Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells

Materials Science and Engineering C 67 (2016) 453–460

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells Xinhui Cui a, Tong Liang b, Changsheng Liu a,b, Yuan Yuan b,⁎, Jiangchao Qian a,⁎ a b

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China

a r t i c l e

i n f o

Article history: Received 28 August 2015 Received in revised form 19 April 2016 Accepted 6 May 2016 Available online 07 May 2016 Keywords: Hydroxyapatite nanoparticles Particle properties Cancer cell Cytotoxicity Uptake Intracellular calcium concentration

a b s t r a c t Three types of hydroxyapatite nanoparticles (HAPNs) were synthesized employing a sonochemistry-assisted microwave method by changing microwave power (from 200 to 300 W) or using calcination treatment: L200 (200 W, lyophilization), L300 (300 W, lyophilization) and C200 (200 W, lyophilization & calcination). Their physiochemical properties were characterized and correlated with cytotoxicity to human gastric cancer cells (MGC803). The major differences among these HAPN preparations were their size and specific surface area, with the L200 showing a smaller size and higher specific surface area. Although all HAPNs inhibited cell proliferation and induced apoptosis of cancer cells, L200 exhibited the greatest toxicity. All types of HAPNs were internalized through energy-dependent pathways, but the L200 nanoparticles were more efficiently uptaken by MGC80-3 cells. Inhibitor studies with dynasore and methyl-β-cyclodextrin suggested that caveolae-mediated endocytosis and, to a much lesser extent, clathrin-mediated endocytosis, were involved in cellular uptake of the various preparations, whereas the inhibition of endocytosis was more obvious for L200. Using fluorescein isothiocyanate-labeled HAPNs and laser-scanning confocal microscopy, we found that all forms of nanoparticles were present in the cytoplasm, and some L200 HAPNs were even found within nuclei. Treatment with all HAPN preparations led to the increase in the intracellular calcium level with the highest level detected for L200. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Hydroxyapatite [HAP, Ca10(PO4)6(OH)2], the main inorganic constituent of bone and teeth, has been extensively used as an artificial bone substitute [1,2]. Recently, hydroxyapatite nanoparticles (HAPNs) have been extensively investigated as delivery vehicles for nucleic acids, proteins or drugs in biological systems [3–5], and HAPNs-based nanomedicines have been developed to improve targeted delivery for cancer therapy [6–9]. Interestingly, HAPNs themselves have been reported to produce cytotoxicity against various human cancer cells, such as hepatoma, gastric cancer, colon cancer, melanoma, breast cancer, and glioma cells [10–13]. They affected cancerous cells specifically but spare healthy matching control cells, as reported between human hepatocellular carcinoma HepG2 cells and normal liver L-02 cells [14], osteosarcoma (MG63) versus osteoblasts [15], respectively. Such an anti-cancer activity has been demonstrated to be related to induction of mitochondrion-mediated apoptosis [10], and the generation of intracellular reactive oxygen species (ROS) [11,13]. It is commonly accepted that basic physicochemical properties of nanoparticulate materials determine cellular uptake and cytotoxicity ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Yuan), [email protected] (J. Qian).

http://dx.doi.org/10.1016/j.msec.2016.05.034 0928-4931/© 2016 Elsevier B.V. All rights reserved.

of nanoparticles. Previous works demonstrated that HAPNs induced a size-dependent cytotoxicity in HepG2 cells [14], and nano-HAP particles with different morphology exerted a greater inhibitory proliferation effect on melanoma cells than micro-sized particles [12]. In spite of these studies, which properties determine cell-material interactions and cytotoxicity of HAPNs in cancer cells, whether arising from cellular uptake and increased cytoplasmic calcium load, are still poorly understood. Reports have shown that size, shape, surface area, and charge of HAPNs have significant effects on biological responses [16,17]. For example, HAPNs with diameters of ~20 nm had the best effect on promotion of cell growth and inhibition of cell apoptosis of osteoblast-like cells than the 80 nm diameter and micro-sized particles [18], high surface area may increase cell-particle interaction and influence ROS generation [19]. The study of Müller et al. showed that particle agglomeration was correlated with particle uptake and cytotoxicity in human macrophages [20]. Motskin et al. proposed the cytoplasmic calcium load to be the cause of cell death, although uptake of HAPNs was governed by the balance of multiple characteristics [21]. Therefore, the biological behavior of HAPNs could be very complicated and it is still not possible to reliably break down the anti-cancer activity of HAPNs to their physicochemical properties, as the properties of nanoparticles are strongly interconnected, and different cell types have different function. Information gained from a broad variety of HAPNs with differential cytotoxicity

