The Physical and Biological Properties of NanoTiO<sub>2</sub> Material

doi:10.4236/msa.2011.27125

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Materials Sciences and Applicatio n, 2011, 2, 940-945

doi:10.4236/msa.2011.27125 Published Online July 2011 (http://www.SciRP.org/journal/msa)

The Physical and Biological Properties of

NanoTiO2 Material

Xiao Feng Pang

Institute of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu; P.R.China and In-

ternational Centre of Materials Physics, Chinese Academy of Sciences, Shengyang, P.R.China.

Email: [email protected]

Received May 28th, 2010; revised June 12th, 2010; accepted May 30th, 2011.

ABSTRACT

The physical and biological properties of TiO2 materials including NanoTiO2, micrometerTiO2 and NanoTiO2 tubes have

been studied using scanning electron and infrared spectrometer, X-ray diffraction instrument as well as 3-(4,5- di-

methylthiazol 2-yl)-2,5 diphenyltetrazo-lium bromide (MTT) colorimetric method, respectively. These materials are pre-

pared by chemical deposition and anode oxidation methods, respectively. The sizes of NanoTiO2 are 80 nm and 1000 nm,

respectively, their infra red properties of absorption are differen t, the characteristic peaks of the former are 1271, 1615,

2957 and 3422 cm–1, the latter are 1645 and 2356 cm–1. The NanoTiO2 tubes can be formed by anode oxidation method,

its diameters are between 50 - 100 nm, different NanoTiO2 tubes contain different components of oxygen and titanium. In

MTT experiment we discover the changes of properties of proliferation of the liver and chick embryo fibroblast cells un-

der influences of NanoTiO2 relative to those of the controlled groups, when small NanoTiO2 suspension is added in these

cultivated liquids of cell, but the influence of NanoTiO2 on the proliferation of the person’s liver cell is still very small,

therefore, the toxicities of N anoTi O 2 co nt ai ni ng 80 nm and 1000 nm to these cells are still first score.

Keywords: NanoTiO2, Micrometer TiO2, NanTiO2 Tube, Infrared and X-Diffraction Spectrum, MTT Method, Cell,

Biological and Physical Property

1. Introduction

At present, the Nanomaterials and Nanotechnology and

Nanoinstrument have been greatly developed and applied

extensively in the industry, agriculture, science and

medicine as well as our living. A lot of Nanomaterials

have been made in bulk in factory and were sold in mar-

kets. In such a case it is very necessary to know whether

these Nanomaterials are safe, or speaking, have toxicity

to the person’s and animal’s health [1-4]. This problem

attracts scientist’s and government’ attentions in the

world. Thus studying the safety or toxicity of the Nano-

materials has important significances in sciences and

applications. In this paper we will study the physical and

biological properties of NanoTiO2.

TiO2 is a kind of crystal, in which one titanium (Ti)

atom combines with six oxygen (O) atoms, one oxygen

atom links again three titanium atoms to form an octagon.

Thus, TiO2 belongs in the inclined crystalline system and

is a semiconductor material. In this structure of crystal

the width of forbidden band between the valence band

and conductive band is between 3.0 eV - 3.2 eV. There-

fore, TiO2 can easily absorb the ultraviolet light with

wavelength of 387 nm and414nm. Just so, single crystal

TiO2 can decompose water and other substances under

action of the light. Hence TiO2 is a good material of

photocatalytic materials. Its photocatalytic theorem can

be described as follows. Under action of incident light

the electrons in the valence band transit into the conduc-

tive band (e–), then the holes (h+) occur in valence band

in TiO2. This reaction is represented by





–+

TiO+ hνTiOe ,h. These electrons and holes

occurred are shifted on the surface of TiO2 under action

of an electric-field and can interact further with other

materials through the following oxidation and reduction

reactions:

h+H OH+H

h+OH OH

,

––

O+e O

–

O+H OOOOH+OH, ,

2 OOOHO+HO 2

––

222

OOOH+H O+eH O+OH,

––

H O+eOH+OH,.

