Targeted delivery and redox activity of folic acid-functionalized

Subscriber access provided by MT ROYAL COLLEGE

Article

Targeted delivery and redox activity of folic acid-functionalized nanoceria in tumor cells James A. Vassie, John M. Whitelock, and Megan S. Lord Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00920 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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 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 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.

Molecular Pharmaceutics 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.

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

Molecular Pharmaceutics

Targeted delivery and redox activity of folic acid-functionalized nanoceria in tumor cells James A. Vassie, John M. Whitelock, Megan S. Lord*

Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia.

*Corresponding author: Megan Lord, Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia.

Abstract Cerium oxide nanoparticles (nanoceria) are promising catalytic nanomaterials that are widely reported to modulate intracellular reactive oxygen species (ROS). In this study, nanoceria synthesized by flame spray pyrolysis and functionalized with a cell-targeting ligand, folic acid (FA). The surface functionalization of nanoceria was stable and FA enhanced the uptake of nanoceria via folate receptors. Internalized nanoceria and FA-nanoceria were localized predominantly in the cytoplasm. FA-nanoceria modulated intracellular ROS to a greater extent than the nanoceria in colon carcinoma cells, but induced ROS in ovarian cancer cells, likely due to their enhanced uptake. Together these data demonstrated that the functionalization of nanoceria with FA modulated their endocytosis and redox activity and may find application in the delivery of anticancer drugs in the future.

Keywords Cerium oxide, nanoceria, folic acid, cellular uptake, cancer cells

Page 1 ACS Paragon Plus Environment

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

Graphical abstract


Page 2 of 31

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


1.

Introduction

Cerium oxide nanoparticles (nanoceria) are being investigated for antioxidant therapy due to their ability to catalytically react with reactive oxygen species (ROS) 1-4. Their unique structure contains a significant number of surface defects in the form of mobile oxygen vacancies 5-6. Cerium ions adjacent to the oxygen vacancies alternate between the trivalent (Ce3+) and tetravalent (Ce4+) oxidation states 6-9. These facile cyclic oxidation states account for the antioxidant properties of nanoceria, allowing them to protect cells against various ROS 1. However, nanoceria are known to generate ROS in acidic environments, such as those found within lysosomes or with high levels of cellular uptake 10. ROS are produced intracellularly by oxidase enzymes during aerobic metabolism and are important messaging molecules in a number of signal transduction pathways 1, 11-12. Under normal conditions the cellular enzymes catalase and superoxide dismutases protect cells against the accumulation ROS 8, 12-14. However, these enzymes are not always effective against the excessive levels of ROS generated by a number of inflammatory pathologies 15-17, including cancer 5, which can then lead to oxidative stress 18. Oxidative stress can result in damage to cells and tissues 12, 19. As a result of the ability of nanoceria to either reduce or enhance intracellular ROS, they are being explored as both antioxidant therapeutics 3, 10, 20 or as inducers of oxidative stress to affect cell function, such as for cancer therapeutics. Nanoceria has been produced using techniques such as supercritical synthesis 21, sol-gel 6, colloidal synthesis 22, and microemulsion processes 23. Some of these techniques employ the use of toxic surfactants or solvents in the manufacturing process 24. Flame spray pyrolysis (FSP) yields metal oxide nanoparticles with monodisperse primary particle sizes, highly reproducible crystallinity and an absence of intraparticle micro-porosity. Furthermore, as FSP is conducted at temperatures above 1900 °C, the final product is devoid of traces of toxic solvents 8, 24-25. Conventional chemotherapeutic agents are effective against, but not always specific to, cancer cells. Therefore, adverse side-effects from chemotherapy are common 26. Targeted nanomedicine offers a way to deliver cancer therapeutics to tumor sites whilst avoiding healthy tissue 27-28. Folic acid (FA) is a 441 Da vitamin that binds to the folate receptor α (FR-α) with high affinity 29. FR-α is a cellsurface receptor which is almost exclusively expressed by cancer cells and widely expressed on various tumor types, particularly ovarian cancer 29-30. Although nanoceria have previously been functionalized with biological molecules including proteins, polymers and glycosaminoglycans to improve their target specificity, therapeutic effects and circulation time 4, 31-33, their functionalization with FA remains relatively unexplored. Indeed, FA-enhanced therapeutic delivery of nanoceria to cancer cells has only recently been demonstrated 34-35, but not using FSPsynthesized nanoceria. The endocytosis of nanoparticles, including nanoceria, is predominantly an energy-dependent process which may be mediated by clathrin, caveolin, actin and/or other pathways 2, 36-37. Given that changes to the physicochemical properties of nanoceria can alter the ways in which cells respond to particle treatments, it is necessary to examine the mechanisms of uptake and intracellular trafficking of FA-functionalized nanoceria. The uptake and ROS scavenging of nanoceria in CD44-expressing human fibroblasts has been reported to be significantly enhanced by surface functionalization with hyaluronan 32. It was, therefore, hypothesized that FA-functionalized nanoceria (FA-nanoceria) have the potential to be more effective scavengers of ROS than nanoceria in FR-α-expressing cells. Hence, the aims of this Page 3 ACS Paragon Plus Environment


