pH-Dependent Activity of Dextran-Coated Cerium Oxide Nanoparticles

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pH Dependent Activity of Dextran Coated Cerium Oxide Nanoparticles on Prohibiting Osteosarcoma Cell Proliferation Ece Alpaslan, Hilal Yazici, Negar Hamedani Golshan, Katherine Ziemer, and Thomas J. Webster ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00194 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

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ACS Biomaterials Science & Engineering

pH Dependent Activity of Dextran Coated Cerium Oxide Nanoparticles on Prohibiting Osteosarcoma Cell Proliferation Ece Alpaslana#, Hilal Yazicia#, Negar H. Golshana, Katherine S. Ziemera and Thomas J. Webstera,b* a

b

Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA

Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia

# Equally contributed (Ece Alpaslan and Hilal Yazici) Corresponding author * Prof. Thomas J. Webster Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA Phone: +1 617 373 6585 E-mail: [email protected]

Mailing address for all authors: 360 Huntington Avenue 313 Snell Engineering Center Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA

1 ACS Paragon Plus Environment


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Abstract Cerium oxide nanoparticles (or nanoceria) have demonstrated great potential as antioxidants in various cell culture models. Despite such promise for reducing reactive oxygen species and ability for surface functionalization, nanoceria has not been extensively studied for cancer applications to date. Herein, we engineered the surface of nanoceria with dextran and observed its activity in the presence bone cancer cells (osteosarcoma cells) at different pH values resembling the cancerous and non-cancerous environment. We found that dextran coated nanoceria was much more effective at killing bone cancer cells at slightly acidic (pH=6) compared to physiological and basic pH values (pH=7 and pH=9). In contrast, minimal toxicity was observed for healthy (noncancerous) bone cells when cultured with nanoceria at pH=6 after one day of treatment in the concentration range of 10-1000 µg/mL. Although healthy bone cancer cell viability decreased after treatment with high ceria nanoparticle concentrations (250-1000 µg/mL) for longer time periods at pH=6 (3 days and 5 days), approximately 2-3 fold higher healthy bone cell viabilities were observed compared to osteosarcoma cell viability at similar conditions. Very low toxicity was observed for healthy osteoblasts cultured with nanoceria for any concentration at any time period at pH=7. In this manner, this study provides the first evidence that nanoceria can be a promising nanoparticle for treating bone cancer without adversely affecting healthy bone cells and, thus, deserves further investigation.

Keywords: Nanoceria, bone cancer, cancer treatment, osteosarcoma, and nanoparticlebased therapy


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1. Introduction Cancer, the number one cause of death in the United States, still suffers from appropriate treatment methods

1

. Current treatment options are surgery, radiotherapy and

chemotherapy. Limited tumor accessibility for resection and the risk of damaging healthy surrounding tissue has been an obstacle for surgery and radiotherapy. However, in chemotherapy, a major issue is failure to accumulate and retain therapeutically relevant 2

drug concentrations at the site of the tumor . For an effective treatment at the tumor site, prolonged exposure of a tumor to sufficiently high drug concentrations is a necessity. However, physiochemical properties like molecular weight or lipophilicity of the compound often end up at sub-therapeutic drug levels at the tumor site. Additionally, most chemotherapeutic agents (such as cisplatin, doxorubicin, and methotrexate) suffer 3

from non-selective toxicity and harm healthy cells as well as cancer cells . Therefore, for cancer treatment in general, a less invasive and more selective treatment regime is needed.

Nanoparticle-based therapies may be the answer as they have been utilized to deliver therapeutic agents to a diseased site and promote the accumulation of therapeutic levels 4

of drugs at the target site . So far, liposomes, nanoparticles based on solid lipids, polymeric nanoparticles, and magnetic nanoparticles have all demonstrated great promise 5

for targeted cancer drug delivery . Among them, liposomes have been approved for clinical use due to their colloidal stability and selectivity of drug accumulation at the tumor site

2, 6

. Such attractive properties of nanocarriers stem from their ability to



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extravasate through the leaky and highly permeable tumor vasculature and accumulate in the tumor interstitium. This phenomenon is called the enhanced permeability and retention (EPR) effect. Various properties of nanoparticles (such as size, shape, surface charge and their dynamic and continuous interactions with components of the 7

vasculature) govern their ability to exhibit the EPR effect .

While promising, many nanoparticles still suffer from cytotoxicity to healthy cells. For these reasons, new carriers, which do not possess such healthy cell cytotoxicity properties, still need to be identified. Cerium oxide nanoparticles (or nanoceria) have enormous potential as antioxidant and radioprotective agents for cancer applications

8-16

.

Of particular interest due to the acidic tumor environment, it has been reported by several groups that nanoceria’s antioxidant properties change with pH since it can switch from Ce+3 to Ce+4

11, 17, 18

. Optimal antioxidant properties have been reported for nanoceria at

physiological pH values, whereas at slightly acidic (more tumor-like) pH values, it acts as an oxidase.

This property of nanoceria may be utilized for cytoprotection towards

healthy cells and be a selective killing agent to cancer cells where the tumor pH is lower than physiological values.

Another remarkable feature of nanoceria is its ability to be a free radical scavenger at physiological pH values which allows for its use as a therapeutic agent to fight against cancer and other diseases due to a reduction in reactive oxygen species (ROS). In cancer, it has been widely reported that ROS can drive both initial cancer development and


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progression. In addition, by causing damage to DNA, ROS production also affects enzymes which normally would combat such free radical production

19, 20

.

Despite such promises noted above, there are several challenges for the use of nanoceria in cancer applications. Nanoceria has a large number of surface defects due to oxygen vacancies at its surface, which exponentially increase per gram of material as particle size decreases

11, 21, 22

. Thus, ceria nanoparticles have a natural tendency to agglomerate when

its surface is not functionalized. Modifying the surface of ceria nanoparticles is not only useful for stability, but it is also a possible approach to increase the circulation time of the nanoparticles in the blood stream and increase water solubility

23

. As one of many

possible surface functional agents, dextran possesses attractive properties since it is a biocompatible, complex polysaccharide, commonly used as a surface modifier for colloidal nanoparticle synthesis due to its high water solubility

12, 24-28

. Moreover, it has

been previously reported that the encapsulation of nanoceria with dextran does not have an impact on its redox properties, but only its surface charge

25, 26

.