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and biological responses in cancer cells will contribute to a more comprehensive understanding of how physicochemical properties relate to biological behavior. In contrast to the conventional chemical precipitation process for synthesis of HAPNs, the sonochemistry-assisted microwave method involves in situ conversion of microwave energy into heat, can thus increase the reaction kinetics and reduce duration of the reaction time [22–24]. In this study, we altered the synthesis condition of the sonochemistry-assisted microwave method to produce three different HAPN preparations. Their physiochemical properties were characterized using transmission electron microscope (TEM), X-ray diffraction (XRD), Raman scattering and dynamic light scattering (DLS). The in vitro investigation was conducted using human gastric cancer cells MGC80-3 since they were more sensitive than other two cancer cells upon HAPN exposure [25]. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Annexin V-FITC/PI double staining, and intracellular calcium were used to analyze the cytotoxic effects of different HAPNs. With FITC (fluorescein isothiocyanate) tagging, particle distribution was observed using confocal laser scanning microscopy (CLSM). Furthermore, we investigated the mechanism of particle uptake in MGC80-3 cells using flow cytometry.

2. Materials and methods 2.1. Preparation and labeling of HAPNs HAPNs were synthesized from Ca(NO3)2·4H2O and (NH4)2HPO4, by sonochemistry-assisted microwave method developed in our lab in a program-controlled sonochemistry-assisted microwave reactor with a reflux cooling condenser (XO-SM50, Nanjing Xianou Instrument Manufacturing Co., Ltd., China) [24]. Single microwave was exerted in the experiment, with the microwave power set at 200 or 300 W. After a reaction time of 30 min, products were collected, washed, dried by lyophilization for 24 h, and then calcined at 550 °C for 5 h if necessary. To visualize the intracellular distribution of nanoparticles, HAPNs were labeled by FITC as previously described [14]. A mixture of HAPNs (0.1 g) and 3-aminopropyltriethoxysilane (AMPTES) (20 mL) was mixed in 100 mL of anhydrous ethanol and stirred at 74 °C for 3 h. Then, FITC (0.05 g) was added and continued to react at 74 °C for another 8 h in the dark. The labeled nanoparticles were centrifuged and rinsed with anhydrous ethanol and deionized water in turn until no visible FITC on the HAPN surfaces, and FITC-labeled HAPNs (FITC-HAPNs) were finally obtained by lyophilization for 24 h. Labeled particles were ground before use.

2.2. Characterization of HAPNs The zeta potential of HAPNs (100 μg mL−1) was determined in deionized water or complete DMEM medium supplemented with 10% serum (cDMEM) using a Zetasizer Nano ZS system (Malvern, Worcestershire, UK) equipped with a standard 633 nm laser. Particle size and morphology were observed by transmission electron microscope (TEM) with a JEOL JEM-2100F electron microscope (Tokyo, Japan) at an acceleration voltage of 120 kV. Phases and structural properties were characterized by X-ray diffraction (XRD) on a Bruker D8 Focus diffractometer (Karlsruhe, Germany) using Cu Kα radiation. The molecular structure and the presence of functional groups were studied using a LabRAM HR Raman spectrometer (Horiba Jobin Yvon, Paris, France), equipped with a deeply depleted thermoelectrically cooled CCD array detector and a high-grade Leica microscope (long working distance objective: 50 ×). The excitation source was 514 nm lines of Ar+ laser. The specific surface area was determined by the Brunauer-Emmett-Teller (BET) method using the data between 0.05 and 0.3.

