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Folic acid-tagged titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells
Accepted Manuscript Folic acid-tagged titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells
Jin-wei Ai, Wei-dong Liu, Bin Liu PII: DOI: Reference:
S0928-4931(16)30894-3 doi: 10.1016/j.msec.2017.03.027 MSC 7535
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
18 August 2016 28 December 2016 3 March 2017
Please cite this article as: Jin-wei Ai, Wei-dong Liu, Bin Liu , Folic acid-tagged titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.027
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ACCEPTED MANUSCRIPT Folic acid-tagged Titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells
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Jin-wei Ai 1, Wei-dong Liu 2*, Bin Liu3,
Department of Orthopedics, Henan University of Chinese Medicine, 450002, China
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Department of Orthopedics, Huai’an First People’s Hospital, Nanjing Medical University,
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Department of Orthopedic Surgery, The Second People’s Hospital of Liaocheng, Shandong,
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223300, China
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252600, China
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Corresponding author: Wei-dong Liu,
Department of Orthopedics, Huai’an First People’s Hospital, Nanjing Medical University, #6 Beijing Road West, Huai'an, Jiangsu, 223300 P. R. China, Tel&Fax: 0086-517-84907287 Email: [email protected]
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Abstract In this study, folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were
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prepared as a suitable alternative to conventional chemotherapeutic agents to treat human
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osteosarcoma. The particle size of TiNP increased marked after polymer assembly on the
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nanoparticles (NP) surface with a spherical morphology. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might
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due to the specific interaction of FA with the folate receptor which is overexpressed in the cancer
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cells. Especially, FA-TiNP treated cells exhibited chromatin condensation, cell shrinkage and membrane blebbing. FA-TiNP showed significantly higher cancer cell apoptosis with nearly
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38% of cells in apoptosis chamber (early and late) compared to only ~16% for TiNP. The higher
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proportion of Annexin V positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2. Importantly, FA-TiNP increased the sub-G0 population
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to ~25% indicating its superior anticancer effect. The results clearly indicated that FA-TiNP
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induced greater reactive oxygen species (ROS) generation that resulted in higher sub-G0 cell population with higher cell apoptosis. FA-TiNP showed a remarkably higher expression of
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cytochrome C (Cyt C) with a marked increase in the expression of cleaved caspase-3 and PARP. Overall, results suggest that surface modification of TiNP with a specific targeting moiety could enhance the chances of having successful therapies for cancer diseases.
Keywords Titanium, nanoparticles, osteosarcoma, folic acid, apoptosis
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1. Introduction Osteosarcoma is a form of cancer that occurs in bone [1]. Osteosarcoma is a most prevalent in
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teenagers and children who are under 30 years of age and a typical bone cancer. To be specific, it
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is more common in women than in men and most common sites are femur and tibia [2,3]. The
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main treatment options includes chemotherapy, surgery and radiotherapy, however unwanted adverse effects following the severe damage to normal cells has been big problem [4,5]. As a
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result, there is a remarkable shift in the cancer treatment strategy. In this study, we have
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attempted a new approach to treat osteosarcoma while to decrease the associated side effects of chemotherapy.
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Recently, nanotechnology has gained increasing attention and has high potential to increase the
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therapeutic efficacy of drug molecules [6]. Several studies have reported the cytotoxic property
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of metal nanoparticles towards cancer cells by virtue of oxidative stress [7]. To be specific, there has been a growing interest in biological application of titanium dioxide nanoparticles (TiNP)
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owing to its high biocompatibility, low toxicity, high photoreactivity, and excellent chemical stability [8,9]. The unique property and high reactivity of TiNP finds its use in various life
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sciences, biomedical and bioengineering field. The promising applications of TiNP include cancer targeting, drug delivery and biosensors. TiNP has been reported to cause DNA damage in goldfish skin cells (GFSk-S1) while it induced apoptosis in human monoblastoid and bronchial epithelial cells by destabilizing lysosomal membrane and lipid peroxidation [10]. Besides, TiNP showed DNA damage and cell apoptosis in lymphocytes, U937 human monoblastoid cells, A549 human alveolar epithelial cells, NRK-52E normal rat kidney cells, and A431 human epidermal
ACCEPTED MANUSCRIPT cells. TiNP acts as a catalyzer to induce cell death in various cancer cells such as lung cancer cells and liver cancer cells by virtue of its high oxidative property. Further studies have showed that TiNP generates hydroxyl radicals and cause cell death in malignant cancer cells [11,12]. The size and shape of TiNP greatly affect its biological performances. For example, NP of 10-20 nm
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size showed more toxicity than that of NP of 60-80 nm and similarly former induced more
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inflammatory reactions compared to that of one which has relatively larger particle size [13].
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Furthermore, it has been reported that the toxicity of TiNP could be reduced by surface
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modification with a polymer layer.
We have realized that if the NP accumulates preferentially in the cancer cells then the efficiency
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of TiO2 will greatly increase. In this regard, surface coverage or conjugation of a targeting
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ligand which has specific affinity to the cell surface receptor will be beneficial. It has been reported that the folate receptor (FR), a glycopolypeptide has high affinity for folic acid
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molecule. The folate receptors have been over expressed in many cancer cells including
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osteosarcoma cells [14]. The FR has unique advantage over other receptors: (a) it binds to small molecules; (b) FR effectively circulates between outer and inner compartments allowing
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internalizing the FA easily; (c) FA is less expressed in normal cells and therefore circulating NP
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could not access the normal cells and preferentially binds to the cancer cells [15,16]. In the present study, we have conjugated FA on the surface of TiNP for the selective binding of cancer cells. The FA will serve as a targeting ligand that can interact with the corresponding receptors on the cancer cells. In this study, we have investigated the anticancer effect of FA-surface modified TiNP towards the osteosarcoma cancers as an alternative treatment strategy. The polymer-modified metal nanoparticles were characterized in terms of particle size and shape. The in vitro cytotoxicity
ACCEPTED MANUSCRIPT analysis was carried out in MG63 human osteosarcoma cells. The anticancer effect of optimized NP was further confirmed by apoptosis analysis (Hoechst 33342 and FACS) and cell cycle analysis. The mechanistic action of TiO2 was studied by Western Blot analysis. 2. Materials and Methods
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2.1.Materials
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Titanium dioxide, folic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,
reagent grade and used without further purification.
