- Email: [email protected]
Magnetite Nanoparticles Functionalized with Vitamin E Analogues: Anticancer Effects
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 3 (2016) 703 – 707
Advances in Functional Materials (Conference 2015), AFM 2015
Magnetite Nanoparticles Functionalized with Vitamin E analogues: Anticancer Effects A. Angulo-Molinaa,b; M.A. Méndez-Rojasc; T. Palacios-Hernándezd; O.E. Contreras-Lópeze; G.A. Hirata-Florese; J.C. Floresf, K.L. Floresf; C. Velázquezb; R. Robles-Zepedab; E. Silva-Campaa; A. Sarabiaa; M. Barboza-Floresa; M. Pedroza-Monteroa*, J.R.Reyes-Leyvaf; J. Hernándezg a
Departamento de Investigación en Física (DIFUS), Universidad de Sonora (UNISON), Hermosillo, Sonora, 83000, México Departamento Químico Biológicas, Universidad de Sonora (UNISON), Hermosillo, Sonora, 83000, México c Universidad de las Américas, Puebla (UDLAP), San Andrés Cholula, Puebla, 72820, México. d Johns Hopkins University (JHU), Baltimore, Maryland 21218, USA. e Centro de Nanociencias y Nanotecnología CNyN-UNAM, Ensenada, B.C., 22860, México. f Centro de Investigación Biomédica de Oriente (CIBIOR), IMSS, HGZ No. 5, Metepec, Puebla, 42730, México. g Centro de Investigación en Alimentación y Desarrollo (CIAD), Km 0.6 Carretera a La Victoria, Hermosillo, Sonora, 83304, México b
Abstract
Magnetite nanoparticles functionalized with alpha-tocopheryl succinate (Nps@α-TOS) increase anticancer activity of this vitamin E analogue. Previously, we reported Nps@α-TOS protect and enhance bioactivity of α-TOS in vitro. Nps@α-TOS selectively affected cervical cancer cells, without toxic effects for normal cells. The nanoparticles were internalized and accumulated around the nucleus. Severe morphological cell changes and lost viability were observed at 72 h post-treatment in contrast with non-effect in normal fibroblasts cells. These results suggested a cytotoxic effect in cancer cells and biocompatibility in normal cells. Further studies are needed in order to evaluate synergic anticancer bioactivity of Nps@α-TOS with other alternative treatments. Copyright © 2014 All rights reserved. © 2016 Elsevier Ltd.Elsevier All rightsLtd. reserved. Selectionand andpeer-review peer-review under responsibility of Conference Committee Members of Advances in Functional Selection under responsibility of Conference Committee Members of Advances in Functional MaterialsMaterials (Conference (Conference2015). 2015). Keywords: magnetite nanoparticles; α-tocopheril succinate, vitamin E analogue, cancer
* Corresponding author. Tel.: +52-622-259-21-56 E-mail address: [email protected]
2214-7853 © 2016 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of Conference Committee Members of Advances in Functional Materials (Conference 2015). doi:10.1016/j.matpr.2016.01.116
704
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
1. Introduction Alpha tocopheryl succinate (α-TOS), one of the most important vitamin E analogues, has been shown to kill cancer cells selectively with low toxicity toward nonmalignant cells [1-2]. This analogue (Fig. 1) is an esterified derivative of α-tocopherol (α-TOH), which suppress cell growth for a wide range of human cancer cells, including breast, prostate and colon cancer cells [3-4]. However, α-TOS show a poor effect in cervical and ovarian cancer cells [4]. In recent years, there has been a growing interest in development of special formulations or multidrug combinations to improve the bioactivity of vitamin E analogues in gynecological cancer cells [4-7]. Magnetite (Fe3O4) nanoparticles as carriers, may have the potential to be used for this purpose [8]. These nanoparticles are considered to be biocompatible and non-cytotoxic [7-9]. Previous in vitro assays by our group suggest that the anticancer activity of α-TOS can be conserved and enhanced when it is chemically linked to a magnetite nanoparticle (labeled as Nps@α-TOS) [10]. When resistant cervix cancer cells are treated with this nanoconjugate, the nanoparticles are internalized occurring lost viability and morphological changes related with cell death [10]. In this work, the synthesis, characterization and in vitro effects of Nps@α-TOS are presented. Firstly, we show synthesis and characterization of Nps@α-TOS [10]. Next, the in vitro biological effect in cancer and normal cells is exposed, remarking the cytotoxic effect, internalization and morphological changes observed [10]. The aim of this work is to show the basis for potential anticancer clinical use of Nps@α-TOS.