With this in mind, nanoceria has been studied for the treatment of breast, pancreas, ovarian and lung cancer cells at different sizes with various surface chemical functionalizations

19, 29-32

. In lung cancer studies, the effect of 20 nm ceria particles in a

concentration and dose dependent manner was analyzed

33

. An increase in ROS

production, which leads to a decrease in antioxidant level of cells, and apoptotic cell death was observed

33

. Specifically, after 72 hours of exposure to 23.3 µg/mL of 20 nm



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cerium oxide nanoparticles, lung cancer cell viability decreased to 53.9%. In ovarian cancer studies, a reduction of the tumor mass was accompanied by attenuation of angiogenesis upon administration of 15-20 nm nanoceria (0.1 mg/kgbdwt) every third day, due to the apoptosis of vascular endothelial cells. In that study, nanoceria was suggested as a novel nanoparticle that can potentially be used as an anti-angiogenic therapeutic agent for treating ovarian cancer

30

. Another novel role for nanoceria was

identified by Wason et al. where nanoceria (5-8 nm) at a 10 µM concentration was used to enhance radiation therapy induced ROS production more than 2 fold and cell death selectively in human pancreatic tumor cells while protecting normal tissues from toxic 32

side effects of radiation therapy depending on environmental acidity .

So far, however, nanoceria has not been tested for treating bone cancer or against osteosarcoma cells. In this study, we specifically focused on primary osteosarcoma, which is a commonly occurring disease with a cure rate of only around 20%

34

.

Osteosarcomas are known to be responsive to some chemotherapeutic agents, such as cisplatin,

doxorubicin,

methotrexate,

and

bisphosphonates

+

ifosfamide,

paclitaxel/gemcitabine/doxorubicin which all suffer from non-selective toxicity

and 3, 35

.

Besides the application of conventional chemotherapeutical agents, recently new chemistries, such as curcumin, and drug encapsulated polymeric nanoparticles have been utilized as a potential bone cancer treatment

3, 36, 37

.

In this study, 0.1 M dextran coated nanoceria was designed as a new cancer treatment agent to kill osteosarcoma cells while protecting healthy osteoblast cellss. In order to 6 ACS Paragon Plus Environment

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achieve this objective, dextran coated sub 5 nm cerium oxide nanoparticles were synthesized and treated with osteosarcoma cells at different pH values (at neutral (pH 7), acidic (pH 3, 5, 6) and basic (pH 9) values) in order to observe nanoceria’s activity in a dose, time and pH dependent manner. The results indicated for the first time that nanoceria is much more effective at killing bone cancer cells at an acidic (pH 6) and neutral (pH 7) environment which mimics the pH of the natural tumor due to its low pH 38, 39

4,

. Such knowledge is helpful for designing nanoceria to selectively kill bone cancer

cells over bone healthy cells.

2. Experimental Section 2.1 Synthesis and Characterization of Ceria Nanoparticles Dextran coated ceria nanoparticles were synthesized according to a modified protocol published from Perez et al. in 0.1 M dextran T-10 (Pharmacosmos, Holback, Denmark) concentrations. Briefly, 1 mL of an aqueous solution of 1 M cerium nitrate (Sigma Aldrich, St Louis, MO) and 2 mL of 0.1 M dextran were mixed and the prepared solutions were added dropwise to 6 mL of a 30% ammonium hydroxide (Sigma Aldrich, St. Louis, MO) solution while stirring for 24 hours at 25 οC. Upon the addition of the precursor into ammonium hydroxide, the solution turned into light yellow. With the formation of stabilized dextran coated cerium oxide nanoparticles, the solution became dark brown. After 24 hours, particles were centrifuged at 4000 rpm for 30 min to remove any debris and any large agglomerates as well as unattached dextran. The final nanoparticle solution was stored in a refrigerator at 4 οC (Figure 1A).



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Before starting any experiments, 1mL of the nanoparticle solution, which was on weighing cups, was dried on a hotplate until all of the water evaporated. Following, all nanoparticle concentrations were calculated based on dried nanoparticle concentrations (weight/mL).

Following nanoparticle synthesis, first, transmission electron microscopy (TEM) (JEM 1010, JEOL) was used to observe particle size, distribution, and morphology at a 80 keV accelerated voltage. Particles were analyzed on a carbon coated cupper grid. Dynamic light scattering (DLS) (Zetasizer, NanoZS, Malvern Instruments) experiments were performed to analyze the particle size and distribution of nanoparticles.

X-ray photoelectron spectroscopy (XPS) was used to determine the valence state of ceria. XPS consists of a dual source, non-monochromated X-ray source (Phi model 04-548) and a hemispherical analyzer (Phi model 10-360). The aluminum anode used in this study produces Al Kα X-rays (1486.6 eV) at 100 W. The system is calibrated using Au 4f and Cu 2p, and has a minimum full width half maximum (FWHM) of 1.2 eV with an 80% Gaussian/Lorentzian distribution at a pass energy of 35.75 eV. Background subtraction was performed using the integrated Shirley method.

The size and surface charge of the synthesized nanoceria particles varied after dispersing them in cell culture media (Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin (P/S) or osteoblast basal medium, supplemental mix, and 1% P/S mentioned as detailed below) at different pH values (pH


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6, 7 and 9). Particle concentration was fixed to 1000 µg/mL. The zeta potential of the particles was measured by DLS (90Plus Zeta, Brookhaven Instruments) and the software provided by the manufacturer. In addition, the size of the nanoceria particles (1000 µg/mL, pH 9.8) were examined in DMEM media supplemented with 0, 1, 2.5, 5, and 10 % FBS and 1% P/S.

UV-visible absorption spectroscopy measurements were performed in order to observe the changes in the oxidation states in solution depending on the pH. Particles were dispersed in PBS at 1000 µg/mL concentrations and the pH of the solution was adjusted to pH 6 and pH 7 with acetic acid (mentioned in detailed below). Spectra for cerium oxide nanoparticles were acquired using SpectraMax M3(MT05412)

at room

temperature in 3 mL cuvettes.

2.2 Cell Culture In this study, two types of cell lines were used. One was an osteosarcoma cell line MG-63 (ATCC CRL-1427) and the other one was an osteoblast non-cancerous cell line (PromoCell, C-12720). Osteosarcoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC® 30-2003™) supplemented with 10% Fetal Bovine Serum (FBS) (ATCC® SCRR-30-2020™) and a 1% penicillin-streptomycin solution (ATCC® 30-2300™).

Healthy osteoblast cells were cultured with osteoblast basal

medium (PromoCell, C-27010) with a supplement mix (PromoCell, C-39615) and a 1% penicillin-streptomycin solution (ATCC® 30-2300™). Both of the cell lines were incubated at 37οC in air supplemented with 5% CO2 until 80% confluency was achieved.