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and RPMI-1640 were purchased from Sigma-Aldrich, China. All other chemicals were of
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2.2.Preparation of Folic acid-coated Titanium Dioxide Nanoparticles (FA-TiNP)
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TiNP was prepared by adding aqueous suspension of TiO2 (2 g) to 30 ml of 1 M HNO3 solution in a dropwise manner and pH of the solution was adjusted to pH 3 (using 1M NaOH) and shaken
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whereby a turbid TiO2 colloids was formed. The colloidal suspension was then centrifuged at
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16000 rpm for 10 min and the supernatant was removed and particles are washed two times with distilled water. After successive washing steps, pellet was collected and dried at 100°C for 2h in
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sample.
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an oven. The resulting powder was placed in a furnace at 400°C for 4 h for the calcination of the
Next, 100 mg of TiNP was dissolved in 25 ml of distilled water and probe sonicated (T25 IKA digital Ultra-Turrax Disperser, USA) for 10 min. Separately, 10 mg of folic acid (FA) was dissolved in 0.1M sodium hydrogen carbonate solution (5 ml) and the pH was adjusted to pH 5.5 using 1 M NaOH solution at room temperature (25°C). The above prepared TiNP suspension (25 ml) was gradually added to FA solution (5 ml) and the solution was gently shaken for 24h at room temperature. The reaction mixture was dialyzed (48h) against distilled water using 3500
ACCEPTED MANUSCRIPT MW cut off dialysis membrane (Spectra/Por, USA) to remove the unreacted FA. The FAconjugated TiO2 NP was collected after freeze drying for 48h. 2.3.Particle size analysis
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The particle size and zeta potential of final NP was measured using Malvern Zetasizer-3000
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(Maler Instruments, U.K). The samples were suitably diluted with distilled water and the
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experiments were repeated in triplicate.
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2.4.Morphology analysis
The morphology of nanoparticle was studied by transmission electron microscope (TEM) using
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(TEM) (JEOL 1200 EXII, JEOL, Japan) operating at an acceleration voltage of 120 Kv. The
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samples were suitably diluted with distilled water. A drop of nanoparticle suspension was placed on the carbon-coated copper grid (200 mesh grids) (Agar Scientific Ltd, UK) and excess liquid
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was removed by filter paper. The particles were counterstained with 2% phosphotungistic acid
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(PTA) and observed in the TEM.
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2.5.Cytotoxicity assay (MTT assay) The reagents (media and serum) were obtained from Gibco (Gibco, Grand Island, NY). Human osteosarcoma cell line (MG-63) was obtained from ATCC, USA. The MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used to measure cellular metabolic potential of treated cells. The MG63 human osteosarcoma cells were purchased from ATCC, USA. The cells were cultured in Roswell Park Memorial Institute (RPMI) culture medium supplemented with 10% of fetal bovine serum (FBS) and 1% of antibiotic mixture. The cells
ACCEPTED MANUSCRIPT were seeded in a 96-well plate and incubated for 24h. The cells were then treated with respective formulations and further incubated for 24h. Next day, cells were washed carefully and treated with MTT solution (20 µl of 5 mg/ml) and incubated for additional 4h. The cells were then exposed with dimethyl sulfoxide (DMSO) to extract the formazan crystal. The absorbance was
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was conducted and measured independently for at least three times.
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then studied at 570 nm using a microplate reader (Biotek, USA) instrument. Each experiment
2.6.Apoptosis assay (Flow cytometer)
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The MG63 cancer cells were incubated in 6-well cell culture plates (Corning®, Sigma-Aldrich) at
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a seeding density of 3×105 cells/well. The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP formulations and further incubated for 24h. The cells were
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carefuly washed two times with ice-cold PBS (Gibco, USA) and harvested. The cells were then
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washed and suspended in a binding buffer as instructed by manufacturer’s protocol (BD Biosciences, USA). The cell suspension was then stained with 1.5 µl of Annexin-V/FITC and 1.5
FACScan
cytometer
and Cell Quest software (FACSCalibur;
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and analyzed using
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µl of PI and incubated for 15 min in dark conditions. The cells were diluted to 1 ml using PBS
BectonDickinson, San Jose, CA).
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2.7.Hoechst 33342 staining The MG63 cancer cells were incubated in 6-well plate at a seeding density of 3×105 cells/well. The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP formulations and further incubated for 24h. Next day, cells were washed and fixed with 2% paraformaldehyde (PFA) (Sigma-Aldrich, China). The cells were again washed and treated with Hoechst 33342 solution (3 µg/ml) (Molecualr ProbesTM, USA) and incubated for 15 min. The
ACCEPTED MANUSCRIPT cells were carefully washed and the images were observed using confocal microscope (Olympus FV1000, Japan). 2.8.Cell cycle analysis
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After cell seeding and overnight incubation, cells were exposed with TiNP and FA-TiNP
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formulations and further incubated for 24h. The cells were then washed with ice-cold PBS and
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fixed with 70% methanol and kept at 4°C for 2h. The cells were then incubated in RNAse free water and incubated with propidium iodide (PI) for 1h at 37°C. The cell cycle analysis was then
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performed using FACScan cytometer and Cell Quest software (FACSCalibur; BectonDickinson,
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San Jose, CA).
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2.9.Western blot analysis
The MG63 cancer cells were incubated in 6-well plate at a seeding density of 3×105 cells/well.