The Physical and Biological Properties of NanoTiO Material941

In these processes a great number of free radicals

of ,and are generated, which have

strong ability of oxidation and can interact and react with

plenty of materials, such as chloroform, PCBS, organic

compounds, formaldehyde. Therefore, the NanoTiO2

have extensively applications in medicine, agriculture

and industry, containing chemical engineering, paint,

paper, plastic, rubber, chemical fiber, electric-appliances,

cosmetics and food packaging [4-7].

OH -

OOOOH

For the NanoTiO2, which is different from that of bulk

TiO2due to its scale to become very small, such as, it has

larger rate of surface area versus volume about 70 m2/g,

its surface energy raises, the ratio of number of atom

between the surface and interior increases, the number of

the coordination of atoms in it is lowered, when com-

pared with that in bulk [3-4]. Thus the physical and

chemical activities as well as unstablities of NanoTiO2

are increased correspondingly. Meanwhile, it is also quite

difficultly resolved in water, but when a lot of NanoTiO2

are collected together, they can combines and concretes

each other to form gluey state in water. These gluey

states of the NanoTiO2 are easily spitted, if some elec-

trolytes are added into this solution, or the PH value of

solution is changed. Meanwhile, it can also form some

new bases or chemical compound, such as, TiOH, K2O-6

TiO2 or K2Ti6O13, etc., with water molecules or ions,

involving OH, NH2, COOH, C = O, through attraction

interaction between the particles with charges on the

surface. Therefore, it is very necessary to investigate

in-depth the physical and biological properties of Nano-

TiO2.

2. Experimental Method

We prepare three kinds of TiO2 materials containing the

NanoscaleTiO2, microscale TiO2 and NanoTiO2 tubes by

using chemical deposition and anode oxidation methods,

respectively. Their features of structures are measured

using Scanning Electron Spectrometer (SEM), respec-

tively, its infrared properties of absorption and

X-diffraction spectra are measured using a Nicolet Nexus

670-FT-IR spectrometer with resolution of 4 cm–1 and

X-ray diffraction spectrometer, respectively. The prolif-

eration and toxicity of NanoTiO2 to the person’s liver

and chick embryo fibroblast (CEF) cells are determined

by MTT colorimetric method [8].

So-called MTT colorimetric method [8] is just a very

effective way checking the states of activity and prolif-

eration of the cells. In this way the coloration substance

used is MTT. The MTT is an abbreviation of 3-(4,

5-dimethylthiazol 2-yl)-2, 5 diphenyltetrazolium bromide,

which is a sort of dye accepting hydrogen atom. In the

mitochondrion of cell, the externally applied yellow te-

trazolium salt 3-(4, 5-dimethylthiazol 2-yl)-2, 5 di-

phenyl-tetrazolium bromide (MTT, Amersco) will be

reduced and become further, under the action of dehy-

drogenase of amber acid, as a blue insoluble formazan

form, which is eventually deposited in the cell after this

reaction. But the dead cell has not this function and effect.

The dimethylsulfoxide (DMSO) added can resolve the

blue insoluble formazan. The quantity of the blue insolu-

ble formazan produced after the resolution is propor-

tional with the number of cell participated in this process.

Thus we can determine indirectly the number of the cells

through measuring the strength of absorption of the light

with determinate wavelengths in this case. The strength

of light can be measured and collected by enzymic im-

munoassay instrument and spectrophotometer. Then we

can determine the number of proliferation and activity of

cells or of biological factors in the cells, thus we can as-

sess the safety or toxicity of NanoTiO2 to the cell, etc.,

according to the toxicology. The advantages of the me-

thod measuring this security or toxicity is fast and accu-

rate, and have higher sensitivity and very good repeat-

ability. Therefore, we here utilize this method to assess

the influences of NanoTiO2 on the proliferation states of

person’s liver cell (L-20) after it interacts with the Nano-

TiO2 by traditional biological technique. The experimen-

tal process in this method is as follows.