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Cells were enzymatically detached from the surface of the T-75 using trypsin (0.5%, Gibco Life Technologies) and collected by centrifugation at 2400 rpm for 3 minutes.

2.3 pH Adjustments To conduct experiments at different pH values, at each nanoparticle concentration (10 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL) the pH of the solution was adjusted to basic (pH 9), neutral (pH 7), and acidic (pH 6, pH 5 and pH 3) values. The pH ranges were decided depending on the characteristic features of nanoceria and the tumor microenvironment. pH 6 is the approximate pH value of the bone tumor 4

microenvironment . pH 7 is the relative value for regular cell growth. pH 9 is also a closer pH value of the synthesized nanoceria. Although pH 5 and pH 3 are very low pH values for regular cell growth, these pH values were also tested due to possible nanoceria free radical scavenger properties that may affect the survival of cells. Among all pH values tested for osteosarcoma cells, pH 6 showed the maximum efficacy in killing them. Thus, the effect of these particles at pH 6 was tested on healthy osteoblast cells as well.

To adjust the pH values, nanoparticles were dispersed in the corresponding cell culture media at each concentration; an aqueous solution of 1 M to 10 M acetic acid (Sigma Aldrich, USA) was added to fix the pH values at the above mentioned acidic pH values. For pH 9, an aqueous solution of 1 M and 10 M sodium hydroxide (Sigma Aldrich, USA) was added dropwise. The pH of the solutions was measured with a pH meter (S230 Seven Compact, Mettler Toledo).


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2.4 Cytotoxicity (MTS) Assays CellTiter 96® AQueous One Solution Cell Proliferation Assays (MTS) were carried out for up to 5 days in culture using DMEM, 10% FBS and a 1% penicillin-streptomycin solution for osteosarcoma cells. For healthy osteoblast cells, MTS assays were also carried out for up to 5 days in culture using osteoblast basal medium with a supplement mix and a 1% penicillin-streptomycin solution. Both of the cell lines were seeded into a 96 well plate at 5,000 cells/well (15,000 cells/cm2) and were allowed to adhere at 37 °C in 5% CO2 for 24 hours. The next day, the cells were treated with dextran coated ceria nanoparticles at 10 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL for each pH value. As a control group, cells were cultured in the pH-adjusted media only (no nanoceria). Following 24 hours of incubation with nanoparticles, 200 µL of media were removed from each well and 200 µL of an MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay, G3581 Promega) reagent (1:5 ratio with cell culture media) was added to each well and incubated for 3 hours. At the end of incubation, a color change from pink to dark brown was observed, and absorbance of each well was measured by a SpectraMax M3 (MT05412) at 490 nm (Figure 1B).

To create a standard curve, serial dilutions of osteosarcoma cells and healthy osteoblast cells were seeded into a 96 well plate (50,000 cells/cm2 to 0 cells/cm2) and were left to equilibrate for 2.5 hours. After cells adhered, 200 µL of an MTS reagent (1:5 ratio with appropriate cell culture media) was added to each well and incubated for 3 hours. Again, the absorbance of the solution was measured at 490 nm (See supporting information).



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Viability graphs were plotted by normalizing the data with respect to control groups from the first day of each experiment. Data of cell viability experiments performed at pH 3 and pH 5 were not reported due to the cell death that occurred in all control groups and nanoceria treated cells due to the very low pH.

2.5. IC-50 Value Calculation IC50 represents the concentration of a drug that is required for 50% inhibition of cell growth/metabolic and/or biochemical function. Here, IC-50 values were calculated and indicated in Table 1 for each pH condition for osteosarcoma cells and pH 6 and 7 for healthy osteoblast cells.

2.6. Reactive Oxygen Species Measurements Reactive oxygen species (ROS) measurements were performed using the Image-iTTM LIVE Green Reactive Oxygen Species Detection Kit (Molecular Probes, I36007) for osteosarcoma and osteoblast cells for one day. Osteosarcoma cells were cultured in 96 well plates at 5,000 cells/well (15,000 cells/cm2) and were allowed to adhere at 37 °C in 5% CO2 for 24 hours using DMEM, 10% FBS, 1% penicillin/streptomycin, whereas healthy osteoblast cells were cultured in osteoblast basal media, supplement mix, and a 1% penicillin/streptomycin solution. The next day, the cells were treated with dextran coated ceria nanoparticles at 250 µg/mL, 500 µg/mL and 1000 µg/mL at pH 6. As a control group, cells were cultured in the pH 6 media only (no nanoceria), as well as regular media at pH 7.4 for comparison. Following 24 hours of incubation with nanoparticles, ROS measurements were performed. The carboxy-H2DCFDA dye was


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dissolved in 50 µL DMSO (provided in the kit) to achieve a 10 mM final concentration. The solution was vortexed until the powder was completely dissolved. 10 mM of a carboxy-H2DCFDA solution was further diluted to 25 µM in a warm Hank’s Balanced Salt Solution (HBSS/Ca/Mg) (Life Technologies, 14025126). After the nanoparticle treatment, cells were washed with warm HBSS/Ca/Mg two times and trypsinized for 15 minutes with 20 µL trypsin (0.083%). Following the trypsinization of the cells, 90 µL of 25 µM carboxy-H2DCFDA was added to each well to label the cells. 100 µL of the cell and dye suspension were transferred into blank 96 well plates and were incubated at 37 °C for 30 min. The fluorescence measurements were taken every 5 minutes for 30 min using a SpectraMax M3 (MT05412) spectrophotometer at 495/529 nm.

2.7 Statistical Analysis Numerical data were analyzed using standard analysis of variance (ANOVA) followed by Student t-tests. All cell experiments were repeated three times in triplicate for all of the nanoparticle concentrations and pH values.

3. Results 3.1 Synthesis and Characterization of Dextran Coated Cerium Oxide Nanoparticles Figure 2 shows the characterization results of the ceria nanoparticles. TEM micrographs of the ceria nanoparticles with the 0.1 M dextran coating demonstrated that particle sizes were 3-4 nm in diameter (Figure 2A) and were spherical in shape. XPS confirmed the presence of Ce. 1 atomic % Ce was detected in the 0.1 M dextran coated cerium oxide samples, suggesting a dextran coating of close to 10 nm around the particles. XPS



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analysis on the dried particles showed that Ce is in the 3+ oxidation state (see Supporting Information Figure S1).