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The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP
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formulations and further incubated for 24h. The cells were carefuly washed two times with icecold PBS and harvested. The proteins were isolated using lysis buffer and quantified using
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bicinchoninic acid assay (BCA) protein assay. The protein samples (30 µg) were subjected to
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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gel. The separated proteins were transferred to Polyvinylidene difluoride (PVDF) membranes for 2 h at 60 V using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA). The membranes were blocked using 5% skimmed milk for 1h and incubated with specific antibodies for 12h (overnight) at 4°C. Next day, membranes were incubated with appropriate peroxidaseconjugated secondary antibodies (dilution ratio, 1:2000) for 2 h. The bands/blots were then visualized using standard x-ray photographic instrument.
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Statistical analysis
All data are expressed as mean±S.D. The statistical analysis was performed using one-way analysis of variance (ANOVA) using Sigma Plot 11.0 software. The level of statistical
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significance chosen was *p < 0.05, unless otherwise stated.
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3. Results and Discussion
Figure 1: Schematic presentation of preparation of folic acid assembled titanium dioxide
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nanoparticles
Although anticancer effect of TiO2 has been reported however no report on osteosarcoma cancer cells is present. Besides, small aerodynamic diameter of TiO2 NP evoked serious concerns on its impact on human health. The ultrafine particles (10-20 nm size) could penetrate deep inside the vital organs and circulatory system by breaching the protective cellular barriers and could produce certain toxicological effects. In the present study, therefore, we have attempted to target
ACCEPTED MANUSCRIPT TiO2 NP to osteosarcoma cancer cells by surface modification with FA (Figure 1). It has been reported that folate receptor is overexpressed in many cancer cells while normal cells does not
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3.1.Physicochemical characterization of FA-TiNP
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express folate receptors [17,18].
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The size distribution and surface charge of TiNP and FA-TiNP was evaluated by dynamic light scattering (DLS) technique. As seen, average particle size of TiNP was 28.5±1.4 nm while the
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particle size significantly increased to 120.7±1.65 nm upon FA conjugation on the NP surface
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(Figure 2). The particle size increase clearly indicates the presence of polymeric layer on the NP surface. Our results revealed that toxicity profile of ultrafine TiNP could be averted by the
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successful polymer conjugation as the particle size increased remarkably. Importantly, particle
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size is in the ideal range for tumor targeting via EPR effect. The FA is therefore expected to play multiple roles in cancer targeting and stability. The FA association on TiNP was further
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confirmed by zeta potential evaluation. The surface charge of TiNP was decreased from strong
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positive charge of 23.4±2.15 mV to -12.4±1.24 mV for FA conjugation on the NP surface. This is due to the fact that negatively charged carboxyl group of FA easily conjugated with positively functionalized TiNP.
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Figure 2: Particle size distributions of TiNP and FA-TiNP using dynamic light scattering
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technique.
The morphology of TiNP and FA-TiNP was assessed by TEM examinations. As shown, bare
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TiNP uniformly distributed on the TEM grid and present as a nanosized and spherical object
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(Figure 3). At specific points, slight agglomeration and aggregation was observed due to the strong interaction of metal NP with each other. On the other hand, FA-TiNP was well-dispersed
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and separated. The presence of polymeric layer on the NP could be visualized as a greyish shell.
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round shape.
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The FA conjugation on the NP surface did not change the particle shape and the present as a
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Figure 3: Particle shape analysis of TiNP and FA-TiNP using transmission electron microscope
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3.2.In vitro cytotoxicity assay
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(TEM).
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The anticancer effect of TiNP and FA-TiNP on the proliferation of MG63 osteosarcoma cancer
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cell was determined by MTT assay (Figure 4).
ACCEPTED MANUSCRIPT Figure 4: Cell survival of MG63 osteosarcoma cells after treatment with TiNP and FA-TiNP and incubated for 24h. The cytotoxicity assay was performed by MTT assay protocol. Error bars indicate the standard error of the mean (SEM) for N = 8 independent experiments As shown, TiNP and FA-TiNP showed a typical concentration-dependent cytotoxic effect in
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osteosarcoma cancer cells. As the concentration of metallic NP increased, cell viability was
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continuously decreased. As expected, FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might due to the specific
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interaction of FA with the folate receptor which is overexpressed in the cancer cells [19]. The FA-TiNP upon interacting with cancer cells were internalized via receptor-mediated endocytosis
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mechanism and resulted in higher concentration in the cancer cells and thereby resulting in
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higher therapeutic effect. The cytotoxic effect of individual NP was further confirmed by calculating the IC50 value. As shown, IC50 value of cancer cells were reduced by 2-fold after
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conjugation of NP with FA (FA-TiNP). It should be noted that at lower concentrations, TiNP did
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not interfere with the MTT-formazan product and started the color intensity (formazan) increased only at higher concentration. In contrast, FA-TiNP interfered with color intensity even at lower
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NP concentration indicating its superior anticancer effect. The enhanced anticancer effect of FA-
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TiNP was mainly attributed to the higher amount of TiO2 NP present in the cancer cells via reorganization of FA to cell surface which allowed it to internalize more quickly than bare TiNP [20].
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Figure 5: (a) Apoptosis analysis of cancer by Hoechst 33342 staining and examined by confocal
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microscope; (b) apoptosis study by Annexin V-FITC/PI staining of MG63 cancer cells. The
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lower left indicates viable cells and upper right quadrants indicates FITC Annexin V binding and for PI uptake. The lower right indicates apoptotic cells. The concentrations of TiNP and FA-
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TiNP used in the study was 50 µg/ml.
3.3.Morphological assessment by Hoechst 33342 staining
ACCEPTED MANUSCRIPT The apoptosis of cancer cells upon treatment with TiNP and FA-TiNP was examined by Hoechst 33342 staining (Figure 5a). The untreated cells maintained its typical morphology without any sign of apoptosis and firmly attached on the cover slip. TiNP and FA-TiNP however induced severe morphological changes which are typical features of cell apoptosis. Especially, FA-TiNP
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treated cells exhibited chromatin condensation, cell shrinkage and membrane blebbing [21].