2. Synthesis As previously it was reported [10], spherical shaped magnetite nanoparticles (Fe3O4) functionalized with α-TOS were prepared using an established co-precipitation method with some modifications [10, 11]. Hexahydrated ferric chloride was used as the metal precursor. Briefly, the Fe (III) precursor was partially reduced to the ferrous ion, Fe (II), using Na2SO3 before alkalinizing with ammonium hydroxide, under strong stirring, and almost immediately yielding a black, magnetic precipitate [10-11]. Then, the precipitate was washed with absolute ethanol, centrifuged and dried at 60oC overnight under vacuum [10]. The dried nanoparticle surface was silanized and functionalized with α-TOS [9-11]. The nanoconjugate was labeled as Nps@α-TOS (Fig. 2). Finally, the nanoparticles were sonicated and dispersed in sterile PBS before use [10].
Fig. 1. Molecular structure of α-Tocopherol and α-Tocopheryl succinate.
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
Fig. 2. Schemes of magnetite nanoparticles functionalized with alpha-tocopheryl succinate. The nanoconjugate was labelled as Nps@α-TOS.
3. Characterization Size, morphology and Selected Area Electron Diffraction (SAED) analyses of Nps@α-TOS were studied with a JEOL Model JEM2010 (Tokyo, Japan) microscope operated at 200 kV. SAED analysis showed the typical crystal structure of magnetite (standard JCPDS card No.19-0629 for Fe3O4). [10]. Magnetic response was evaluated by exposing the nanoparticles to a strong magnetic field generated by a permanent ceramic Neodymium magnet (Fig. 3).
Fig. 3. Characterization of Nps@α-TOS: (A) Tyndall effect was observed when the laser beam was scattered by the nanoparticles of the colloidal system; (B) Nps@α-TOS showing a spherical- like morphology of 15 nm average size (TEM). (C) SAED analysis showed the crystal structure of magnetite (JCPDS card No.19-0629 for Fe3O4). (D) The nanoparticles respond intensely to the application of an external magnetic field.
4. Cell internalization
Internalization of functionalized nanoparticles was evaluated in SiHa cells. The cells were seeded at a density of 1.5 x 105 cells per well (Lab-TekII Chamber slides, Nalge Nunc Inc). After overnight, the cells were exposed to Nps@αTOS labelled with FITC (Nps@α-TOS@FITC) [9, 10] at 0, 5, 40, and 80µg/mL concentrations [10]. Chamber slides were incubated at 37o C, 5% CO2 for 72 h. The medium was removed and the cells were washed with PBS and fixed with 1:1 (vol/vol) methanol-acetone solution by 30 min followed by washing in PBS-Tween 0.05%. Cells were
705
706
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
counterstained with propidium iodide (PI) for 5 min to detect dead cells and observed in confocal microscopy (Nikon D-Eclipse C1 confocal laser scanning microscope). Both, 488 and 533 nm wavelength lasers were used to excite FITC and PI, respectively [10]. Nps@α-TOS@FITC was observed in green colour around the nucleus. After 72 h of incubation period, Nps@α-TOS@FITC were distributed at different Z positions inside of the nucleus (Fig. 4 A, B). Cells in which the nucleus is red (A) were stained with propidium iodide (PI) and can be scored as dead. PI generally is excluded from viable cells, only the dead cells are permeable to it. Additionally, it was possible to observe condensed chromatin in dead cells.
