Core-Shell Structures of Upconversion Nanocrystals Coated with Silica for Near Infrared Light Enabled Optical Imaging of Cancer Cells

Optical imaging of cancer cells using near infrared (NIR) light is currently an active area of research, as this spectral region directly corresponds to the therapeutic window of biological tissues. Upconversion nanocrystals are photostable alternatives to conventional fluorophores. In our work, we have prepared upconversion nanocrystals of NaYF4:Yb/Er and encapsulated them in silica to form core-shell structures. The as-prepared core-shell nanostructures have been characterized for their structure, morphology, and optical properties using X-ray diffraction, transmission electron microscopy coupled with elemental mapping, and upconversion luminescence spectroscopy, respectively. The cytotoxicity examined using cell viability assay indicated a low level of toxicity of these core-shell nanostructures. Finally, these core-shell nanostructures have been utilized as photostable probes for NIR light enabled optical imaging of human breast cancer cells. This work paves the way for the development of advanced photostable, biocompatible, low-toxic core-shell nanostructures for potential optical imaging of biological cells and tissues.


Introduction
Optical imaging is a useful technique to visualize the structural and functional evolution of biological materials with negligible invasion, and without disturbing the usual metabolic activities [1][2][3]. Luminescent labels are required for optical imaging of biological materials, since most biomolecules do not have measurable fluorescent signals [4][5][6]. In biomedical research there is an enormous demand for fluorescent probes which emit light at particular wavelengths and can be detected under in vivo and in vitro conditions through fluorescence microscopy [7,8]. Despite sensitivity and other benefits, traditional fluorescent molecular probes have a number of limitations, such as low penetration depth of excitation light, high toxicity due to the longer exposure needed at shorter wavelengths, auto-fluorescence and strong light scattering from biological tissues that cause low signal to noise ratio (SNR), and photo-bleaching [9,10]. These undesirable properties of conventional organic fluorophores motivate researchers to look for alternatives in luminescent materials, such as quantum dots, carbon-based nanostructures (e.g., nanodiamonds, carbon dots), and upconverting phosphors. For instance, near infrared (NIR) active inorganic luminescent materials can potentially overcome the limitations of traditional fluorescent molecular probes [11].
In recent years, different research groups have prepared NIR active luminescent nanoparticles and efficiently used them for optical imaging; amongst these, considerable attention has been devoted to

NaYF 4 :Yb/Er Nanocrystals
Lanthanide (Yb and Er) doped NaYF 4 nanocrystals were prepared through a microwave assisted synthesis route as earlier reported by our group [58]. In the typical synthesis procedure, YCl 3 (0.8 mmol), YbCl 3 (0.18 mmol) and ErCl 3 (0.02 mmol) were mixed with a solution mixture of 8 mL of ethanol (EtOH), NaOH (7.5 mmol) dissolved in 2 mL of water, 2 mL acetic acid (AcOH) under constant magnetic stirring at room temperature (RT). Lastly, 2 mL water containing 4 mmol of NH 4 F was added drop wise to the above solution mixture. After 30 min of stirring at room temperature, the entire reaction mixture was moved to a microwave reaction vessel (20 mL) and tightly sealed to heat for 3 h at 150 • C with continuous stirring of 600 rpm. Then the complete system was allowed to cool to 40 • C. Last, of all, the products were rinsed with ethanol and water (7:3 v/v) mixture three times and collected through centrifugation to eliminate any unreacted leftover precursors. Then the UCNC were dried in vacuum at 60 • C.

UCNC@SiO 2 Core-Shell Nanostructures
UCNC@SiO 2 core-shell nanostructures synthesis was carried out as per a previous report with some modifications [59]. In brief, 20 mg of UCNC were properly dispersed in 20 mL ethanol and 2 mL of TritonX-100 was added to the above solution while stirring. Stirring was continued for 30 min, then 0.42 mL of NH 4 OH was added and allowed to stir for another 30 min. Lastly, 0.051 mL of tetraethyl orthosilicate (TEOS) was added and allowed it to stir for 6 h. Then silica coated UCNC core-shell nanostructures were collected through centrifugation. Finally, the obtained core-shell nanostructures were dispersed in ethanol and kept at 4 • C in refrigerator for future use.

