
Though there are many cell imaging methods, non-invasive and
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Fig. 10 (A) Schematic representation of synthesis of highly water-dispersible
TiO2 nanoparticles and loading of DOX via non-covalent complexation (TiO2/
DOX) or covalent conjugation (TiO2-DOX). (B) Illustration of the effect of loading
mode on the intracellular location of DOX. Most of TiO2-DOX was distributed in
the cytoplasm (a), while in the case of TiO2/DOX, the main fraction of released
DOX was found inside the nuclei (b). (Obtained from ref. 13).
targeted detection of molecular biomarkers is absolutely
promising, as they can allow early stage diagnosis, better
understanding of the functional characteristics of disease,
predict the prognosis, and evaluate the treatment efficacy,
resulting in significant improvement in the total disease manage-
ment. Since nanosized materials exhibit specific behavior in biolo-
gical environment, such as prolonging the circulation time in the
body compared to small molecules, penetrating into tissues,
accumulating in tumor tissue, applying nanoobjects for prepara-
tion of advanced cell imaging agents is very advantageous.95,96
During the past few years, TiO2 has gained lots of researchers’
interests due to its unique chemical active surface in many
potential applications. With the advancement of nanotechnology,
the fluorescent dye or magnetic resonance contrast agents labelled
TiO2 nanoparticles, nanotubes or nanoprobes have been success-
fully prepared for cell imaging through fluorescent analysis or MRI.
Moreover, compared with X-ray and other medical imaging
methods, fluorescence and MRI are both non-invasive or
minimally invasive methods for in vivo imaging, indicating that
these prepared TiO2 nano-structures may have a wonderful
prospect for cell imaging in the future.
K. C. Wu et al.14 has synthesized mesoporous titania nano-
particles (MTNs) with excellent biocompatibility (LC50 E
400 mg mL 1) and a large surface area (ca. 237.3 m2 g 1), and
they further functionalized MTNs with a phosphate-containing
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Fig. 12 A merged fluorescence confocal image of FMN–MTNs (green dots) in

BT-20 cancer cells where the nuclei were stained with DAPI (blue spots).
(Obtained from ref. 14).
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P. Chandran et al.15 has reported their development of a

novel magnetic nano-contrast agent (nano-CA) based on Gd3+
doped amorphous TiO2 of size B25 nm. Quantitative T1 mapping
of phantom samples using a 1.5 T clinical MR imaging system
revealed that the amorphous phase of Gd3+ doped TiO2 (GTN)
has the highest r1 relaxivity which is B2.5 fold higher than the
commercially used CA Magnevistt. The crystalline (anatase)
samples formed by air annealing at 250 1C and 500 1C showed
significant reduction in r1 values and MR contrast, which is
attributed to the loss of proton-exchange contribution from the
adsorbed water and atomic re-arrangement of Gd3+ ions in
the crystalline host lattice (Fig. 13). The study reveals that
GTN is considered as a promising candidate for molecular
receptor targeted MR imaging. However, imaging at time
periods in excess of 24 hours after delivery is generally not
possible for Gd-based contrast agents because of their rapid
clearance, so the approaches to prolong retention of contrast
agents in the cells is desirable. T. Paunesku et al.97 investigated
that their prepared nanoconjugates composed of TiO2 nano-
particles and a Gd contrast agent, showed superior to the free
contrast agent of the same formulation with regard to intracellular

Fig. 11 (A) Schematic illustration of the fabrication of HER-HNP-CPT. (B) Cell

viability of SK-BR-3 human breast cancer cells (a) and RAW264.7 mouse macro-
phage cells (b) incubated with CPT, HNP, HER-HNP, HNP-CPT, and HER-HNP-CPT
for 24 h. (Obtained from ref. 94).

fluorescent molecule (flavin mononucleotide; FMN). When

BT-20 cells were incubated with FMN–MTNs for 4 h, the green
fluorescence that resulted from FMN-MTNs was visualized in the
cytoplasm, indicated that the FMN molecules remained inside the
MTNs without much leaching (Fig. 12). FMN–MTNs performed as
a satisfactory intracellular bioimaging agent. Furthermore,
Fig. 13 T1 weighted phantom NMR images of undoped TiO2 (TN), Gd3+ doped
the specific antibody (CEA) expressed by cancer cells can be TiO2 (GTN), GTN air annealed at 250 1C (GTN-250) and GTN air annealed at
conjugated with these fluorescent contrast modified TiO2 particles 500 1C (GTN-500). Water and 0.1 mM Gd(NO3)3 are taken as the controls.
as well, in order to track cancer cells precisely.83 (Obtained from ref. 15).