Fig. 4. Overlaying confocal microscopy images of SiHa cervical cancer cells after 72 h incubation with Nps@α-TOS@FITC at 40 μg/mL and counterstained with propidium iodide (IP) to detect dead cells. The cells (A and B) were observed at different Z positions (100x) showing Nps@αTOS@FITC in green colour distributed at diferentes zones of nucleus. A) Dead cells, where the red color allow to be scored as dead. These cells contained clearly condensed chromatin. Bar= 10 μm
5. Cell viability The human cervix cancer cell line SiHa, resistant to α-TOS, as well as non-malignant cell line of mouse fibroblasts were cultured at 37o C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM). The medium was supplemented with 5% FBS (fetal bovine serum), 1% penicillin-streptomycin and 1% glutamine. Viability was determined using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) colorimetric assay. The cells were plated at a density of 1 x 10 4 cells/well in a 96-wells plate and incubated in a humidified 5% CO2 at 37o C. The concentrations evaluated were 0, 2.5, 5, 10, 20, 40, and 80 µg/mL of Nps@α-TOS for 24, 48 and 72 h. After incubation time, MTT solution (5 mg/mL in PBS pH 7.4) was added and incubated at 37 o C for 4 h. Then, the formazan crystals formed were dissolved with 100 µL of acidified isopropanol and the absorbance was read at 550 nm. All the tests were performed by triplicate. The cell viability was calculated as: % Cell viability = (Absorbance of sample well/absorbance of control well) × 100 [10]. After 72 h exposure normal cells were not affected by α-TOS and Nps pure as well. The viability in fibroblasts was affected <10% with all the evaluated dose of Nps@α-TOS. Only in the highest concentration was observed lost viability, but this effect was <20%. These result indicated biocompatibility [10]. The viability and morphology of SiHa cells was not affected by α-TOS or pure Nps (data not show), confirming that SiHa cells are not sensitive [1]. However, SiHa cells become susceptible with Nps@α-TOS (IC50=65.29 μg/mL) in a dose and time dependent [10]. This suggested that the anticancer activity of α-TOS was protected or enhanced when it is functionalized to magnetite nanoparticles (Fig. 5). Additionally, Nps@-α-TOS was selective in cancer cells, affecting only SiHa cells but not fibroblast cells. The mechanism of Nps@-αTOS bioactivity remain to be evaluated [10].
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
707
Fig. 5. The viability in cervix cancer cells (SiHa cells) was not affected by Np or the vitamin E analogue α-TOS alone. SiHa cells become susceptible just with Nps@α-TOS, where this vitamin E analogue is functionalized to magnetite nanoparticles. This result is very interesting because SiHa cells is a resistant cancer cell to α-TOS. The functionalization protected the bioactivity.
6. Conclusion Physical and chemical characterization of Nps@α-TOS is important for detailing the biological and side effects. Anticancer activity of α-TOS in resistant cervical cancer cells was improved when this vitamin E analogue was functionalized to magnetite nanoparticles. Nps@α-TOS has shown anticancer effect in SiHa cancer cells without toxicity effects on normal cells. Further studies are needed in order to evaluate mechanism and synergic bioactivity of Nps@α-TOS with other alternative treatments.
Acknowledgements Authors are grateful to the staff from CIBIOR, science students from UDLAP and DIFUS. This study was supported by the SEP-CONACYT (Fondo de Investigación Científica Básica) Grant No. 154602. CIBIOR was supported by funds from the Mexican Institute for Social Security (CTFIS/10RD/12/2011). References [1] A. Angulo-Molina, J. Reyes-Leyva, A. López-Malo, J. Hernández. Nutr Cancer. 66 (2014) 167-176. [2] Hahn T, Polansczyk MJ, Borodovsky A, Ramanathapuram LV, Akporiaye ET, Ralph SJ. Curr Pharm Biotechnol. 14 (2013) 357[3] Dong LF, Freeman R, Liu J, Zobalova R, Marin-Hernandez A, Stantic M, et al. Clin Cancer Res. 15 (2009) 1593-1600. [4] K. Anderson, M. Simmons-Menchaca, K.A. Lawson, J. Atkinson, B.G. Sanders, K. Kline. Cancer Res. 64 (2004) 4263-4269. 376 [5] Y. Ma, L. Huang, C. Song, X. Zeng, G. Liu, L. Mei. Polymer. 51 (2010) 5952–5959. [6] J. Turanek, X.F. Wang, P. Knotigova, S. Koudelka, L.F. Dong, E. Vrublova, et al. Toxicol Appl Pharmacol. 237 (2009) 249-257 [7] M. Tomasetti, E. Strafella, S. Staffolani, L. Santarelli, J. Neuzil and R. Guerrieri. Brit J Cancer. 102 (2010) 1224-1234 [8] D. Baba, Y. Seiko, T. Nakanishi, H. Zhang, A. Arakaki, T. Matsunaga, et al. Coll Surf B Biointerfaces. 95 (2012) 254-257. [9] Y. Zhang and J. Zhang. J of Coll Interface Sci. 283 (2005) 352-357. [10] A. Angulo-Molina, M.A. Méndez-Rojas, T. Palacios-Hernández, O.E. Contreras López, G.A. Hirata-Flores, et al. J Nanopart Res. 16 (2014) 2528-2536. [11] S. Qu, H. Yang, D. Ren, S. Kan, G. Zou, D. Lo, et al. J Colloid Interface Sci. 215 (1999)190-192.
