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Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation
Accepted Manuscript Title: Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation Authors: Anees A Ansari, Maqsood A Siddiqui, Aslam Khan, N. Ahmad, M. Alam, Ahmed Mohamed El-Toni, Abdulaziz A. Al-Khedairy PII: DOI: Reference:
S0927-7757(19)30357-7 https://doi.org/10.1016/j.colsurfa.2019.04.049 COLSUA 23393
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
10 January 2019 28 February 2019 17 April 2019
Please cite this article as: Ansari AA, Siddiqui MA, Khan A, Ahmad N, Alam M, El-Toni AM, Al-Khedairy AA, Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), https://doi.org/10.1016/j.colsurfa.2019.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation
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Anees A Ansari1*, Maqsood A Siddiqui2,3, Aslam Khan1, N. Ahmad4, M. Alam4, Ahmed Mohamed El-Toni1, Abdulaziz A. Al-Khedairy3 1 King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia 2Al-Jeraisy Chair for DNA Research, Zoology Department, College of Science, King Saud University, Riyadh-11451, Saudi Arabia 3Zoology Department, College of Science, King Saud University, Riyadh-11451 Saudi Arabia 4 Department of Chemistry, King Saud University, Riyadh 11451, Saudi Arabia
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Corresponding author Email: [email protected], [email protected] Tel.:+966-11-4676838; Fax.: +966-11-467662
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Graphical Abstract:
Highlights:
We fabricated aqueous dispersible spherical shaped SiO2@Tb(OH)3 nanospheres. Nanospheres revealed excellent photoluminescent properties. 1
Highly biocompatible and low toxicity over a wide range of concentrations.
Abstract Spherical shaped SiO2@Tb(OH)3 nanospheres (core-shell NSs) with an average grain size of 300-350 nm were successfully synthesized at a large scale via sol-gel process with subsequent heat treatment. XRD results demonstrated the non-crystalline nature of the sample even after
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coating with terbium hydroxide. TEM and SEM images clearly showed that the prepared sample was highly monodispersed, perfectly spherical in shape, and mesoporous with narrow size
distribution (an average crystalline size of 350 nm). XRD, TGA, TEM, SEM, and EDX results demonstrated that the terbium hydroxide layer was effectively grafted over the surface of the
silica spheres, which was further verified using FTIR, UV/Visible, and emission spectral results. The emission spectrum of core-shell NSs demonstrates that Tb3+ is strongly bonded covalently to
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the silica framework with surface anchored oxygen atoms, thus preventing the accumulation of
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Tb3+ ions to give an efficient luminescence. The emission spectrum was dominated by 5D4→4F5
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of the Tb3+ ion located in the green region at 2.88 eV along with broad emission of the silica framework. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide and
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neutral red uptake assays on MCF-7 and A-549 cells clearly illustrated the high biocompatibility and non-toxic nature of the samples. Because of the high specific surface area, mesoporous
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nature, and presence of abundant hydroxyl groups on the surface of the particles, these materials
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can be applied for tagging bioactive molecules or for loading high quantity anti-cancer drugs for biomedical applications.
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Keyword: Terbium hydroxide, Silica nanospheres, Optical properties, Luminescence properties, Biocompatibility, Cytotoxicity
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1. Introduction Currently, nano-scale luminescent lanthanide materials have become one of the most fascinating areas of research in modern science and technology because of their outstanding
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chemical, physical, and luminescent properties such as multiple absorption-emission transitions in the whole visible and NIR region, stark splitting, high quantum efficiency, long decay time, superior photo-chemical stability, low autofluorescence, biocompatibility, and non-toxicity [1-9]. These unique features make them ideal candidates for applications in the various fields of clinical sciences such as bio-imaging/bio-labeling, optical detection of analytes, finger printing, magnetic resonance imaging, and photodynamic therapy and in designing of optical probes and 2
biosensors [10-21]. These nanostructured materials must be photo-chemically stable, aqueoussoluble, biocompatible, and non-toxic in nature to be used in clinical sciences. However, most of the luminescent lanthanide nanomaterials are hydrophobic in nature [22-31]. In addition, another major disadvantage of these materials is the weaker emission intensity compared to the parent uncoated luminescent material because multi-photon relaxation pathways suppress the
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luminescence efficiency and quantum yields and reduce molar extinction coefficients of the surface coated nanostructured materials [2, 25-27, 32-37]. To overcome these drawbacks, alternate synthesis methods have been developed to improve the emission efficiency as well as the photo-chemical stability, aqueous dispersibility, colloidal stability, biocompatibility, and non-cytotoxicity if the luminescent nanomaterials and therefore, this has become an exciting research topic. Thus, there is an urgent need to either modify the present techniques for surface
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coating of nanoparticles by addition of appropriate functional groups or to adopt some alternative
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synthesis process during the fabrication of nanostructured materials that can enhance their solubility and colloidal stability.
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Till date, hexagonal, cubic, rod-shaped, planar, spherical, octahedral, pyramidal, tube-
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shaped, and irregular spherical luminescent lanthanide nano-materials have been successfully fabricated using several techniques and have been applied in photonic based bio-applications
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[22, 24, 25, 35, 38-40]. Among them, spherical shaped luminescent lanthanide micro/nano
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phosphor materials are extensively used due to their attractive features including better photoluminescence (PL) high definition, much improved screen packing, distinct large effective densities, high specific surface areas, huge inner space, and low scattering of light. These novel
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features of the spherical shaped luminescent materials are highly advantageous in many photonic-based applications such as in development of photonic devices, optical bio-
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probe/assays, and optical biosensors and in bio-detection. Therefore, it is essential to develop alternate methods that can enhance the PL performance along with the aqueous dispersibility
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with colloidal stability, biocompatibility, and low toxicity of the spherical shaped phosphoric materials. To control the morphology of the phosphoric materials, various methods have been adopted such as hydrothermal/solvothermal method, thermal decomposition, co-precipitation, micro-emulsion, application of microwaves, sol-gel chemical method, and homogeneous coprecipitation using a weak base (urea, thiourea, hydrazine, and trimethylamine) [41-45]. 3
Among them, the sol-gel method is a low-cost, non-hazardous, eco-friendly, low temperature, well-organized, and effective process for preparation of nano-materials. Silica nanospheres prepared using established sol-gel methods have been successfully applied in areas including drug delivery and biolabeling/biodetection due to their uniform spherical shape and stable mesoporous structure. Because of abundant surface anchored silanol groups, the silica
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surface can easily be functionalized with fluorescent materials to improve their optical properties for further uses in clinical and biomedical sciences. Previously, nanomaterials of spherical shape with controlled structure, high mesoporous structure, low toxicity, and high aqueous dispersibility of silica-spheres have been successfully applied as phosphor supports (or capping agent) or cell imaging materials. Lin and his co-workers fabricated silica supported sub-micron SiO2@Y2SiO5:Eu, SiO2@Y2SiO5:Ce/Tb[46], SiO2@YVO4:Eu[47], SiO2@YVO4:Dy/Sm [48]
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spherical particles. Yang et al. and a few other research groups utilized silica spheres as a
labeling/bio imaging applications [14, 24, 49-59].
