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Article
Rational design of cancer-targeted BSA protein nanoparticles as radiosensitizer to overcome cancer radioresistance Yanyu Huang, Yi Luo, Wenjie Zheng, and Tianfeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/am505246w • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 18, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Rational design of cancer-targeted BSA protein nanoparticles as radiosensitizer to overcome cancer radioresistance Yanyu Huang, Yi Luo, Wenjie Zheng, Tianfeng Chen*
Department of Chemistry, Jinan University, Guangzhou 510632, China
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ABSTRACT
Radiotherapy displays curative potential for cervical cancer management, but radioresistance occurs during long-term therapy. To overcome this limitation, tumor-targeted nanotechnology has been proposed to enhance the radiosensitivity of solid tumors. Herein, we used biocompatible bovine serum albumin nanoparticles (BSANPs) as payloads of organic selenocompound (PSeD) with folate (FA) as targeting ligand to fabricate cancer-targeted nanosystem. The combination of PSeD and BSANPs endowed the nanosystem with higher light absorption and ROS generation owing to their properties of surface plasomon resonance (SPR) effect, heavy metal effect, high refractive index and nanoparticulate interfacial effect. The combined treatment drastically increased the ROS overproduction, VEGF/VEGFR2 inactivation and inhibition of XRCC-1-mediated repair of DNA damage, thus triggered G2/M phase arrest and apoptosis. Taken together, our findings demonstrate the utility of FA-BSANPs as a promising radiosensitizer to improve cancer radiotherapy.
KEYWORDS: Bovine serum albumin nanoparticles, cancer-targeted nanosystem, folate, radiosensitization, radiotherapy
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INTRODUCTION Radiotherapy has been extensively applied for noninvasive cancer treatment. Clinically， high energy X-ray or γ-ray are used in radiotherapy to suppress tumor proliferation through causing DNA damage or free radical damage in tumor sites.1-3 However, due to the differences in individual radiosensitivity and the limitation of irradiative dosage, some patients may suffer cancer recurrence and have poor therapeutic effect after radiotherapy. Besides, irradiative side effects caused by the poor discrimination between normal and tumor surrounding tissues limit the clinical implementation of radiotherapy. Moreover, cancer cells in lung, cervix et al undergoing radiation may trigger underlying molecular repair processes and finally lead to radioresistance.4, 5 Therefore, the development of new effective protocol for enhancing tumor radiosensitivity without compromising normal cell viability becomes an urgent priority. Currently, the combined use of chemotherapy and radiotherapy is considered as effective strategy for radiosensitization. Recently, chemical therapeutics such as cisplatin, paclitaxel have been developed to be used as radiosensitizers to enhance cancer cells’ sensitivity to radiation for lung, cervical, gastric, head and neck cancers .6-8 Selenium (Se) is a trace element with essential benefit for humans and animals. Numerous studies have shown that selenocompounds have excellent chemopreventive and chemotherapeutic effects in cancer treatment.9, 10Among them, organoselenium compounds show higher absorption efficacy, better anticancer activity and lower toxicity comparing to inorganic Se forms in our previous studies.11-14 Among these organic selenadiazole compounds, phenylbenzo [1, 2, 5] selenadiazole derivatives (PSeD), are identified to have strong anticancer abilities against different kinds of cancer and have little effect on normal cells. What’s more, PSeD as semiconducting material has metalloid properties, e.g. surface plasomon resonance
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(SPR) effect, heavy metal effect and high refractive index which facilitate for light absorption.15 Therefore, PSeD is considered to be promising radiosensitizer due to its high optical absorption. Despite this potency, the poor water-soluble ability and weak selective cytotoxicities of PSeD block its effective and precise accumulation in tumor sites. To achieve high therapeutic outcomes, high dosage of free PSeD is needed, which can inevitably result in undesirable toxicity. Therefore, there has been intense interest in developing new types of drug delivery vehicles of PSeD for radiosensitization without compromising systemic viability. Nanotechnology is considered as a promising strategy for cancer diagnostics owing to its smart, sophisticated and multifunctional applications in tumor-targeted drug delivery and drug localization.16-20 Nanoparticles provide customizable and selective drug delivery in tumor sites able to ferry effective amount of chemotherapeutic agents while sparing normal cells.21-23 Among them, albumin nanoparticles are considered as one of the biodegradable nanopayloads with ease functionalization for cancer therapy.24, 25 Indeed, Sufeng Zhang et al found out that bovine serum albumin nanoparticles (BSANPs)/ poly (ethyleneglycol) modified polyethylenimine (PEI–PEG) as carrier of BMP-2 significantly enhanced de novo bone formation and drastically reduced PEI-caused NP toxicity.26 Moreover, Vaishakhi Mohanta et al and Lili Xie et al respectively reported that surface decoration of chitosan on BSANPs contributed to drug-controlled release in response to pH changes.27, 28 Recently, researchers found out that the targeted ligand conjugation conferred the albumin nanoparticles to remarkable cytotoxicities against various cancer cells or even in xenografted nu/nu mouse.29 However, tumor-targeting BSANPs delivery system used as chemical sensitizing agent for radiosensitization has been marginal.