study were to investigate the activity of FA-nanoceria in FR-α-expressing cells in terms of cell proliferation, endocytosis, intracellular trafficking and modulation of ROS.

2.

Materials and Methods

Chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) unless stated otherwise.

2.1. Synthesis and characterization of nanoceria functionalized with APTES and FITC Nanoceria were synthesized using FSP as described previously 38 with liquid precursor and sheath gas flow rates of 5 L/min each. The nanoceria were found to have a Brunauer-Emmett-Teller equivalent particle diameter of 7 nm, and a diameter of 12 nm by X-ray diffraction analysis. Nanoceria were functionalized with (3-aminopropyl)triethoxysilane (APTES) as described previously 37. APTES-nanoceria were functionalized with the fluorophore, fluorescein isothiocyanate isomer I (FITC) as described previously 39. Before doing so, the level of APTES surface functionalization was measured by thermogravimetric analysis (TGA; see section 2.1.1) to determine the correct amount of FITC required per unit mass of APTES-nanoceria to substitute 50% of the primary amine functional groups on the APTES-nanoceria. APTES-nanoceria and FITC-nanoceria were functionalized with FA. Before doing so, the level of APTES and FITC surface functionalization was measured by TGA to determine the correct amount of FA required per unit mass of APTES-nanoceria and FITC-nanoceria to substitute 100% and 50% of the primary amine functional groups on the APTES-nanoceria and FITC-nanoceria, respectively. FA was conjugated to the surface amine groups via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) coupling with the carboxyl groups (primarily γ carboxyls) of FA. For the known masses of FA, the amounts of EDC and NHS required for a minimum molar ratio of FA:EDC:NHS of 1:1.2:2 was used to ensure that EDC was in excess with respect to FA and NHS was in excess with respect to EDC. The FA, EDC and NHS were added to 10 mL of anhydrous DMF and purged with N2. The reaction vessel was stirred for 6 h at 45 °C in an oil bath on a magnetic stirring plate. A known amount of APTES-nanoceria or FITC-nanoceria was added to 15 mL of anhydrous DMF and purged with N2. The reaction vessel was sonicated at 45 °C for 3 h until the particles were suspended and the vessel was then transferred to a magnetic stirring plate. EDC/NHS-activated FA was added to the particle suspensions, purged with N2 and then stirred at 45 °C in an oil bath for 24 h. After the reaction was completed, the FA-nanoceria and FAFITC-modified nanoceria (FA-FITC-nanoceria) were washed, dried and stored in the same manner as the APTES-nanoceria and FITC-nanoceria.