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Fig. 14 Visualization of rat mesenchymal stem cell suspensions: (A) magnetic
resonance image of vials with TiO2@RhdGd labeled cells (left) and control
unlabelled ones (right); (B) fluorescence from TiO2@RhdGd labeled cells (left)
and control unlabelled ones (right). (Obtained from ref. 16).
accumulation, retention, and subcellular localization. Even 48 hours
after treatment, the concentration of Gd in nanoconjugate-treated
cells was 1000-fold higher than in cells treated with contrast
agent alone.
The combination of various cell imaging methods into one
agent gives rise to multimodal imaging probes, ensuring the full
utilization of advantages of each particular imaging technique.
Recently, multimodal imaging probes have excited interests of
many researchers. I. Řehoř et al.16 prepared a new multimodal
imaging-therapeutic nanoprobe TiO2@RhdGd, and verified that
TiO2@RhdGd can successfully be used for in vitro and in vivo cell
tracking by means of fluorescence microscopy and MRI (Fig. 14).
The probe core consists of a TiO2 nanoparticle, and its surface is
covered with Gd(III) chelates responsible for contrast in MRI and
fluorescent rhodamine B (Rhd) derivatives for contrast in fluores-
cence microscopy. Furthermore, it was shown that the probe
TiO2@RhdGd can be changed into a cancer cell killer upon UV
light irradiation owing to the photocatalysis of TiO2.
3.4 Biosensors for biological assay
The design and fabrication of simple and sensitive electrochemical
biosensors have been of high interest for their widespread applica-
tions such as drug discovery, disease diagnosis, environmental
monitoring and food safety. However, the miniaturization
trend of electrochemical biosensors makes the utilization of
them still challenging because of the limited active surface area
of microelectrodes and the low detection signal.98
Among all the materials for biosensors, one promising
candidate is TiO2 because of its good biocompatibility, relatively
high conductivity, environmentally benign nature, chemical
and thermal stability. Until now, TiO2 in various forms,
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such as nanoparticles,99,100 nanotubes,101,102 nanosheets,103
three-dimensional macroporous matrices,98,104 and sol–gel
matrices,105 have been extensively applied in developing bio-
sensors. These prepared TiO2 matrices, provided a promising
interface to assemble different kinds of proteins (antibody,
enzyme), are promising matrices for the fabrication of electro-
chemical biosensors. Many results showed that these biosensors
can qualitatively and quantitatively analyze biological molecules,
such as tumor makers (CEA, AFP), glucose.106–108
It has been confirmed that the morphology and structure of
TiO2 nanomaterials have an important effect on the property of
the immobilized enzyme, which determines their efficacy in
biosensors.109 In order to achieve controlled fabrication of new
morphologies of TiO2 nanostructures with high performance,
Q. Xie et al.17 synthesized nanosheet-based TiO2 microspheres
(Fig. 15A) with a hollow core–shell structure. With the unique
‘‘trumpet’’-shaped pores between the nanosheets, the adsorbed
enzyme easily becomes immobilized on the inner core of
the microspheres, and the substrate has easy access to the
immobilized enzyme in the hollow interior of the core–shell
structure, therefore concentration of substrate and enzyme
in this confined region will increase the chance of effective
collisions between them. Further researches showed that the
biosensor based on the nanosheet-based hollow core–shell
structure of the TiO2 microspheres displayed good performance
for the detection of H2O2, with both a low detection limit of
0.05 mM and a wide linear range of 0.4–140 mM, as well as a
fast response and excellent long-term stability (Fig. 15B and C).
Hence, such structure should be a promising biosensor for
biological analysis.
However, the complexity of the human proteome requires
the development of sophisticated multiplexed technologies for
more appropriate methods of analysis, such as biochips and
microarrays, which can offer a valid approach to interrogate the
proteome by multiplexing the information at a reasonable cost.
R. Carbone et al.18 presented a novel cell-based microarray
for phenotype screening on primary and cancer cells based
on the localized reverse infection by retroviruses. The high-
performance coating for protein binding surfaces of the novel
microarray was made up of systematic nanostructured titanium
oxide film (ns-TiO2), synthesized by supersonic cluster beam
deposition (SCBD). The retroviral cell array was validated by
overexpression of GFP reporter genes in primary and cancer
cells additionally (Fig. 16), and the results demonstrated that
ns-TiO2 may serve as reliable substrate for biochips in analytical
protein microarray application.