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platform for functionalization of luminescent materials and examined their potential in bio-
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In this article, we demonstrated the successful surface functionalization of a Tb(OH)3
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shell over monodispersed mesoporous SiO2 core-NSs, forming well-defined core-shell structures. The formation of the Tb(OH)3 shell over the surface of SiO2 core-NSs makes them
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water dispersible and able to form stable colloids over a period of time without any observable
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sedimentation or precipitation. Synthesized NSs were characterized using state-of-art experimental techniques including X-ray diffraction, transmission electron microscopy, energy dispersive X-ray spectroscopy/field emission-scanning electron microscopy, thermogravimetric Brunauer
Emmett
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analysis,
Teller
(BET)
analysis,
Raman
spectroscopy,
and
PL
spectrophotometry. Furthermore, MTT assay and neutral red uptake (NRU) assay were
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employed using a concentration-based method for investigating the toxicity and cell viability of
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the deigned core-shell NSs.
2. Experimental set up 2.1.Materials Analytical-grade terbium nitrate hexahydrate (99.9%, Alfa Aesar, Germany), tetraethylorthosilicate (TEOS), ethanol, NaOH and cetyltrimethylammonium bromide (CTAB) were 4
analytical grade and used directly as received (Sigma-Aldrich, U.K). Milli-Q (Millipore, Bedford, USA) water was used for synthesis and characterization. 2.2. Synthesis of terbium hydroxide coated silica NSs In a typical synthesis reaction, 2 M aqueous solution of sodium hydroxide and 1 g CTAB
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were mixed in 250 ml dist. water and the solution mixture was kept on a hot plate with constant mechanical stirring at 80°C. Then, 0.5 ml of TEOS was slowly injected into the vigorously stirred hot solution mixture and allowed to react on a hot plate for 2 h [24, 56, 60, 61]. After that, a hot aqueous dissolved solution of 0.5 mg terbium nitrate hexahydrate was introduced into the vigorously stirring reaction mixture. The reaction was allowed to proceed for another 2 h for complete complexation on the surface of silica spheres. The resulting white precipitate was
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separated using centrifugation, washed with dist. water and dried overnight in an oven at 60°C.
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2.3. Characterization X-Ray diffraction pattern (XRD) was carried out using Rigaku-Dmax 25 diffractometer
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equipped with Cu Kα (1.5406Ǻ) radiations. Thermogravimetric analysis (TGA) was performed
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from TGA/DTA Mettler Toledo AG, Analytical CH-8603 Schwerzenbach, Switzerland). Morphological images were obtained using scanning electron microscope (SEM, JEOL JSM
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7600F) operating at 5-10 kV, and transmission electron microscope (TEM). Both of these techniques were equipped with energy dispersive x-ray analysis (EDX, FETEM, 2100F, JEOL,
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Japan) operating at an accelerating voltage 200 kV. Surface chemistry was examined by BET method by using the Barrett-Joyner-Halenda (BJH) model. Infrared spectra were measured from
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Vortex 80 (Bruker, USA) spectrometer using KBr pellet method. Optical absorption spectra were recorded from Cary 60 UV/Visible (Agilent Technologies, USA) spectrophotometer in the range
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of 200-900 nm. Raman and PL spectra were obtained using a Horiba Synapse (Horiba, JobinYvon Edison, USA) spectrophotometer. A slit width of 100 microns was employed, ensuring a
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spectral resolution higher than 1 cm-1. Particle size distribution curve was measured by diffusion light scattering technique using a dynamic light scattering (DLS; Malvern ZetaSizer, ZS, UK).
2.4. MTT assay MTT assay is a colorimetric assay for evaluating viable cells or cytotoxicity of a given material. The test is based on the enzymatic reduction of the lightly colored tetrazolium salt to its 5
formazan which is intense purple-blue color. MTT assay was carried out for measuring the toxic potentiality of the designed core-shell NSs using Mossman et al method with minor modification [62]. 2.5. Neutral red uptake assay Natural red uptake assay is based on the detection of viable cells via the uptake of the
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natural red dye, such as a eurhodin dye that stains lysosomes in viable cells [63]. Repetto et al. method was employed for measuring the neutral red uptake assay [63]. MCF-7 and A549 cell lines were used for both the assays.
3. Results and Discussion 3.1. Chemical composition and thermal stability The X-ray diffraction pattern was obtained to determine the chemical composition and
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crystallinity of the synthesized nano-products. As shown in Fig. 1a, a broad plane with a high-
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intensity band at 2 = 25° is observed in both the XRD diffraction patterns, which is the characteristic peak of amorphous silica [JCPDS card No.29-0085] [47, 56, 64-67]. As seen in
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Fig. 1a, no reflection plane is observed in the core-shell nanostructure even after drying at
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300°C, which indicates that the surface-anchored terbium hydroxide is non-crystalline in nature. However, in case of the core-shell samples, a few broad reflection planes were seen, which could
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be due to ultra-small crystalline nature of terbium hydroxide. These observations are consistent
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with the previously published reports [47, 65-67]. Thermogravimetric analysis was performed to examine the thermal stability of the designed core and core-shell NSs between room temperature to 900°C in a nitrogen-rich atmosphere. As shown in Fig. 1b, the TGA curve of core-NSs
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demonstrates the first decomposition at around 160°C with 4.1% weight loss, which can be attributed to the surface adsorbed water or organic moieties. In the second step, decomposition
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started at 120°C accompanied by continuous weight loss with an increase in temperature till 900°C and a final weight loss of 13%. This indicated the slow decomposition of silica and
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conversion of the remaining silica into silicate. A similar trend was observed in the TGA curve of core-shell NSs. Initial weight loss started at 160°C, and later, the curve showed a sluggish weight loss up to 900°C. This implies that at first, hydroxyl groups are decomposed over the surface of core-shell NSs and the remaining silica is converted into silicate (Fig. 1b). These observations are in agreement with previous literature [38, 44]. It is interesting to note that we 6
observed a similar decomposition trend in both samples, suggesting that silica spheres are coated with the amorphous form of terbium hydroxide. 3.2. Morphological structure and surface chemistry The morphology of the samples was inspected using TEM and SEM micrographs. TEM
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micrographs in Fig. 2a-d clearly indicate that the prepared core-shell structures are composed of uniform, monodisperse, and spherical nanostructures with a mean diameter of 340 nm. These perfect spherical particles were prepared using the sol-gel method. They were distributed homogeneously throughout the image, and mesopores were created among these spherical coreshell structures, as shown by many light spots observed in Fig. 2b&c. We expected that in the presence of a strong base (NaOH), terbium nitrate will decompose rapidly at elevated temperatures. The decay of terbium nitrate releases precipitating cations swiftly and
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heterogeneously into the reaction system. This leads to uncontrolled growth of nucleation
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(terbium cations) on the silica NSs. As seen in Fig. 2a&b, each silica-sphere is completely
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covered with terbium hydroxyl molecules. It is worth noticing that the core and the metal shell of each sphere can be clearly distinguished due to the different electron penetrability between the
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amorphous core and semi-crystalline terbium hydroxide shell-structure. Notably, the core-shell NSs are covered with the semi-crystalline terbium hydroxide shell rendering the core-shell NSs