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Generally, expression of folate receptors (FARs) on normal cells is low, but the demand for FARs increases when the cells undergo cellular activation, proliferation, and cells under malignant transformation.30 Hela cervical cells overexpressing FARs are considered as radioresistant malignant cancer cells because they express pro-survival cytokines such as X-ray repair cross complementing protein 1 (XRCC-1), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) to favor tumor progression and acquire treatment resistance after radiation.31, 32 Therefore, in this study, we chose folate (FA) as targeting ligand to fabricate cancer-targeted BSANPs formulation as drug delivery system of PSeD and meanwhile, assessed its selective cytotoxicity as an important indicator of being a potential radiosensitizer. Besides, Hela cervical cells were selected as X-ray-resistant cell model to determine the sensitizing capacity of FA-BSANPs in radiotherapy and the underlying action mechanisms.
EXPERIMENTAL METHODS Materials Phenylbenzo [1, 2, 5] selenadiazole derivatives (PSeD) was synthetized as previously described.13, 14 Folate (FA), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), glutaraldehyde 25% solution were purchased from Aladdin. Bovine serum albumin (BSA, fraction V, purity 95-99%), MTT and PI were obtained from Sigma-Aldrich. Bicinchoninic acid (BCA) kit was obtained from Thermo Fisher Scientific Inc.. TUNEL assay kit was purchased from Roche Applied Science. DMEM medium and fetal bovine serum (FBS) were purchased from NQBB International Biological Corporation. Milli-Q water was used in all experiments. Radiotherapy instrument (Elekta Precise linear accelerator)
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Preparation of FA-BSANPs BSANPs was prepared using desolvation technique as previously described with little modification.29 Briefly, 40 mg of BSA powder dissolved in 2 mL of Milli-Q water was titrated to pH 8.2 using 0.1 N sodium hydroxide (NaOH), followed by dropwise addition of 8 mL ethanol under stirring (800 rpm) at a rate of 1mL/min. After the desolvation process, the nanoparticles were formed. Then 23.5 uL of 8% glutaraldehyde was slowly added and was allowed to stir for 24 h to cause particle crosslinking. To remove free BSA and glutaraldehyde, the prepared nanoparticles were purified by 2 rounds of centrifugation (12000 rpm, 20 min). The pellet was redispersed to the original volume in water under ultrasonication over 10 min. For FA conjugation, FA was activated with EDC. Briefly, 20 mg/mL of FA solution (dissolved in 0.1N NaOH solution) was first reacted with EDC solution at a molar ratio of 1:5. The reaction was performed in dark with constant stirring for 2 h. Subsequently, the activated FA solution was dropwise added to BSANPs solution to form FA-conjugate BSANPs. Finally, PSeD solution (300 uL, 12.5 mg/mL) was added to FA-conjugated BSANPs under constant stirring at a time period of 24 h for incorporating PSeD. The FA-BSANPs was purified by 3 rounds of centrifugation (12000 rpm, 20 min) to removed unloaded PSeD, FA and EDC. The Se content in the nanosystem was calculated by ICP-AES method. The whole process was performed at room temperature. Characterization of FA-BSANPs The morphology of FA-BSANPs was determined by transmission electron microscope (TEM, Hitachi H-7650), scanning electron microscope (SEM, EX-250 system, horiba) and atomic force microscope (AFM, Bioscope Catylyst Nanoscope-V). Fourier transform infrared spectroscopy (FTIR, Equinox 55 IR spectrometer) was used to characterize the structure information of FA-