ScienceDirect Materials Today: Proceedings 3 (2016) 703 – 707
Advances in Functional Materials (Conference 2015), AFM 2015
Magnetite Nanoparticles Functionalized with Vitamin E analogues: Anticancer Effects A. Angulo-Molinaa,b; M.A. Méndez-Rojasc; T. Palacios-Hernándezd; O.E. Contreras-Lópeze; G.A. Hirata-Florese; J.C. Floresf, K.L. Floresf; C. Velázquezb; R. Robles-Zepedab; E. Silva-Campaa; A. Sarabiaa; M. Barboza-Floresa; M. Pedroza-Monteroa*, J.R.Reyes-Leyvaf; J. Hernándezg a
Departamento de Investigación en Física (DIFUS), Universidad de Sonora (UNISON), Hermosillo, Sonora, 83000, México Departamento Químico Biológicas, Universidad de Sonora (UNISON), Hermosillo, Sonora, 83000, México c Universidad de las Américas, Puebla (UDLAP), San Andrés Cholula, Puebla, 72820, México. d Johns Hopkins University (JHU), Baltimore, Maryland 21218, USA. e Centro de Nanociencias y Nanotecnología CNyN-UNAM, Ensenada, B.C., 22860, México. f Centro de Investigación Biomédica de Oriente (CIBIOR), IMSS, HGZ No. 5, Metepec, Puebla, 42730, México. g Centro de Investigación en Alimentación y Desarrollo (CIAD), Km 0.6 Carretera a La Victoria, Hermosillo, Sonora, 83304, México b
Abstract
Magnetite nanoparticles functionalized with alpha-tocopheryl succinate (Nps@α-TOS) increase anticancer activity of this vitamin E analogue. Previously, we reported Nps@α-TOS protect and enhance bioactivity of α-TOS in vitro. Nps@α-TOS selectively affected cervical cancer cells, without toxic effects for normal cells. The nanoparticles were internalized and accumulated around the nucleus. Severe morphological cell changes and lost viability were observed at 72 h post-treatment in contrast with non-effect in normal fibroblasts cells. These results suggested a cytotoxic effect in cancer cells and biocompatibility in normal cells. Further studies are needed in order to evaluate synergic anticancer bioactivity of Nps@α-TOS with other alternative treatments. Copyright © 2014 All rights reserved. © 2016 Elsevier Ltd.Elsevier All rightsLtd. reserved. Selectionand andpeer-review peer-review under responsibility of Conference Committee Members of Advances in Functional Selection under responsibility of Conference Committee Members of Advances in Functional MaterialsMaterials (Conference (Conference2015). 2015). Keywords: magnetite nanoparticles; α-tocopheril succinate, vitamin E analogue, cancer
* Corresponding author. Tel.: +52-622-259-21-56 E-mail address: [email protected]
2214-7853 © 2016 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of Conference Committee Members of Advances in Functional Materials (Conference 2015). doi:10.1016/j.matpr.2016.01.116
704
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
1. Introduction Alpha tocopheryl succinate (α-TOS), one of the most important vitamin E analogues, has been shown to kill cancer cells selectively with low toxicity toward nonmalignant cells [1-2]. This analogue (Fig. 1) is an esterified derivative of α-tocopherol (α-TOH), which suppress cell growth for a wide range of human cancer cells, including breast, prostate and colon cancer cells [3-4]. However, α-TOS show a poor effect in cervical and ovarian cancer cells [4]. In recent years, there has been a growing interest in development of special formulations or multidrug combinations to improve the bioactivity of vitamin E analogues in gynecological cancer cells [4-7]. Magnetite (Fe3O4) nanoparticles as carriers, may have the potential to be used for this purpose [8]. These nanoparticles are considered to be biocompatible and non-cytotoxic [7-9]. Previous in vitro assays by our group suggest that the anticancer activity of α-TOS can be conserved and enhanced when it is chemically linked to a magnetite nanoparticle (labeled as Nps@α-TOS) [10]. When resistant cervix cancer cells are treated with this nanoconjugate, the nanoparticles are internalized occurring lost viability and morphological changes related with cell death [10]. In this work, the synthesis, characterization and in vitro effects of Nps@α-TOS are presented. Firstly, we show synthesis and characterization of Nps@α-TOS [10]. Next, the in vitro biological effect in cancer and normal cells is exposed, remarking the cytotoxic effect, internalization and morphological changes observed [10]. The aim of this work is to show the basis for potential anticancer clinical use of Nps@α-TOS.