Instrumentation
NaYF 4 :Yb/Er UCNC synthesis was done by using Anton Paar Monowave 300 microwave synthesizer (New Delhi, India). For the collection of powder X-ray diffraction data, Rigaku Smart Lab 9 KW powder X-ray diffractometer (PXRD, New Delhi, India) was used. The transmission electron microscope (TEM) of FEI Tecnai G2 20 S-twin (New Delhi, India) operating at 200 kV was used to acquire the morphology of the nanostructures. Energy dispersive X-ray analysis (EDAX) and elemental mapping were also performed using the same TEM instrument. For upconversion luminescence (UCL) measurements, 980 nm laser diode (CW, 500 mW, purchased from Optochem International, New Delhi, India) equipped Cary Eclipse fluorescence spectrophotometer was used. For UCL measurements, 1 mg of samples (UCNC and UCNC@SiO 2 ) was dispersed in 1 mL of Milli-Q water, followed by 5 min of sonication. Subsequently, the sample was transferred to a 1 mL cuvette and measurements were performed. Leica SP5 STED/MP microscope (New Delhi, India) was used for cell imaging, wherein for excitation, a Ti:sapphire femtosecond pulse laser (Chameleon Ulta, Coherent Inc., Santa Clara, CA, USA) at 980 nm was used. In this case, a standard PMT detector and a 63× oil immersion objective (Leica) was used for imaging the cells.

Cell Viability Assessment
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) cell viability assay was used to measure cell toxicity. MDA-MB-231, Human breast cancer cells, were developed in a 96 well microplate (7000 cells/well) in cell growth medium (DMEM) and then incubated at 37 • C, 5% CO 2 for 24 h. From each well, the growth medium was replaced with the addition of UCNC and UCNC@SiO 2 , which was prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer (pH 7.4). Next, 5, 10, 25 µg mL −1 concentration of particles were added to the cell media and incubated for 48 h. Then 10 µL of WST-1 was added to each well and incubated again for 2 h at 37 • C, 5% CO 2 . After the incubation, the 96 well plate was read by a Varioskan Flash Multimode Reader (Thermo Scientific Inc., Waltham, MA, USA) to get the absorbance at 440 nm. Averaged absorbance readings were plotted and the viable cells were correlated with the absorbance of each concentration of particles.

Cancer Cell Imaging
The MDA-MB-231 cells were plated over glass coverslips using cell media (DMEM) at 5% CO 2 and 37 • C. Cells (7 × 10 4 cells mL −1 ) were placed on a fresh coverslip and left to stick for 24 h. Later the cells were cleaned with phosphate buffer solution (PBS) then incubated with UCNC and UCNC@SiO 2 (5 µg mL −1 dispersed in DMEM) for 48 h at 5% CO 2 and 37 • C. Then 4% paraformaldehyde was used to fix the cells and cell sample was mounted using VECTASHIELD ® with DAPI for optical microscopy. TCS SP5 MP (Multi-photon, Leica microsystems), HyD detectors, 100× (HCX PL APO CS 100.0×/1.40 OIL) oil objective and LASAF software (Leica application suite) consisting multi-photon microscopy setup was used for imaging. For excitation, Ti-sapphire femtosecond pulse at 980 nm (1.07 W) was used while performing multi-photon microscopy. The green and red emissions were collected in 520-570 nm and 630-680 nm, respectively.

Results and Discussion
The synthesis of UCNC@SiO 2 and their application in cancer cell imaging is schematically demonstrated in Scheme 1.