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BSANPs in the range of 400- 4000 cm-1. The size distribution and zeta potential of particles were monitored by Malvern Zetasizer Software. Cell culture and determination of cell viability In this article, Hela cervical carcinoma cells, MCF-7 breast adenocarcinoma cells, A375 melanoma cells and L02 human hepatic cells were human cell lines, which were applied from American Type Culture Collection (ATCC, Manassas, Virginia). These cell lines were maintained in DMEM medium with 100 units/mL penicillin, 10% fetal bovine serum and 50 units/mL streptomycin at 37 °C in 5% CO2 incubator. FA-BSANPs-caused cell proliferative inhibition against various cell lines was measured by clonogenic assay.33 and MTT assay.34 Determination of FA-BSANPs cellular uptake and intracellular trafficking Intracellular uptake of FA-BSANPs in Hela and L02 cells was quantified by determining Se concentration using ICP-MS method. Briefly, Hela and L02 cells were treated with different concentrations of FA-BSANPs with or without radiation (8 Gy) for different time periods， followed by cell digestion and redispersion. After that, the cells were counted, lysed using hydrogen nitrate (HNO3) and perchloric acid (HClO4) mixture (HNO3 : HClO4=3:1). Finally, the lysis solution was diluted with water to a volume of 10 mL. ICP-MS determination was performed to detect Se concentration, which quantified the uptake efficiency of internalized nanoparticles. The intracellular trafficking of FA-BSANPs in Hela and L02 cells was monitored using fluorescence imaging technique under fluorescence microscope (IX51, Olympus).35 Folate competing assay The binding efficiency of FARs between FA-BSANPs and excessive FA was compared as previously described.35 The internalized FA-BSANPs was quantified by the fluorescence
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intensity from PSeD under fluorescence microplate reader (Bio-Tek) (excitation wavelength at 482 nm and emission wavelength at 526 nm ). In vitro drug release of FA-BSANPs Four copies of FA-BSANPs radiated with 0 and 8 Gy were respectively dispersed in PBS solution at a concentration of 1 mg/mL at acidic lysosomal (pH 5.3) and neutral blood (pH 7.4) environments. The solutions were kept in dark at 37 °C to imitate in vivo environment. 250 µL of buffer was taken out and fresh buffer at same volume was supplied. The release rate of PSeD was represented by its fluorescence intensity using fluorescence microplate reader (excitation wavelength at 482 nm and emission wavelength at 526 nm ). Flow cytometry The cell cycle distribution after the treatment of FA-BSANPs and X-ray was measured by flow cytometric analysis.36 The cells incubated with FA-BSANPs and X-ray were digested, collected and centrifuged at a speed of 1500 rpm for 10 min. The harvested cells were fixed with 70% ethanol at -20°C overnight followed by PI staining. The DNA contents were quantified by the fluorescence intensity of PI. The cell samples were analyzed on a Beckman Coulter Epics XL MCL flow cytometer (Beckman Coulter, Inc, Miami, FL). The DNA histogram represented the cell number in G0/G1, S, and G2/M phases, while the sub-G1 peak quantified the hypodiploid DNA number in apoptotic cells. The DNA contents of the cell samples were analyzed by MultiCycle software (Phoenix Flow Systems, San Diego, CA); TUNEL & DAPI staining assay FA-BSANPs-caused DNA fragmentation was confirmed by TUNEL assay in Hela cells. Briefly, the cells attached in 2-cm dishes were fixed and permeabilized with 3.7% formaldehyde and 0.1% Triton-X solution respectively. After that, TUNEL working buffer and DAPI were