In contrary to XPS results performed on dried particles immediately after synthesis, UVvisible absorption spectroscopy measurements showed the presence of Ce (IV) oxides at pH 6 and pH 7 in the PBS solution as a single peak was obtained around 300 nm

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(Supporting Information Figure S2).

The hydrodynamic diameter of the particle was measured as 40 nm when it was dispersed in a PBS solution (i.e. 0% FBS) at 1000 µg/mL. Then, an increase in the hydrodynamic diameter was observed as the FBS concentration increased to 1% (85 nm in diameter) and 2.5% (79 nm in diameter). Further increasing the FBS concentration to 5% and 10% showed a declining trend in the particle size, where it was measured to be around 65 nm for both concentrations. These findings can be interpreted as the formation of a protein corona around the particles, especially at lower FBS concentrations at short times. Regardless, particle size was still measured to be below 100 nm (Figure 2B).

After observing the effect of the FBS amount on the diameter of the nanoparticles, pH dependent particle sizes were also examined. As shown in Figure 2C, particle size changed with respect to the pH of the solution. In an acidic environment (pH 6), particle size was 29 nm and at a physiological pH (pH 7), particle size was 45 nm. Lastly, in a basic environment (pH 9), particles were 50 nm in diameter.


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To observe the effect of pH on the surface charge of the 0.1M dextran coated cerium oxide nanoparticles, zeta potential measurements were performed. The zeta potential of the 0.1 M dextran coated ceria nanoparticles was 0.58 mV with a deviation of +/- 2.27 mV at physiological pH values (pH 7) (Figure 2D). It was observed that at different pH values, the surface charge of the particles changed as well. At pH 6 in healthy osteoblast and osteosarcoma media, the surface charge of the particles were 16.68 mV and 17.52 mV, respectively. At pH 9, in osteosarcoma media, the zeta potential was measured as 6.32 mV. The surface charge of the nanoceria was positive at pH 6 and pH 9 and near a neutral zeta potential at pH 7.

3.2 Cellular Responses to Dextran Coated Nanoceria Treatments at Different pH Values 3.2.1 0.1 M Dextran Coated Nanoceria Toxicity on Osteosarcoma Cells at pH 9 As shown in Figure 3A, at the end of the first day, osteosarcoma cell viability did not change at any ceria nanoparticle concentration (10 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL) compared to the control group at pH 9. After 3 days, at low nanoparticle concentrations (10-250 µg/mL), no toxic effect of nanoceria was observed. Slight increases in cell viability were obtained due to cell proliferation. However, cell viability when treated with 500 µg/mL was statistically less compared to the control group (160% compared to 140%, respectively). After 5 days, a very similar trend in terms of cell viability at low nanoparticle concentrations (10-100 µg/mL) was observed as after 3 days. For higher concentrations, cell viability when treated with 250 µg/mL ceria nanoparticles reduced to 140% and when treated with 1000 µg/mL, cell viability reduced to 75%. After day 3, a drastic change was observed at 500 µg/mL, but



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the cytotoxic effect of nanoceria shifted to lower concentrations of 250 µg/mL at the end of the 5th day (cell density graphs are shown in Figure S3A in the Supporting Information section).

3.2.2 0.1 M Dextran Coated Nanoceria Toxicity on Osteosarcoma Cells at pH 7 Osteosarcoma cell viability also showed a time and dose dependent response to nanoceria treatments at pH 7. At the end of the first day, cell viability was not significantly different at any nanoparticle concentration (10 µg/mL to 1000 µg/mL) compared to the control, when cells were treated with media at pH 7. A similar cell response was observed for pH 9 as described above. After 3 days of treatment, cell viability decreased to 180% when exposed to 500 µg/mL and then gradually decreased to 125% when exposed to 1000 µg/mL dextran coated ceria nanoparticles. After 5 days, osteosarcoma cells showed a statistically significant decrease compared to the control group when they were treated with 250 µg/mL ceria nanoparticles. Cell viability gradually decreased from 425% to 80% when exposed to 1000 µg/mL ceria nanoparticles compared to controls (Figure 3B) (cell density graphs are shown in Figure S3B in the Supporting Information section).

3.2.3 0.1 M Dextran Coated Nanoceria Toxicity on Osteosarcoma Cells at pH 6 An enhancement in nanoceria’s cytotoxicity was observed as the pH was reduced to below physiological pH values and closer to that of tumor values. After 1 day of nanoparticle treatment at pH 6, cell viability decreased to 75% when exposed to 250 µg/mL of ceria nanoparticles; when exposed to 500 µg/mL of ceria nanoparticles, cell viability decreased to 55% and at 1000 µg/mL, cell viability decreased to 50% (Figure 4).


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The most significant observation at pH 6 involved a shorter treatment time at a certain concentration for killing osteosarcoma cells. In addition, a lower dose of nanoparticles was shown to have a higher efficiency in killing osteosarcoma cells at pH 6 compared to pH 9 and pH 7 after 1 day of treatment. After 3 days, a statistically significant reduction in cell viability was observed at 100 µg/mL and no cell growth was observed at 1000 µg/mL of ceria nanoparticles. A similar trend was observed after 5 days of treatment at 100 µg/mL and 1000 µg/mL of ceria nanoparticles as shown in Figure 4 (cell density graphs are shown in Figure S4 in the Supporting Information section).

3.2.4 0.1 M Dextran Coated Nanoceria Toxicity on Healthy Osteoblast Cells at pH 6 and pH 7 As shown in the previous sections, the most effective pH for killing osteosarcoma cells 4

was pH 6, which is the approximate pH of the bone tumor microenvironment . In order to utilize nanoceria as a therapeutic agent for bone cancer at pH 6, observing the response of healthy osteoblasts to the same conditions is crucial. For this reason, osteoblasts were also treated with dextran coated cerium oxide nanoparticles at pH 6. After the first day of treatment, no statistical difference was observed at any nanoparticle concentration (10 µg/mL to 1000 µg/mL) compared with the control group. After 3 days of treatment, cell viability for each ceria nanoparticle concentration was roughly half of the control group and no statistical difference was observed between them. As Figure 5 indicates, statistically significant decreases were observed between 250 µg/mL to 1000 µg/mL in a time dependent manner. After 5 days of treatment, cell viability dropped from 60% (at 10



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µg/mL) to 18% (at 1000 µg/mL) as shown in Figure 5 (cell density graphs are shown in Figure S5 in the Supporting Information section).