2. Synthesis As previously it was reported [10], spherical shaped magnetite nanoparticles (Fe3O4) functionalized with α-TOS were prepared using an established co-precipitation method with some modifications [10, 11]. Hexahydrated ferric chloride was used as the metal precursor. Briefly, the Fe (III) precursor was partially reduced to the ferrous ion, Fe (II), using Na2SO3 before alkalinizing with ammonium hydroxide, under strong stirring, and almost immediately yielding a black, magnetic precipitate [10-11]. Then, the precipitate was washed with absolute ethanol, centrifuged and dried at 60oC overnight under vacuum [10]. The dried nanoparticle surface was silanized and functionalized with α-TOS [9-11]. The nanoconjugate was labeled as Nps@α-TOS (Fig. 2). Finally, the nanoparticles were sonicated and dispersed in sterile PBS before use [10].
Fig. 1. Molecular structure of α-Tocopherol and α-Tocopheryl succinate.
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
Fig. 2. Schemes of magnetite nanoparticles functionalized with alpha-tocopheryl succinate. The nanoconjugate was labelled as Nps@α-TOS.
3. Characterization Size, morphology and Selected Area Electron Diffraction (SAED) analyses of Nps@α-TOS were studied with a JEOL Model JEM2010 (Tokyo, Japan) microscope operated at 200 kV. SAED analysis showed the typical crystal structure of magnetite (standard JCPDS card No.19-0629 for Fe3O4). [10]. Magnetic response was evaluated by exposing the nanoparticles to a strong magnetic field generated by a permanent ceramic Neodymium magnet (Fig. 3).
Fig. 3. Characterization of Nps@α-TOS: (A) Tyndall effect was observed when the laser beam was scattered by the nanoparticles of the colloidal system; (B) Nps@α-TOS showing a spherical- like morphology of 15 nm average size (TEM). (C) SAED analysis showed the crystal structure of magnetite (JCPDS card No.19-0629 for Fe3O4). (D) The nanoparticles respond intensely to the application of an external magnetic field.
4. Cell internalization
Internalization of functionalized nanoparticles was evaluated in SiHa cells. The cells were seeded at a density of 1.5 x 105 cells per well (Lab-TekII Chamber slides, Nalge Nunc Inc). After overnight, the cells were exposed to Nps@αTOS labelled with FITC (Nps@α-TOS@FITC) [9, 10] at 0, 5, 40, and 80µg/mL concentrations [10]. Chamber slides were incubated at 37o C, 5% CO2 for 72 h. The medium was removed and the cells were washed with PBS and fixed with 1:1 (vol/vol) methanol-acetone solution by 30 min followed by washing in PBS-Tween 0.05%. Cells were
705
706
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
counterstained with propidium iodide (PI) for 5 min to detect dead cells and observed in confocal microscopy (Nikon D-Eclipse C1 confocal laser scanning microscope). Both, 488 and 533 nm wavelength lasers were used to excite FITC and PI, respectively [10]. Nps@α-TOS@FITC was observed in green colour around the nucleus. After 72 h of incubation period, Nps@α-TOS@FITC were distributed at different Z positions inside of the nucleus (Fig. 4 A, B). Cells in which the nucleus is red (A) were stained with propidium iodide (PI) and can be scored as dead. PI generally is excluded from viable cells, only the dead cells are permeable to it. Additionally, it was possible to observe condensed chromatin in dead cells.