TiO2 biosensors can not only be applied as substrate for
detecting biological molecules, but also be used in cell-capture
assay. According to a up-to-date research, N. Zhang et al.19
prepared an electrospun TiO2 nanofiber-based cell capture
assay. The advantages of using electrospun TiO2 nanofibers
(TiNFs) include (i) the dimension and packing density of TiNFs
can be precisely controlled; (ii) TiNFs can be deposited onto any
given substrates (i.e., silicon or glass) using a well established
experimental setup; and (iii) it is feasible to explore the use of a
variety of organic materials that can be engineered onto a cell
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Fig. 15 (A) Schematic illustration of the hollow core–shell structure of the
nanosheet-based TiO2 microspheres. (B) The biosensor performance of the
HRP/TiO2/Nafion/GC electrode was studied by the amperometric current–time
method. When H2O2 was added to the stirred PBS, the reduction current
increased rapidly and 95% of the steady-state current was obtained within 3 s
(C) Plot of steady-state current vs. H2O2 concentration. (Obtained from ref. 17).
capture substrate. The enhanced local topographic interactions
between the horizontally packed TiNFs deposited substrates and
extracellular matrix scaffolds, in addition to anti-EpCAM/EpCAM
biological recognition, contributed to the significantly enhanced
capture efficiency (Fig. 17A). Using these TiNFs-deposited sub-
strates, it is possible to capture circulating tumor cells (CTCs)
from the blood samples of colorectal and gastric cancer patients
(Fig. 17B). CTCs is of vital importance to the early diagnosis and
longitudinal monitoring of cancer in clinic, therefore the TiNFs-
deposited substrate has great potential application.
3.5 Genetic engineering
During the lifetime, organisms often acquire unwanted foreign
or mutated DNA that may negatively affect their health.
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Fig. 16 Retroviral microarrays with the application of nanostructured TiO2 for
the overexpression of EGFP-fused proteins; (A) 10  immunofluorescence panel
of a 10  24 array of MCF10A cells infected by PINCO and PINCO GFP-NPM vectors
in an alternate fashion. (B) 10  immunofluorescence panel of a 12  24 array of
U2OS cells infected by the same vectors in an alternate fashion. Blue, cell nuclei
(DAPI staining); green, GFP fluorescence. (Obtained from ref. 18).
Traditional modes of diagnosis are often unable to detect
the presence of deleterious DNA; in addition, conventional
treatments do not address the underlying cause of the
diseases—changes at the level of the genome—and therefore
do not discriminate well between target and healthy cells,
resulting in the low therapeutic efficacy of such diseases.
Consequently, imaging and elimination of unwanted genes and
gene products have been major goals of molecular biology over the
past few decades.110–112 Genetic engineering is a group of techniques
used for direct genetic modification of organisms or population
of organisms using recombination of DNA. With the rapid
development of genetic engineering, now it is possible to alter
directly a genome and insert or remove a chunk of DNA,
indicating the bright future of gene therapy for human beings.
TiO2 has been demonstrated as a candidate tool for genetic
engineering in recent years. T. Paunesku and his co-researchers
have conducted a series of work on the preparation of TiO2–
DNA nanocomposites. In 2003, they synthesized TiO2–DNA
nanocomposites, made up of DNA oligonucleotides attached
to 45 Å TiO2 nanoparticles (Fig. 18).11 The new TiO2–DNA
nanocomposites not only retain the intrinsic photocatalytic
capacity of TiO2 and the bioactivity of the oligonucleotide
DNA (covalently attached to the TiO2 nanoparticle), but also
possess the chemically and biologically unique new property of
a light-inducible nucleic acid endonuclease, opening the door
to a new tool for gene therapy. It has also been investigated that
these TiO2–DNA nanocomposites can be specifically retained in
mitochondria or nucleoli (Fig. 19).20 Moreover, the linking of
biomoleculars with TiO2 was also achieved by using the catechol
moiety, dopamine, which facilitates hole transfer across the
interface, establishing efficient crosstalk between the biomole-
cular and metal oxide nanoparticles.113–115 Excitingly, these TiO2
nanoparticles decorated with DNA oligonucleotides are able to
specifically cleave mutated genomic DNA in a sequence-specific
and inducible manner. The targeting activity is accomplished via
oligonucleotide hybridization to an intracellular organelle’s
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Fig. 17 (A) (a) Electrospinning and calcination was employed to prepare TiO2
nanofibers (TiNFs) coated on a silicon wafer. (b) Biotinylated epithelial-cell
adhesion-molecule antibody (Anti-EpCAM) grafted flat Si/TiNFs alternately patterned
substrate (top), FDA fluorescence micrograph of target cells captured on flat and
patterned substrate (bottom). (c) SEM images of target cells captured on flat Si
substrate (top) and on TiNFs patterned substrate (bottom). (B) CTC enumeration
results obtained from 0.5 mL blood samples of colorectal cancer patients (red
columns) and gastric cancer patients (blue columns). (Obtained from ref. 19).