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Besides, FA-TiNP treated cells showed bright blue fluorescence indicating the high apoptosis
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potential of this nanoformulation.
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3.4.Apoptosis assay by flow cytometer
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The apoptosis potential of TiNP and FA-TiNP was further investigated by Annexin-V/FITC and PI staining (Figure 5b). It has been reported that cells with both FITC Annexin V and PI negative
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is considered viable while cells with both FITC Annexin V and PI positive is considered
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necrotic. And cells with FITC Annexin V positive and PI negative are considered apoptotic. As seen, no apoptosis was observed in untreated control cells and present in the viable chamber. The
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TiNP treated cells however showed increased apoptosis of osteosarcoma cells. Consistent with
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Hoechst staining assay, FA-TiNP showed significantly higher cancer cell apoptosis with nearly 38% of cells in apoptosis chamber (early and late) compared to only ~16% for TiNP. The higher
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proportion of Annexin V-FITC positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2 that might increase the intracellular level of metal NP and thereby higher therapeutic effect [22-24].
3.5.Cell cycle analysis
ACCEPTED MANUSCRIPT In general, metal NP exhibits the anticancer effect by inducing the reactive oxygen species (ROS) which then induce the cancer cell apoptosis. The ROS-mediated cell apoptosis was further
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studied by means of cell cycle analysis via FACS analysis (Figure 6).
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Figure 6: Cell cycle analysis of MG63 cancer cells after treatment with TiNP and FA-TiNP.
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As seen, untreated cells did not show any peaks in the sub-G0 phase while treatment with TiNP induced nearly ~14% of cells in sub-G0 phase. Importantly, FA-TiNP increased the sub-G0
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population to ~25% indicating its superior anticancer effect. The results clearly indicated that
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FA-TiNP induced greater ROS generation that resulted in higher sub-G0 cell population with higher cell apoptosis [25-26]. FA-TiNP induces morphological changes typical of cell death, and
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a marked increase in the sub-G1 population in DNA histogram, indicating apoptosis. The modification of folic acid on the NP surface remarkably promoted the uptake of TiO2 nanoparticles in MG63 cells, on which the folate receptors are overexpressed. Results clearly suggest that the folic acid modified TiNP is practically useful for the cancer therapy since most of the cancer cells are rich of folic acid receptor on their surface.
ACCEPTED MANUSCRIPT 3.6.Western blot analysis As mentioned before, TiO2 actively involved in the generation of intracellular ROS and could result in DNA damage and apparent cell death. In the present study, mechanisms of cell death
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were examined by Western blot analysis (Figure 7).
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Figure 7: Western blot analysis of MG63 cancer cells. Caspase-3, cytochrome C, and PARP was
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examined as a prototype protein.
The ROS generation will damage the mitochondrial membrane which will initiate the apoptosis
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process. Upon mitochondrial damage, cytochrome C (Cyt C) will be released into the cytoplasm
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and induce the cell apoptosis by beginning the intrinsic pathway. As seen, FA-TiNP showed a remarkably higher expression of Cyt C compared to that of control indicating its superior anticancer effect. The apoptosis is a programmed cell death pathways which is of immense importance in health and disease. The mitochondrial dysfunction will result in the release of Cyt C in the cytoplasmic region that will initiate the apoptosis cascade. Therefore change in the mitochondrial function is considered as early event in apoptosis. As seen, FA-TiNP consequently increased the expression of cleaved caspase-3 indicating that Cyt C induced a caspase-dependent
ACCEPTED MANUSCRIPT cell death pathway. Caspases, a family of aspartic acid-specific proteases, are the major effectors of apoptosis. Followed by the initiation of caspase-3, PARP was also significantly upregulated indicating the superior anticancer efficacy of formulations. It has been reported that TiO2 could produce ROS such as hydroxyl radical, superoxide anion, and hydrogen peroxide. The
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generation of ROS will result in the degradation of multiple cellular organelles and cellular
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shown as downstream modulators of p53-dependent apoptosis
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membrane which will eventually cause cell death by apoptosis. Therefore, ROS have been
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4. Conclusion
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In summary, folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were successfully prepared as a suitable alternative to treat human osteosarcoma. The particle size of
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TiNP increased marked after polymer assembly on the nanoparticles (NP) surface with a
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spherical morphology. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might due to the specific interaction of FA with
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the folate receptor which is overexpressed in the cancer cells. Especially, FA-TiNP treated cells
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exhibited chromatin condensation, cell shrinkage and membrane blebbing. FA-TiNP showed significantly higher cancer cell apoptosis with nearly 38% of cells in apoptosis chamber (early
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and late) compared to only ~16% for TiNP. The higher proportion of Annexin V-FITC positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2. Importantly, FA-TiNP increased the sub-G0 population to ~25% indicating its superior anticancer effect. The results clearly indicated that FA-TiNP induced greater ROS generation that resulted in higher sub-G0 cell population with higher cell apoptosis. FA-TiNP showed a remarkably higher expression of Cyt C with a marked increase in the expression of cleaved
ACCEPTED MANUSCRIPT caspase-3 and PARP. Overall, results suggests that surface modification of TiNP with a specific
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targeting moiety could enhance the chances of having successful therapies for cancer diseases.
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Acknowledgement
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The study was supported from the funding grant of Nanjing Medical University, China.
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Competing Interest
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The authors report no conflict of interest.