Cell Viability Assessment
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) cell viability assay was used to measure cell toxicity. MDA-MB-231, Human breast cancer cells, were developed in a 96 well microplate (7000 cells/well) in cell growth medium (DMEM) and then incubated at 37 °C, 5% CO2 for 24 h. From each well, the growth medium was replaced with the addition of UCNC and UCNC@SiO2, which was prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer (pH 7.4). Next, 5, 10, 25 µ g mL −1 concentration of particles were added to the cell media and incubated for 48 h. Then 10 µ L of WST-1 was added to each well and incubated again for 2 h at 37 °C , 5% CO2. After the incubation, the 96 well plate was read by a Varioskan Flash Multimode Reader (Thermo Scientific Inc., Waltham, MA, USA) to get the absorbance at 440 nm. Averaged absorbance readings were plotted and the viable cells were correlated with the absorbance of each concentration of particles.

Cancer Cell Imaging
The MDA-MB-231 cells were plated over glass coverslips using cell media (DMEM) at 5% CO2 and 37 °C. Cells (7 × 10 4 cells mL −1 ) were placed on a fresh coverslip and left to stick for 24 h. Later the cells were cleaned with phosphate buffer solution (PBS) then incubated with UCNC and UCNC@SiO2 (5 µ g mL −1 dispersed in DMEM) for 48 h at 5% CO2 and 37 °C . Then 4% paraformaldehyde was used to fix the cells and cell sample was mounted using VECTASHIELD ® with DAPI (type, city, state code if USA or Canada, country) for optical microscopy. TCS SP5 MP (Multi-photon, Leica microsystems, type, city, state code if USA or Canada, country), HyD detectors, 100× (HCX PL APO CS 100.0×/1.40 OIL, type, city, state code if USA or Canada, country) oil objective and LASAF software (Leica application suite) consisting multi-photon microscopy setup was used for imaging. For excitation, Ti-sapphire femtosecond pulse at 980 nm (1.07 W) was used while performing multi-photon microscopy. The green and red emissions were collected in 520-570 nm and 630-680 nm, respectively.