Page 4 of 31

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


2.1.1 TGA of nanoceria A thermogravimetric analyzer 2950HR V5.4A operating in an N2 atmosphere with a heating rate of 5 °C min−1 between 20 and 1,000 °C was used to measure the mass of the molecules conjugated to the surface of the nanoceria. By applying the formula, ܰ =

௑ேಲ ఘ௏ ெೈ

, where ܺ is the percentage weight

loss due to the conjugate, ܰ஺ is Avogadro’s number, ߩ is the density of the nanoparticle, ܸ is the volume of one nanoparticle and ‫ܯ‬ௐ is the molecular weight of the conjugate, the number of molecules, ܰ , on each nanoparticle was quantified.

2.1.2 Attenuated total internal reflectance-Fourier transform infrared spectrophotometry (ATRFTIR) A Perkin Elmer Spotlight 400 ATR-FTIR was used to study changes in the surface chemical structure of the nanoceria following functionalization with APTES and FA.

2.2 Culture of human ovarian and colon cancer cells The human ovarian adenocarcinoma cell line, SKOV3, and the human colon carcinoma cell line, WiDr, were obtained from ATCC and cultured in standard RPMI-1640 and high-glucose DMEM culture media respectively, with both media containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified incubator (5% CO2/95% air atmosphere at 37 °C).

2.3 Cell proliferation analysis SKOV3 and WiDr cells were seeded in 96-well tissue culture polystyrene plates at a density of 2.5×104 cells/well or 7.5×103 cells/well in 200 µL of RPMI or phenol red-free DMEM containing 10% (v/v) FBS and antibiotics, respectively. Triplicate wells were prepared for each treatment. Cells were incubated overnight prior to the addition of 50 µg/mL of nanoceria or FA-nanoceria suspended in medium. Cell proliferation was analyzed at 24, 48, and 72 h post-treatment using the CellTiter 96® AQueous One Solution MTS reagent (Promega, Madison, USA). The MTS reagent was added to the cell cultures 4 h prior to measurement of the absorbance at 490 nm. Cells exposed to medium only were used as a positive control for the assay. Background absorbance readings were also obtained for each of the treatments and controls in the absence of cells to compensate for the effects of the nanoceria and media on MTS absorbance. Relative absorbance values were calculated by subtracting the background absorbance readings. The percentage of cell number relative to the positive control was quantified by dividing the relative absorbance values of each treatment by the relative absorbance value of the positive control.

2.4 Stability of FITC and FA conjugation The stability of the conjugations of FITC and FA to the APTES-nanoceria was investigated by FLIM. Using a MicroTime 200 fluorescence microscope (PicoQuant), the fluorescence lifetimes (the time between photon absorption and emission) of SKOV3 cells treated with 50 µg/mL of FITC-nanoceria or FA-FITC-nanoceria for 4 h were measured and compared with the lifetimes of RPMI solutions containing FITC (2.12 µM) or FA (1.83 µM), which were at the same concentration as FITC and FA conjugated to the particles at 50 µg/mL (as determined by TGA). PBS was used as Page 5 ACS Paragon Plus Environment


a control for autofluorescence. The data was acquired and analyzed using SymphoTime software (PicoQuant). Phasor (phase vector) analysis of the fluorescence lifetime of each pixel was performed using SimFCS software (Laboratory for Fluorescence Dynamics, University of California, Irvine) to calculate the coordinates g= mcos(φ) and s= msin(φ) as defined in Digman et al. 40. In this method of analysis the Fourier transforms of the fluorescence decay curves were calculated and the results were plotted on a two-dimensional complex plane, thereby providing a graphical representation of the measured fluorescence lifetimes.