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hydrophilic so that they can be easily dispersed in water and form stable and clear colloidal
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solutions. In the core-shell structure, silica is light grey in color, whereas the metal shell exhibits dark black color in spot like forms. As demonstrated in Fig. 2c&d, the surface of core-shell NSs
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is rough and has distinct punctuations, proving the formation of porous structured SiO2@Tb(OH)3 NSs. The SEM image in Fig. 2e&f shows that the morphology of the core-shell
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NSs is monodisperse, uniform, spherical-shaped, and rough-surfaced with narrow size distribution and an average grain size of ~350 nm, which is consistent with the FE-TEM results. The DLS measurement was performed to measure the particle size distribution of the prepared
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luminescent NSs. The size of the particle obtained by DLS studies (Fig. 2g) was found to be ~385 nm, which is consistent with the results obtained by TEM and SEM. Energy dispersive Xray (EDX) analysis demonstrates the chemical constituents of the designed core-shell NSs. The EDX analysis in Fig. 3a-e clearly shows all of the expected chemical elements such as Si, Tb, and O in the core-shell nanostructure. 7
The surface features of the nanomaterials were measured using the BET-N2-adsorptiondesorption isotherm. Fig. 4 illustrates the N2-adsorption-desorption isotherm of Tb(OH)3 grafted silica NSs along with blank silica NSs. Here, the isotherm of the silica NSs is used only for comparison. As observed in Fig. 4, Tb-functionalized NSs exhibit a typical IV-type isotherm and H1-hysteresis loop, which is a known characteristic feature of mesoporous materials. The BET-
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isotherm showed that the BET specific surface area, pore size, and pore volume were 49.93 m2/g, 6.2505 nm, and 0.1694 cm3/g, respectively, for the terbium functionalized NSs, whereas those for the silica NSs were 165.7767 m2/g, 16.3176 nm, and 1.2323 cm3/g, respectively. The surface properties of the Tb-functionalized NSs are significantly suppressed as compared to their respective pure SiO2 NSs. It has been shown that silica NSs have abundant surface silanol (SiOH) groups, and these silanol groups are easily available for covalent bonding with terbium
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hydroxide in aqueous media. Thus, a layer of Tb(OH)3 is successfully grafted over the surface of
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silica NSs through covalent bonding. These surface grafted Tb(OH)3 molecules block the interstitial spaces because of the high density of Tb ions and as a result, they occupy the pore
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cavity and suppress the surface properties, as seen in the SEM, TEM, Raman, and PL spectral
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results.
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3.3. Optical properties The FTIR spectra were recorded to determine the surface purity and attached organic
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functional groups on the core and core-shell NSs. As illustrated in Fig. 5, a diffused band between 2800 to 3800 cm-1 along with a medium intensity band at 1645 cm-1 was observed in
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both spectra due to symmetrical and asymmetrical stretching and the bending vibrational mode of physically and chemically attached hydroxyl(-OH) and silanol (Si-OH) groups, respectively 1
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[24, 56, 68]. A high intensity band in the middle of the infrared spectrum is observed at 1200 cmalong with medium intensity bands located at 956 and 805 cm-1, and these can be assigned to
the Si-O-Si, Si-O, and Si-OH vibrational modes, respectively, which originate from amorphous
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silica [25, 56, 69]. Another strong intensity peak at 494 cm-1 attributed to the phonon bonds of metal oxide (Si-O-Tb) network [55, 69]. Optical properties and colloidal stability of the designed nanomaterials were investigated using the UV/visible absorption spectra. Fig. 6 displays the absorption spectra of core and coreshell NSs dispersed in aqueous media at room temperature. In Fig. 6, we demonstrate a 8
comparative analysis of the absorption spectra of core-shell NSs along with bare silica core-NSs and terbium nitrate hexahydrate in an aqueous environment. As shown in Fig. 6, the spectral edge of core-shell NSs is significantly uplifted in the UV/visible region than their respective bare core-NSs due to the 4f8→4f75d absorption transition of Tb3+ [56, 70, 71]. It implies that terbium hydroxide is effectively coated over the surface of core-NSs. The 4f-electrons of terbium ions
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can easily interact with surface anchored hydroxyl groups through hydrogen bonding, making the nanomaterials aqueous dispersible.
Fig. 7 represented the Raman spectrum of the core-shell NSs evaluating the Raman active modes of silica and terbium hydroxide. It can be seen from the spectrum that the Raman active modes are observed at 295, 390, 738, 1224, 1298, 1383, 1446, and 1626 cm-1 along with a broad high-intensity band in between 2850 and 2877 cm-1, which are typical Raman active Ag, Eg, and
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Fg modes of terbium hydroxide [72-74] and the silica network [75]. These Raman active modes
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are consistent with those reported in previous studies [72, 73, 75]. The occurrence of these Raman active modes verified the successful formation of the SiO2@Tb(OH)3 core-shell nano-
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structure of the designed material.
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To ensure the surface functionalization of terbium hydroxide over the silica core-NSs, we recorded the PL spectra of the core-shell NSs. The PL spectrum shows a remarkable high-
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intensity emission band in the visible region, which covered the whole visible region, causing the
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formation of amorphous silica [66, 67, 76]. The emission spectrum of the core-shell NSs under excitation at 3.82eV (325 nm) is composed of various lines peaking at 2.38 eV (521 nm), 2.288 eV (543 nm), 2.1 eV (590 nm), 2.01 eV (614 nm), 1.89 eV (653 nm), and 1.77 eV (701 nm),
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which can be contributed to the 5D4→7F6, 5D4→7F5, 5D4→7F4, 5D4→7F3,5D4→7F2, and 5D4→7F0 transitions, respectively, which are all 4f-4f intra-configurational transitions of Tb3+ ion (Fig. 8)
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[56, 77, 78]. The most prominent transition in the emission spectra was observed in the green region, located at 2.288 eV (5D4→7F5), which is the most chemically sensitive in the spectrum
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and hence provides more information about the chemical environment surrounding the Tb3+. Therefore, it is a so-called hypersensitive transition and factual thumbprint of the characteristic emission transitions of 4f-4f lines of the Tb3+ ions that are altered in their intensity during the formation of new chemical bonds between Tb3+ ion and organic chelating agents. In comparison to the hypersensitive transition, other emission transitions are weak in their sensitivity and show low intensity in the spectrum because of the shielding effect of 4f-subshell by 5s25p6 electrons 9
[25, 56, 68]. Additionally, the occurrence of these weak efficiency emission peaks at higher wavelengths can be attributed to the low phonon energy of the Tb-O-Si bond (494 cm-1) because the multi-photon relaxation by the Tb-O vibration is not able to diminish the intensity of the emission band, as observed from the FTIR spectra in Fig. 5. As shown in Fig. 8, the luminescence spectrum is highly uplifted and revealed significant broadening in all emission
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peak widths and shapes, caused due to the influence of the amorphous silica core NSs. The possible explanation is that 4f-electrons of surface coated Tb(III) ions interact with the silica core producing an abundant, non-bridging oxygen network. This silica network altered the bond angle, bond length, and symmetry of the crystal field, inducing these emission transitions and resulting in characteristic broadening in 4f-4f transitions. We expected that the Tb (III) ion in the current form is of low symmetry in the Si framework and this structured environment increases
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the emission intensity of the hypersensitive (5D4→7F5) transition. It also reflects the formation of
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new chemical bonds or energy transfer from silica molecules to 4f -electrons of Tb3+ ions, altering the emission peak intensity and broadening of hypersensitive transition. As seen in Fig.
