World Journal of Nano Science and Engineering, 2012, 2, 25-31
http://dx.doi.org/10.4236/wjnse.2012.21005 Published Online March 2012 (http://www.SciRP.org/journal/wjnse)
25
Biocompatibility of Porous Spherical Calcium Carbonate
Microparticles on Hela Cells
Yaran Zhang1, Ping Ma2, Yao Wang3, Juan Du1, Qi Zhou1, Zhihong Zhu4, Xu Yang1*, Junlin Yuan1*
1Laboratory of Environmental Sciences, College of Life Sciences, Central China Normal University, Wuhan, China
2Basic Medical College, Xianning University, Xianning, China
3School of Life Sciences, Fudan University, Shanghai, China
4Nanotechnology Institute, College of Physical Science and Technology, Central China Normal University, Wuhan, China
Received November 30, 2011; revised December 12, 2011; accepted January 2, 2012
ABSTRACT
Recently there has been a wide concern on inorganic nanoparticles as drug delivery carriers. CaCO3 particles have
shown promising potential for the development of carriers for drugs, but little research had been performed regarding
their safe dosage for maximizing the therapeutic activity without harming biosystems. In this study, we assessed the
biological safety of porous spherical CaCO3 microparticles on Hela cells. The reactive oxygen species (ROS), glu-
tathione (GSH), carbonyl content in proteins (CCP), DNA-protein crosslinks (DPC) and cell viability were measured.
Results showed that with the exposure concentration increase, ROS and CCP in Hela cells presented a significant in-
crease but GSH contents in Hela cells and cell viability showed a significant decrease respectively compared with the
control. DPC coefficient ascended, but no statistically significant changes were observed. The results indicated that po-
rous spherical CaCO3 microparticles may induce oxidative damage to Hela cells. But compared with other nanomateri-
als, porous spherical CaCO3 appeared to have good biocompatibility. The results implied that porous spherical calcium
carbonate microparticles could be applied as relatively safe drug vehicles, but with the caveat that the effect of high
dosages should not be ignored when attempting to maximize therapeutic activity by increasing the concentration.
Keywords: Calcium Carbonate Microparticles; Hela Cells; Biological Safety
1. Introduction
During the past decades, inorganic nanoparticles as drug
delivery carriers have attracted much attention in modern
pharmaceutical and medication area. Many inorganic ma-
terials, such as calcium phosphate [1], colloidal gold [2],
carbon nanotubes [3], silicon [4], iron oxide [5] and lay-
ered double hydroxide (LDH) [6] have been studied.
In addition to the conventional applications in tooth-
pastes and cosmetics, paper industry, and water treatment
as filtering materials, CaCO3 particles have also shown
potentiality for the development of carriers for drugs [7-
12]. Y. Ueno [10] et al. incorporated betamethasone phos-
phate (BP) and erythropoietin into nano-CaCO3 particles,
which were chemically stable and released very slowly.
Chaoyang Wang [11] et al. loaded amorphous ibuprofen
in the pores of the CaCO3 microparticles which had a
rapider release in the gastric fluid and a slower release in
the intestinal fluid. Caiyu Peng [12] et al. prepared car-
boxymethyl cellulose (CMC)-doped CaCO3 microparti-
cles with an average diameter of 5 μm and coated them
by chitosan and alginate multilayers. These particles could
spontaneously load positively charged doxorubicin (DOX)
molecules whose releasing from the CaCO3 microparti-
cles could be sustained to more than 150 h. Moreover,
the multilayer coating could lower down the release amount
of the loaded DOX within the same incubation time.
With the development of new drug delivery systems,
both the positive and negative sides of nanotechnology
appeared. The need to maximize therapeutic activity may
lead to negative side effects. They could exhibit unex-
pected toxicity to living organisms [13-15]. It had been
mentioned in some reports that CaCO3 microparticles
was relatively safe drug carriers, but little research had
been performed regarding its safe dosage for maximizing
the therapeutic activity without harming biosystems.
Currently, oxidative stress is a well-defined paradigm
to explain toxic effects induced by nanomaterials [16].
The generation of reactive oxygen species (ROS) and the
formation of oxidative stress are the best developed mo-
del to explain the toxic effects of nanomaterials [17].
Oxidative stress refers to a state in which glutathione
(GSH) is depleted while oxidized glutathione accumu- *Corresponding authors.
C
opyright © 2012 SciRes. WJNSE

Y. R. ZHANG ET AL.
26
lates. In addition, DNA-protein crosslinks (DPC) can also
be used as biomarkers for assessing group modifications
on DNA and protein and can be regarded as a signal of
cancer [18,19]. Thus, in the present study, the reactive
oxygen species, DNA-protein crosslinks and cell viabil-
ity were measured to access the biological safety of po-
rous calcium carbonate microparticles on Hela cells.