Results and Discussion
The synthesis of UCNC@SiO2 and their application in cancer cell imaging is schematically demonstrated in Scheme 1. To study structural and phase properties, UCNC and UCNC@SiO2 were characterized using X-ray diffraction technique and the obtained patterns are shown in Figure 1 [60]. UCNC@SiO2 diffraction peaks were also obtained at the same 2θ position where the core (UCNCs) peaks were evidenced without any additional peaks, indicating that the formation of core-shell nanostructures does not alter the crystallinity of the core UCNC, and also do not lead to the Scheme 1. Schematic illustration of upconversion nanocrystals (UCNC)@SiO 2 synthesis and their application in cancer cell imaging.
To study structural and phase properties, UCNC and UCNC@SiO 2 were characterized using X-ray diffraction technique and the obtained patterns are shown in Figure 1 [60]. UCNC@SiO 2 diffraction peaks were also obtained at the same 2θ position where the core (UCNCs) peaks were evidenced without any additional peaks, indicating that the formation of core-shell nanostructures does not alter the crystallinity of the core UCNC, and also do not lead to the formation of any additional compounds.
In accordance with the amorphous nature of the silica shell around UCNC, the peaks corresponding to SiO 2 could not be evidenced.
Micromachines 2018, 9, x FOR PEER REVIEW 5 of 12 formation of any additional compounds. In accordance with the amorphous nature of the silica shell around UCNC, the peaks corresponding to SiO2 could not be evidenced. To investigate the morphology and size of the UCNC and core-shell nanostructures, TEM images were acquired (shown in Figure 2). Figure 2a shows the TEM image of microwave assisted synthesized NaYF4:Yb/Er nanocrystals. These particles are of spherical shape with an average size of approximately 25 nm. Figure 2b shows a high-resolution TEM (HRTEM) image of UCNC, which reveals the cubic phase of NaYF4 with lattice distance of 0.19 nm, also confirming the d-spacing for (220) Miller indices of the material. The selected area electron diffraction (SAED) patterns presented in Figure 2c clearly shows the diffraction rings corresponding to the cubic NaYF4 lattice. The thin silica layer coated on the UCNC to form core-shell nanostructures can be clearly seen in Figure 2d. The energy dispersive X-ray analysis (EDAX) of the core-shell nanostructures shown in Figure 2e clearly indicates the existence of all the constituent elements. In addition, the existence of all integral elements such as, sodium (Na), ytterbium (Yb), yttrium (Y), erbium (Er), fluoride (F), silicon (Si), and oxygen (O) was confirmed by TEM elemental mapping of UCNC@SiO2 core-shell nanostructures and is presented in Figure 3. To investigate the morphology and size of the UCNC and core-shell nanostructures, TEM images were acquired (shown in Figure 2). Figure 2a shows the TEM image of microwave assisted synthesized NaYF 4 :Yb/Er nanocrystals. These particles are of spherical shape with an average size of approximately 25 nm. Figure 2b shows a high-resolution TEM (HRTEM) image of UCNC, which reveals the cubic phase of NaYF 4 with lattice distance of 0.19 nm, also confirming the d-spacing for (220) Miller indices of the material. The selected area electron diffraction (SAED) patterns presented in Figure 2c clearly shows the diffraction rings corresponding to the cubic NaYF 4 lattice. The thin silica layer coated on the UCNC to form core-shell nanostructures can be clearly seen in Figure 2d. The energy dispersive X-ray analysis (EDAX) of the core-shell nanostructures shown in Figure 2e clearly indicates the existence of all the constituent elements. In addition, the existence of all integral elements such as, sodium (Na), ytterbium (Yb), yttrium (Y), erbium (Er), fluoride (F), silicon (Si), and oxygen (O) was confirmed by TEM elemental mapping of UCNC@SiO 2 core-shell nanostructures and is presented in Figure 3. formation of any additional compounds. In accordance with the amorphous nature of the silica shell around UCNC, the peaks corresponding to SiO2 could not be evidenced. To investigate the morphology and size of the UCNC and core-shell nanostructures, TEM images were acquired (shown in Figure 2). Figure 2a shows the TEM image of microwave assisted synthesized NaYF4:Yb/Er nanocrystals. These particles are of spherical shape with an average size of approximately 25 nm. Figure 2b shows a high-resolution TEM (HRTEM) image of UCNC, which reveals the cubic phase of NaYF4 with lattice distance of 0.19 nm, also confirming the d-spacing for (220) Miller indices of the material. The selected area electron diffraction (SAED) patterns presented in Figure 2c clearly shows the diffraction rings corresponding to the cubic NaYF4 lattice. The thin silica layer coated on the UCNC to form core-shell nanostructures can be clearly seen in Figure 2d. The energy dispersive X-ray analysis (EDAX) of the core-shell nanostructures shown in Figure 2e clearly indicates the existence of all the constituent elements. In addition, the existence of all integral elements such as, sodium (Na), ytterbium (Yb), yttrium (Y), erbium (Er), fluoride (F), silicon (Si), and oxygen (O) was confirmed by TEM elemental mapping of UCNC@SiO2 core-shell nanostructures and is presented in Figure 3.   