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Graphical Abstract
ACCEPTED MANUSCRIPT Highlights 1. Folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were prepared to
target osteosarcoma
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2. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to
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that of bare TiNP
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3. FA-TiNP showed a remarkably higher expression of cytochrome C (Cyt C) with a
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marked increase in other proteins
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4. Surface modification of TiNP with a specific targeting moiety could enhance the chances
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of having successful therapies for cancer diseases
Jin-wei Ai, Wei-dong Liu, Bin Liu PII: DOI: Reference:
S0928-4931(16)30894-3 doi: 10.1016/j.msec.2017.03.027 MSC 7535
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
18 August 2016 28 December 2016 3 March 2017
Please cite this article as: Jin-wei Ai, Wei-dong Liu, Bin Liu , Folic acid-tagged titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.027
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ACCEPTED MANUSCRIPT Folic acid-tagged Titanium dioxide nanoparticles for enhanced anticancer effect in osteosarcoma cells
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Jin-wei Ai 1, Wei-dong Liu 2*, Bin Liu3,
Department of Orthopedics, Henan University of Chinese Medicine, 450002, China
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Department of Orthopedics, Huai’an First People’s Hospital, Nanjing Medical University,
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Department of Orthopedic Surgery, The Second People’s Hospital of Liaocheng, Shandong,
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3
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223300, China
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252600, China
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Corresponding author: Wei-dong Liu,
Department of Orthopedics, Huai’an First People’s Hospital, Nanjing Medical University, #6 Beijing Road West, Huai'an, Jiangsu, 223300 P. R. China, Tel&Fax: 0086-517-84907287 Email: [email protected]
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Abstract In this study, folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were
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prepared as a suitable alternative to conventional chemotherapeutic agents to treat human
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osteosarcoma. The particle size of TiNP increased marked after polymer assembly on the
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nanoparticles (NP) surface with a spherical morphology. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might
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due to the specific interaction of FA with the folate receptor which is overexpressed in the cancer
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cells. Especially, FA-TiNP treated cells exhibited chromatin condensation, cell shrinkage and membrane blebbing. FA-TiNP showed significantly higher cancer cell apoptosis with nearly
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38% of cells in apoptosis chamber (early and late) compared to only ~16% for TiNP. The higher
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proportion of Annexin V positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2. Importantly, FA-TiNP increased the sub-G0 population
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to ~25% indicating its superior anticancer effect. The results clearly indicated that FA-TiNP
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induced greater reactive oxygen species (ROS) generation that resulted in higher sub-G0 cell population with higher cell apoptosis. FA-TiNP showed a remarkably higher expression of
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cytochrome C (Cyt C) with a marked increase in the expression of cleaved caspase-3 and PARP. Overall, results suggest that surface modification of TiNP with a specific targeting moiety could enhance the chances of having successful therapies for cancer diseases.
Keywords Titanium, nanoparticles, osteosarcoma, folic acid, apoptosis
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1. Introduction Osteosarcoma is a form of cancer that occurs in bone [1]. Osteosarcoma is a most prevalent in
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teenagers and children who are under 30 years of age and a typical bone cancer. To be specific, it
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is more common in women than in men and most common sites are femur and tibia [2,3]. The
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main treatment options includes chemotherapy, surgery and radiotherapy, however unwanted adverse effects following the severe damage to normal cells has been big problem [4,5]. As a
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result, there is a remarkable shift in the cancer treatment strategy. In this study, we have
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attempted a new approach to treat osteosarcoma while to decrease the associated side effects of chemotherapy.
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Recently, nanotechnology has gained increasing attention and has high potential to increase the
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therapeutic efficacy of drug molecules [6]. Several studies have reported the cytotoxic property
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of metal nanoparticles towards cancer cells by virtue of oxidative stress [7]. To be specific, there has been a growing interest in biological application of titanium dioxide nanoparticles (TiNP)
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owing to its high biocompatibility, low toxicity, high photoreactivity, and excellent chemical stability [8,9]. The unique property and high reactivity of TiNP finds its use in various life
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sciences, biomedical and bioengineering field. The promising applications of TiNP include cancer targeting, drug delivery and biosensors. TiNP has been reported to cause DNA damage in goldfish skin cells (GFSk-S1) while it induced apoptosis in human monoblastoid and bronchial epithelial cells by destabilizing lysosomal membrane and lipid peroxidation [10]. Besides, TiNP showed DNA damage and cell apoptosis in lymphocytes, U937 human monoblastoid cells, A549 human alveolar epithelial cells, NRK-52E normal rat kidney cells, and A431 human epidermal
ACCEPTED MANUSCRIPT cells. TiNP acts as a catalyzer to induce cell death in various cancer cells such as lung cancer cells and liver cancer cells by virtue of its high oxidative property. Further studies have showed that TiNP generates hydroxyl radicals and cause cell death in malignant cancer cells [11,12]. The size and shape of TiNP greatly affect its biological performances. For example, NP of 10-20 nm
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size showed more toxicity than that of NP of 60-80 nm and similarly former induced more
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inflammatory reactions compared to that of one which has relatively larger particle size [13].
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Furthermore, it has been reported that the toxicity of TiNP could be reduced by surface
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modification with a polymer layer.
We have realized that if the NP accumulates preferentially in the cancer cells then the efficiency
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of TiO2 will greatly increase. In this regard, surface coverage or conjugation of a targeting
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ligand which has specific affinity to the cell surface receptor will be beneficial. It has been reported that the folate receptor (FR), a glycopolypeptide has high affinity for folic acid
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molecule. The folate receptors have been over expressed in many cancer cells including
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osteosarcoma cells [14]. The FR has unique advantage over other receptors: (a) it binds to small molecules; (b) FR effectively circulates between outer and inner compartments allowing
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internalizing the FA easily; (c) FA is less expressed in normal cells and therefore circulating NP
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could not access the normal cells and preferentially binds to the cancer cells [15,16]. In the present study, we have conjugated FA on the surface of TiNP for the selective binding of cancer cells. The FA will serve as a targeting ligand that can interact with the corresponding receptors on the cancer cells. In this study, we have investigated the anticancer effect of FA-surface modified TiNP towards the osteosarcoma cancers as an alternative treatment strategy. The polymer-modified metal nanoparticles were characterized in terms of particle size and shape. The in vitro cytotoxicity
ACCEPTED MANUSCRIPT analysis was carried out in MG63 human osteosarcoma cells. The anticancer effect of optimized NP was further confirmed by apoptosis analysis (Hoechst 33342 and FACS) and cell cycle analysis. The mechanistic action of TiO2 was studied by Western Blot analysis. 2. Materials and Methods
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2.1.Materials
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Titanium dioxide, folic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,
reagent grade and used without further purification.