2.3. Cells and cell culture Human gastric cancer cells (MGC80-3) and normal liver cells (L-02) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (Gibco, Carlsbad, USA) supplemented with 10% newborn calf serum (NBCS) and 1% antibiotics (100 units mL−1 penicillin and 100 μg mL− 1 streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. 2.4. Cell viability assay The viability of cells exposed to HAPNs was assessed by the MTT assay. Cells were seeded into 96-well plates at a density of 3 × 103 cells per well and allowed to attach for 24 h. Then, the medium in the wells was replaced with fresh medium containing HAPNs (31– 1000 μg mL−1) and the cells were treated for 72 h. Thirty microlitres of MTT solution (5 mg mL−1) was added to each well and the cells were further incubated for another 4 h at 37 °C. Then, culture medium was removed and 200 μL of dimethylsulphoxide (DMSO) was added to each well. Plates were incubated for 10 min at room temperature to dissolve formazan crystals, and optical density at 492 nm was detected using a M2 microplate reader (Molecular Devices, Sunnyvale, USA) and cell viability was expressed as percentage of the control cells without HAPN treatment. 2.5. Detection of nuclear morphology Cells were treated by different HAPN preparations at a concentration of 500 μg mL−1. After 48 h, cells were rinsed twice with PBS and fixed by 3.7% paraformaldehyde for 20 min. The cell nucleus was then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 25 °C. The nuclear morphology was observed under a TE2000-U inverted fluorescence microscope (Nikon, Tokyo, Japan) with the excitation and emission wavelength of 330–380 nm and 430–460 nm, respectively. 2.6. Annexin V-FITC/PI apoptosis assay Annexin V-FITC/PI assay kit (Kaiji Bioengineering Institute, Nanjing, China) was used to identify normal, apoptotic, and necrotic cells. MGC80-3 cells were seeded in 6-well plates, and treated with 250 or 500 μg mL−1 of HAPNs for 48 h. After incubation, the Annexin V and PI staining were performed as per manufacturer's instructions. Flow cytometry analysis was carried out using FACS Aria™ flow cytometer (Becton Dickinson, USA) at an emission wavelength of 530 nm for Annexin V and 585 nm for PI. The gated MGC80-3 cells were then plotted for Annexin V-FITC and PI in a 2-way dot plot to assess percentage of apoptotic MGC80-3 cells. 2.7. Cellular uptake and localization of HAPNs Cellular uptake of HAPNs was determined via flow cytometric measurement. The side scattering of light in flow cytometry is proportional to the cellular granularity and therefore has been proposed as a measure for cellular nanoparticle uptake [26]. MGC80-3 cells were seeded in 6well plates, and treated with 250 μg mL− 1 of HAPNs for 1, 2, 3, 4, 6, and 8 h at 37 °C or 4 °C, respectively. After exposure, cells were detached by trypsin, washed and resuspended in PBS. Side scatter (SSc) was measured by flow cytometry in 1 × 104 cells per sample. The mean SSc for each sample was calculated in FlowJo software ver. 7.6.2 (Tree Star, USA) and normalized against the vehicle control. To analyze the uptake pathways, cells were pre-incubated with specific inhibitors: methyl-β-cyclodextrin (MβCD) or dynasore (Sigma-Aldrich, USA) diluted in serum free medium at the concentration of 10 mM or 80 μM, for 30 min or 1 h, respectively. Thereafter, cells were

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treated with HAPNs (250 μg mL−1) for 3 h, and processed for uptake determination as described above. To observe intracellular distribution of HAPNs, MGC80-3 cells (2 × 104) were seeded in 20 mm glass bottom culture dishes, incubated with different FITC-HAPN preparations at a concentration of 500 μg mL− 1. After 20 h treatment, the medium was aspirated and cells were washed twice with PBS to eliminate floating HAPNs. Then, the cells were stained with DAPI and observed using an A1R confocal laser scanning microscopy (Nikon, Tokyo, Japan). All images were taken under a 60 × oil-immersion objective. DAPI was excited with a 404-nm laser and the resulting fluorescence was collected between 425 and 475 nm. Green luminescent emissions from FITC were excited at the wavelength of 488 nm, and the emission wavelengths were ranged from 500 nm to 550 nm. There was no interference between these two channels. The scanning mode was in sequential frame.

The XRD patterns of different preparation of HAPNs (Fig. 2A) showed the same characteristic peaks at 2θ regions of 26°, 29°, 32°– 34°, 40°, and 46°–54°, which are consistent with that of standard hydroxyapatite (JCPDS, No. 09-0432), indicating the three different preparations were phase pure hydroxyapatite with no obvious variation in their phase composition. Raman spectra of the HAPNs were also taken. As shown in Fig. 2B, the strongest Raman active υ1 PO4 mode appeared in the spectrum of all samples at 958 cm−1, with the most intense peak observed for C200; other weak bands observed at 426 cm−1, 582 cm−1, and 1042 cm−1 were assigned to υ2, υ4, and υ3 bending modes of O\\P\\O in −1 due to the symmetric PO3− 4 , respectively. A weak band at 3571 cm stretching mode of the OH− was not observed in the spectra of L200 and L300, probably because of poor crystallization of the crystallites.