2.5 FR-α expression 2.5.1 Microscopy analysis Cells were seeded into 8-well 1.5 borosilicate LabTek™ II plates (Thermo Scientific, Rochester, NY, USA) at a density of 105 cells/well in 400 µL of medium containing 10% (v/v) FBS and antibiotics. Duplicate wells were prepared for each cell line. The cells were incubated overnight before being stained for FR-α. After incubating overnight the cells were washed with DPBS and fixed in 4% (w/v) paraformaldehyde and 1% (w/v) sucrose in DPBS for 15 min at room temperature (RT), away from light. After fixation, the cells were washed twice in DPBS and permeabilized with 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 2 mM HEPES, 0.5% (v/v) Triton X-100 in 50 ml deionized water, pH 7.2 for 5 min at 4 °C. After permeabilization, the cells were washed twice in 0.05% (w/v) Tween 20 in DPBS (PBS-T) and blocked for 1 h in blocking solution (2% (w/v) bovine serum albumin (BSA) and 4% (v/v) goat serum in PBS-T). The cells were then washed once with PBS-T before being treated with an anti-FR-α antibody (clone #548908, R&D Systems, Noble Park, VIC, Australia; 10 µg/mL) diluted in 200 µL of blocking solution for 2 h at RT, away from light. For each sample, a no primary antibody control was used in order to measure non-specific fluorescence. After 2 h, the cells were washed three times with PBS-T for 5 min before being treated with AlexaFluor® 488-conjugated secondary IgG antibodies (Life Technologies, Scoresby, VIC, Australia; 1:500) diluted in 200 µL of blocking solution for 1 h at RT, away from light. After 1 h, the cells were washed three times with PBS-T for 5 min, before their nuclei were counterstained with Hoechst 33342 (Life Technologies, Scoresby, VIC, Australia; 10 µg/mL) for 10 min. After 10 min, the cells were washed three times with PBS-T for 5 min, before their lipid structures were counterstained for 30 min with CellMask™ Orange Plasma membrane stain (Life Technologies, Scoresby, VIC, Australia; 2.5 µg/mL) diluted in 200 µL of blocking solution, away from light. After 30 min, the cells were washed three times with DPBS for 5 min. Images were taken with an epifluorescence microscope (Zeiss Axioskop Mot Mat 2, Zeiss, Australia) operating on a 100× oil objective and filter cubes with excitation/emission peaks of 350-365/445-450 nm, 440-470/525-550 nm and 525-550/605-670 nm and processed using Zeiss ZEN 2012 (Zeiss, Australia) and FIJI Is Just ImageJ 1.49n. Four spatial regions were imaged per well.

2.5.2 Flow cytometry analysis Cells were removed from cell culture plates by brief treatment with trypsin so as not to remove cell surface receptors and were fixed in 4% (w/v) paraformaldehyde in DPBS for 15 min at RT, away from light. After fixation, the cells were centrifuged and washed twice in DPBS and permeabilized as in section 2.5.1. After permeabilization, the cells were centrifuged and washed twice in PBS-T and blocked for 1 h in blocking solution (2% (w/v) BSA in PBS-T). The cells were then centrifuged Page 6 ACS Paragon Plus Environment

Page 6 of 31

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


and washed once with PBS-T before being treated with the anti- FR-α antibody (2.5 µg/106 cells) or IgG isotype control antibody (Life Technologies, Scoresby, VIC, Australia, 0.5 µg/106 cells) diluted in blocking solution for 2 h at RT, away from light. After 2 h, the cells were centrifuged and washed three times with PBS-T for 5 min before being treated with AlexaFluor® 594-conjugated secondary antibodies (Life Technologies, Scoresby, VIC, Australia; 1.5 µg/106 cells) diluted in blocking solution for 1 h at RT, away from light. After 1 h, the cells were centrifuged and washed three times with PBS-T for 5 min. The cells were then centrifuged and resuspended in DPBS prior to analysis by flow cytometry. For each sample, data was acquired for 104 gated events using a flow cytometer (BD FACSort) by measuring fluorescence along with the number of cells. Two runs were performed per replicate. Data were analyzed using FCS Express 4 (De Novo Software).

2.6 Nanoparticle uptake analysis Cells were seeded into 24-well plates at a density of 2.5×105 cells/well (1.3×105 cells/cm2) in 1 mL of medium containing 10% (v/v) FBS and antibiotics. Triplicate wells were prepared for each treatment. The cells were incubated overnight at 37 °C in 5% (v/v) CO2 before being treated for 4 h with medium containing 10% (v/v) FBS and antibiotics, or with 50 µg/mL of FITC-nanoceria or FA-FITC-nanoceria dispersed in medium containing 10% (v/v) FBS and antibiotics. Live cells were prepared for flow cytometry analysis as described in section 2.5.2 and data acquired for forward and side scatter.