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and RPMI-1640 were purchased from Sigma-Aldrich, China. All other chemicals were of
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2.2.Preparation of Folic acid-coated Titanium Dioxide Nanoparticles (FA-TiNP)
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TiNP was prepared by adding aqueous suspension of TiO2 (2 g) to 30 ml of 1 M HNO3 solution in a dropwise manner and pH of the solution was adjusted to pH 3 (using 1M NaOH) and shaken
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whereby a turbid TiO2 colloids was formed. The colloidal suspension was then centrifuged at
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16000 rpm for 10 min and the supernatant was removed and particles are washed two times with distilled water. After successive washing steps, pellet was collected and dried at 100°C for 2h in
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sample.
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an oven. The resulting powder was placed in a furnace at 400°C for 4 h for the calcination of the
Next, 100 mg of TiNP was dissolved in 25 ml of distilled water and probe sonicated (T25 IKA digital Ultra-Turrax Disperser, USA) for 10 min. Separately, 10 mg of folic acid (FA) was dissolved in 0.1M sodium hydrogen carbonate solution (5 ml) and the pH was adjusted to pH 5.5 using 1 M NaOH solution at room temperature (25°C). The above prepared TiNP suspension (25 ml) was gradually added to FA solution (5 ml) and the solution was gently shaken for 24h at room temperature. The reaction mixture was dialyzed (48h) against distilled water using 3500
ACCEPTED MANUSCRIPT MW cut off dialysis membrane (Spectra/Por, USA) to remove the unreacted FA. The FAconjugated TiO2 NP was collected after freeze drying for 48h. 2.3.Particle size analysis
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The particle size and zeta potential of final NP was measured using Malvern Zetasizer-3000
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(Maler Instruments, U.K). The samples were suitably diluted with distilled water and the
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experiments were repeated in triplicate.
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2.4.Morphology analysis
The morphology of nanoparticle was studied by transmission electron microscope (TEM) using
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(TEM) (JEOL 1200 EXII, JEOL, Japan) operating at an acceleration voltage of 120 Kv. The
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samples were suitably diluted with distilled water. A drop of nanoparticle suspension was placed on the carbon-coated copper grid (200 mesh grids) (Agar Scientific Ltd, UK) and excess liquid
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was removed by filter paper. The particles were counterstained with 2% phosphotungistic acid
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(PTA) and observed in the TEM.
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2.5.Cytotoxicity assay (MTT assay) The reagents (media and serum) were obtained from Gibco (Gibco, Grand Island, NY). Human osteosarcoma cell line (MG-63) was obtained from ATCC, USA. The MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used to measure cellular metabolic potential of treated cells. The MG63 human osteosarcoma cells were purchased from ATCC, USA. The cells were cultured in Roswell Park Memorial Institute (RPMI) culture medium supplemented with 10% of fetal bovine serum (FBS) and 1% of antibiotic mixture. The cells
ACCEPTED MANUSCRIPT were seeded in a 96-well plate and incubated for 24h. The cells were then treated with respective formulations and further incubated for 24h. Next day, cells were washed carefully and treated with MTT solution (20 µl of 5 mg/ml) and incubated for additional 4h. The cells were then exposed with dimethyl sulfoxide (DMSO) to extract the formazan crystal. The absorbance was
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was conducted and measured independently for at least three times.
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then studied at 570 nm using a microplate reader (Biotek, USA) instrument. Each experiment
2.6.Apoptosis assay (Flow cytometer)
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The MG63 cancer cells were incubated in 6-well cell culture plates (Corning®, Sigma-Aldrich) at
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a seeding density of 3×105 cells/well. The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP formulations and further incubated for 24h. The cells were
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carefuly washed two times with ice-cold PBS (Gibco, USA) and harvested. The cells were then
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washed and suspended in a binding buffer as instructed by manufacturer’s protocol (BD Biosciences, USA). The cell suspension was then stained with 1.5 µl of Annexin-V/FITC and 1.5
FACScan
cytometer
and Cell Quest software (FACSCalibur;
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and analyzed using
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µl of PI and incubated for 15 min in dark conditions. The cells were diluted to 1 ml using PBS
BectonDickinson, San Jose, CA).
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2.7.Hoechst 33342 staining The MG63 cancer cells were incubated in 6-well plate at a seeding density of 3×105 cells/well. The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP formulations and further incubated for 24h. Next day, cells were washed and fixed with 2% paraformaldehyde (PFA) (Sigma-Aldrich, China). The cells were again washed and treated with Hoechst 33342 solution (3 µg/ml) (Molecualr ProbesTM, USA) and incubated for 15 min. The
ACCEPTED MANUSCRIPT cells were carefully washed and the images were observed using confocal microscope (Olympus FV1000, Japan). 2.8.Cell cycle analysis
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After cell seeding and overnight incubation, cells were exposed with TiNP and FA-TiNP
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formulations and further incubated for 24h. The cells were then washed with ice-cold PBS and
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fixed with 70% methanol and kept at 4°C for 2h. The cells were then incubated in RNAse free water and incubated with propidium iodide (PI) for 1h at 37°C. The cell cycle analysis was then
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performed using FACScan cytometer and Cell Quest software (FACSCalibur; BectonDickinson,
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San Jose, CA).