2.8. Measurement of intracellular calcium ion concentration

Cells were incubated with the three different HAPN preparations for 72 h, and cell viability decreased significantly as a function of dosage levels in MGC80-3 cells (Fig. 3A). Whereas, no proliferatory inhibition was observed in L-02 cells even at the concentration of 1000 μg mL−1 (Fig. 3B). The cytotoxicity was most pronounced for cells exposed to L200, where viability fell to 53% at the concentration of 250 μg mL−1 and decreased to 17% at 1000 μg mL−1 (Fig. 3A). C200 and L300 also induced a significant but relatively low toxicity, reducing viability to 75% at 250 μg mL−1, to 47% and 40% at 1000 μg mL−1.

Fluo-3 AM was used to determine intracellular calcium ion concentration ([Ca2 +]i). MGC80-3 cells grown in 96-well plates were incubated with 4 μM Fluo-3 AM (Beyotime Institute of Biotechnology, Haimen, China) in DMSO (1:1000) in HEPES Hank's Salt Solution (HHSS) containing BSA (1 mg mL−1) at 37 °C for 30 min, and protected from light. Subsequently, cells were washed twice with fresh HHSS containing BSA, and then incubated for another 30 min. Next, fluorescence was measured with a M2 microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. 2.9. Statistical analysis All experiments were carried out in triplicates unless otherwise indicated. Data obtained were expressed as mean ± standard deviation. Significant difference between two groups was evaluated using the oneway analysis of variance followed by Dunnett's post hoc test. Differences were considered statistically significant when p b 0.05. 3. Results 3.1. Characterization of HAPNs Three different preparations of HAPNs which refer to as L200 (200 W, lyophilization), L300 (300 W, lyophilization) and C200 (200 W, lyophilization & calcination) were synthesized using sonochemistry-assisted microwave method by regulating only one reaction parameter as shown in Table 1. When dispersed in the complete DMEM medium supplemented with 10% serum (cDMEM), L200 showed the smallest hydrodynamic diameter of 184 nm. All HAPN preparations were negatively charged in cDMEM, and the L200 had a slightly lower Zeta potential of −4.2 mV. Based on BET measurements, the L200 had 1.5 times more surface area than C200 and L300. TEM image revealed that the L200 were in a short rod shape, with the average size of about 50 nm in length, 15 nm in width (Fig. 1A). When this precipitate was calcined, the particle shape changed to a long rod-shaped morphology with a diameter of about 30 nm and length of about 100 nm (Fig. 1B). However, the L300 exhibited a needle-like shape, increased to about 150 nm in length and 20 nm in diameter after the microwave power was increased to 300 W (Fig. 1C).

3.2. Varying cytotoxicity of different HAPN preparations on MGC80-3 cells

3.3. Cellular apoptosis induced by different HAPN preparations MGC80-3 cells were exposed to HAPNs at 500 μg mL−1 for 48 h, and then stained with DAPI to check the nuclear morphology. It was seen that that untreated cells (Fig. 4A(i)) contained round nuclei with homogeneous chromatin and exhibited less bright blue color. However, cells treated with HAPNs, particularly with the L200, contained nuclei that were condensed and/or fragmented into pieces (Fig. 4A(ii–iv)). To further quantify the cell apoptosis upon exposure to different HAPN preparations, cells were stained by Annexin V-FITC/PI double staining and analyzed by flow cytometry. In the control group without HAPN treatment, almost no apoptotic cells were detected (Fig. 4B). On the contrary, MGC80-3 cells exposed to all HAPN preparations showed an increase in the percentage of apoptotic cells. The most significant increase was observed for L200 treatment, the percentages of necrosis (5%) and early apoptosis (11%) increased to 9% and 16% when increasing HAPNs concentration from 250 to 500 μg mL−1, with a substantial increase of late apoptosis from 16% to 27%, implying that late apoptosis might mostly affect the viability of MGC80-3. 3.4. Cellular uptake and intracellular distribution In order to evaluate nanoparticle uptake or binding to the MGC80-3 cells, the cellular side scattering of light by flow cytometry after exposure to HAPNs was measured at the moderately toxic concentration of 250 μg mL−1. As shown in Fig. 5A, saturation type time-dependent uptake was observed for all types of HAPNs at 37 °C. When comparing the side scatter (SSc) after exposure to different preparations, a statistical significant difference was observed. L200 seemed to have a much higher uptake efficiency than C200 and L300, reaching the maximum (254% of the control) at about 3 h. While for C200 and L300, the maximum SSc