In addition to pH 6 results, the effect of nanoceria on healthy osteoblasts at pH 7 (as a model of the physiological pH environment) was also evaluated (Figure S6). The results indicated that nanoceria at pH 7 did not show any toxicity after 1 day of treatment to healthy osteoblast cells. After 3 and 5 days of nanoceria treatment, a significant decrease (albeit small) in healthy osteoblast viability was observed for 250 µg/mL. Compared to nanoceria treatment at pH 6 to healthy osteoblast cells, at pH 7, a lower toxicity was observed. In general, little to no toxicity was observed for healthy osteoblasts exposed to nanoceria at any nanoparticle concentration or time. At pH 6, after 3 and 5 days of treatment, a significant decrease started at the lowest nanoparticle concentration, which was 10 µg/mL.

As reported here for bone tumor applications, for all pH conditions, the toxicity of the nanoceria needs to be evaluated not only based on dose but also in a time dependent manner on both healthy osteoblasts and osteosarcoma cells.

3.3 ROS Generation of Dextran Coated Ceria Nanoparticles on Osteosarcoma and Osteoblast Cells at pH 6

According to the highest efficiency in killing osteosarcoma cells as shown in Figure 4, the dose range of nanoparticles studied in this experiment was limited to between 250


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µg/mL to 1000 µg/mL at one day. Two control groups (pH 7.4 and pH 6) were used in this experiment. To understand the effect of acetic acid for the generation of ROS, the control group at pH 6 was added to the experimental data set. As shown in Figure 6, there was no ROS generation at pH 6.

Generation of ROS was observed at 250 and 500 µg/mL nanoceria concentrations with a similar trend (150%) when cultured with osteosarcoma cells. At 1000 µg/mL, a drastic change in ROS generation was examined around 180%. In healthy osteoblast cultures, there was no significant ROS generation at any ceria concentration (Figure 6).

4. Discussion Even though nanosized materials have been utilized for a multitude of applications over several decades, toxicologists are still investigating their potentially hazardous health effects

9, 40

. The toxicity of nanoparticles are dependent on several factors including the

nature and chemical composition of the nanoparticle core, synthesis process and unreacted reactants, size, shape, crytallinity, surface reactibility, solubility in aqueous media, and degree of aggregation among many other factors

13, 41, 42

.

In terms of reducing their toxicity and enhancing their circulation time, surface coatings are now a prerequisite for nanoparticles. However, it is difficult to control the response of collodial nanoparticles to their enviroment due to the complexity of biological fluids. The nanoparticles are very sensitive to conditions like pH, ionic strength, concentration and temperature

43

. Therefore, it is important to characterize the nanoparticles in 19 ACS Paragon Plus Environment


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biological fluids. When nanoparticles are dispersed in biological fluids, two different hypotheses have been reported. One possible scenario is that particles may be surrounded with a protein corona and other bio-macromolecules. Another possibility is that they may aggregate in kinetically driven processes through the formation of clusters

44, 45

.

The role of serum in the cell culture media on particle stability was investigated here by measuring the particle size by DLS at different FBS concentrations (Figure 2B). These experiments were performed because it is anticipated that the proteins and the other macromolecules in cell culture media might be active towards the surface of the nanoparticle. The results demonstrated that nanoparticle size in various amounts of FBS was larger than their non-serum containing samples. As indicated in the literature, it was assumed here that the change in diameter of the nanoparticles can be explained by the formation of a protein corona around the particles, although this would need to be the focus of future studies

44, 45

.

Besides the physical and chemical properties of nanoparticles, reactive oxygen species (ROS) generation is one of the most well known methods related to nanoparticle toxicity 9, 46

. The exact mechanism of ROS generation by nanoparticles still remains unclear, but

it is known that nanoparticles of various sizes and chemistries attack the mitochondria (a redox active organelle of cells) which may change ROS production, and possibly cause 9

an alteration in antioxidant activity . Of course, this mechanism of toxicity is being exploited

for cancer

cell

treatment

to

increase their death

without

using

chemotherapeautic agents that also kill healthy cells.


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Nanoparticle interactions with cells in vitro need to be evaluated to understand their mechanism of biological influence induced by nanoparticles

46

. Here, 0.1 M dextran

coated sub 5 nm cerium oxide nanoparticles were synthesized in a 30% ammonium hydroxide solution, when they were dispersed in cell culture media at various pH values and concentrations (see Supporting Information: Table 1). It is believed that the catalytic activity of nanoceria stems from oxygen defects or delocalized electron density on the surface of the particles

47, 48

. Low reduction potential of the Ce+3/Ce+4 (~1.52 V) redox

couple makes regeneration of the surface and catalytically active sites feasible for extended time periods

47

. Thus, by altering the pH of the environment (i.e., ion balance),

a change in the catalytic activity can be created.

Initially, the cytotoxicity of nanoceria on osteosarcoma cells was studied at 5 different pH values: pH 3, pH 5, pH 6, pH 7, and pH 9. Results from pH 3 and 5 were not reported, because, including the control groups, none of the cells were alive at any nanoparticle concentration. When first day cytotoxicity results were compared at pH 7 and 9, it was observed that there were no significant differences compared to controls at any concentration. However, at pH 6, cell death started at a 250 µg/mL dextran coated cerium oxide concentrations for the first day. At the end of 3 days of nanoparticle treatment, the IC-50 value for pH 7 was 1000 µg/mL and was greater than 1000 µg/mL for pH 9 (Table 1). After 5 days, it was found to be greater than 250 µg/mL for both pH 7 and 9. 3 days after treating osteosarcoma cells at pH 6, the IC- 50 value was found to be greater than 50 µg/mL; after 5 days this value was 100 µg/mL. Among all of the pH 21 ACS Paragon Plus Environment


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values, nanoceria showed the most toxic behavior at a pH of 6, meaning the highest numbers of osteosarcoma cell death.

After determinig an optimal pH value that killed bone cancer cells when exposed to ceria nanoparticles, we then determined the cytotoxicity of ceria nanoparticles against healthy bone cells. At the end of the first day of nanoceria treatment at pH 6, there was no significant difference in healthy bone cell viability compared to controls at any nanoparticle concentration (10 µg/mL – 1000 µg/mL). Cell viability started to decrease for healthy bone cells as well as after 3 days of treatment, but was much less compared to bone cancer cells; even though bone cancer cells grew much faster compared to healthy bone cells. 3 days after treating osteoblasts at pH 6, no difference among nanoparticle concentrations was observed. When osteosarcoma cells were treated at the same condition, this value was found to be greater than 50 µg/mL. After 5 days, at pH 6, the osteosarcoma IC-50 value was found to be 100 µg/mL, whereas it was greater than 500 µg/mL for healthy osteoblasts (Table 1). This provides the first data of an optimal concentration of nanoceria (between 100 and 500 µg/mL) for the selective killing of osteosarcome over healthy osteoblasts.