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added for cell staining. The morphology of stained cells was observed under a fluorescence microscope (EVOS FL auto, Life Technologies). Determination of reactive oxygen species (ROS) generation Changes of intracellular ROS generation caused by FA-BSANPs with or without radiation were evaluated using dihydroethidium (DHE, Beyotime) and DCFDA probe respectively. Briefly, Hela cells pre-treated with different concentration of FA-BSANPs for 2 h were irradiated at a dosage of 0, 8 Gy respectively. At the end of radiation, the cells were stained with 10 µM DHE at final concentration and DCFDA of at 37°C for 30 min. The cells rinsed with PBS twice were incubated in fresh PBS. The fluorescence intensity of DHE and DCF (excitation and emission wavelength at 300 nm / 610 nm and 488 nm / 525 nm respectively) quantified the intracellular ROS level. For the determination of extracellular ROS generation caused by FABSANPs and free PSeD, DHE staining assay was conducted in PBS without cells. Western blot analysis The expression of proteins involved in signaling pathways after combined treatment of FABSANPs and radiation in Hela cells and L02 cells were determined by western blot analysis. Briefly, the cells were lysed with RIPA buffer and the total proteins were obtained from supernatant after centrifugation (12000 rpm) at 4°C. The proteins were quantified using BCA assay. After that, SDS-PAGE was performed with equivalent amount of proteins loading. Subsequently, the separated proteins were transferred to PVDF membranes, blocked with 5% non-fat milk, incubated with primary antibodies and second antibodies-conjugated horseradish peroxidase, and were finally visualized on X-ray films.
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Statistical analysis All experiments were performed at least for 3 times and the data in this study was expressed as means ± S.D. Statistical significance of the data were analyzed by SPSS statistical program (Version 13, SPSS Inc., Chicago, IL). Difference between two groups was calculated by twotailed Student’s t-test. Variation with P
Article
Rational design of cancer-targeted BSA protein nanoparticles as radiosensitizer to overcome cancer radioresistance Yanyu Huang, Yi Luo, Wenjie Zheng, and Tianfeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/am505246w • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 18, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Rational design of cancer-targeted BSA protein nanoparticles as radiosensitizer to overcome cancer radioresistance Yanyu Huang, Yi Luo, Wenjie Zheng, Tianfeng Chen*
Department of Chemistry, Jinan University, Guangzhou 510632, China
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ABSTRACT
Radiotherapy displays curative potential for cervical cancer management, but radioresistance occurs during long-term therapy. To overcome this limitation, tumor-targeted nanotechnology has been proposed to enhance the radiosensitivity of solid tumors. Herein, we used biocompatible bovine serum albumin nanoparticles (BSANPs) as payloads of organic selenocompound (PSeD) with folate (FA) as targeting ligand to fabricate cancer-targeted nanosystem. The combination of PSeD and BSANPs endowed the nanosystem with higher light absorption and ROS generation owing to their properties of surface plasomon resonance (SPR) effect, heavy metal effect, high refractive index and nanoparticulate interfacial effect. The combined treatment drastically increased the ROS overproduction, VEGF/VEGFR2 inactivation and inhibition of XRCC-1-mediated repair of DNA damage, thus triggered G2/M phase arrest and apoptosis. Taken together, our findings demonstrate the utility of FA-BSANPs as a promising radiosensitizer to improve cancer radiotherapy.