Fig. 18 Atomic force microscopy image of TiO2–oligonucleotide nanocompo-
sites hybridized with long (phage) DNA. This image covers a 1 mm  1 mm area
and was obtained with a Digital Instruments Multimode AFM operated in air
using a tapping mode. The width of the DNA is known to be 20 Å. (Obtained
from ref. 11).
matching DNA sequence. When photoactivated, a charge separa-
tion occurs within the TiO2 nanoparticle, finally resulting in
knocking off the defective genes effectively, indicating the
therapeutic activity.
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Fig. 19 (A)TEM image of 100 nm thin section of a MCF7/WS8 cells electro-
porated with nucleolus specific nanoconjugate; (a) cell cross section; (b) TEM
detail with two nanoconjugate rich spots. (B) TEM image of a 100 nm thin section
of a PC12 cell electroporated by a mitochondria specific nanoconjugate. (a) cell
with several mitochondria containing nanoconjugate. (b) detailed image of two
adjacent mitochondria. (Obtained from ref. 20).
Though TiO2 nanocomposites linked with DNA oligonucleo-
tides have been demonstrated to be excellent gene knockout
devices, the targeting efficiency and intracellular retention of
TiO2–DNA nanocomposite may be lowered by cellular factors,
such as degradation by intracellular nucleases. In order to solve
these potential problems and improve the stability of hybridi-
zation with target sequences, E. M. B. Brown. et al.116 developed
a peptide nucleic acid (PNA)–TiO2 nanoconjugate. PNAs are
synthetic DNA analogs, resistant to degradation by cellular
enzymes. PNA–TiO2 nanoconjugates can hybridize to target
ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA
under physiological-like ionic and temperature conditions,
enabling rapid, inexpensive, sequence-specific concentration
of nucleic acids in vitro, providing essential benefits for genetic
engineering.
In order to simultaneously detect TiO2–DNA nanocompo-
sites in cells, P. J. Endres et al.117 prepared a Gd(III)-modified
DNA TiO2 semiconducting nanoparticles. The labeled particles
presented to be retained at specific locations inside cells by the
conjugated DNA oligonucleotides hybridizing to intracellular
targets, while being non-invasively monitored by MR imaging,
therefore possibly allowing in vivo detection of tumors. Addi-
tionally, TiO2–DNA nanocomposites can also be labeled with
dyes, to be observed through optical fluorescence microscopy.
K. T. Thurn et al.118 modified the TiO2–DNA nanocomposites
directly with Alizarin Red S (ARS), then transfected ARS-labeled
TiO2–DNA nanoconjugates into PC-3M cells, and successfully
visualized them (Fig. 20).
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Fig. 20 Confocal microscopy image of PC-3M cells transfected with ARS-labeled
TiO2–DNA nanoconjugates. (a) the presence of ARS labeled nanoconjugates
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within the nucleus (1); (b) the presence of ARS labeled nanoconjugates
within in the cytoplasm (2). The nucleus was dyed with Hoechst. (Obtained
from ref. 118).
4. Conclusion and outlooks
Among various oxide semiconductor photocatalysts, TiO2 has
proven to be the most widely used, due to its strong oxidizing
power, long-term photostability, low toxicity and ease of avail-
ability. In order to emphasize the vital importance of TiO2,
which is promising to benefit human health, the primary
studies on biomedical applications of TiO2 during the past
decade, such as PDT for cancer, drug delivery system, cell
imaging, biosensor, and genetic engineering, have been
summarized in this review. Viewing that significant efforts have
been spent on the modifications of TiO2 to improve the efficacy
and expand the applications of TiO2, the numerous techniques
and the applications of TiO2 nanocomposites have been
reviewed in this paper as well. Other biomedical applications
of TiO2 are also beneficial to our health. For example, with the
prolonged life span, the needs for medical implants, which are
used to replace or act as a fraction of the whole biological
structure, have also increased. Owing to its biocompatibility,
TiO2-blasted implants (Astra Techt) have been widely used in
clinic, particularly in orthopedics and dental implant procedures,
giving hope to millions of patients.119–122 The ROS induced by
photoactivated TiO2 can kill not only cancer cells, but also
many pathogenic organisms, such as bacteria and fungi.