2. Experimental Detail
2.1. Cell Culture and Exposure to CaCO3
Particles
Hela cells were cultured in RPMI 1640 medium (GIBCO)
supplemented with 10% newborn bovine serum (NBS,
GIBCO), penicillin and streptomycin (each at 100 mU/
mL) at 37˚C in a humidified atmosphere containing 5%
CO2. CaCO3 were dispersed in a complete culture me-
dium with 10% NBS at final concentrations of 50, 100,
200 and 400 μg/mL. Before exposure to cells, all suspend-
sions were sonicated for 40 min. Cells were then inocu-
lated into the medium containing different concentrations
of nanomaterials and incubated at 37˚C in a humidified
atmosphere containing 5% CO2 for 12 h.
2.2. Intracellular ROS Measurement
The formation of intracellular ROS was measured by
monitoring the changes in 2’,7’-dichlorofluorescein-diace-
tate (DCFH-DA, Calbiochem) fluorescence. This mem-
brane permeable dye enters the cell where intracellular
esterases cleave off the diacetate group and the resulting
DCFH retained in the cytoplasm and oxidized to DCF by
ROS. A 10 mM DCFH-DA stock solution (in dimethyl
sulfoxide) was diluted 1000-fold in PBS to yield a 10 µM
working solution. After 12 h exposure to CaCO3 suspen-
sion, cells in 96-well plate were washed twice with phos-
phate buffered saline (PBS, pH 7.4) to remove the
CaCO3 microparticals and then incubated with 150 μL
DCFH-DA working solution at 37˚C for 30 min. Fluo-
rescence was determined using a fluorescence reader
(FLx 800, Multi-Detection Microplate Reader, Bio-Tek
Instrument Inc., USA) with excitation and emission at
485 nm and 528 nm, respectively [20].
2.3. Intracellular GSH Measurement
The concentration of GSH in cells was measured by
5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, Sigma) me-
thod. After 12 h exposure to CaCO3 suspension, cells in
6-well plate were suspended in 1 mL of PBS, frozen at
–20˚C and thawed at 37˚C for three circles. Centrifuged
at 10,000 rpm for 10 min and collected the supernatant.
200 μL of supernatant were mixed with 50 μL of 10%
trichloroacetic acid (TCA). Centrifuged at 10,000 rpm
for 5 min and collected the supernatant. 50 μL of the su-
pernatant was obtained and placed into 96-well plate,
which was reacted with 150 μL of DTNB (60 μg/mL) for
5 min in dark place. The intracellular GSH reacted with
DTNB to give TNB. The total TNB formed was deter-
mined by measureing the absorption at 412 nm with a mi-
croplate spectro-photometer (Power Wave XS, Bio-Tek
Instrument Inc., USA).
2.4. DNA and Protein Crosslink (DPC) Assay
KCl-SDS method was used to detect DNA damage and
the DPC content induced by CaCO3 in the Hela cell line.
After 12 h exposure to CaCO3 suspension, cells were har-
vested by centrifugation at 6000 rpm for 3 min, resus-
pended in 0.5 mL of PBS, and lysed using 0.5 mL of 2%
SDS solution and gentle vortexing. Proteins were pre-
cipitated by KCl solution and digested by proteinase K
(Amrsco). The methods used here were based on Liu and
Collins’s report [21,22] with minor modification. Fluo-
rescence was measured using a fluorescence spectro-
fluorimeter (F-4500 Microplate Scanning Spectropho-
tometer Operator, Bio-Tek Instrument Inc., USA) with
excitation and emission at 350 nm and 450 nm, respect-
tively, for measuring DNA and DPC. The sample DNA
contents were determined quantitatively via a DNA stan-
dard curve generated from calf thymus DNA. The DPC
coefficient was calculated as a ratio of the percentage of
the DNA involved in DPC over the percentage of the
DNA involved in DPC plus the unbound DNA fraction.
2.5. Carbonyl Content in Proteins (CCP) Assay
2,4-dinitrophenylhydrazine (2, 4-DNPH, Sigma) col-
orimetry was employed to detect CCP in Hela cells. Af-
ter 12 h exposure to CaCO3 suspensions, cell proteins
were collected by freezing cells at –20˚C for 1 h and then
melted at 37˚C for three cycles, dispersed into suspension
by minishaker, and then centrifugated at 3000 rpm for 5
min. The method followed Shiou-sheng Chen’s report [23].
The absorbance of the samples at 370 nm was measured
and the CCP was calculated by using Beer’s Law.