Table 1 HAPNs synthesized with different reaction conditions. HAPNs

Microwave power (W)

Calcination

L200 C200 L300

200 200 300

No 550 °C, 5 h No

TEM

Hydrodynamic size in cDMEM

Average width (nm)

Average length (nm)

Average size (nm)

Zeta potential (mV)

15 30 20

50 100 150

184 320 221

−4.2 −1.9 −2.0

Specific surface area (m2 g−1)

69.2 ± 3.6 47.5 ± 3.5 47.7 ± 4.7

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Fig. 1. TEM images of HAPNs L200 (A), C200 (B), and L300 (C).

was detected at 8 h, which was 177% and 171% of the control, respectively. The passive attachment to the cell membrane was measured at 4 °C. Clearly, the level of SSc was much lower than that detected at 37 °C for all HAPN preparations, suggesting the uptake of HAPNs was mainly through an energy-dependent process. Next, we evaluated the pathways for cellular entry of different HAPN preparations with known biochemical inhibitors of major endocytotic pathways: clathrin-mediated endocytosis and lipid-raft/caveolae-mediated endocytosis. Dynasore is a small molecule GTPase inhibitor selective for dynamin that plays a critical role in clathrin/caveolinmediated endocytosis, and methyl-β-cyclodextrin (MβCD) is a specific inhibitor of caveolin-mediated endocytosis [27,28]. The data shown in Fig. 5B revealed significant, although incomplete inhibition of HAPNs internalization by MβCD, with the L200 having a stronger (56%) decrease in uptake, relative to untreated control cells, compared with a 32% and 38% inhibition of uptake for the C200 and L300, respectively. However, a weak reduction of HAPNs uptake was observed after dynasore treatment, with SSc being reduced by 27%, 12%, and 10% respectively for L200, C200, and L300. Therefore, MβCD was more effective than dynasore in inhibiting uptake of HAPNs in MGC80-3 cells, suggesting a vital role for caveolin-mediated endocytosis on the uptake of different HAPN preparations. After incubation for 24 h, CLSM analyses showed that most of the green FITC signals could be seen at the perinuclear region in the cytoplasm, as indicated by arrowheads in Fig. 6. More interestingly, most particles were observed in the cytoplasm for the C200 and L300, while some L200 HAPNs could also be found within nuclei as confirmed by the overlapping of fluorescence intensity profiles of FITC and DAPI (Fig. 6A). 3.5. Effect of HAPNs on intracellular calcium levels The HAPN dissolution has been observed within lysosomes [21], and we have found that the rise in calcium level correlated well with the HAPN-induced cytotoxicity in different cancer cells [29]. We thus examined the intracellular calcium ion concentration ([Ca2+]i) in MGC80-3

cells after incubation with three types of HAPNs at a concentration of 250 μg mL−1 (Fig. 7). It could be seen that exposure to all HAPN preparations caused an increase of fluorescent intensity in manifestation of [Ca2 +]i status in MGC80-3 cells, and the highest [Ca2 +]i value was seen for L200. The [Ca2+]i-level steadily increased between 6 and 24 h post-incubation, with observed increases of 80%, 43% and 47%, respectively, for L200, C200 and L300. After this time point, [Ca2+]i dropped, but was still higher than that of the untreated control at 36 h. 4. Discussion Although HAPNs have been reported to exhibit cytotoxicity to several types of human cancer cells, including MGC80-3 cells [12,13,25, 30], the effect of the physiochemical properties of HAPNs on their cytotoxicity and the mechanism involved have been poorly understood so far. In this study, we synthesized 3 different hydroxyapatite nanoparticles with variation of only one reaction parameter using a programcontrolled sonochemistry-assisted microwave reactor. All these HAPNs shared the same composition, had the similar crystallinity and zeta potential close to neutral, despite the different size and specific surface area. They inhibited cell proliferation and induced apoptosis of MGC80-3 cells, but cytotoxic effects differed significantly between preparations, with the L200 being the most toxic. Such a differential cytotoxicity of HAPN preparations was closely related to cellular uptake and the elevated intracellular calcium levels. This study may prove useful in choosing appropriate HAPN candidates for various biomedical applications utilizing highly controlled synthesis techniques. It has been reported that the physicochemical properties of HAPNs have significant effects on their biological responses, and the cytotoxicity of HAPNs is cell-type specific as well [17–19,31–33]. The anti-cancer efficacy of HAPNs was also size-dependent, as demonstrated in malignant melanoma cells [12]. Our previous result also demonstrated sizedependent cytotoxic effects of HAPNs on HepG2 cells in the order 45 N 26 N 78 N 175 nm [14,17]. Here, L200 exhibited the most significant cytotoxicity among the HAPNs we prepared, and the major differences in physico-chemical properties between L200 and the other two