2.1. Cell Growth

The person’s liver and primary chick embryo fibroblast

(CEF) cells are prepared, their secondary cultures are

grown in the 5% CO2 enriched incubator with tempera-

ture of 37˚C. The person’s liver and CEF cells were

grown in RPMI1 640(Hyclone,American) supplemented

with 5% fetal calf serum (FCS, Biological Industries,

BaiAn, China). Microscopic inspection to them verifies

that the cells are not contaminated from third passage,

and so forth.

2.2. The Nanotio2 is Added into the Group

Solutions

In our experiments, 4 × 105 cells per well are seeded in

60-well micro-culture plates and allowed to continually

grow. The 60-well cells are added into the 100 μL/well

foster liquids containing the fetal calf serum in which the

1 mL/250 mL insulin liquid is included. The NanoTiO2s

are added into these wells to study the influences of the

NanoTiO2 on the proliferation behavior of the person’s

liver and CEF cells. In this experiment these cells are

separated as controlled and experimental groups, which

are all 30 well. The NanoTiO2s are assigned in the fol-

lowing rule. (a) the first or controlled group is 30 wells,

which the 15 mL/ well foster liquid without the fetal calf

serum is added into; (b) the second or experiment group

1 has 10 wells, in which the 5 μL NanoTiO2 suspension

The Physical and Biological Properties of NanoTiO Material

942 2

and 10 μL foster liquid without the fetal calf serum are

added, the concentration of NanoTiO2 achieves 43

μg/mL; (c) the third or experiment group 2 contains 10

wells, in which there is 10 μL NanoTiO2 suspension and

5uL foster without fetal calf serum, the concentration of

the NanoTiO2 achieves 86 ug/mL; (d) the fourth or ex-

periment group 3 contains 10 wells, in which 15 uL

NanoTiO2 suspension is added, the concentration of the

NanoTiO2 achieves 129 μg/mL. The above four groups

are all placed into the CO2 enriched incubator with 37˚C

to develop about 24 hours. The cell proliferation was

evaluated after 24 h.

2.3. Measurement of the Proliferation of the

Person’s Liver Cell

We observe and measure the proliferation of the person’s

liver cell (L-20) in above conditions by the MTT method

[8-10] and calculate the proliferation rate of cell by using

these experimental data.. In the calculation we should

firstly measure the increased values of mitochondrial

dehydrogenase activity as the number of cells increases.

In such a case, 100 uL/ well MTT solution is prepared in

PBS (5 mg/mL) and further diluted (10%v/v) in RPMI

1640. The cell growth medium is aspirated. In this case

the 100 μL of MTT solution are added into each well in

the above four groups. The cells are then further incu-

bated for 4 h at 37˚C Excess of MTT solution is removed,

after this the 100 μL/well DMSO is added into dissolve

the blue crystals formed in the cells. The absorption

strength of the DMSO solution and values of optical

density (OD) of each well to the incident light with wa-

velength of 570 nm for the controlled and experimental

groups are measured spectrophotometrically by DG3022

enzymicimmunoassay instrument, respectively. Finally

we can find out the cell proliferation rate (CPR) for the

person’s liver cell by using the experimental data and the

formula: CPR = [(Fexp – Fcon)/Fcon] × 100%, here Fexp is

the value of optical density of experimental group, Fcon is

the value of optical density of controlled group.

3. Experimental Results and Discussion

3.1. Properties of Microscale TiO2 and NanoTiO2

Tubes

The structures of two kind of TiO2 using SEM are shown

in Figure 1(a) and (b), respectively. This figure shows

that the NanoTiO2 is 80nm, other is 1000nm. We collect

their spectra of infrared absorption by 670 FT-IR instru-

ment, which is shown in Figure 2. We see from this fig-

ure that their infrared absorptions are different not only

the strengths and frequencies of peaks of absorption but

also the amounts of peaks, the new peaks at 2422, 2956

and 1271 cm–1 occur in the NanoTiO2, they are the char-

(a)

(b)

Figure 1. The images of SEM of TiO2 powder. (a)The image

of NanoTiO2 powder; (b) The image of micrometerTiO2

powder.

materal 2

2356

1271

1645

materal 1

2957

3422

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Absorbance

1000 2000 3000

cm–1

Figure 2. The spectra of infrared absorption for NanoTiO2

(material1) and micro TiO2 (material 2).