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2.9.Western blot analysis
The MG63 cancer cells were incubated in 6-well plate at a seeding density of 3×105 cells/well.
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The cells were incubated overnight. The cells were then treated with TiNP and FA-TiNP
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formulations and further incubated for 24h. The cells were carefuly washed two times with icecold PBS and harvested. The proteins were isolated using lysis buffer and quantified using
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bicinchoninic acid assay (BCA) protein assay. The protein samples (30 µg) were subjected to
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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gel. The separated proteins were transferred to Polyvinylidene difluoride (PVDF) membranes for 2 h at 60 V using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA). The membranes were blocked using 5% skimmed milk for 1h and incubated with specific antibodies for 12h (overnight) at 4°C. Next day, membranes were incubated with appropriate peroxidaseconjugated secondary antibodies (dilution ratio, 1:2000) for 2 h. The bands/blots were then visualized using standard x-ray photographic instrument.
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Statistical analysis
All data are expressed as mean±S.D. The statistical analysis was performed using one-way analysis of variance (ANOVA) using Sigma Plot 11.0 software. The level of statistical
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significance chosen was *p < 0.05, unless otherwise stated.
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3. Results and Discussion
Figure 1: Schematic presentation of preparation of folic acid assembled titanium dioxide
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nanoparticles
Although anticancer effect of TiO2 has been reported however no report on osteosarcoma cancer cells is present. Besides, small aerodynamic diameter of TiO2 NP evoked serious concerns on its impact on human health. The ultrafine particles (10-20 nm size) could penetrate deep inside the vital organs and circulatory system by breaching the protective cellular barriers and could produce certain toxicological effects. In the present study, therefore, we have attempted to target
ACCEPTED MANUSCRIPT TiO2 NP to osteosarcoma cancer cells by surface modification with FA (Figure 1). It has been reported that folate receptor is overexpressed in many cancer cells while normal cells does not
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3.1.Physicochemical characterization of FA-TiNP
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express folate receptors [17,18].
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The size distribution and surface charge of TiNP and FA-TiNP was evaluated by dynamic light scattering (DLS) technique. As seen, average particle size of TiNP was 28.5±1.4 nm while the
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particle size significantly increased to 120.7±1.65 nm upon FA conjugation on the NP surface
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(Figure 2). The particle size increase clearly indicates the presence of polymeric layer on the NP surface. Our results revealed that toxicity profile of ultrafine TiNP could be averted by the
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successful polymer conjugation as the particle size increased remarkably. Importantly, particle
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size is in the ideal range for tumor targeting via EPR effect. The FA is therefore expected to play multiple roles in cancer targeting and stability. The FA association on TiNP was further
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confirmed by zeta potential evaluation. The surface charge of TiNP was decreased from strong
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positive charge of 23.4±2.15 mV to -12.4±1.24 mV for FA conjugation on the NP surface. This is due to the fact that negatively charged carboxyl group of FA easily conjugated with positively functionalized TiNP.
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Figure 2: Particle size distributions of TiNP and FA-TiNP using dynamic light scattering
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technique.
The morphology of TiNP and FA-TiNP was assessed by TEM examinations. As shown, bare
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TiNP uniformly distributed on the TEM grid and present as a nanosized and spherical object
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(Figure 3). At specific points, slight agglomeration and aggregation was observed due to the strong interaction of metal NP with each other. On the other hand, FA-TiNP was well-dispersed
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and separated. The presence of polymeric layer on the NP could be visualized as a greyish shell.
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round shape.
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The FA conjugation on the NP surface did not change the particle shape and the present as a
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Figure 3: Particle shape analysis of TiNP and FA-TiNP using transmission electron microscope
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3.2.In vitro cytotoxicity assay
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(TEM).
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The anticancer effect of TiNP and FA-TiNP on the proliferation of MG63 osteosarcoma cancer
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cell was determined by MTT assay (Figure 4).
ACCEPTED MANUSCRIPT Figure 4: Cell survival of MG63 osteosarcoma cells after treatment with TiNP and FA-TiNP and incubated for 24h. The cytotoxicity assay was performed by MTT assay protocol. Error bars indicate the standard error of the mean (SEM) for N = 8 independent experiments As shown, TiNP and FA-TiNP showed a typical concentration-dependent cytotoxic effect in
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osteosarcoma cancer cells. As the concentration of metallic NP increased, cell viability was
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continuously decreased. As expected, FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might due to the specific
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interaction of FA with the folate receptor which is overexpressed in the cancer cells [19]. The FA-TiNP upon interacting with cancer cells were internalized via receptor-mediated endocytosis
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mechanism and resulted in higher concentration in the cancer cells and thereby resulting in
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higher therapeutic effect. The cytotoxic effect of individual NP was further confirmed by calculating the IC50 value. As shown, IC50 value of cancer cells were reduced by 2-fold after
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conjugation of NP with FA (FA-TiNP). It should be noted that at lower concentrations, TiNP did
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not interfere with the MTT-formazan product and started the color intensity (formazan) increased only at higher concentration. In contrast, FA-TiNP interfered with color intensity even at lower
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NP concentration indicating its superior anticancer effect. The enhanced anticancer effect of FA-
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TiNP was mainly attributed to the higher amount of TiO2 NP present in the cancer cells via reorganization of FA to cell surface which allowed it to internalize more quickly than bare TiNP [20].
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Figure 5: (a) Apoptosis analysis of cancer by Hoechst 33342 staining and examined by confocal
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microscope; (b) apoptosis study by Annexin V-FITC/PI staining of MG63 cancer cells. The
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lower left indicates viable cells and upper right quadrants indicates FITC Annexin V binding and for PI uptake. The lower right indicates apoptotic cells. The concentrations of TiNP and FA-
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TiNP used in the study was 50 µg/ml.