Upconversion luminescence (UCL) of UCNC@SiO2 core-shell nanostructures was measured at room temperature by excitation at 980 nm wavelength through infrared continuous laser. Two Upconversion luminescence (UCL) of UCNC@SiO 2 core-shell nanostructures was measured at room temperature by excitation at 980 nm wavelength through infrared continuous laser. Two bands have been observed in the UCL spectra of UCNC@SiO 2 at 658 nm and 544 nm, which is shown in Figure 4a. These upconversion emissions are the results of Er 3+ ions electronic transitions from 4 F 9/2 → 4 I 15/2 and 4 S 3/2 → 4 I 15/2 , respectively, and the transitions are schematically shown in Figure 4b [61]. bands have been observed in the UCL spectra of UCNC@SiO2 at 658 nm and 544 nm, which is shown in Figure 4a. These upconversion emissions are the results of Er 3+ ions electronic transitions from 4 F9/2 → 4 I15/2 and 4 S3/2 → 4 I15/2, respectively, and the transitions are schematically shown in Figure 4b [61]. Before performing cell imaging studies, cytotoxicity experiments were performed using a cell proliferation assay (WST-1). The cell toxicity induced by nanoparticulate imaging probes is mainly associated with their concentration and their surface functionalization [62,63]. In our cell viability experiment, DMSO was used as a positive control, as it is a known cell growth inhibitor and thus behaves like an effective toxin. Therefore, DMSO (10%) was mixed with the DMEM (cell growth media) as a toxin (positive control), while as a negative control, untreated cells (merely DMEM) were used. The untreated UCNC@SiO2 core-shell nanostructures were measured in variable concentrations up to 25 μg mL −1 . The cell proliferation assay was used to evaluate the effect of UCNC@SiO2 core-shell nanostructures on the proliferation of human breast cancer cells (MDA-MB-231) after 48 h. The cell viability data is shown in Figure 5. The cell proliferation with UCNC@SiO2 did not induce significant toxicity at lower concentrations, and the proliferation of untreated cells is comparable. Nevertheless, a reduction in cell proliferation was noticed with the regular augmentation of UCNC@SiO2 nanostructure concentrations in cell growth media.  Before performing cell imaging studies, cytotoxicity experiments were performed using a cell proliferation assay (WST-1). The cell toxicity induced by nanoparticulate imaging probes is mainly associated with their concentration and their surface functionalization [62,63]. In our cell viability experiment, DMSO was used as a positive control, as it is a known cell growth inhibitor and thus behaves like an effective toxin. Therefore, DMSO (10%) was mixed with the DMEM (cell growth media) as a toxin (positive control), while as a negative control, untreated cells (merely DMEM) were used. The untreated UCNC@SiO 2 core-shell nanostructures were measured in variable concentrations up to 25 µg mL −1 . The cell proliferation assay was used to evaluate the effect of UCNC@SiO 2 core-shell nanostructures on the proliferation of human breast cancer cells (MDA-MB-231) after 48 h. The cell viability data is shown in Figure 5. The cell proliferation with UCNC@SiO 2 did not induce significant toxicity at lower concentrations, and the proliferation of untreated cells is comparable. Nevertheless, a reduction in cell proliferation was noticed with the regular augmentation of UCNC@SiO 2 nanostructure concentrations in cell growth media.
Micromachines 2018, 9, x FOR PEER REVIEW 7 of 12 bands have been observed in the UCL spectra of UCNC@SiO2 at 658 nm and 544 nm, which is shown in Figure 4a. These upconversion emissions are the results of Er 3+ ions electronic transitions from 4 F9/2 → 4 I15/2 and 4 S3/2 → 4 I15/2, respectively, and the transitions are schematically shown in Figure 4b [61]. Before performing cell imaging studies, cytotoxicity experiments were performed using a cell proliferation assay (WST-1). The cell toxicity induced by nanoparticulate imaging probes is mainly associated with their concentration and their surface functionalization [62,63]. In our cell viability experiment, DMSO was used as a positive control, as it is a known cell growth inhibitor and thus behaves like an effective toxin. Therefore, DMSO (10%) was mixed with the DMEM (cell growth media) as a toxin (positive control), while as a negative control, untreated cells (merely DMEM) were used. The untreated UCNC@SiO2 core-shell nanostructures were measured in variable concentrations up to 25 μg mL −1 . The cell proliferation assay was used to evaluate the effect of UCNC@SiO2 core-shell nanostructures on the proliferation of human breast cancer cells (MDA-MB-231) after 48 h. The cell viability data is shown in Figure 5. The cell proliferation with UCNC@SiO2 did not induce significant toxicity at lower concentrations, and the proliferation of untreated cells is comparable. Nevertheless, a reduction in cell proliferation was noticed with the regular augmentation of UCNC@SiO2 nanostructure concentrations in cell growth media.  