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8, an observed hypersensitive (5D4→7F5) transition in the green region demonstrates remarkable
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intensity enhancement than those of the pure complexes and different types of nano-scale host matrices [77-84]. This novel luminescence intensity of the 5D4→7F5 transition could be applied
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in photonic based biomedical applications and laser-based technologies. To calculate the
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emission quantum yield, we used a previously reported method [2, 78, 85] for the core-shell NS sample which involved monitoring the 325-nm excitation wavelength using He-Ne laser at room temperature (Rhodamine 6G dissolved in alcohol was used as a calibration standard). The
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emission quantum yield was found to be 35%.
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3.4. MTT assay
MTT assay was performed to analyze the biocompatibility and toxic nature of the
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designed samples. The induced cytotoxic effects of the core and core-shell NSs on MCF-7 and A-549 cells are shown in Fig. 9a&b. A concentration-dependent cytotoxic response was observed in MCF-7 and A549 cells exposed to 2-200 μg/ml of core and core-shell NSs for 24 h. The cell viability percentage was recorded as 90% and 77% in cells treated with core NSs and 81% and 64% in core-shell NSs at 100 and 200 μg/ml, respectively, in MCF-7 cells compared to untreated control (Fig. 9a). However, the cell viability percentage was recorded as 90%, 82%, 10
and 70% in the core NS treated cells and 80%, 66% and 44% in core-shell NSs treated A-549 cells at 50, 100 and 200 μg/ml, respectively, compared to the untreated control (Fig. 9b). When the concentration of the core-shell NSs in both cell lines was below 50 μg/ml, a remarkably high cell viability was observed. Although, significant suppression in cell viability was observed when the concentration of core-shell NSs was increased, it could be due to high mesoporosity
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and large surface area of the NSs. However, additional of cytotoxicity on the nature of the surface covered functional group cannot be ruled out. It is also obvious from previous reports that there might be aspects contributing to cytotoxicity such as particle size (diameter) and surface functionalization of the materials [86-88]. The cytotoxicity observed when the cells were treated with the core-shell NSs in a dose-dependent manner might be due to the growth of abundant surface anchored hydroxyl groups [87, 89]. It is implied that these OH radicals are
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responsible for the production of reactive oxygen species, which are accountable for cell
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apoptosis [24, 86, 89].
red uptake assay is
the lysosomes of viable
cells,
based on the accumulation of neutral
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Neutral
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3.5. NRU assay
while
MTT
is based
on
the
red dye in
reduction of yellow
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tetrazolium MTT reagent by viable cells to purple formazan. It is important to verify the results
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of MTT assay with another experimental method. Thus, we performed the NRU assay. The results of the NRU assay are presented in Fig. 10a&b. The NRU assay also revealed a concentration-dependent cytotoxicity in MCF-7 and A-549 cells exposed to core and core-shell
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NSs at 2-200 μg/ml. The percentage decrease in cell viability at 50, 100 and 200 μg/ml is recorded as 90%, 83%, and 76% in the core and 84%, 76% and 61% in core-shell NSs exposed
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MCF-7 cells, respectively (Fig. 10a). However, the percentage decrease in the cell viability at 50, 100 and 200 μg/ml are recorded as 88%, 81% and 65% in the core and 76%, 63% and 41% in
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core-shell NSs exposed A-549 cells, respectively (Fig. 10b). As shown in Fig. 10a&b, the coreshell NSs at a concentration of 50 μg/ml demonstrate a remarkably high cell viability, which gets suppressed after increasing the concentration.
4. Conclusions 11
An effective and simple synthesis process was developed for the preparation of highly biocompatible, well monodispersed, spherical shaped, mesoporous, terbium hydroxide surface functionalized SiO2@Tb(OH)3 core-shell nanostructures. The TEM and SEM images exhibit the successful surface functionalization of the silica core via terbium hydroxide, which was verified using the EDX, FTIR, UV/vis, FT-Raman and PL spectra. The luminescence spectra verified that
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the emission transitions of Tb3+ ions were highly perturbed due to the covalent bonding of the terbium hydroxide layer with the silica network present on the surface of NSs to form the mesoporous cell surrounding the silica NSs. The highly ordered nanopores were preserved with the terbium hydroxide layer, and the O-Si-O and Si-OH groups in the framework structures were rearranged into new Si-O-Tb-OH groups by the interactions of Tb3+ ions with the outer O atoms. The dominant emission due to the 5D4→7F5 transition along with a diffused emission band of
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silica (570 nm, 2.14 eV) was located in the center of the emission spectrum confirming the
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existence of Tb3+ in a low symmetry environment within the silica framework structure. Therefore, the surface functionalization of luminescent terbium hydroxide over the silica spheres
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was successfully elucidated by changing the interaction between the Tb (III) ions and silica
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framework structures. While our results showed good cell viability at low concentrations, increasing the dose of the NSs causes reduction in cell viability due to the effects of the surface
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environment. Hence, the core-shell NSs with good luminescent properties in the visible region,
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excellent solubility, biocompatibility, and non-toxicity at low concentrations could be suitable candidates for the development of FRET-based biosensor/biolabeling and for high quantity
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loading of anti-cancer drugs for drug delivery/release.
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Acknowledgment: The authors extend their appreciation to the Deanship of Scientific Research at King Saud University, Riyadh for funding this work through Research Group RG-1438-094.
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Conflict of interest: The authors declare that there are no conflicts of interest.
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Figure 1. (a)XRD pattern and (b) TGA analysis of core and core-shell NSs. Figure 2. TEM micrographs of (a) low magnified TEM image (b) single particle show the coating of terbium hydroxide (c) size measured of single particle (d) cross section image (inset shows the high magnified cross section image). (e) low resolution SEM image and (f) high resolution SEM image of core-shell NSs. (g) Particle size distribution curve of core-shell NSs dispersed in water showing an average of 385 nm diameter. Figure 3. SEM micrographs of (a) high magnified electron image (b)EDX of Si element(c) EDX of Oxygen (d) EDX of terbium ion (e) line mapping of each elements in single particle of NSs. Figure 4. (a)BET isotherm and (b) Pore-size distribution of core and core-shell NSs. Figure 5. FTIR spectra of core and core-shell NSs. Figure 6. UV/Visible spectra of core, terbium nitrate hexahydrate and core-shell NSs. Figure 7. FT-Raman spectrum of the core-shell NSs. Figure 8. Photoluminescence spectrum of the core-shell NSs. Figure 9.(a) Percent cell viability by MTT assay in human breast cancer cell line (MCF-7) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure 9.(b) Percent cell viability by MTT assay in human lung epithelial cell line (A-549) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure10.(a) Percent cell viability by neutral red uptake assay in human breast cancer cell line (MCF-7) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure10.(b) Percent cell viability by neutral red uptake assay in human lung epithelial cell line (A-549) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments.