Fig. 2. XRD patterns (A) and Raman spectrum (B) of HAPNs L200, C200, and L300.

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Fig. 3. Cell viability of MGC80-3 (A) and L-02 (B) cells treated with different HAPNs at 31–1000 μg mL−1 for 72 h. *Statistically significant difference as compared to the corresponding controls (p b 0.05 for each).

preparations were their size and specific surface area, L200 were smaller and had a higher specific surface area. This effect could originate from a size-dependent uptake rate, and also from an increasing reactive surface area of smaller nanoparticles. As the size of a nanoparticle decreases, a greater proportion of its atoms or molecules will be exposed, and the increase in surface area may create discontinuous crystal planes that increase the number of structural defects, thus establishing specific surface groups that function as reactive sites. On the other hand, in case cytotoxicity takes place in the intracellular space, it may be correlated with nanoparticle uptake. Compared to C200 and L300, the smaller particles L200 were internalized more rapidly and efficiently by MGC80-3 cells. Therefore, it can be considered that cytotoxicity of the HAPN preparations was related to their uptake efficiency in MGC80-3 cells. This is consistent with results reported in a previous study for different hydroxyapatite nano and microparticles in human monocyte-derived macrophages [21]. Numerous studies have attempted to address the role of physicochemical properties on nanoparticle uptake, and it has been reported that the degree of particle uptake appeared to be governed by physical properties, surface chemistry, the adsorbed protein corona under biological conditions, agglomeration of nanoparticles, and the cell type used as well [34]. In the case of HAPNs, the uptake was also particleand cell-type-dependent. Positively charged HAPNs were found to be easier to internalize into MC3T3-E1 cells [16], and a dependency of uptake on agglomeration was suggested [20]. Motskin et al. studied the uptake of hydroxyapatite nano and microparticles by human macrophage cells, but indicated that uptake was governed by the balance of multiple characteristics such as shape, charge, size and surface area [21].

To understand how HAPNs could influence cancer cells and to obtain information about their cytotoxic, potential knowledge about the route of uptake is important. Prior studies have imaged HAPNs uptake in macrophages [21], osteoblasts [16] and other cell types [31], and suggested the clathrin-mediated endocytosis pathway for needle-shaped HAPNs in liver cancer cells [35], but to our knowledge no study investigated uptake pathway of HAPNs with known endocytosis inhibitors for further understandings. MβCD, a specific inhibitor of caveolae-mediated endocytosis, strongly decreased internalization of all HAPNs in MGC80-3 cells, indicating that the caveolin-mediated pathway represented the major mechanism for different HAPNs. Dynasore inhibits the GTPase activity of dynamin, a protein that is essential for the formation of clathrin coated vesicles [36]. Here, dynasore inhibited internalization of L200 by 27% at 3 h, showing that clathrin-mediated endocytosis was a secondary pathway for internalization of L200 in MGC80-3 cells, but internalization of C200 and L300 was minimally inhibited by dynasore. These experiments strongly suggested that caveolae-mediated endocytosis and, to a much lesser extent, clathrin-mediated endocytosis, were involved in all HAPNs uptake in MGC80-3 cells. Note that none of the inhibitors led to complete inhibition of internalization, whereas the inhibition was more obvious for L200 compared to C200 and L300. This observation most likely indicated the role of clathrin- and caveolin-independent pathways for internalization, as heterogeneity of nanoparticle surfaces and dispersion always requires multiple uptake pathways to be involved. For instance, at higher concentrations the HAPNs were found sequestered within a large convoluted membranebound surface-connected compartment (SCC) [37], the formation of

Fig. 4. (A) Morphological changes in the nuclear chromatin of MGC80-3 cells untreated (i) or treated with L200 (ii), C200 (iii) or L300 (iv) at 500 μg mL−1 for 48 h. Cells were stained with DAPI to visualize nuclear morphology. Scale bar: 30 μm. (B) Apoptosis and necrosis ratios obtained from Annexin V-FITC/PI double staining in cells treated with HAPNs for 48 h. The results are representative of three independent experiments.