Fig. 4. Overlaying confocal microscopy images of SiHa cervical cancer cells after 72 h incubation with Nps@α-TOS@FITC at 40 μg/mL and counterstained with propidium iodide (IP) to detect dead cells. The cells (A and B) were observed at different Z positions (100x) showing Nps@αTOS@FITC in green colour distributed at diferentes zones of nucleus. A) Dead cells, where the red color allow to be scored as dead. These cells contained clearly condensed chromatin. Bar= 10 μm
5. Cell viability The human cervix cancer cell line SiHa, resistant to α-TOS, as well as non-malignant cell line of mouse fibroblasts were cultured at 37o C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM). The medium was supplemented with 5% FBS (fetal bovine serum), 1% penicillin-streptomycin and 1% glutamine. Viability was determined using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) colorimetric assay. The cells were plated at a density of 1 x 10 4 cells/well in a 96-wells plate and incubated in a humidified 5% CO2 at 37o C. The concentrations evaluated were 0, 2.5, 5, 10, 20, 40, and 80 µg/mL of Nps@α-TOS for 24, 48 and 72 h. After incubation time, MTT solution (5 mg/mL in PBS pH 7.4) was added and incubated at 37 o C for 4 h. Then, the formazan crystals formed were dissolved with 100 µL of acidified isopropanol and the absorbance was read at 550 nm. All the tests were performed by triplicate. The cell viability was calculated as: % Cell viability = (Absorbance of sample well/absorbance of control well) × 100 [10]. After 72 h exposure normal cells were not affected by α-TOS and Nps pure as well. The viability in fibroblasts was affected <10% with all the evaluated dose of Nps@α-TOS. Only in the highest concentration was observed lost viability, but this effect was <20%. These result indicated biocompatibility [10]. The viability and morphology of SiHa cells was not affected by α-TOS or pure Nps (data not show), confirming that SiHa cells are not sensitive [1]. However, SiHa cells become susceptible with Nps@α-TOS (IC50=65.29 μg/mL) in a dose and time dependent [10]. This suggested that the anticancer activity of α-TOS was protected or enhanced when it is functionalized to magnetite nanoparticles (Fig. 5). Additionally, Nps@-α-TOS was selective in cancer cells, affecting only SiHa cells but not fibroblast cells. The mechanism of Nps@-αTOS bioactivity remain to be evaluated [10].
A. Angulo-Molina et al. / Materials Today: Proceedings 3 (2016) 703 – 707
707
Fig. 5. The viability in cervix cancer cells (SiHa cells) was not affected by Np or the vitamin E analogue α-TOS alone. SiHa cells become susceptible just with Nps@α-TOS, where this vitamin E analogue is functionalized to magnetite nanoparticles. This result is very interesting because SiHa cells is a resistant cancer cell to α-TOS. The functionalization protected the bioactivity.
6. Conclusion Physical and chemical characterization of Nps@α-TOS is important for detailing the biological and side effects. Anticancer activity of α-TOS in resistant cervical cancer cells was improved when this vitamin E analogue was functionalized to magnetite nanoparticles. Nps@α-TOS has shown anticancer effect in SiHa cancer cells without toxicity effects on normal cells. Further studies are needed in order to evaluate mechanism and synergic bioactivity of Nps@α-TOS with other alternative treatments.
Acknowledgements Authors are grateful to the staff from CIBIOR, science students from UDLAP and DIFUS. This study was supported by the SEP-CONACYT (Fondo de Investigación Científica Básica) Grant No. 154602. CIBIOR was supported by funds from the Mexican Institute for Social Security (CTFIS/10RD/12/2011). References [1] A. Angulo-Molina, J. Reyes-Leyva, A. López-Malo, J. Hernández. Nutr Cancer. 66 (2014) 167-176. [2] Hahn T, Polansczyk MJ, Borodovsky A, Ramanathapuram LV, Akporiaye ET, Ralph SJ. Curr Pharm Biotechnol. 14 (2013) 357[3] Dong LF, Freeman R, Liu J, Zobalova R, Marin-Hernandez A, Stantic M, et al. Clin Cancer Res. 15 (2009) 1593-1600. [4] K. Anderson, M. Simmons-Menchaca, K.A. Lawson, J. Atkinson, B.G. Sanders, K. Kline. Cancer Res. 64 (2004) 4263-4269. 376 [5] Y. Ma, L. Huang, C. Song, X. Zeng, G. Liu, L. Mei. Polymer. 51 (2010) 5952–5959. [6] J. Turanek, X.F. Wang, P. Knotigova, S. Koudelka, L.F. Dong, E. Vrublova, et al. Toxicol Appl Pharmacol. 237 (2009) 249-257 [7] M. Tomasetti, E. Strafella, S. Staffolani, L. Santarelli, J. Neuzil and R. Guerrieri. Brit J Cancer. 102 (2010) 1224-1234 [8] D. Baba, Y. Seiko, T. Nakanishi, H. Zhang, A. Arakaki, T. Matsunaga, et al. Coll Surf B Biointerfaces. 95 (2012) 254-257. [9] Y. Zhang and J. Zhang. J of Coll Interface Sci. 283 (2005) 352-357. [10] A. Angulo-Molina, M.A. Méndez-Rojas, T. Palacios-Hernández, O.E. Contreras López, G.A. Hirata-Flores, et al. J Nanopart Res. 16 (2014) 2528-2536. [11] S. Qu, H. Yang, D. Ren, S. Kan, G. Zou, D. Lo, et al. J Colloid Interface Sci. 215 (1999)190-192.