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S0927-7757(19)30357-7 https://doi.org/10.1016/j.colsurfa.2019.04.049 COLSUA 23393
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
10 January 2019 28 February 2019 17 April 2019
Please cite this article as: Ansari AA, Siddiqui MA, Khan A, Ahmad N, Alam M, El-Toni AM, Al-Khedairy AA, Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), https://doi.org/10.1016/j.colsurfa.2019.04.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Luminescent surface-functionalized mesoporous core-shell nanospheres and their cytotoxicity evaluation
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Anees A Ansari1*, Maqsood A Siddiqui2,3, Aslam Khan1, N. Ahmad4, M. Alam4, Ahmed Mohamed El-Toni1, Abdulaziz A. Al-Khedairy3 1 King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia 2Al-Jeraisy Chair for DNA Research, Zoology Department, College of Science, King Saud University, Riyadh-11451, Saudi Arabia 3Zoology Department, College of Science, King Saud University, Riyadh-11451 Saudi Arabia 4 Department of Chemistry, King Saud University, Riyadh 11451, Saudi Arabia
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Corresponding author Email: [email protected], [email protected] Tel.:+966-11-4676838; Fax.: +966-11-467662
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Graphical Abstract:
Highlights:
We fabricated aqueous dispersible spherical shaped SiO2@Tb(OH)3 nanospheres. Nanospheres revealed excellent photoluminescent properties. 1
Highly biocompatible and low toxicity over a wide range of concentrations.
Abstract Spherical shaped SiO2@Tb(OH)3 nanospheres (core-shell NSs) with an average grain size of 300-350 nm were successfully synthesized at a large scale via sol-gel process with subsequent heat treatment. XRD results demonstrated the non-crystalline nature of the sample even after
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coating with terbium hydroxide. TEM and SEM images clearly showed that the prepared sample was highly monodispersed, perfectly spherical in shape, and mesoporous with narrow size
distribution (an average crystalline size of 350 nm). XRD, TGA, TEM, SEM, and EDX results demonstrated that the terbium hydroxide layer was effectively grafted over the surface of the
silica spheres, which was further verified using FTIR, UV/Visible, and emission spectral results. The emission spectrum of core-shell NSs demonstrates that Tb3+ is strongly bonded covalently to
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the silica framework with surface anchored oxygen atoms, thus preventing the accumulation of
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Tb3+ ions to give an efficient luminescence. The emission spectrum was dominated by 5D4→4F5
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of the Tb3+ ion located in the green region at 2.88 eV along with broad emission of the silica framework. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide and
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neutral red uptake assays on MCF-7 and A-549 cells clearly illustrated the high biocompatibility and non-toxic nature of the samples. Because of the high specific surface area, mesoporous
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nature, and presence of abundant hydroxyl groups on the surface of the particles, these materials
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can be applied for tagging bioactive molecules or for loading high quantity anti-cancer drugs for biomedical applications.
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Keyword: Terbium hydroxide, Silica nanospheres, Optical properties, Luminescence properties, Biocompatibility, Cytotoxicity
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1. Introduction Currently, nano-scale luminescent lanthanide materials have become one of the most fascinating areas of research in modern science and technology because of their outstanding
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chemical, physical, and luminescent properties such as multiple absorption-emission transitions in the whole visible and NIR region, stark splitting, high quantum efficiency, long decay time, superior photo-chemical stability, low autofluorescence, biocompatibility, and non-toxicity [1-9]. These unique features make them ideal candidates for applications in the various fields of clinical sciences such as bio-imaging/bio-labeling, optical detection of analytes, finger printing, magnetic resonance imaging, and photodynamic therapy and in designing of optical probes and 2
biosensors [10-21]. These nanostructured materials must be photo-chemically stable, aqueoussoluble, biocompatible, and non-toxic in nature to be used in clinical sciences. However, most of the luminescent lanthanide nanomaterials are hydrophobic in nature [22-31]. In addition, another major disadvantage of these materials is the weaker emission intensity compared to the parent uncoated luminescent material because multi-photon relaxation pathways suppress the
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luminescence efficiency and quantum yields and reduce molar extinction coefficients of the surface coated nanostructured materials [2, 25-27, 32-37]. To overcome these drawbacks, alternate synthesis methods have been developed to improve the emission efficiency as well as the photo-chemical stability, aqueous dispersibility, colloidal stability, biocompatibility, and non-cytotoxicity if the luminescent nanomaterials and therefore, this has become an exciting research topic. Thus, there is an urgent need to either modify the present techniques for surface
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coating of nanoparticles by addition of appropriate functional groups or to adopt some alternative
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synthesis process during the fabrication of nanostructured materials that can enhance their solubility and colloidal stability.
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Till date, hexagonal, cubic, rod-shaped, planar, spherical, octahedral, pyramidal, tube-
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shaped, and irregular spherical luminescent lanthanide nano-materials have been successfully fabricated using several techniques and have been applied in photonic based bio-applications
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[22, 24, 25, 35, 38-40]. Among them, spherical shaped luminescent lanthanide micro/nano
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phosphor materials are extensively used due to their attractive features including better photoluminescence (PL) high definition, much improved screen packing, distinct large effective densities, high specific surface areas, huge inner space, and low scattering of light. These novel
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features of the spherical shaped luminescent materials are highly advantageous in many photonic-based applications such as in development of photonic devices, optical bio-
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probe/assays, and optical biosensors and in bio-detection. Therefore, it is essential to develop alternate methods that can enhance the PL performance along with the aqueous dispersibility
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with colloidal stability, biocompatibility, and low toxicity of the spherical shaped phosphoric materials. To control the morphology of the phosphoric materials, various methods have been adopted such as hydrothermal/solvothermal method, thermal decomposition, co-precipitation, micro-emulsion, application of microwaves, sol-gel chemical method, and homogeneous coprecipitation using a weak base (urea, thiourea, hydrazine, and trimethylamine) [41-45]. 3
Among them, the sol-gel method is a low-cost, non-hazardous, eco-friendly, low temperature, well-organized, and effective process for preparation of nano-materials. Silica nanospheres prepared using established sol-gel methods have been successfully applied in areas including drug delivery and biolabeling/biodetection due to their uniform spherical shape and stable mesoporous structure. Because of abundant surface anchored silanol groups, the silica
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surface can easily be functionalized with fluorescent materials to improve their optical properties for further uses in clinical and biomedical sciences. Previously, nanomaterials of spherical shape with controlled structure, high mesoporous structure, low toxicity, and high aqueous dispersibility of silica-spheres have been successfully applied as phosphor supports (or capping agent) or cell imaging materials. Lin and his co-workers fabricated silica supported sub-micron SiO2@Y2SiO5:Eu, SiO2@Y2SiO5:Ce/Tb[46], SiO2@YVO4:Eu[47], SiO2@YVO4:Dy/Sm [48]
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spherical particles. Yang et al. and a few other research groups utilized silica spheres as a
labeling/bio imaging applications [14, 24, 49-59].