KEYWORDS: Bovine serum albumin nanoparticles, cancer-targeted nanosystem, folate, radiosensitization, radiotherapy
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INTRODUCTION Radiotherapy has been extensively applied for noninvasive cancer treatment. Clinically， high energy X-ray or γ-ray are used in radiotherapy to suppress tumor proliferation through causing DNA damage or free radical damage in tumor sites.1-3 However, due to the differences in individual radiosensitivity and the limitation of irradiative dosage, some patients may suffer cancer recurrence and have poor therapeutic effect after radiotherapy. Besides, irradiative side effects caused by the poor discrimination between normal and tumor surrounding tissues limit the clinical implementation of radiotherapy. Moreover, cancer cells in lung, cervix et al undergoing radiation may trigger underlying molecular repair processes and finally lead to radioresistance.4, 5 Therefore, the development of new effective protocol for enhancing tumor radiosensitivity without compromising normal cell viability becomes an urgent priority. Currently, the combined use of chemotherapy and radiotherapy is considered as effective strategy for radiosensitization. Recently, chemical therapeutics such as cisplatin, paclitaxel have been developed to be used as radiosensitizers to enhance cancer cells’ sensitivity to radiation for lung, cervical, gastric, head and neck cancers .6-8 Selenium (Se) is a trace element with essential benefit for humans and animals. Numerous studies have shown that selenocompounds have excellent chemopreventive and chemotherapeutic effects in cancer treatment.9, 10Among them, organoselenium compounds show higher absorption efficacy, better anticancer activity and lower toxicity comparing to inorganic Se forms in our previous studies.11-14 Among these organic selenadiazole compounds, phenylbenzo [1, 2, 5] selenadiazole derivatives (PSeD), are identified to have strong anticancer abilities against different kinds of cancer and have little effect on normal cells. What’s more, PSeD as semiconducting material has metalloid properties, e.g. surface plasomon resonance
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(SPR) effect, heavy metal effect and high refractive index which facilitate for light absorption.15 Therefore, PSeD is considered to be promising radiosensitizer due to its high optical absorption. Despite this potency, the poor water-soluble ability and weak selective cytotoxicities of PSeD block its effective and precise accumulation in tumor sites. To achieve high therapeutic outcomes, high dosage of free PSeD is needed, which can inevitably result in undesirable toxicity. Therefore, there has been intense interest in developing new types of drug delivery vehicles of PSeD for radiosensitization without compromising systemic viability. Nanotechnology is considered as a promising strategy for cancer diagnostics owing to its smart, sophisticated and multifunctional applications in tumor-targeted drug delivery and drug localization.16-20 Nanoparticles provide customizable and selective drug delivery in tumor sites able to ferry effective amount of chemotherapeutic agents while sparing normal cells.21-23 Among them, albumin nanoparticles are considered as one of the biodegradable nanopayloads with ease functionalization for cancer therapy.24, 25 Indeed, Sufeng Zhang et al found out that bovine serum albumin nanoparticles (BSANPs)/ poly (ethyleneglycol) modified polyethylenimine (PEI–PEG) as carrier of BMP-2 significantly enhanced de novo bone formation and drastically reduced PEI-caused NP toxicity.26 Moreover, Vaishakhi Mohanta et al and Lili Xie et al respectively reported that surface decoration of chitosan on BSANPs contributed to drug-controlled release in response to pH changes.27, 28 Recently, researchers found out that the targeted ligand conjugation conferred the albumin nanoparticles to remarkable cytotoxicities against various cancer cells or even in xenografted nu/nu mouse.29 However, tumor-targeting BSANPs delivery system used as chemical sensitizing agent for radiosensitization has been marginal.
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Generally, expression of folate receptors (FARs) on normal cells is low, but the demand for FARs increases when the cells undergo cellular activation, proliferation, and cells under malignant transformation.30 Hela cervical cells overexpressing FARs are considered as radioresistant malignant cancer cells because they express pro-survival cytokines such as X-ray repair cross complementing protein 1 (XRCC-1), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) to favor tumor progression and acquire treatment resistance after radiation.31, 32 Therefore, in this study, we chose folate (FA) as targeting ligand to fabricate cancer-targeted BSANPs formulation as drug delivery system of PSeD and meanwhile, assessed its selective cytotoxicity as an important indicator of being a potential radiosensitizer. Besides, Hela cervical cells were selected as X-ray-resistant cell model to determine the sensitizing capacity of FA-BSANPs in radiotherapy and the underlying action mechanisms.