2.7 FA competition assay The role of FR-α in the endocytosis of FA-FITC-nanoceria was investigated by flow cytometry and FA competition. Cells were seeded at a density of 2.5×105 cells/well (1.3×105 cells/cm2) into 24well tissue culture plates in 1 mL of standard RPMI (containing 2.3 µM of FA; standard RPMI) or RPMI containing 200 µM of FA (high FA RPMI) . Triplicate wells were prepared for each treatment. The cells were incubated overnight at 37 °C in 5% (v/v) CO2 before being treated for 4 h with standard RPMI or high FA RPMI with or without 50 µg/mL of FITC-nanoceria or FA-FITCnanoceria. After 4 h, live cells were prepared for flow cytometry as described in section 2.5.2 and fluorescence measured along with the number of cells. Two runs were performed per replicate. Average cell fluorescence was normalized to the fluorescence of each treatment (medium or medium containing nanoceria) and the normalized fluorescence of untreated cells was subtracted to allow direct comparison between treatments.

2.8 Intracellular trafficking of nanoceria The intracellular trafficking nanoceria was investigated using confocal microscopy by analyzing colocalisation of particles with clathrin, caveolin-1 and lysosomes. For particle colocalization with clathrin and caveolin-1, cells were seeded into 8-well borosilicate plates at a density of 105 cells/well in 400 µL of medium. Duplicate wells were prepared for each treatment. The cells were incubated overnight before being treated for 4 h with medium, or with 50 µg/mL of FITC-nanoceria or FA-FITC-nanoceria suspended in fresh medium. For particle colocalization with lysosomes, cells were seeded into 8-well borosilicate plates at a density of 8×104 cells/well in 400 µL of medium. Duplicate wells were prepared for each treatment. The cells were incubated overnight before being



treated for 1, 4 and 24 h with medium, or with 50 µg/mL of FITC-nanoceria or FA-FITC-nanoceria suspended in fresh medium. Particle treatments were timed to finish simultaneously. For cells to be stained for clathrin or caveolin-1, after 4 h, the cells were fixed and prepared analyzed by confocal microscopy as described in section 2.5.1 using anti-clathrin heavy chain antibody (ab21679, Abcam, Melbourne, VIC, Australia; 1 µg/mL) or anti-caveolin-1 (ab2910, Abcam, Melbourne, VIC, Australia; 2 µg/mL) antibodies. Lipid structures in the cells were counterstained for 30 min with CellMask™ Deep Red Plasma membrane stain (Life Technologies, Scoresby, VIC, Australia; 2.5 µg/mL) diluted in 200 µL of blocking solution, away from light. After 30 min, the cells were washed three times with DPBS for 5 min. For cells to be stained for lysosomes, after 1, 4 and 24 h of treatment with particles, the cells were washed with DPBS and stained with LysoTracker® Red DND-99 (Life Technologies, Scoresby, VIC, Australia; 50 nM) diluted in DPBS for 30 min, away from light. The cells were then washed with DPBS and fixed for 15 min, away from light. The cells were washed again and counterstained with CellMask™ Deep Red Plasma membrane stain as described above. Images were taken with a multiphoton laser scanning confocal microscope (Zeiss LSM 780, Zeiss, Australia) operating on a 100 × oil objective and processed using Zeiss ZEN 2012 (Zeiss, Australia) and FIJI Is Just ImageJ 1.49n. The intracellular optical sections were 1 µm thick and imaged at a zoom factor of 1 with 8 bits per pixel. Four spatial regions were imaged per well. Particles that were colocalized with the cytoplasm were considered internalized. Using the cytoplasm as a guide, colocalization was quantified at two regions of interest per image using the Manders’ colocalisation coefficients (MCCs), which provides a measure of co-occurrence of two channels independent of signal proportionality. Just Another Colocalisation Plugin within Image J was used to determine the MCC. Colocalization was reported as the fraction of internalized particles that had colocalized with clathrin, caveolin-1 or lysosomes after thresholding background fluorescence in both fluorescence channels. The pixel intensity threshold values were set within the plugin and due to spatial variation in the background fluorescence levels, a single threshold value was not applied to all images, however the threshold applied to all data sets was 19 ± 2.1 and 24 ± 2.2 for the green and red channels, respectively. Brightness and contrast of the images were adjusted for visual presentation.