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platform for functionalization of luminescent materials and examined their potential in bio-
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In this article, we demonstrated the successful surface functionalization of a Tb(OH)3
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shell over monodispersed mesoporous SiO2 core-NSs, forming well-defined core-shell structures. The formation of the Tb(OH)3 shell over the surface of SiO2 core-NSs makes them
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water dispersible and able to form stable colloids over a period of time without any observable
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sedimentation or precipitation. Synthesized NSs were characterized using state-of-art experimental techniques including X-ray diffraction, transmission electron microscopy, energy dispersive X-ray spectroscopy/field emission-scanning electron microscopy, thermogravimetric Brunauer
Emmett
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analysis,
Teller
(BET)
analysis,
Raman
spectroscopy,
and
PL
spectrophotometry. Furthermore, MTT assay and neutral red uptake (NRU) assay were
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employed using a concentration-based method for investigating the toxicity and cell viability of
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the deigned core-shell NSs.
2. Experimental set up 2.1.Materials Analytical-grade terbium nitrate hexahydrate (99.9%, Alfa Aesar, Germany), tetraethylorthosilicate (TEOS), ethanol, NaOH and cetyltrimethylammonium bromide (CTAB) were 4
analytical grade and used directly as received (Sigma-Aldrich, U.K). Milli-Q (Millipore, Bedford, USA) water was used for synthesis and characterization. 2.2. Synthesis of terbium hydroxide coated silica NSs In a typical synthesis reaction, 2 M aqueous solution of sodium hydroxide and 1 g CTAB
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were mixed in 250 ml dist. water and the solution mixture was kept on a hot plate with constant mechanical stirring at 80°C. Then, 0.5 ml of TEOS was slowly injected into the vigorously stirred hot solution mixture and allowed to react on a hot plate for 2 h [24, 56, 60, 61]. After that, a hot aqueous dissolved solution of 0.5 mg terbium nitrate hexahydrate was introduced into the vigorously stirring reaction mixture. The reaction was allowed to proceed for another 2 h for complete complexation on the surface of silica spheres. The resulting white precipitate was
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separated using centrifugation, washed with dist. water and dried overnight in an oven at 60°C.
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2.3. Characterization X-Ray diffraction pattern (XRD) was carried out using Rigaku-Dmax 25 diffractometer
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equipped with Cu Kα (1.5406Ǻ) radiations. Thermogravimetric analysis (TGA) was performed
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from TGA/DTA Mettler Toledo AG, Analytical CH-8603 Schwerzenbach, Switzerland). Morphological images were obtained using scanning electron microscope (SEM, JEOL JSM
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7600F) operating at 5-10 kV, and transmission electron microscope (TEM). Both of these techniques were equipped with energy dispersive x-ray analysis (EDX, FETEM, 2100F, JEOL,
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Japan) operating at an accelerating voltage 200 kV. Surface chemistry was examined by BET method by using the Barrett-Joyner-Halenda (BJH) model. Infrared spectra were measured from
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Vortex 80 (Bruker, USA) spectrometer using KBr pellet method. Optical absorption spectra were recorded from Cary 60 UV/Visible (Agilent Technologies, USA) spectrophotometer in the range
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of 200-900 nm. Raman and PL spectra were obtained using a Horiba Synapse (Horiba, JobinYvon Edison, USA) spectrophotometer. A slit width of 100 microns was employed, ensuring a
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spectral resolution higher than 1 cm-1. Particle size distribution curve was measured by diffusion light scattering technique using a dynamic light scattering (DLS; Malvern ZetaSizer, ZS, UK).
2.4. MTT assay MTT assay is a colorimetric assay for evaluating viable cells or cytotoxicity of a given material. The test is based on the enzymatic reduction of the lightly colored tetrazolium salt to its 5
formazan which is intense purple-blue color. MTT assay was carried out for measuring the toxic potentiality of the designed core-shell NSs using Mossman et al method with minor modification [62]. 2.5. Neutral red uptake assay Natural red uptake assay is based on the detection of viable cells via the uptake of the
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natural red dye, such as a eurhodin dye that stains lysosomes in viable cells [63]. Repetto et al. method was employed for measuring the neutral red uptake assay [63]. MCF-7 and A549 cell lines were used for both the assays.
3. Results and Discussion 3.1. Chemical composition and thermal stability The X-ray diffraction pattern was obtained to determine the chemical composition and
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crystallinity of the synthesized nano-products. As shown in Fig. 1a, a broad plane with a high-
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intensity band at 2 = 25° is observed in both the XRD diffraction patterns, which is the characteristic peak of amorphous silica [JCPDS card No.29-0085] [47, 56, 64-67]. As seen in
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Fig. 1a, no reflection plane is observed in the core-shell nanostructure even after drying at
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300°C, which indicates that the surface-anchored terbium hydroxide is non-crystalline in nature. However, in case of the core-shell samples, a few broad reflection planes were seen, which could
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be due to ultra-small crystalline nature of terbium hydroxide. These observations are consistent
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with the previously published reports [47, 65-67]. Thermogravimetric analysis was performed to examine the thermal stability of the designed core and core-shell NSs between room temperature to 900°C in a nitrogen-rich atmosphere. As shown in Fig. 1b, the TGA curve of core-NSs
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demonstrates the first decomposition at around 160°C with 4.1% weight loss, which can be attributed to the surface adsorbed water or organic moieties. In the second step, decomposition
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started at 120°C accompanied by continuous weight loss with an increase in temperature till 900°C and a final weight loss of 13%. This indicated the slow decomposition of silica and
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conversion of the remaining silica into silicate. A similar trend was observed in the TGA curve of core-shell NSs. Initial weight loss started at 160°C, and later, the curve showed a sluggish weight loss up to 900°C. This implies that at first, hydroxyl groups are decomposed over the surface of core-shell NSs and the remaining silica is converted into silicate (Fig. 1b). These observations are in agreement with previous literature [38, 44]. It is interesting to note that we 6
observed a similar decomposition trend in both samples, suggesting that silica spheres are coated with the amorphous form of terbium hydroxide. 3.2. Morphological structure and surface chemistry The morphology of the samples was inspected using TEM and SEM micrographs. TEM
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micrographs in Fig. 2a-d clearly indicate that the prepared core-shell structures are composed of uniform, monodisperse, and spherical nanostructures with a mean diameter of 340 nm. These perfect spherical particles were prepared using the sol-gel method. They were distributed homogeneously throughout the image, and mesopores were created among these spherical coreshell structures, as shown by many light spots observed in Fig. 2b&c. We expected that in the presence of a strong base (NaOH), terbium nitrate will decompose rapidly at elevated temperatures. The decay of terbium nitrate releases precipitating cations swiftly and
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heterogeneously into the reaction system. This leads to uncontrolled growth of nucleation
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(terbium cations) on the silica NSs. As seen in Fig. 2a&b, each silica-sphere is completely
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covered with terbium hydroxyl molecules. It is worth noticing that the core and the metal shell of each sphere can be clearly distinguished due to the different electron penetrability between the
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amorphous core and semi-crystalline terbium hydroxide shell-structure. Notably, the core-shell NSs are covered with the semi-crystalline terbium hydroxide shell rendering the core-shell NSs