EXPERIMENTAL METHODS Materials Phenylbenzo [1, 2, 5] selenadiazole derivatives (PSeD) was synthetized as previously described.13, 14 Folate (FA), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), glutaraldehyde 25% solution were purchased from Aladdin. Bovine serum albumin (BSA, fraction V, purity 95-99%), MTT and PI were obtained from Sigma-Aldrich. Bicinchoninic acid (BCA) kit was obtained from Thermo Fisher Scientific Inc.. TUNEL assay kit was purchased from Roche Applied Science. DMEM medium and fetal bovine serum (FBS) were purchased from NQBB International Biological Corporation. Milli-Q water was used in all experiments. Radiotherapy instrument (Elekta Precise linear accelerator)
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Preparation of FA-BSANPs BSANPs was prepared using desolvation technique as previously described with little modification.29 Briefly, 40 mg of BSA powder dissolved in 2 mL of Milli-Q water was titrated to pH 8.2 using 0.1 N sodium hydroxide (NaOH), followed by dropwise addition of 8 mL ethanol under stirring (800 rpm) at a rate of 1mL/min. After the desolvation process, the nanoparticles were formed. Then 23.5 uL of 8% glutaraldehyde was slowly added and was allowed to stir for 24 h to cause particle crosslinking. To remove free BSA and glutaraldehyde, the prepared nanoparticles were purified by 2 rounds of centrifugation (12000 rpm, 20 min). The pellet was redispersed to the original volume in water under ultrasonication over 10 min. For FA conjugation, FA was activated with EDC. Briefly, 20 mg/mL of FA solution (dissolved in 0.1N NaOH solution) was first reacted with EDC solution at a molar ratio of 1:5. The reaction was performed in dark with constant stirring for 2 h. Subsequently, the activated FA solution was dropwise added to BSANPs solution to form FA-conjugate BSANPs. Finally, PSeD solution (300 uL, 12.5 mg/mL) was added to FA-conjugated BSANPs under constant stirring at a time period of 24 h for incorporating PSeD. The FA-BSANPs was purified by 3 rounds of centrifugation (12000 rpm, 20 min) to removed unloaded PSeD, FA and EDC. The Se content in the nanosystem was calculated by ICP-AES method. The whole process was performed at room temperature. Characterization of FA-BSANPs The morphology of FA-BSANPs was determined by transmission electron microscope (TEM, Hitachi H-7650), scanning electron microscope (SEM, EX-250 system, horiba) and atomic force microscope (AFM, Bioscope Catylyst Nanoscope-V). Fourier transform infrared spectroscopy (FTIR, Equinox 55 IR spectrometer) was used to characterize the structure information of FA-
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BSANPs in the range of 400- 4000 cm-1. The size distribution and zeta potential of particles were monitored by Malvern Zetasizer Software. Cell culture and determination of cell viability In this article, Hela cervical carcinoma cells, MCF-7 breast adenocarcinoma cells, A375 melanoma cells and L02 human hepatic cells were human cell lines, which were applied from American Type Culture Collection (ATCC, Manassas, Virginia). These cell lines were maintained in DMEM medium with 100 units/mL penicillin, 10% fetal bovine serum and 50 units/mL streptomycin at 37 °C in 5% CO2 incubator. FA-BSANPs-caused cell proliferative inhibition against various cell lines was measured by clonogenic assay.33 and MTT assay.34 Determination of FA-BSANPs cellular uptake and intracellular trafficking Intracellular uptake of FA-BSANPs in Hela and L02 cells was quantified by determining Se concentration using ICP-MS method. Briefly, Hela and L02 cells were treated with different concentrations of FA-BSANPs with or without radiation (8 Gy) for different time periods， followed by cell digestion and redispersion. After that, the cells were counted, lysed using hydrogen nitrate (HNO3) and perchloric acid (HClO4) mixture (HNO3 : HClO4=3:1). Finally, the lysis solution was diluted with water to a volume of 10 mL. ICP-MS determination was performed to detect Se concentration, which quantified the uptake efficiency of internalized nanoparticles. The intracellular trafficking of FA-BSANPs in Hela and L02 cells was monitored using fluorescence imaging technique under fluorescence microscope (IX51, Olympus).35 Folate competing assay The binding efficiency of FARs between FA-BSANPs and excessive FA was compared as previously described.35 The internalized FA-BSANPs was quantified by the fluorescence