acteristic peaks of NanoTiO2. However, in micrometer

TiO2 there is only a small new peak at 2355 cm–1. Only

the peak at 1645 cm–1 is same for The NanoTiO2 and

The Physical and Biological Properties of NanoTiO2 Material

943

micrometerTiO2. Obviously, this is due to the changes of

structure and sizes of molecules of TiO2, the new peaks

in Figure 2 indicate that there are many new radicals or

base groups in NanoTiO2. This is just the feature of Na-

nomaterials.

and titanium are shown in Figure 5, where Figure 5a is

the result obtained from solution of phosphoric acid and

hydrofluoric acid in which the weight ratio of oxygen

and titanium are 17.54% and 82.46%, respectively, their

volume ratios are 38.91% and 61.09%, respectively,

Figure 5b is the result obtained from solution of hydro-

fluoric acid in which the weight ratio of oxygen and

titanium are 24.22% and 75.78%, respectively, their

volume ratios are 48.90% and 51.10%, respectively.

These results manifest that not only different NanoTiO2

tubes can be obtained by different conditions in the an-

ode oxidation but also the products of NanoTiO2 tubes

manufactured by this method are very pure and contain

not impurities.

We also use SEM to measure the feature and size of

NanoTiO2 tubes formed by anode oxidation method,

which are shown in Figures 3-4, which are born from the

surface of titanium alloy in the solutions of hydrofluoric

acid as well as phosphoric acid and hydrofluoric acid,

respectively. From these figures we see clearly the oc-

currences of a great number of Nanotubes, which are

distributed densely and nonuniformly on the surface,

their sizes of diameter are different and between 50 nm

and 100 nm. On the other hand, we determine the solubility of the

NanoTiO2 in some liquids . Experiments show that the

NanoTiO2 is insoluble in the unorganic ( as D-Han’k

liquid) and organic (as DMSO) solutions and alcohols

(95%) and t1 640 foster liquid without fetal calf serum,

etc.

In the meanwhile, we measure the X-diffraction spec-

trum of NanoTiO2 tubes using X-ray diffraction instru-

ment. These results for different components of oxygen

3.2. The Biological Properties of the NanoTiO2

We measured the values of optical density (OD) in

MTT experiment of liver cell and changes of prolifera-

tion of CEF by MTT method [8].

a) The OD values for the controlled group (30 wells)

obtained are as follows,

0.37, 0.31, 0.41, 0.46, 0.54, 0.45, 0.44, 0.45, 0.42,

0.43, 0.50, 0.43, 0.51, 0.54, 0.41, 0.39, 0.52, 0.47, 0.46,

o.31, 0.54, 0.38, 0.42, 0.47, 0.34, 0.41, 0.42, 0.44, 0.46.

Therefore, the average value of the OD of the con-

trolled group is 0.43 + 0.05

b) The OD values for the experimental groups are as

follows. The OD values of the experimental group 1 (10

wells) are 0.41, 0.44, 0.41, 0.62, 0.35, 0.42, 0.77, 0.49,

0.54, 0.45. Thus, its average value of OD is 0.49 + 0.09,

its PRC value is +13.95%.

Figure 3. The images of SEM of NanoTiO2 tube formed in

the solution of hydrofluoric acid.

The OD values of the experimental group 2 (10 wells)

are 0.44, 0.59, 0.40, 0.56, 0.40, 0.46, 0.32, 0.20, 0.43,

0.35. Then its average value of OD is 0.42 + 0.08, its

PRC value is –2.32%.

The OD values of the experimental group 3 (10 wells)

are 0.45, 0.44, 0.42, 0.46, 0.46, 0.40, 0.45, 0.58, 0.35,

0.32.Then its average value of OD is 0.43 + 0.05, its PRC

value is 0.0%.

3.3. Discussion of the Results

From above results we can find out the average value of

OD for the experimental group which is 0.45 + 0.07, its

CPR value is +0.02%. This shows that the NanoTiO2 in

fluences not basically the proliferation of the person’s liver

cell (L-20).