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hydrophilic so that they can be easily dispersed in water and form stable and clear colloidal
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solutions. In the core-shell structure, silica is light grey in color, whereas the metal shell exhibits dark black color in spot like forms. As demonstrated in Fig. 2c&d, the surface of core-shell NSs
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is rough and has distinct punctuations, proving the formation of porous structured SiO2@Tb(OH)3 NSs. The SEM image in Fig. 2e&f shows that the morphology of the core-shell
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NSs is monodisperse, uniform, spherical-shaped, and rough-surfaced with narrow size distribution and an average grain size of ~350 nm, which is consistent with the FE-TEM results. The DLS measurement was performed to measure the particle size distribution of the prepared
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luminescent NSs. The size of the particle obtained by DLS studies (Fig. 2g) was found to be ~385 nm, which is consistent with the results obtained by TEM and SEM. Energy dispersive Xray (EDX) analysis demonstrates the chemical constituents of the designed core-shell NSs. The EDX analysis in Fig. 3a-e clearly shows all of the expected chemical elements such as Si, Tb, and O in the core-shell nanostructure. 7
The surface features of the nanomaterials were measured using the BET-N2-adsorptiondesorption isotherm. Fig. 4 illustrates the N2-adsorption-desorption isotherm of Tb(OH)3 grafted silica NSs along with blank silica NSs. Here, the isotherm of the silica NSs is used only for comparison. As observed in Fig. 4, Tb-functionalized NSs exhibit a typical IV-type isotherm and H1-hysteresis loop, which is a known characteristic feature of mesoporous materials. The BET-
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isotherm showed that the BET specific surface area, pore size, and pore volume were 49.93 m2/g, 6.2505 nm, and 0.1694 cm3/g, respectively, for the terbium functionalized NSs, whereas those for the silica NSs were 165.7767 m2/g, 16.3176 nm, and 1.2323 cm3/g, respectively. The surface properties of the Tb-functionalized NSs are significantly suppressed as compared to their respective pure SiO2 NSs. It has been shown that silica NSs have abundant surface silanol (SiOH) groups, and these silanol groups are easily available for covalent bonding with terbium
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hydroxide in aqueous media. Thus, a layer of Tb(OH)3 is successfully grafted over the surface of
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silica NSs through covalent bonding. These surface grafted Tb(OH)3 molecules block the interstitial spaces because of the high density of Tb ions and as a result, they occupy the pore
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cavity and suppress the surface properties, as seen in the SEM, TEM, Raman, and PL spectral
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results.
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3.3. Optical properties The FTIR spectra were recorded to determine the surface purity and attached organic
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functional groups on the core and core-shell NSs. As illustrated in Fig. 5, a diffused band between 2800 to 3800 cm-1 along with a medium intensity band at 1645 cm-1 was observed in
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both spectra due to symmetrical and asymmetrical stretching and the bending vibrational mode of physically and chemically attached hydroxyl(-OH) and silanol (Si-OH) groups, respectively 1
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[24, 56, 68]. A high intensity band in the middle of the infrared spectrum is observed at 1200 cmalong with medium intensity bands located at 956 and 805 cm-1, and these can be assigned to
the Si-O-Si, Si-O, and Si-OH vibrational modes, respectively, which originate from amorphous
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silica [25, 56, 69]. Another strong intensity peak at 494 cm-1 attributed to the phonon bonds of metal oxide (Si-O-Tb) network [55, 69]. Optical properties and colloidal stability of the designed nanomaterials were investigated using the UV/visible absorption spectra. Fig. 6 displays the absorption spectra of core and coreshell NSs dispersed in aqueous media at room temperature. In Fig. 6, we demonstrate a 8
comparative analysis of the absorption spectra of core-shell NSs along with bare silica core-NSs and terbium nitrate hexahydrate in an aqueous environment. As shown in Fig. 6, the spectral edge of core-shell NSs is significantly uplifted in the UV/visible region than their respective bare core-NSs due to the 4f8→4f75d absorption transition of Tb3+ [56, 70, 71]. It implies that terbium hydroxide is effectively coated over the surface of core-NSs. The 4f-electrons of terbium ions
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can easily interact with surface anchored hydroxyl groups through hydrogen bonding, making the nanomaterials aqueous dispersible.
Fig. 7 represented the Raman spectrum of the core-shell NSs evaluating the Raman active modes of silica and terbium hydroxide. It can be seen from the spectrum that the Raman active modes are observed at 295, 390, 738, 1224, 1298, 1383, 1446, and 1626 cm-1 along with a broad high-intensity band in between 2850 and 2877 cm-1, which are typical Raman active Ag, Eg, and
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Fg modes of terbium hydroxide [72-74] and the silica network [75]. These Raman active modes
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are consistent with those reported in previous studies [72, 73, 75]. The occurrence of these Raman active modes verified the successful formation of the SiO2@Tb(OH)3 core-shell nano-
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structure of the designed material.
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To ensure the surface functionalization of terbium hydroxide over the silica core-NSs, we recorded the PL spectra of the core-shell NSs. The PL spectrum shows a remarkable high-
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intensity emission band in the visible region, which covered the whole visible region, causing the
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formation of amorphous silica [66, 67, 76]. The emission spectrum of the core-shell NSs under excitation at 3.82eV (325 nm) is composed of various lines peaking at 2.38 eV (521 nm), 2.288 eV (543 nm), 2.1 eV (590 nm), 2.01 eV (614 nm), 1.89 eV (653 nm), and 1.77 eV (701 nm),
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which can be contributed to the 5D4→7F6, 5D4→7F5, 5D4→7F4, 5D4→7F3,5D4→7F2, and 5D4→7F0 transitions, respectively, which are all 4f-4f intra-configurational transitions of Tb3+ ion (Fig. 8)
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[56, 77, 78]. The most prominent transition in the emission spectra was observed in the green region, located at 2.288 eV (5D4→7F5), which is the most chemically sensitive in the spectrum
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and hence provides more information about the chemical environment surrounding the Tb3+. Therefore, it is a so-called hypersensitive transition and factual thumbprint of the characteristic emission transitions of 4f-4f lines of the Tb3+ ions that are altered in their intensity during the formation of new chemical bonds between Tb3+ ion and organic chelating agents. In comparison to the hypersensitive transition, other emission transitions are weak in their sensitivity and show low intensity in the spectrum because of the shielding effect of 4f-subshell by 5s25p6 electrons 9
[25, 56, 68]. Additionally, the occurrence of these weak efficiency emission peaks at higher wavelengths can be attributed to the low phonon energy of the Tb-O-Si bond (494 cm-1) because the multi-photon relaxation by the Tb-O vibration is not able to diminish the intensity of the emission band, as observed from the FTIR spectra in Fig. 5. As shown in Fig. 8, the luminescence spectrum is highly uplifted and revealed significant broadening in all emission
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peak widths and shapes, caused due to the influence of the amorphous silica core NSs. The possible explanation is that 4f-electrons of surface coated Tb(III) ions interact with the silica core producing an abundant, non-bridging oxygen network. This silica network altered the bond angle, bond length, and symmetry of the crystal field, inducing these emission transitions and resulting in characteristic broadening in 4f-4f transitions. We expected that the Tb (III) ion in the current form is of low symmetry in the Si framework and this structured environment increases
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the emission intensity of the hypersensitive (5D4→7F5) transition. It also reflects the formation of
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new chemical bonds or energy transfer from silica molecules to 4f -electrons of Tb3+ ions, altering the emission peak intensity and broadening of hypersensitive transition. As seen in Fig.