Based on optical properties and cell toxicity investigations of UCNC@SiO 2 , in vitro imaging of human breast cancer cells was performed by incubating them with 10 µg mL −1 UCNC@SiO 2 core-shell nanostructures for 48 h, and the resulting images are presented in Figure 6. To investigate the intracellular location of UCNC in the cancer cells, their nucleus was stained with DAPI ( Figure 6a). It can clearly be seen (Figure 6b,c) that a cluster of cells displayed bright signal in both the green and red channels, upon an excitation with 980 nm multi-photon excitation, which demonstrates that the UCNC@SiO 2 core-shell nanostructures are capable of internalization into the human breast cancer cells. UCL emission of core-shell nanostructures was mainly localized in the cytoplasm of the cells, and we did not observe luminescence in the nucleus. The overlay image is presented in Figure 6d. The UCNC@SiO 2 core-shell nanostructures showed sufficiently good cell viability and efficacy of cellular labeling for optical imaging applications even without further surface functionalization, which could be readily applied on to the silica coating for promoting further cellular uptake or other functions [64]. Importantly, auto-fluorescence of MDA-MB-231 cells can be removed by excitation at longer wavelengths, i.e., 980 nm laser. Consequently, UCNC@SiO 2 is capable of multi-photon excitation with longer wavelengths, which, in turn, allows less scattering, deep penetration, low tissue absorption, and low autofluorescence for imaging of biological samples [65,66]. approximately 100% cell proliferation. The UCNC@SiO2 treated cells have shown concentration-dependent cell proliferation i.e. the higher the particle concentration, the lower the cell proliferation. The measured cell viability for 5 μg mL −1 , 10 μg mL −1 , and 25 μg mL −1 UCNC@SiO2 treated cells were approximately 90%, 80%, and 70%, respectively.
Based on optical properties and cell toxicity investigations of UCNC@SiO2, in vitro imaging of human breast cancer cells was performed by incubating them with 10 μg mL −1 UCNC@SiO2 core-shell nanostructures for 48 h, and the resulting images are presented in Figure 6. To investigate the intracellular location of UCNC in the cancer cells, their nucleus was stained with DAPI ( Figure  6a). It can clearly be seen (Figure 6b,c) that a cluster of cells displayed bright signal in both the green and red channels, upon an excitation with 980 nm multi-photon excitation, which demonstrates that the UCNC@SiO2 core-shell nanostructures are capable of internalization into the human breast cancer cells. UCL emission of core-shell nanostructures was mainly localized in the cytoplasm of the cells, and we did not observe luminescence in the nucleus. The overlay image is presented in Figure  6d. The UCNC@SiO2 core-shell nanostructures showed sufficiently good cell viability and efficacy of cellular labeling for optical imaging applications even without further surface functionalization, which could be readily applied on to the silica coating for promoting further cellular uptake or other functions [64]. Importantly, auto-fluorescence of MDA-MB-231 cells can be removed by excitation at longer wavelengths, i.e., 980 nm laser. Consequently, UCNC@SiO2 is capable of multi-photon excitation with longer wavelengths, which, in turn, allows less scattering, deep penetration, low tissue absorption, and low autofluorescence for imaging of biological samples [65,66].

Conclusions
In summary, we have synthesized NaYF 4 :Yb/Er upconversion nanocrystals and coated them with silica to procedure core-shell nanostructures. Powder X-ray diffraction pattern of UCNC@SiO 2 core-shell structures confirms that the core UCNC are in cubic phase. TEM images show that the core-shell nanostructures are predominantly spherical in shape comprising a thin silica shell around the UCNC core of about 25 nm size. The presence of all the constituent elements was confirmed through EDAX and elemental mapping studies. Upconversion photoluminescence measurements proved that the UCNC@SiO 2 core-shell nanostructures display very good upconversion luminescence. Cell viability experiments through MTT assay shows low cytotoxicity of the nanostructure. Finally, we have demonstrated the bioimaging capability of the UCNC@SiO 2 core-shell nanostructures through NIR light enabled optical imaging of breast cancer cells. This work opens the way for the development of low-toxic photostable alternatives to traditional fluorophores for bioimaging applications.