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intensity from PSeD under fluorescence microplate reader (Bio-Tek) (excitation wavelength at 482 nm and emission wavelength at 526 nm ). In vitro drug release of FA-BSANPs Four copies of FA-BSANPs radiated with 0 and 8 Gy were respectively dispersed in PBS solution at a concentration of 1 mg/mL at acidic lysosomal (pH 5.3) and neutral blood (pH 7.4) environments. The solutions were kept in dark at 37 °C to imitate in vivo environment. 250 µL of buffer was taken out and fresh buffer at same volume was supplied. The release rate of PSeD was represented by its fluorescence intensity using fluorescence microplate reader (excitation wavelength at 482 nm and emission wavelength at 526 nm ). Flow cytometry The cell cycle distribution after the treatment of FA-BSANPs and X-ray was measured by flow cytometric analysis.36 The cells incubated with FA-BSANPs and X-ray were digested, collected and centrifuged at a speed of 1500 rpm for 10 min. The harvested cells were fixed with 70% ethanol at -20°C overnight followed by PI staining. The DNA contents were quantified by the fluorescence intensity of PI. The cell samples were analyzed on a Beckman Coulter Epics XL MCL flow cytometer (Beckman Coulter, Inc, Miami, FL). The DNA histogram represented the cell number in G0/G1, S, and G2/M phases, while the sub-G1 peak quantified the hypodiploid DNA number in apoptotic cells. The DNA contents of the cell samples were analyzed by MultiCycle software (Phoenix Flow Systems, San Diego, CA); TUNEL & DAPI staining assay FA-BSANPs-caused DNA fragmentation was confirmed by TUNEL assay in Hela cells. Briefly, the cells attached in 2-cm dishes were fixed and permeabilized with 3.7% formaldehyde and 0.1% Triton-X solution respectively. After that, TUNEL working buffer and DAPI were
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added for cell staining. The morphology of stained cells was observed under a fluorescence microscope (EVOS FL auto, Life Technologies). Determination of reactive oxygen species (ROS) generation Changes of intracellular ROS generation caused by FA-BSANPs with or without radiation were evaluated using dihydroethidium (DHE, Beyotime) and DCFDA probe respectively. Briefly, Hela cells pre-treated with different concentration of FA-BSANPs for 2 h were irradiated at a dosage of 0, 8 Gy respectively. At the end of radiation, the cells were stained with 10 µM DHE at final concentration and DCFDA of at 37°C for 30 min. The cells rinsed with PBS twice were incubated in fresh PBS. The fluorescence intensity of DHE and DCF (excitation and emission wavelength at 300 nm / 610 nm and 488 nm / 525 nm respectively) quantified the intracellular ROS level. For the determination of extracellular ROS generation caused by FABSANPs and free PSeD, DHE staining assay was conducted in PBS without cells. Western blot analysis The expression of proteins involved in signaling pathways after combined treatment of FABSANPs and radiation in Hela cells and L02 cells were determined by western blot analysis. Briefly, the cells were lysed with RIPA buffer and the total proteins were obtained from supernatant after centrifugation (12000 rpm) at 4°C. The proteins were quantified using BCA assay. After that, SDS-PAGE was performed with equivalent amount of proteins loading. Subsequently, the separated proteins were transferred to PVDF membranes, blocked with 5% non-fat milk, incubated with primary antibodies and second antibodies-conjugated horseradish peroxidase, and were finally visualized on X-ray films.
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Statistical analysis All experiments were performed at least for 3 times and the data in this study was expressed as means ± S.D. Statistical significance of the data were analyzed by SPSS statistical program (Version 13, SPSS Inc., Chicago, IL). Difference between two groups was calculated by twotailed Student’s t-test. Variation with P