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8, an observed hypersensitive (5D4→7F5) transition in the green region demonstrates remarkable
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intensity enhancement than those of the pure complexes and different types of nano-scale host matrices [77-84]. This novel luminescence intensity of the 5D4→7F5 transition could be applied
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in photonic based biomedical applications and laser-based technologies. To calculate the
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emission quantum yield, we used a previously reported method [2, 78, 85] for the core-shell NS sample which involved monitoring the 325-nm excitation wavelength using He-Ne laser at room temperature (Rhodamine 6G dissolved in alcohol was used as a calibration standard). The
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emission quantum yield was found to be 35%.
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3.4. MTT assay
MTT assay was performed to analyze the biocompatibility and toxic nature of the
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designed samples. The induced cytotoxic effects of the core and core-shell NSs on MCF-7 and A-549 cells are shown in Fig. 9a&b. A concentration-dependent cytotoxic response was observed in MCF-7 and A549 cells exposed to 2-200 μg/ml of core and core-shell NSs for 24 h. The cell viability percentage was recorded as 90% and 77% in cells treated with core NSs and 81% and 64% in core-shell NSs at 100 and 200 μg/ml, respectively, in MCF-7 cells compared to untreated control (Fig. 9a). However, the cell viability percentage was recorded as 90%, 82%, 10
and 70% in the core NS treated cells and 80%, 66% and 44% in core-shell NSs treated A-549 cells at 50, 100 and 200 μg/ml, respectively, compared to the untreated control (Fig. 9b). When the concentration of the core-shell NSs in both cell lines was below 50 μg/ml, a remarkably high cell viability was observed. Although, significant suppression in cell viability was observed when the concentration of core-shell NSs was increased, it could be due to high mesoporosity
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and large surface area of the NSs. However, additional of cytotoxicity on the nature of the surface covered functional group cannot be ruled out. It is also obvious from previous reports that there might be aspects contributing to cytotoxicity such as particle size (diameter) and surface functionalization of the materials [86-88]. The cytotoxicity observed when the cells were treated with the core-shell NSs in a dose-dependent manner might be due to the growth of abundant surface anchored hydroxyl groups [87, 89]. It is implied that these OH radicals are
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responsible for the production of reactive oxygen species, which are accountable for cell
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apoptosis [24, 86, 89].
red uptake assay is
the lysosomes of viable
cells,
based on the accumulation of neutral
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Neutral
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3.5. NRU assay
while
MTT
is based
on
the
red dye in
reduction of yellow
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tetrazolium MTT reagent by viable cells to purple formazan. It is important to verify the results
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of MTT assay with another experimental method. Thus, we performed the NRU assay. The results of the NRU assay are presented in Fig. 10a&b. The NRU assay also revealed a concentration-dependent cytotoxicity in MCF-7 and A-549 cells exposed to core and core-shell
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NSs at 2-200 μg/ml. The percentage decrease in cell viability at 50, 100 and 200 μg/ml is recorded as 90%, 83%, and 76% in the core and 84%, 76% and 61% in core-shell NSs exposed
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MCF-7 cells, respectively (Fig. 10a). However, the percentage decrease in the cell viability at 50, 100 and 200 μg/ml are recorded as 88%, 81% and 65% in the core and 76%, 63% and 41% in
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core-shell NSs exposed A-549 cells, respectively (Fig. 10b). As shown in Fig. 10a&b, the coreshell NSs at a concentration of 50 μg/ml demonstrate a remarkably high cell viability, which gets suppressed after increasing the concentration.
4. Conclusions 11
An effective and simple synthesis process was developed for the preparation of highly biocompatible, well monodispersed, spherical shaped, mesoporous, terbium hydroxide surface functionalized SiO2@Tb(OH)3 core-shell nanostructures. The TEM and SEM images exhibit the successful surface functionalization of the silica core via terbium hydroxide, which was verified using the EDX, FTIR, UV/vis, FT-Raman and PL spectra. The luminescence spectra verified that
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the emission transitions of Tb3+ ions were highly perturbed due to the covalent bonding of the terbium hydroxide layer with the silica network present on the surface of NSs to form the mesoporous cell surrounding the silica NSs. The highly ordered nanopores were preserved with the terbium hydroxide layer, and the O-Si-O and Si-OH groups in the framework structures were rearranged into new Si-O-Tb-OH groups by the interactions of Tb3+ ions with the outer O atoms. The dominant emission due to the 5D4→7F5 transition along with a diffused emission band of
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silica (570 nm, 2.14 eV) was located in the center of the emission spectrum confirming the
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existence of Tb3+ in a low symmetry environment within the silica framework structure. Therefore, the surface functionalization of luminescent terbium hydroxide over the silica spheres
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was successfully elucidated by changing the interaction between the Tb (III) ions and silica
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framework structures. While our results showed good cell viability at low concentrations, increasing the dose of the NSs causes reduction in cell viability due to the effects of the surface
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environment. Hence, the core-shell NSs with good luminescent properties in the visible region,
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excellent solubility, biocompatibility, and non-toxicity at low concentrations could be suitable candidates for the development of FRET-based biosensor/biolabeling and for high quantity
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loading of anti-cancer drugs for drug delivery/release.
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Acknowledgment: The authors extend their appreciation to the Deanship of Scientific Research at King Saud University, Riyadh for funding this work through Research Group RG-1438-094.
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Conflict of interest: The authors declare that there are no conflicts of interest.
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Figure 1. (a)XRD pattern and (b) TGA analysis of core and core-shell NSs. Figure 2. TEM micrographs of (a) low magnified TEM image (b) single particle show the coating of terbium hydroxide (c) size measured of single particle (d) cross section image (inset shows the high magnified cross section image). (e) low resolution SEM image and (f) high resolution SEM image of core-shell NSs. (g) Particle size distribution curve of core-shell NSs dispersed in water showing an average of 385 nm diameter. Figure 3. SEM micrographs of (a) high magnified electron image (b)EDX of Si element(c) EDX of Oxygen (d) EDX of terbium ion (e) line mapping of each elements in single particle of NSs. Figure 4. (a)BET isotherm and (b) Pore-size distribution of core and core-shell NSs. Figure 5. FTIR spectra of core and core-shell NSs. Figure 6. UV/Visible spectra of core, terbium nitrate hexahydrate and core-shell NSs. Figure 7. FT-Raman spectrum of the core-shell NSs. Figure 8. Photoluminescence spectrum of the core-shell NSs. Figure 9.(a) Percent cell viability by MTT assay in human breast cancer cell line (MCF-7) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure 9.(b) Percent cell viability by MTT assay in human lung epithelial cell line (A-549) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure10.(a) Percent cell viability by neutral red uptake assay in human breast cancer cell line (MCF-7) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments. Figure10.(b) Percent cell viability by neutral red uptake assay in human lung epithelial cell line (A-549) exposed to SiO2 and SiO2@Tb(OH)3 at 2-200 mg/ml for 24 h. Results are presented as mean+SD from three independent experiments.
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