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Fig. 5. (A) Time course of HAPNs uptake in MGC80-3 cells determined by side scatter analysis. Cells were exposed to HAPNs (250 μg mL−1) at 37 °C or 4 °C, and the side scatter was recorded by flow cytometry to indicate binding/uptake of the nanoparticles. The side scatter of treated cells was normalized to that of untreated controls. The results are representative of three independent experiments. (B) Effect of MβCD and Dynasore on internalization of HAPNs in MGC80-3. The cells were pretreated with different inhibitors, MβCD (10 mM) for 30 min or dynasore (80 μM) for 1 h, respectively, and then incubated with HAPNs at 250 μg mL−1 for another 3 h. The side scatter of treated cells was normalized to the control cells without inhibitor pretreatment. *Statistically significant difference as compared to the corresponding controls (p b 0.05 for each).

this SCC was governed by nanoparticle agglomeration, but small agglomerates could be taken up by other mechanisms than the SCC [20]. Once taken up by cells, intracellular localization is a key factor in determining the cytotoxicity of nanoparticles. HAPNs were reported to elude the phagocytic pathway and enter the nuclei of macrophages [21]. We showed in a previous study that HAPNs in the range of 20–80 nm could be translocated into the nuclei of HepG2 cells with a distinct size-mediated manner, and correspondingly induced a sizedependent cytotoxicity, while HAPNs spared normal L-02 cells which showed little or no nuclear presence of nanoparticles [14]. Such a nuclear targeting of L200 might contribute to their greater toxicity in MGC80-3 cells observed in this study, and this might be attributed to the smaller size of L200, as smaller particles could readily pass through the nuclear pore complex. If nanomaterials may gain access to the cell nucleus where the genetic information and the transcription machinery reside, they might directly interact with the DNA or DNA-related proteins, causing damage. Treatment with HAPNs was reported to cause the retrogressive change of nucleolus in MG63 cells [15], induce DNA damage in rat macrophages [38] and human breast cancer cells [13]. In fact, nuclear-targeted drug delivery is expected to kill cancer cells more directly and efficiently, and several kinds of nuclear localization signals (NLSs) have been introduced to decorate the surfaces of

nanoparticles to enhance nuclear import [39–41]. If intranuclear translocation could be correlated to the physicochemical properties of HAPNs, we can design nanoparticles with enhanced nuclear internalization and cytotoxicity in cancer cells. Thus, HAPNs may be expected to efficiently target the nucleus and deliver anticancer drugs, such as doxorubicin, into cancer cells, providing significantly enhanced anticancer efficiency resulting from the combination effect of HAPNs and chemotherapeutics. Previously, increased intracellular calcium levels have been reported in different cancer cells after exposure to HAPNs, including MGC80-3 cells, and a correlation between the degree of cytotoxicity and [Ca2+]i increase was observed [25]. Here, a good correlation was also seen between the rise in [Ca2+]i level and cytotoxicity of different HAPN preparations. The increase of intracellular calcium concentration after exposure to HAPNs may be due to the particle dissolution in the acidic environment of the lysosome where the pH ranges from 4.8 to 6.0 [42], as hydroxyapatite particles were observed in various states of dissolution within the lysosomal compartments in macrophages [21]. Treatment of MGC80-3 cells with different HAPNs led to a differential [Ca2+]i increase. This may be caused by the varying uptake efficiency among different HAPN preparations, as MGC80-3 cells exposed to L200 showed the highest particle load. This effect could also originate

Fig. 6. Intracellular distribution of different HAPNs within MGC80-3 cells. Fluorescent micrographs of MGC80-3 cells treated with FITC-labeled L200 (A), C200 (B), or L300 (C), at 500 μg mL−1 for 20 h. Confocal microscopy was used to visualize the fluorescence of the internalized FITC-HAPNs (FITC) and nuclei (DAPI). The corresponding bright field images as well as the merged images of all three channels were presented (merged). Arrowheads indicated examples of HAPN signals located in cytoplasm, intensity profiles through arrows were also represented (right panels). Scale bar: 20 μm.