2.6. MTT Assay
The MTT assay was applied to investigate the cytotoxic-
ity of CaCO3 particles. Cells were seeded in a 96-well
plate at an initial density of 1 × 104 cells/well, incubated
for 24 h, exposed to different concentrations of materials
for 36 h, and then 20 μL of MTT solution (5 mg/mL) was
added to each well. After incubation at 37˚C in the dark
for 4 h, the medium was removed, 150 μL of DMSO was
added, and the absorbance at 570 nm was measured with
a microplate spectrophotometer (Power Wave XS, Bio-
Tek Instrument Inc., USA).
Cell viability was calculated using the Equation (1):






test control
Cell viability % 100%AA (1)

Y. R. ZHANG ET AL. 27
2.7. Data Analysis
Averages and standard deviations were based on 5 sam-
ples and all tests performed in triplicate. Test data for
statistical treatment were performed by software Origin
version 6.0. Results were evaluated statistically using the
analysis of variance (ANOVA) followed by Tukey test. P
values less than 0.05 were considered to be statistically
significant.
3. Results and Discussion
3.1. Characterization of CaCO3 Particals
The toxicity of a nanomaterial is closely related to its
size, shape and structure [24], so it is necessary to char-
acterize material before the toxicity tests. As it is shown
in the SEM image (Figure 1), the CaCO3 particals is
spherical with a diameter of 2 μm. The surface of the
particles is very rough and consists of large number of
nanometer-sized pores and channels. The specific mor-
phology would offer a unique opportunity to capture bio-
macromolecules such as drugs which can permeate into
the particles, or by the affinity to the carbonate surface,
enabling very high substrate loading.
3.2. ROS Generation
ROS generation and oxidative stress are regarded as the
best developed paradigms to explain the toxic effects of
nanomaterials. ROS is a natural byproduct of the normal
metabolism in cells. A proper amount of ROS could func-
tion as a second messager in the signal transduction of
healthy cells [25]. However, excessive ROS damages bio-
molecules, triggers the apoptosis pathway, and even fur-
ther induces cell death.
Assessment of ROS generation following 12 h of ex-
posure to CaCO3 microparticles (0, 50, 100, 200, and 400
μg/mL) showed that the DCF-fluorescence intensity
which represented the concentration of ROS increased
slowly in a concentration-dependent manner (Figure 2).
When the concentrations of CaCO3 microparticles were
Figure 1. Scanning electronic microscopy (SEM) image of
CaCO3 particals.
Figure 2. DCF-fluorescence intensity of Hela cells after 12 h
exposure to CaCO3 suspensions. (*p < 0.05, compared with
control).
more than 200 μg/mL, the fluorescence intensity was
significantly enhanced (200, and 400 μg/mL, p < 0.05,
compared with the control), which indicated that CaCO3
microparticles may introduce excess ROS which may
cause damage to cells at certain higher concentrations.
The surface chemistry and reactivity of nanometer-
sized pores and channels are important considerations for
ROS production. The interaction of active sites with mo-
lecular dioxygen may possibly lead to the formation of
the additional ROS through dismutation or Fenton chem-
istry [26]. The toxic effects induced by nanoparticles via
a mechanism involving the ROS generation had been
reported in previous studies. Several nanoparticles such
as Fe2O3 [27], TiO2 [28], ZnO [29] and MnO2 [30]
nanoparticles were found to introduce irregular ROS levels
in living organisms, subsequently leading to the toxicity.
However, compared with these nanoparticles CaCO3 need
even higher concentration to introduce excess ROS.
3.3. Intracellular GSH Content
Reduced glutathione (GSH) is the major free sulfhydryl
groups containing molecule in cells and is involved in
detoxification of xenobiotics, removal of ROS and main-
tenance of oxidation state of protein sulfhydryl groups. It
is the key antioxidant presenting in most of the cells.
Cells under normal conditions have natural ability to sca-
venge ROS effects. However, under conditions of excess
ROS production, the natural antioxidant defenses, such
as glutathione (GSH) and antioxidant enzymes may be
overwhelmed [31].
To further explore the relationship between oxidative
stress and CaCO3 concentration, the GSH contents in
Hela cells were studied. As shown in Figure 3, with the
increase of CaCO3 concentration, GSH contents in Hela
cells showed a tendency to ascend at ≤100 μg/mL and

Y. R. ZHANG ET AL.
28
descend >100 μg/mL. While at 200 μg/mL resulted in a
significant decrease (p < 0.05). The data here suggested
that, with ROS increase, the antioxidant defense system
may be triggered and this effect may reach the maximum
at 100 μg/mL. However, antioxidant defense system may
not be destroyed if CaCO3 concentration was <200 μg/mL.