In addition to data for healthy osteoblasts and osteosarcoma cells at pH 6, the IC-50 values at a physiological pH (pH7) for cancer and healthy cells were compared. After 3 and 5 days of treatment for healthy osteoblast cells at pH 7, IC-50 values were observed well above 1000 µg/mL. However, in the presence of osteosarcoma cells after 5 and 3 days of treatment, the IC-50 values were 250 µg/mL and 1000 µg/mL, respectively.


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Although pH 6 was found to be an efficient condition for killing osteosarcoma cells in the presence of ceria, siginificant differences were found at pH 7 for selective killing cancer over healthy bone cells in a dose and time dependent manner.

Previously, Perez et al. reported optimal antioxidant activity at physiological and basic pH values, whereas at acidic pH, nanoceria acted like an oxidase. In other words, the higher cytotoxic activity of nanoceria at lower pH values was explained as ‘oxidase-like’ activity

13

. In Figure 6, osteosarcoma cells also showed an increase in ROS generation

when treated with a 250-1000 µg/mL nanoparticle dose range at pH 6 as opposed to nonsiginficant changes in ROS generation for healthy osteoblast cells. Thus, a higher toxic activity of nanoceria at pH values of 6 may also be explained by an enhanced ‘oxidaselike’ activity in nanoceria. Although acidic cellular environments lead to a preferential uptake of nanoceria into cancer cells, the mechanism of uptake is still controversial. Wason et al. explained the activity of nanoceria as the producer of H2O2 in an acidic environment (similar with the cancer microenvironment) and as a scavenger of H2O2 in a neutral environment

32

.

Another factor related with cytotoxicity is cellular uptake and sub-cellular localization of the particles. This localization depends on surface charge of the nanoparticles, charge of the cellular membrane (negatively charged) and pH of the cellular organelles such as lysosomes (which are acidic) and cytoplasma (which is at a neutral pH in normal cells) 23, 24

13,

. Due to the negative charge of the cell membrane, highly positively charged

nanoparticles will attack the cell membrane intensively through electrostatic interactions. 23 ACS Paragon Plus Environment


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Due to this possible interation, zeta potential studies were performed here. In the current studies, the zeta potential of the 0.1 M dextran coated ceria nanoparticles was measured at 0.58 mV with a deviation of +/- 2.27 mV at physiological pH values (Figure 2D). At pH 9, in osteosarcoma media, the zeta potential was measured at 6.32. At acidic pH values (pH 6), the zeta potential of the particles increased up to 17 mV, both in osteosarcoma and osteoblast media. The highly positive surface charge of the particles at pH 6 may enhance the cellular uptake of the nanoparticles compared to other pH values. In addition to the change in surface charge, a change in size was also observed in a pH dependent manner (Figure 2C). Since the particles are smaller in diameter at pH 6, their cellular uptake may be easier than their larger counterparts at pH 7 and pH 9, although this remains to be studied.

In this study, the cytotoxic effect of nanoceria on killing osteosarcoma cells was demonstrated for the first time. Previously, different sizes and surface coatings for nanoceria have been studied for lung, pancreas, breast and ovarian cancer cells

13, 29, 32

showing various level of toxicity. However, as previously declared by Park et al. and others, different cell types or cellular microenvironments have a various effects on the cytotoxicity exerted by the same nanoparticles

9, 13, 25, 31

. The exact mechanism of

cytotoxicity is not fully understood at this moment. However, there are several differences in cellular physiology that may be related with nanoceria’s antioxidant effects that have yet to be exploited.

5. Conclusions 24 ACS Paragon Plus Environment

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This research mainly focused on the cytotoxic effect of 0.1 M dextran coated cerium oxide nanoparticles on osteosarcoma and healthy osteoblast cells based on a dose and time dependent manner at different pH values (pH 3, 5, 6, 7, and 9). 0.1 M Dextran coated sub 5 nm cerium oxide nanoparticles were synthesized by an alkaline based precipitation method. Among all of the pH values, dextran coated nanoceria enhanced killing of osteosarcoma cells at pH of 6. Nanoceria was found to be much less toxic to healthy osteoblasts at pH 6. An efficient dose range after 3 days of treatment of osteosarcoma cells was between 250 µg/mL – 1000 µg/mL whereas it exerted minimum toxicity to healthy osteoblasts at the same concentration.

Acknowledgements

Dr. Hilal Yazici is supported by the TÜBĐTAK (The Scientific and Technological Research Council of Turkey) – BĐDEB, International Postdoctoral Research Scholarship Programme (Programme Number: 2219).

The authors also thank Northeastern

University for funding and William Fowle for his help with transmission electron microscopy images.

Supporting Information Information is provided on XPS analyses and UV-visible absorption spectra of cerium oxide nanoparticles, MTS results based on cell density calculations, osteoblast viability after ceria treatment at pH 7, and a table of pH correlation to nanoparticle concentration. This material is available free of charge via the Internet at http://pubs.acs.org.



