Monitoring the cellular uptake of Silica-Coated CdSe / ZnS quantum dots by Time lapse Confocal Laser Scanning Microscopy

© 2018 Heba ElSayed ElZorkany et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Taher A. Salaheldin, Mostafa Elsayed Center for Nanotechnology Research, British University in Egypt. E-mail: Taher.salah @ bue.edu.eg Monitoring the cellular uptake of Silica-Coated CdSe/ZnS quantum dots by Time lapse Confocal Laser Scanning Microscopy


INTRODUCTION
Quantum dots (QDs) are zero-dimensional nanoparticles in which charge carriers in the system are confined in all dimensions (Medintz and Hildebrandt, 2014).QDs are a very interesting nanomaterial with unique characteristics, that could help in many clinical and pharmaceutical purposes (Peuschel et al., 2016).
Due to its intrinsic photophysical properties, QDs are promising tools for many biological applications.Recently, QDs have even been tested for multicolor optical barcodes (Kim et al., 2016), visualizing in vitro protein movements, microbiological labels, detection of dual DNA targets (Cui et al., 2016).The optical properties of QDs are much suited to imaging purposes than those of molecular fluorophores (Thomas, 2015).However, organic fluorophores have limited usage in long-term imaging as it has broad absorption/emission profiles in addition to the low photobleaching thresholds (Medintz and Hildebrandt, 2014).
QDs show interesting advantages for lots of applications.They exhibit a high fluorescence yield especially cadmium based QDs.This kind of QDs started to play a role in molecular pathology and malignant-tumor biomarkers (Massey et al., 2015).However, QDs also possess a serious disadvantage manifested in the high toxicity (Oh et al., 2016).Recently, QDs showed distinct cell damage, resulting from ROS receptor generation and lysosome increment inside the cell (Liu et al., 2017), the thing that led researchers to make further surface modifications to them.Most of surface modifications cause a complete or partial loss of photoluminescence (PL) (Hoshino et al., 2011).Growing a silica shell around the QDs is a very special type of surface modification, as it doesn't only increase the PL of the QDs but also it becomes biocompatible rather than it enhances the uptake of QDs by biological cells (Dhawan and Sharma, 2010).
In the present study, we have synthesized silica coated CdSe/ZnS nanocrystals (CdSe/ZnS-SiO 2 NCs) that have been tested for imaging purposes.Therefore, the uptake of CdSe/ ZnS-SiO 2 inside live HepG2 cells was monitored for eight hours by Confocal Laser Scanning Microscopy (CLSM).The results showed a very fast uptake and stability of the CdSe/ZnS-SiO 2 NCs inside HepG2 cells.

Synthesis of quantum dots
Size-tunable The synthesis of CdSe capped with trioctylphosphine oxide (CdSe-TOPO) nanoparticles QDs was synthesized as previously described (Peng and Peng, 2001).In a typical synthesis, trioctylphosphine selenide was prepared by dissolving 0.15 g Se in 4 mL of TOP, at 200°C in a three-necked flask under N 2 atmosphere until Se is completely dissolved.
In another three-necked, 0.17 g CdO and 2.67 ml oleic acid was ignitedto 200-250°C under an argon gas until the red color disappeared.A mixture of 1.94 g TOPO and 1.94 g HDA were added to the reaction vessel, and the temperature was further increased to 300°C.At this temperature, the vessel was removed and trioctylphosphine selenide solution was swiftly inoculated into the reaction mixture under vigorous stirring, which resulted in an instantaneous nucleation and growth of the nanoparticles.

Formation of ZnS shell on CdSe QDs
Formation of ZnS shell on CdSe was applied as previously described (Ibrahim et al., 2014).Typically, in a threenecked flask 0.9 g TOPO and 0.9 g HDA were loaded and heated up to 170°C under magnetic stirring and N 2 gas.Later, CdSe was added in 1-octadecene solution.Then, a premixed solution containing shell components in 2 ml TOP (1 ml Zn(Et) 2 , and 220 μl (TMS) 2 S 1.0 M in hexane) was added dropwise into the reaction mixture in a periodof 10 min.The reaction mixture was then stirred for one hour at 170°C, then left for cooling.The synthesized CdSe/ZnS NCs were purified and washed by precipitation via the addition of 5:1 ethanol: CdSe/ZnS NCs.The precipitate was collected by centrifugation at 3000 rpm for 5min.The washing process was repeated three times then the precipitate re-dissolved in a minimum amount of chloroform.