Compared with other nanomaterials, CaCO3 appeared to
have good biocompatibility. For example, with only 10
μg/mL concentration, nano-SiO2 [32] could induce sig-
nificant ROS increase and GSH depletion, and nano-Ag
[33] can produce some harmful effects to the antioxidant
defense system at 50 μg/mL.
3.4. DPC Assay
As shown in Figure 4, In the DPC assay, with the CaCO3
concentration increased, DPC coefficient ascended, but
no statistically significant changes were observed for all
CaCO3 exposures compared with the control.
Exposure of cells to CaCO3 microparticles resulted in
the generation of ROS (Figure 2). These ROS may be
Figure 3. GSH content of Hela cells after 24 h exposure to
CaCO3 suspensions. (*p < 0.05, compared with control).
Concentration (μg/mL)
Figure 4. DNA and protein crosslink assay of Hela cells
after 12 h exposure to CaCO3 suspensions.
localized within a short distance of each other and of the
DNA. Many of ROS, including the extremely reactive
hydroxyl radical, will be generated at high levels within
small discrete regions known as spurs, blobs, and short
tracks [34], which can react with DNA or protein and
create crosslinks between proteins and DNA.
The covalent crosslinking of proteins to DNA was ex-
pected to interrupt DNA metabolic processes such as
replication, repair, recombination, transcription, chroma-
tin remodeling, etc. Unfortunately, the biological cones-
quences of DPCs were hampered by the fact that no
agent exclusively induced these lesions in genomic DNA
[35]. Nonetheless, several studies had reported that the
induction of DPCs by many agents correlated with ge-
netic damage such as sister chromatid exchanges (SCEs),
transformation, and cytotoxicity [36-40]. Thus, DPCs
may contribute to the genotoxic effects. The results indi-
cated that CaCO3 particles with concentration of less
than 400 μg/mL may have no mutagenicity and genetic
toxicity.
3.5. CCP Assay
The measurement of CCP showed that the carbonyl con-
tent in proteins in Hela cells increased in a concentra-
tion-dependent manner (Figure 5), and 400 μg/mL of
CaCO3 treatment resulted in a significant increase of
carbonyl contents in proteins (p < 0.05) which indicated
that CaCO3 microparticles may have harmful effects on
these biomacromolecules at the highest concentrations
tested.
Oxidative stress and reactive oxygen species (ROS)
play an important role in the progression of a number of
human diseases [41]. The generation of ROS may occur
through a large number of physiological or nonphysi-
ological processes, which include their generation as
Figure 5. Carbonyl content in proteins (CCP) of Hela cells
after 24 h exposure to CaCO3 suspensions. (*p < 0.05, com-
pared with control).

Y. R. ZHANG ET AL. 29
by-products of normal cellular metabolism, primarily in
the mitochondria. Excess ROS may damage all types of
biological molecules. Oxidative damages to proteins, lip-
ids, or DNA may all be seriously deleterious and may take
place concomitantly [42].
3.6. Cytotoxicity of CaCO3 Microparticles
Drug delivery systems require biocompatible inorganic
matrices that permit safe retention as well as controlled
drug delivery. CaCO3 appear to fit these conditions.
To evaluate the CaCO3 mcroparticles induced cyto-
toxicity, an MTT assay was conducted to determine the
viability of the treated Hela cells. As shown in Figure 6,
even at the concentration of 400 μg/mL, the viabilities of
the treated cells were 89.6%. Compared with MnO2 [30]
at the same concentration the viabilities of Hela cells
were only 32.45%. The results showed that porous sphe-
rical CaCO3 microparticles had only a little cytotoxicity
and could be applied as relatively safe drug vehicles.
4. Conclusion
Results showed that at the high level of CaCO3 exposure,
DCF-fluorescence intensity and carbonyl content in pro-
teins（CCP） presented a significant increase compared with
the control, DNA and protein crosslinks (DPC) coeffi-
cient were also ascended, but no statistically significant
changes were observed. Furthermore, GSH contents in
Hela cells and cell viability were showed a significant
decrease compared with the control. But compared with
other nanomaterials, CaCO3 appeared to have good bio-
compatibility. Therefore this paper implied that CaCO3
microparticles could be applied as relatively safe basic
materials for drug vehicles, but with the caveat that the
effects of high dosages should not be ignored when at-
Figure 6. Results of MTT assay of Hela cells after 36 h ex-
posure to CaCO3 suspensions. (*p < 0.05, compared with
control).
tempting to maximize therapeutic activity by increasing
the concentration.
5. Acknowledgements
This work was financially supported by a grant from the
National Natural Science Foundation of China (Nos.
50802032, 20877202 and 20907075).
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