Consequently, TiO2 is also considered as effective antimicrobial
drugs.123–125 In addition, TiO2 illuminated by UV could increase
the chemotherapeutic drug accumulation in targeted cancer
cells, inhibiting the multiple drug resistance, which is a major
obstacle to the successful outcome of chemotherapy.126
Furthermore, it has been reported that TiO2 can also be
irradiated by ultrasound, resulting in its application as a novel
material to treat cancer and infection.127–129
In spite of the promising biomedical applications of TiO2,
undoubtedly there are still some challenges that need to be
tackled as soon as possible, in order to make TiO2 benefit
human beings to the upmost in the near future.
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(i) Among all the biomedical applications, only TiO2-blasted
implants have been successfully applied in clinic, especially in
orthopedics and dentistry, while most of the investigations are
confined in vivo and in vitro. Additionally, many scientists have
focused on the modifications of this semiconductor material to
improve the efficacy and extend the applications. The results
demonstrated that some of these modifications are effective,
however, comparative studies between different methods are
rarely performed, so until now there has no consensus on the
best. In the 21st century, Translational Medicine is bound to
become the new concept and the new direction in the medical
field.130 Therefore, it is imperative for investigators to develop
controlled and standardized researches in order to select out
the optimum TiO2 or TiO2 nanocomposites, and then perform
researches in vivo and in vitro, with the reasonable cooperation
of doctors, nurses, and pharmacists. Only in that way can TiO2
ultimately be widely and successfully applied in clinic.
(ii) Compared with the validity, the safety issue of TiO2 is
even more important when accessing whether it could be used
for human beings or not. The ROS generation of illuminated
TiO2 is the basis of its biomedical applications, especially in
PDT and genetic engineering. However, every coin has two
sides, the ROS generation has also been suggested for the
cytotoxicity of TiO2 nanoparticles. Hitherto, previous investiga-
tions have documented that TiO2 might have potential adverse
impact on human health, particularly harming the lung, brain
and skin.131–134 Therefore, the potential toxic effects of TiO2 and its
nanocomposites on human health and the mechanisms under-
lining the processes warrant further studies, and how to reduce the
side effects should also be deeply concerned in the future.
(iii) The application of TiO2 in PDT may be the most
extensively reported researches, however, there are some
limitations for its practical use in the future. Generally speaking,
UV is not the best means of excitation in photodynamic therapy,
with a shallow penetration depth and a low content in the light.
In addition, considering excessive exposure to UV is a main risk
in the development of skin cancer, it is even unsafe to treat
patients using UV.135,136 Until now, great efforts have been
invested to extend the optical absorption of TiO2 to the visible
light region, which provides the basis for the TiO2 in treating
diseases of skin and natural orifices in the future. Compared
with UV and visible light, infrared ray and laser have a deep
penetration depth into tissues, both of which have been applied
in the clinic, especially in cancer treatment.137,138 Whether TiO2
can also be also illuminated by infrared ray and laser is quite
interesting and needs to be demonstrated. If so, we can apply
TiO2 as a promising approach to treat diseases which locate
relatively deeper. To our knowledge, we have not found many
studies about this, so we suggest more researches relevant to
this area to make TiO2 practically useful.
(iv) Some new concepts should be proposed to synthesize a
new class of nanocomposites featuring diagnostic and thera-
peutic properties. These multifunctional nanocomposites have
potential applications in the early diagnosis and effective
treatment of diseases. To our delight, the rapid progress in
science and technology has paved the way for the preparation of
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such multifunctional TiO2 nanocomposites. With cautious
optimism, we look forward to the advent of successful TiO2
nanocomposites, which can find out the targeted cells or
tissues accurately in the early stage of diseases, bind with and
wipe out the targeted cells precisely with high proficiency.
(v) Gene therapy can not only be regarded as a modality in
the treatment of genetic diseases, but also be applied as an
treatment for other diseases, such as cancer and cardiovascular
disease,139–141 which are the two leading causes of death for human
beings. The improvement of gene therapy is largely dependent
on the development of the genetic engineering tools. TiO2 can
be used as a light-inducible gene knock-out device, which may
be an excellent tool for gene therapy. However, more efforts
Published on 28 January 2013 on http://pubs.rsc.org | doi:10.1039/C3CP43938K
need to be made in this new research field, and there is more
work in progress than has been reported.
It is worthwhile to indicate that there is a bright future that
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lies ahead for TiO2, which may make a great contribution to
mankind. However, many researches from different subjects
should co-operate together through reasonable communication
in order to realise more achievements.
Acknowledgements
This work was financially supported by National Natural Science
Foundation of China (30973968), International Cooperation
Project of China (30810103910), New Hundred Talents Program
of Shanghai (XBR2011066).
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