2.9 Reactive oxygen species analysis The level of ROS in cells exposed to nanoceria or FA-nanoceria relative to untreated cells was measured every 24 h over a 72 h period using the intracellular peroxide-dependent oxidation of the ROS indicator, 2′,7′–dichlorodihydrofluorescein diacetate (DCFH-DA). Cells were seeded into 24well tissue culture plates at a density of 2.5×105 cells/well in 1 mL of media. Triplicate wells were prepared for each treatment. The cells were incubated overnight prior to the addition of 50 µg/mL nanoceria suspended in fresh media. Cells exposed to medium only were analyzed at each of the time points to determine the basal level of ROS in the cells. Control groups were not probed with DCFH-DA in order to measure and gate background fluorescence and side scatter. At each time point, cells were removed from the plates by trypsinization and centrifuged at 1,400 rpm for 5 min. The supernatant was removed and the cells were resuspended in 1 mL of medium and were incubated with 10 µM DCFH-DA for 30 min at 37 °C before being centrifuged and then washed with DPBS. The cells were then centrifuged and resuspended in fresh medium prior to analysis by Page 8 ACS Paragon Plus Environment

Page 8 of 31

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


flow cytometry as described above. Data were presented as mean fold change in DCF signal compared to cells exposed to medium only after correction for the level of background fluorescence.

2.10 Statistical analysis Statistical significances between treatments were compared in GraphPad Prism 6 by using a twoway analysis of variance (ANOVA), Tukey’s post-hoc test was applied after establishing normality using the Shapiro-Wilk test and that the variance for each condition was equal using an F-test. Results with a p-value of less than 0.05 were considered statistically significant.

3.

Results

3.1 Characterization of functionalized nanoceria The functionalization of nanoceria with FA through the organosilane linker, APTES, was verified by ATR-FTIR (Fig. 1). The broad peaks at 3,400 cm−1 in nanoceria and all of their derivatives corresponded to the O−H symmetric stretching from their surface hydroxyl groups. Successful modification of nanoceria with APTES was also confirmed by the appearance of N−H bending at 1,554 cm–1 from the amide bond and CH2 stretching at ~3,000 cm–1. The introduction of silane onto the surface of the nanoceria was also confirmed by the bands at 1,113 and 1,048 cm–1, assigned to the Si−O−Si and Si−O−C bonds, respectively. In order for APTES to attach to a surface hydroxyl group, at least one of its three ethoxy groups must undergo hydrolysis. The Si−O−C band indicated that not all of the ethoxy groups on APTES had undergone hydrolysis due to the insufficient amount of water available for the reaction. The conjugation of FA to APTES-nanoceria was confirmed by the presence of a peak at 1,440 cm–1, which represented the COO asymmetric stretching of the unmodified carboxyl group on FA. Nanoceria were functionalized with FITC to enable analysis of uptake and localization in subsequent imaging studies and with FA to facilitate enhanced uptake in FR-α-expressing cells. TGA analyses indicated that 16% of the surface hydroxyl groups on the nanoceria were modified with APTES, which was equivalent to approximately 338 APTES groups per nanoparticle (Table 1). Furthermore, APTES-nanoceria were functionalized with approximately 33 FITC groups or 104 FA groups per nanoparticle, and FITC-nanoceria were functionalized with approximately 27 FA groups per nanoparticle.

3.2 Cellular interactions with nanoceria The effect of nanoceria and FA-nanoceria on SKOV3 and WiDr cell viability was analyzed over a period of 72 h (Fig. 2). The number of both SKOV3 and WiDr cells exposed to nanoceria or FAnanoceria over the 72 h analysis period was not significantly (p

Targeted delivery and redox activity of folic acid-functionalized

Recommend Documents