Figure 4. The images of SEM of NanoTiO2 tube formed in

the solution of phosphoric acid and hydrofluoric ac id.

The Physical and Biological Properties of NanoTiO Material

944 2

(a)

(b)

Figure 5. X-diffraction spectra of NanoTiO2 tubes with different components of oxygen and titanium in different conditions.

In order to verify above result we further study and

measure the changes of the proliferation behavior of the

chick embryo fibroblast (CEF) cell by above MTT me-

thod [8-10] , when the NanoTiO2 of 80 nm and the TiO2

of 1000 nm are added into these cells, respectively. We

obtain their OD values and find correspondingly out the

relative cell proliferation rate (RCPR) of chick embryo

fibroblast by the formula, (Fexp/Fcon) × 100%. The OD

values and corresponding RCPR values and evaluation of

their toxicity scores in the cases of interaction times of

24 hours and 48 hours are shown in table 1 and 2 for the

six samples in the two groups, respectively, where 100%

group denotes this case in which the NanoTiO2 samples

are not added into the water, the 50% group denotes that

the 50% water is added into the sample, 25% group de-

notes that 75% water is added into the sample. From the

table 1 and 2 we see that the degrees of toxicity for the

chick embryo fibroblast cell are all first score. This

shows that the toxicities of both the NanoTiO2 and TiO2

of 1000 nm to the cells are all lower. Therefore, the Na-

noTiO2 is safe in the living systems.

4. Conclusions

In this paper we investigated the physical and biological

properties of TiO2 materials including NanoTiO2, microme-

terTiO2 and NanoTiO2 tubes using scanning electron and

infrared spectrometer and X-ray diffraction instrument as

well as MTT colorimetric method, respectively.

The Physical and Biological Properties of NanoTiO Material945

Table 1. The OD values, Relative cell proliferation Rate

(RCPR) and toxicity Score of CEF cell after 24 hours.

Groups 24hours

groups OD value(X+S)RCPR (%) Score

100% T1 0.298±0.006, 98.3 1

50% T1 0.298±0.006, 98.8 1

25% T1 0.2995±0.007 99 1

100% T2 0.276±0.007 91.4 1

50% T2 0.286±0.003 94.7 1

25% T2 0.294±0.009 97.4 1

Control

group 0.302±0.009 100 0

Table 2. The OD values, Relative cell proliferation Rate

(RCPR) and toxicity Score of CEF cell after 48 hours.

Groups 48hours

groups OD value(X+S)RCPR (%) Score

100% T1 0.309±0.01 90.6 1

50% T1 0.320±0.003 93.8 1

25% T1 0.327±0.008 95.9 1

100% T2 0.307±0.007 90.0 1

50% T2 0.319±0.003 93.5 1

25% T2 0.325±0.008 995.3 1

Control

gronp 0.341±0.009 100 0

These materials used are prepared by chemical deposi-

tion and anode oxidation methods, respectively. The siz-

es of NanoTiO2 are checked using scanning electron

spectrometer and 80nm and 1000 nm, respectively, di-

ameters of the NanoTiO2 tubes are between 50 - 100 nm.

The infrared properties of absorption for the NanoTiO2

and micrometerTiO2 are different, the characteristic

peaks of the former are 1271 cm–1, 1615 cm–1, 2957 cm–1

and 3422 cm–1, the latter are 1645 cm–1 and 2356 cm–1.

Meanwhile, we can obtain different components of oxy-

gen and titanium in the NanoTiO2 tubes in different con-

ditions by the anode oxidation method. In MTT experi-

ment we discover the changes of properties of prolifera-

tion of the liver and chick embryo fibroblast cells under

influences of NanoTiO2 relative to those of the controlled

groups, when small NanoTiO2 suspension is added in

these cultivated liquids of cell, but the influence of Na-

noTiO2 on the proliferation of the person’s liver cell is

still very small, therefore, the toxicities of NanoTiO2

containing 80 nm and 1000 nm to these cells are still first

score.

5. Acknowledgements

The authors would like to acknowledge the National

“973” project of China for financial support (grate No:

2007CB936103)

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