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REFERENCES: (1) Siegel, R.; Ma, J.; Zou, Z., Jemal, A. Cancer statistics, 2014. CA: A Cancer Journal for Clinicians. 2014, 64, 9-29. (2) Chertok, B.; Moffat, B. A.; David, A. E.; Yu, F. Q.; Bergemann, C.; Ross, B. D., et al. Iron oxide nanoparticles as a drug delivery vehicle for mri monitored magnetic targeting of brain tumors. Biomaterials. 2008, 29, 487-96. (3) Dhule, S. S.; Penfornis, P.; He, J. B.; Harris, M. R.; Terry, T.; John, V., et al. The combined effect of encapsulating curcumin and c6 ceramide in liposomal nanoparticles against osteosarcoma. Molecular Pharmaceutics. 2014, 11, 417-27. (4) Barua, S., Mitragotri, S. Challenges associated with penetration of nanoparticles across cell and tissue barriers: A review of current status and future prospects. Nano Today. 2014, 9, 223-43. (5) Wilczewska, A. Z.; Niemirowicz, K.; Markiewicz, K. H., Car, H. Nanoparticles as drug delivery systems. Pharmacological Reports. 2012, 64, 1020-37. (6) Wang, A. Z.; Langer, R., Farokhzad, O. C. Nanoparticle delivery of cancer drugs. Annual Review of Medicine, Vol 63. 2012, 63, 185-98. (7) Palakurthi, S.; Yellepeddi, V. K., Vangara, K. K. Recent trends in cancer drug resistance reversal strategies using nanoparticles. Expert Opinion on Drug Delivery. 2012, 9, 287-301. (8) Alili, L.; Sack, M.; Karakoti, A. S.; Teuber, S.; Puschmann, K.; Hirst, S. M., et al. Combined cytotoxic and anti-invasive properties of redox-active nanoparticles in tumor– stroma interactions. Biomaterials. 2011, 32, 2918-29. (9) Park, E.-J.; Choi, J.; Park, Y.-K., Park, K. Oxidative stress induced by cerium oxide nanoparticles in cultured beas-2b cells. Toxicology. 2008, 245, 90-100. (10) Bouzigues, C.; Gacoin, T., Alexandrou, A. Biological applications of rare-earth based nanoparticles. ACS Nano. 2011, 5, 8488-505. (11) Karakoti, A. S.; Monteiro-Riviere, N. A.; Aggarwal, R.; Davis, J. P.; Narayan, R. J.; Self, W. T., et al. Nanoceria as antioxidant: Synthesis and biomedical applications. JOM. 2008, 60, 33-7. (12) Lord, M. S.; Tsoi, B.; Gunawan, C.; Teoh, W. Y.; Amal, R., Whitelock, J. M. Antiangiogenic activity of heparin functionalised cerium oxide nanoparticles. Biomaterials. 2013, 34, 8808-18. (13) Asati, A.; Santra, S.; Kaittanis, C., Perez, J. M. Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles. ACS Nano. 2010, 4, 5321-31. (14) Sack, M.; Alili, L.; Karaman, E.; Das, S.; Gupta, A.; Seal, S., et al. Combination of conventional chemotherapeutics with redox-active cerium oxide nanoparticles—a novel aspect in cancer therapy. Molecular cancer therapeutics. 2014, 13, 1740-9. (15) Alili, L.; Sack, M.; von Montfort, C.; Giri, S.; Das, S.; Carroll, K. S., et al. Downregulation of tumor growth and invasion by redox-active nanoparticles. Antioxidants & redox signaling. 2013, 19, 765-78. (16) Das, S.; Dowding, J. M.; Klump, K. E.; McGinnis, J. F.; Self, W., Seal, S. Cerium oxide nanoparticles: Applications and prospects in nanomedicine. Nanomedicine. 2013, 8, 1483-508.


Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17) Asati, A.; Santra, S.; Kaittanis, C.; Nath, S., Perez, J. M. Oxidase-like activity of polymer-coated cerium oxide nanoparticles. Angewandte Chemie-International Edition. 2009, 48, 2308-12. (18) Dowding, J. M.; Das, S.; Kumar, A.; Dosani, T.; McCormack, R.; Gupta, A., et al. Cellular interaction and toxicity depend on physicochemical properties and surface modification of redox-active nanomaterials. ACS nano. 2013, 7, 4855-68. (19) Wason, M. S., Zhao, J. Cerium oxide nanoparticles: Potential applications for cancer and other diseases. American Journal of Translational Research. 2013, 5, 126-31. (20) Waris, G., Ahsan, H. Reactive oxygen species: Role in the development of cancer and various chronic conditions. Journal of carcinogenesis. 2006, 5. (21) Campbell, C. T., Peden, C. H. F. Chemistry - oxygen vacancies and catalysis on ceria surfaces. Science. 2005, 309, 713-4. (22) Dunnick, K. M.; Pillai, R.; Pisane, K. L.; Stefaniak, A. B.; Sabolsky, E. M., Leonard, S. S. The effect of cerium oxide nanoparticle valence state on reactive oxygen species and toxicity. Biological trace element research. 2015, 1-12. (23) Patil, S.; Sandberg, A.; Heckert, E.; Self, W., Seal, S. Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zeta potential. Biomaterials. 2007, 28, 4600-7. (24) Asati, A.; Santra, S.; Kaittanis, C.; Nath, S., Perez, J. M. Oxidase-like activity of polymer-coated cerium oxide nanoparticles. Angewandte Chemie International Edition. 2009, 48, 2308-12. (25) Perez, J. M.; Asati, A.; Nath, S., Kaittanis, C. Synthesis of biocompatible dextrancoated nanoceria with ph-dependent antioxidant properties. Small. 2008, 4, 552-6. (26) Zhai, Y.; Zhou, K.; Xue, Y.; Qin, F.; Yang, L., Yao, X. Synthesis of water-soluble chitosan-coated nanoceria with excellent antioxidant properties. Rsc Advances. 2013, 3, 6833-8. (27) Yazici, H.; Alpaslan, E., Webster, T. J. The role of dextran coatings on the cytotoxicity properties of ceria nanoparticles toward bone cancer cells. JOM. 2015, 67, 804-10. (28) Karakoti, A.; Kuchibhatla, S. V.; Babu, K. S., Seal, S. Direct synthesis of nanoceria in aqueous polyhydroxyl solutions. The Journal of Physical Chemistry C. 2007, 111, 17232-40. (29) Lin, W.; Huang, Y.-w.; Zhou, X.-D., Ma, Y. Toxicity of cerium oxide nanoparticles in human lung cancer cells. International Journal of Toxicology. 2006, 25, 451-7. (30) Giri, S.; Karakoti, A.; Graham, R. P.; Maguire, J. L.; Reilly, C. M.; Seal, S., et al. Nanoceria: A rare-earth nanoparticle as a novel anti-angiogenic therapeutic agent in ovarian cancer. Plos One. 2013, 8. (31) Gao, Y.; Chen, K.; Ma, J.-l., Gao, F. Cerium oxide nanoparticles in cancer. Oncotargets and Therapy. 2014, 7, 835-40. (32) Wason, M. S.; Colon, J.; Das, S.; Seal, S.; Turkson, J.; Zhao, J. H., et al. Sensitization of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ros production. Nanomedicine-Nanotechnology Biology and Medicine. 2013, 9, 558-69. (33) Hussain, S.; Al-Nsour, F.; Rice, A. B.; Marshburn, J.; Yingling, B.; Ji, Z., et al. Cerium dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood monocytes. Acs Nano. 2012, 6, 5820-9.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

