Supporting Information
Different Inhibitory Effect and Mechanism of
Hydroxyapatite Nanoparticles on Normal Cells and
Cancer Cells In Vitro and In Vivo
Yingchao Han,† Shipu Li,*,†, ‡ Xianying Cao,†,§ Lin Yuan,†,# Youfa Wang,† Yixia Yin,† Tong Qiu,†
Honglian Dai† and Xinyu Wang†
†Biomedical Materials and Engineering Center, Wuhan university of Technology, 122 Luoshi
Road, Wuhan, 430070, P.R. China
‡State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, 122
Luoshi Road, Wuhan, 430070, P.R. China
Corresponding Author
* Shipu Li, e-mail: lishipu46@126.com
Present Addresses
§ Key Laboratory of Tropic Biological Resources of Ministry of Education, Hainan University,
58, Renmin Avenue, Haikou, 570228, P.R. China
# College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Renai Rd.,
Suzhou, 215123, P.R. China
1
Methods
Preparation of HAP particles with averaged particle size of 170 nm. 170nm-HAP particles
were prepared as the following32. The saturated Ca(OH)2 aqueous solution was rapidly poured
into Ca(H2PO4)2·H2O aqueous solution while intensively stirring. BSA with a concentration of 4
g L-1 was added into the suspension and then the turbid suspension was intensively stirred for a
few minutes. After irradiated for 8min by a high-intensity ultrasonic probe, the transparent
colloidal precursors were obtained. After freeze-drying process, the freeze-dried precursors were
calcined at 600°C for 1h to get HAP particles. Before interaction with cells, the powders were
ultrasonically dispersed in distilled water with addition of sodium heparin.
Preparation of HAP particles with averaged particle size of 289 nm. The preparation
process of 289nm-HAP particles was shown as the following33. First, Ca(NO3)2·4H2O,
C6H8O7·H2O and (NH4)2HPO4 were completely dissolved in deionized water. The pH value of
solution was adjusted to 2–3 by HNO3. Then the reaction beaker was heated at 70–80 °C under
vigorous stirring. After the gel formed, it was put into drying cabinet at 110–120 °C immediately
to get dried gel. Finally, the dried gel was calcined at high temperature to get HAP particles.
Before interaction with cells, the powders were ultrasonically dispersed in distilled water with
addition of sodium heparin.
Preparation of Eu-nHAP. The Eu-nHAP was synthesized by co-precipitation method.
According to the molar ratios of Eu/(Eu+Ca)=2% and (Eu+Ca)/P=1.67, the aqueous solution of
Ca2+ and Eu3+ by mixing Ca(NO3)2·4H2O and Eu(NO3)3·6H2O aqueous solutions was poured
into (NH4)2HPO4 aqueous solution under vigorous stirring. The pH value of the suspension was
adjusted to about 10 by NH3·H2O and then the precipitation was obtained by centrifugation and
2
washed by distilled water. After wash thrice, the precipitation was re-dispersed in distilled water
and treated by same process to the preparation of nHAP to get Eu-nHAP.
Results
Characterization of HAP particles.
The XRD patterns (Figure S1) demonstrate the polycrystalline structures assigned to HAP.
Moreover, XRD pattern of nHAP shows the broadened diffraction peaks and the merged broad
peak of three major peaks (211, 112, 300), indicating the nanocrystalline nature (calculated
crystalline sizes of 39.5 nm in D002 and 13.3 nm in D310) and the lower degree of crystallinity
(0.52 of Xc). The 170nm-HAP also shows some nanosize nature (calculated crystalline sizes of
60.7 nm in D002 and 25.8 nm in D310) with enhanced crystallinity (0.79 of Xc), although
diffraction peaks are enhanced a little. The sharp and distinct diffraction peaks of 290nm-HAP
verify the well-crystallization (0.86 of Xc) with the loss of nanosize nature.
Figure S1. XRD patterns of nHAP (a), 170nm-HAP (b) and 290nm-HAP (c).
FT-IR spectra (Fig. S2) also display the characteristics of HAP, for example, the vibrational
bands at 962 cm-1, 474 cm-1, 1091 and 1049 cm-1, 602 and 571 cm-1 attributed to the ν1, ν2, ν3, ν4
vibrational bands of phosphate group; the small sharp peaks at 3571 cm-1 and 631 cm-1 assigned
to the O−H stretching and bending vibrations in the crystal structure of HAP. Moreover, the
3
strength of peaks at 3571 cm-1 and 631 cm-1 is increased gradually from nHAP to 170nm-HAP
and 290nm-HAP, also indicating the enhanced crystallinity degree of HAP.
Figure S2. FT-IR patterns of nHAP (a), 170nm-HAP (b) and 290nm-HAP (c).
TEM image (Fig. S3a) shows that nHAP consists of short rod-like particles of 10–20 nm ×
40–70 nm and similar spherical particles of 10–30 nm. 170nm-HAP (Fig. S3b) mainly comprises
similar spherical (100-150 nm) and short rod-like (80-120 nm×150-220 nm) particles. 290nmHAP (Figure S3c) shows a similar spherical shape with a diameter of 0.2-0.3 μm.
Figure S3. TEM images of nHAP (a), 170nm-HAP (b) and 290nm-HAP (c).
The specific surface areas of nHAP, 170nm-HAP and 290nm-HAP are determined by the
BET-N2 adsorption method to be 139.7 m2/g, 10.9 m2/g and 3.9 m2/g respectively. According to
4
the formula, d=6/ρ∙s, the diameters are calculated to be about 13.6 nm, 174.5 nm and 487.7 nm
respectively, which are well consistent with the TEM results.
Adsorption of BSA, DNA and RNA on HAP.
Table S1 Adsorption of BSA, DNA and RNA on HAP ( x ± SE, n=3).
Average size of HAP (nm)
Amount
(g-1 HAP)
60
170
290
BSA (mg)
55.47±1.69***
42.40±2.00*
33.33±1.51
DNA (g)
1.12±0.02***
1.36±0.01***
0.43±0.01*
RNA (mg)
4.93±0.20***
12.87±1.07***
8.03±0.33***
* and *** represent p<0.05 and p<0.001, respectively.
Effect of nHAP on the activity of SDH and SOD.
Table S2 The effect of nHAP on the activity of succinate dehydrogenase (SDH) and
superoxidase dismutase (SOD) in mitochondria ( x ± SE, n=3-5).
Enzymic activity
(U/mg protein)
nHAP concentrations (g L-1)
0
0.07
0.14
0.28
0.56
SDH
5.13±0.24
4.25±0.12*
4.05±0.12*
3.78±0.22**
2.79±0.21**
SOD
4.26±0.12
ND
4.21±0.05
4.15±0.11
4.15±0.25
ND means not determined. * and ** represent p<0.05 and p<0.01, respectively.
5