3.3.Morphological assessment by Hoechst 33342 staining
ACCEPTED MANUSCRIPT The apoptosis of cancer cells upon treatment with TiNP and FA-TiNP was examined by Hoechst 33342 staining (Figure 5a). The untreated cells maintained its typical morphology without any sign of apoptosis and firmly attached on the cover slip. TiNP and FA-TiNP however induced severe morphological changes which are typical features of cell apoptosis. Especially, FA-TiNP
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treated cells exhibited chromatin condensation, cell shrinkage and membrane blebbing [21].
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Besides, FA-TiNP treated cells showed bright blue fluorescence indicating the high apoptosis
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potential of this nanoformulation.
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3.4.Apoptosis assay by flow cytometer
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The apoptosis potential of TiNP and FA-TiNP was further investigated by Annexin-V/FITC and PI staining (Figure 5b). It has been reported that cells with both FITC Annexin V and PI negative
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is considered viable while cells with both FITC Annexin V and PI positive is considered
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necrotic. And cells with FITC Annexin V positive and PI negative are considered apoptotic. As seen, no apoptosis was observed in untreated control cells and present in the viable chamber. The
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TiNP treated cells however showed increased apoptosis of osteosarcoma cells. Consistent with
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Hoechst staining assay, FA-TiNP showed significantly higher cancer cell apoptosis with nearly 38% of cells in apoptosis chamber (early and late) compared to only ~16% for TiNP. The higher
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proportion of Annexin V-FITC positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2 that might increase the intracellular level of metal NP and thereby higher therapeutic effect [22-24].
3.5.Cell cycle analysis
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studied by means of cell cycle analysis via FACS analysis (Figure 6).
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Figure 6: Cell cycle analysis of MG63 cancer cells after treatment with TiNP and FA-TiNP.
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As seen, untreated cells did not show any peaks in the sub-G0 phase while treatment with TiNP induced nearly ~14% of cells in sub-G0 phase. Importantly, FA-TiNP increased the sub-G0
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population to ~25% indicating its superior anticancer effect. The results clearly indicated that
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FA-TiNP induced greater ROS generation that resulted in higher sub-G0 cell population with higher cell apoptosis [25-26]. FA-TiNP induces morphological changes typical of cell death, and
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a marked increase in the sub-G1 population in DNA histogram, indicating apoptosis. The modification of folic acid on the NP surface remarkably promoted the uptake of TiO2 nanoparticles in MG63 cells, on which the folate receptors are overexpressed. Results clearly suggest that the folic acid modified TiNP is practically useful for the cancer therapy since most of the cancer cells are rich of folic acid receptor on their surface.
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were examined by Western blot analysis (Figure 7).
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Figure 7: Western blot analysis of MG63 cancer cells. Caspase-3, cytochrome C, and PARP was
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examined as a prototype protein.
The ROS generation will damage the mitochondrial membrane which will initiate the apoptosis
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process. Upon mitochondrial damage, cytochrome C (Cyt C) will be released into the cytoplasm
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and induce the cell apoptosis by beginning the intrinsic pathway. As seen, FA-TiNP showed a remarkably higher expression of Cyt C compared to that of control indicating its superior anticancer effect. The apoptosis is a programmed cell death pathways which is of immense importance in health and disease. The mitochondrial dysfunction will result in the release of Cyt C in the cytoplasmic region that will initiate the apoptosis cascade. Therefore change in the mitochondrial function is considered as early event in apoptosis. As seen, FA-TiNP consequently increased the expression of cleaved caspase-3 indicating that Cyt C induced a caspase-dependent
ACCEPTED MANUSCRIPT cell death pathway. Caspases, a family of aspartic acid-specific proteases, are the major effectors of apoptosis. Followed by the initiation of caspase-3, PARP was also significantly upregulated indicating the superior anticancer efficacy of formulations. It has been reported that TiO2 could produce ROS such as hydroxyl radical, superoxide anion, and hydrogen peroxide. The
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generation of ROS will result in the degradation of multiple cellular organelles and cellular
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shown as downstream modulators of p53-dependent apoptosis
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membrane which will eventually cause cell death by apoptosis. Therefore, ROS have been
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4. Conclusion
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In summary, folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were successfully prepared as a suitable alternative to treat human osteosarcoma. The particle size of
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TiNP increased marked after polymer assembly on the nanoparticles (NP) surface with a
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spherical morphology. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to that of bare TiNP. The reason might due to the specific interaction of FA with
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the folate receptor which is overexpressed in the cancer cells. Especially, FA-TiNP treated cells
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exhibited chromatin condensation, cell shrinkage and membrane blebbing. FA-TiNP showed significantly higher cancer cell apoptosis with nearly 38% of cells in apoptosis chamber (early
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and late) compared to only ~16% for TiNP. The higher proportion of Annexin V-FITC positive cells for FA-TiNP treated group was mainly attributed to the higher intracellular uptake of the TiO2. Importantly, FA-TiNP increased the sub-G0 population to ~25% indicating its superior anticancer effect. The results clearly indicated that FA-TiNP induced greater ROS generation that resulted in higher sub-G0 cell population with higher cell apoptosis. FA-TiNP showed a remarkably higher expression of Cyt C with a marked increase in the expression of cleaved
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targeting moiety could enhance the chances of having successful therapies for cancer diseases.
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Acknowledgement
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The study was supported from the funding grant of Nanjing Medical University, China.
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Competing Interest
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The authors report no conflict of interest.
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Graphical Abstract
ACCEPTED MANUSCRIPT Highlights 1. Folic acid surface modified-Titanium dioxide nanoparticles (FA-TiNP) were prepared to
target osteosarcoma
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2. FA-TiNP exhibited a superior anticancer effect in osteosarcoma cancer cells compared to
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that of bare TiNP
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3. FA-TiNP showed a remarkably higher expression of cytochrome C (Cyt C) with a
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marked increase in other proteins
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4. Surface modification of TiNP with a specific targeting moiety could enhance the chances
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of having successful therapies for cancer diseases