(34) Morton, S. W.; Shah, N. J.; Quadir, M. A.; Deng, Z. J.; Poon, Z., Hammond, P. T. Osteotropic therapy via targeted layer-by-layer nanoparticles. Advanced Healthcare Materials. 2014, 3, 867-75. (35) Marina, N.; Gebhardt, M.; Teot, L., Gorlick, R. Biology and therapeutic advances for pediatric osteosarcoma. Oncologist. 2004, 9, 422-41. (36) Chang, R.; Sun, L. L., Webster, T. J. Short communication: Selective cytotoxicity of curcumin on osteosarcoma cells compared to healthy osteoblasts. International Journal of Nanomedicine. 2014, 9, 461-5. (37) Dhule, S. S.; Penfornis, P.; Frazier, T.; Walker, R.; Feldman, J.; Tan, G., et al. Curcumin-loaded gamma-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma. Nanomedicine-Nanotechnology Biology and Medicine. 2012, 8, 440-51. (38) Yang, S.; Chen, D. Y.; Li, N. J.; Mei, X.; Qi, X. X.; Li, H., et al. A facile preparation of targetable ph-sensitive polymeric nanocarriers with encapsulated magnetic nanoparticles for controlled drug release. Journal of Materials Chemistry. 2012, 22, 25354-61. (39) Gerweck, L. E., Seetharaman, K. Cellular ph gradient in tumor versus normal tissue: Potential exploitation for the treatment of cancer. Cancer Research. 1996, 56, 1194-8. (40) Lewinski, N.; Colvin, V., Drezek, R. Cytotoxicity of nanoparticles. Small. 2008, 4, 26-49. (41) Verma, A., Stellacci, F. Effect of surface properties on nanoparticle–cell interactions. Small. 2010, 6, 12-21. (42) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P., et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009, 8, 543-57. (43) Qi, L.; Fresnais, J.; Muller, P.; Theodoly, O.; Berret, J.-F., Chapel, J.-P. Interfacial activity of phosphonated-peg functionalized cerium oxide nanoparticles. Langmuir. 2012, 28, 11448-56. (44) Safi, M.; Courtois, J.; Seigneuret, M.; Conjeaud, H., Berret, J.-F. The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles. Biomaterials. 2011, 32, 9353-63. (45) Ould-Moussa, N.; Safi, M.; Guedeau-Boudeville, M.-A.; Montero, D.; Conjeaud, H., Berret, J.-F. In vitro toxicity of nanoceria: Effect of coating and stability in biofluids. Nanotoxicology. 2014, 8, 799-811. (46) Lord, M. S.; Jung, M.; Teoh, W. Y.; Gunawan, C.; Vassie, J. A.; Amal, R., et al. Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line u937. Biomaterials. 2012, 33, 7915-24. (47) Das, S.; Chigurupati, S.; Dowding, J.; Munusamy, P.; Baer, D. R.; McGinnis, J. F., et al. Therapeutic potential of nanoceria in regenerative medicine. MRS Bulletin. 2014, 39, 976-83. (48) Cafun, J. D.; Kvashnina, K. O.; Casals, E.; Puntes, V. F., Glatzel, P. Absence of ce3+ sites in chemically active colloidal ceria nanoparticles. Acs Nano. 2013, 7, 1072632.


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Figure Legends:

Figure 1: (A) 0.1 M Dextran coated cerium oxide nanoparticle (DCN) synthesis and (B) Schematic of DCN treatment against bone cancer and healthy cells.

Figure 2: (A) TEM micrograph of 0.1 M DCN; (B) Size of DCN in DMEM supplemented with 0, 1, 2.5, 5, and 10 % FBS and 1% P/S; (C) Size of DCN in the presence of osteosarcoma culture media (DMEM supplemented with 10% FBS and 1% P/S) at different pH values; and (D) Zeta potential of DCN at different pH values. 1

denotes that particles were dispersed in osteosarcoma media, 2denotes that particles were

dispersed in osteoblast basal medium (PromoCell, C-27010) with a supplement mix (PromoCell, C-39615).

Figure 3: Toxicity effect of 0.1 M DCN against osteosarcoma cells at various concentrations after 1, 3 and 5 days of treatment at (A) pH 9 and (B) pH 7. Data = mean +/- SEM and *p < 0.05 compared to controls at the same time period and **p < 0.05 at the same DCN concentration at the same time period. Control groups are only cells treated with media at (A) pH 9 and (B) pH 7.

Figure 4: Toxicity effect of 0.1 M DCN against osteosarcoma cells at various concentrations after 1, 3 and 5 days of treatment at pH 6. Data = mean +/- SEM and *p < 0.05 compared to controls at the same time period and **p < 0.05 at the same DCN concentration at the same time period. Control groups are only cells treated with media at pH 6. 29 ACS Paragon Plus Environment


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Figure 5: Toxicity effect of 0.1 M dextran DCN against osteoblast cells at various concentrations after 1, 3 and 5 days of treatment at pH 6. Data = mean +/- SEM and *p < 0.05 compared to controls at the same time period and **p < 0.05 at the same DCN concentration at the same time period. Control groups are cells treated with media at pH 6.

Figure 6: ROS generation of 0.1 M dextran DCN against osteosarcoma and healthy osteoblast cells at various concentrations after 1 day of treatment at pH 6. Data = mean +/- SEM and *p < 0.05 compared to controls and **p < 0.05 compared to 1000 µg/mL. Control groups are cells treated with media at pH 6.

Table 1: IC-50 values of osteosarcoma cells at pH 6, 7, and 9 and healthy osteoblast cells at pH 6 and pH 7 after 3 and 5 days of nanoparticle treatment.


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Table of Contents Use Only pH Dependent Activity of Dextran Coated Cerium Oxide Nanoparticles on Prohibiting Osteosarcoma Cell Proliferation Ece Alpaslan, Hilal Yazici, Negar H. Golshan, Katherine S. Ziemer and Thomas J. Webstera

+16 mV

H+

H+

H+

H+ Tumor Mass

+ ++ + + + +

H

H

+ ++ + + + +

particles delivered > pH 6 H+

1000 µg/mL

1000 µg/mL

> 250 µg/mL

> 250 µg/mL

Osteoblast

pH 6

pH 6

pH 7

> 50 µg/mL

ND

> 1000 µg/mL

100 µg/mL

> 500 µg/mL

> 1000 µg/mL

ACS Paragon Plus Environment

Page 38 of Figure 6 38


ROS Generation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

250 Osteosarcoma 200

*

Osteoblast

150

** *

** *

100 50 0 Control (pH 7.4)

Control (pH 6)

1000

500

Nanoparticle Concentration (µg/mL)

ACS Paragon Plus Environment

250

pH-Dependent Activity of Dextran-Coated Cerium Oxide Nanoparticles

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