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5. Conclusions

Fig. 7. Time course of [Ca2+]i fluorescence intensities in MGC80-3 cells exposed to HAPNs (250 μg mL−1). The relative fluorescent intensity was normalized to the value obtained with cells untreated by HAPNs. *Statistically significant difference as compared to the corresponding controls (p b 0.05 for each).

from an increasing reactive surface area of L200, for the dissolution rate constant of nanomaterials depends on the surface area of particles under certain conditions [43]. Calcium plays a crucial role in virtually all cellular processes including cell proliferation and apoptosis. The cytoplasmic Ca2 + is tightly regulated to maintain at a low level of ~100 nM in resting cells [44]. Elevated [Ca2+]i levels can be toxic, but the presence of excessive calcium ions in the cytosol has a positive role in facilitating endosomal escape, cytosolic stability and high nuclear uptake of DNA through the nuclear pore complex [45], as the movement of the nuclear pore complex (NPC) central plug is associated with the Ca2+-dependent mechanism [46]. In our study, treatment with L200 triggered the most significant increase of [Ca2+]i level, nuclear localization, and cytotoxicity. This suggested that smaller size and higher specific area might enhance cellular uptake and subsequent dissolution of HAPNs, which could be the main cause of cytotoxicity. In this study, we synthesized three different HAPN preparations by regulating only one reaction parameter, and attempted to address the role of particle characteristics on their cytotoxicity in cancer cells. It seemed that the cytotoxicity of HAPNs to cancer cells was sizedependent. Unfortunately, heat treatment and increase of the microwave power altered the particle shape, crystallinity and number of surface hydroxyl group as well. In the study of Zhao et al., they demonstrated that needle- and plate-shaped HAPNs induced the most significant cell death compared to sphere and rod particles, but nanoparticles showed different hydrodynamic sizes, zeta potentials, and specific surface areas [17]. When Chen et al. investigated the role of surface charge on the uptake and biocompatibility of HAPNs with osteoblast cells, they found that the zero uptake of neutral nanoparticles could not be explained only by surface charge, as the shape and size might also play a role [16,47,48]. Therefore, the correlation of HAPN properties with their cytotoxicity is not straightforward, as many of these properties are mutually connected, it is hard to synthesize nanoparticles in which only one parameter can be varied. For HAPNs generated via chemical precipitation method, process parameters such as temperature, pH, Ca/P ratio, starting solution concentration, and reactant addition rate have been reported to affect particle characteristics including purity, crystallinity, and morphology [47,48]. Although our results, together with other previous studies, are still limited to help prediction of certain physicochemical properties on the cellular behavior of HAPNs, they can provide us with very useful information. With more elaborate synthesis, reproducible HAPNs of controlled physicochemical properties can be obtained, and used to explore correlations between HAPN properties and their cytotoxicity to cancer cells. Then, it may be possible to tailor HAPN characteristics for specific biomedical applications, including treatment of cancers by enhancing its cytotoxicity or in combination with other anticancer agents.

Using sonochemistry-assisted microwave method, by varying only one reaction condition, we synthesized three HAPN preparations: L200, C200, and L300, with similar composition, crystallinity and surface charge, but different size. These HAPNs could all inhibited cell proliferation and induced apoptosis in human gastric cancer MGC80-3 cells, but the smallest particles L200 with the highest specific surface area exhibited the most significant toxic effects. The uptake of all HAPNs studied was through energy-dependent processes, mainly via a caveolinmediated pathway, while the uptake of L200 was also through dynamin-dependent clathrin-mediated endocytosis. All forms of HAPNs were present in the cytoplasm, and a few L200 were even translocated to the nucleus. Toxicity correlates well with the amount of particle uptake and the rise in [Ca2+]i, suggesting that calcium release and interference with intracellular calcium homeostasis might be the main cause of toxicity. These data may provide us with useful information to understand how physicochemical properties of HAPNs relate to their anti-cancer behavior and how designs of those properties could be used to optimize their utility for therapeutic use and safety.

Acknowledgments We appreciate the financial support from the National Basic Research Program of China (973 Program, 2012CB933600), the National Natural Science Foundation of China (31271010), and Program for New Century Excellent Talents in University (NCET-11-0639). The authors are cordially thankful to Professor Yifan Han and Dr. Like Ouyang for the Raman measurements.

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