Silica coating of QDs
To be suitable for imaging purpose the prepared core/ shell QDs should be transferred from organic to aqueous media, so, the prepared hydrophobic ZnS/CdSe were overcoated with Silica-based using the procedure of Vibin et al. (Vibin et al., 2014).Typically, a mixture of TOPO capped ZnS/CdSe QDs in 400 μL tetrahydrofuran and 150 μL TEOS was under sonication for 30 min, in an inert atmosphere.This mixture was added to premixed solution of 1 mL Igepal CO-520 in 10 mLcyclohexane and stirred for 30 min.Ammonia solution (1 mL, 33 wt.%) was added dropwise, and the stirring was continued for one day.The CdSe/ZnS-SiO 2 NCs were purified by repeatedly washing with dry propanol, ethanol, and water.Later on, the surface modified hydrophilic CdSe/ZnS-SiO 2 NCs were re-dispersed in sterile PBS for biological evaluation tastes.NCs were stored in the dark at room temperature.

QDs characterization
TEM images, XRD and various spectroscopic and microscopic techniques were employed to characterize morphology, structure, crystallinity, size, absorption and emission of the prepared CdSe, CdSe/ZnS and CdSe/ZnS-SiO 2 NCs.

Spectroscopy analysis
The UV-Vis spectroscopy measurements of all synthesized QDs samples were recorded on Cary 5000 (UV-Vis-NIR spectrophotometer, Varian, Australia) in the wavelength ranging from 400 to 800 nm.PL was collected by Cary Eclipse spectrofluorometer (Varian, Australia).All Spectroscopic measurements were performed at room temperature.

Transmission electron microscope (TEM)
High-resolution transmission electron microscope (HR-TEM, Tecnai G20, FEI, Netherland) was used for imaging.A drop of a dilute sample solution deposited on an amorphous carbon coated-copper grid.Bright field imaging mode was performed using Eagle CCD camera at electron accelerating voltage 200 kV using lanthanum hexaboride (LaB6) electron source gun.

Dynamic light scattering
Dynamic light scattering (DLS) measurements were performed using the Zetasizer nano-ZS equipped with 633 nm laser (Malvern Instruments Ltd, UK).The analysis yields the size distribution of the particles inside the sample and polydispersity index.The hydrodynamic particle diameter is available via the Stokes-Einstein equation.To transfer the weighted hydrodynamic particle sizes distribution to number %, a Mie correction is used to consider the size-dependent extinction coefficient (Finsy and De Jaeger, 1991).Each run was replicated three times to ensure data consistency.

X-ray diffraction analysis
All synthesized NCs samples were dried and the X-ray diffraction (XRD) patterns of the dry powder of each sample were obtained starting from 2θ = 4° to 80° in a continuous scan mode in steps of 0.02° via Philips Panlytical X'pert Pro X-ray diffractometer using Cu Ka (1.54059 A°) radiation, and X-ray generator operating at 45 kV and 30 mA.

Cell culture
Liver hepatocellular carcinoma (HepG2) cells were obtained from the Egyptian Organization for Biological Products and Vaccines (VACSERA).Cells were cultured in RPMI-1640 media supplemented with 10% FBS and then incubated in 5% CO 2 at 37˚C.

Mitochondrial activity
For assessing the cytotoxicity effect of Si-CdSe/ZnS NCs on HepG2 cells, the cell proliferation reagent WST-1 was used to evaluate the mitochondrial activity (Pardo et al., 2017).Briefly, 5 × 10 4 cell/well were loaded in a 96-well plate 18 h after seeding, the old media was removed and new media containing CdSe/ZnS-SiO 2 NCs with different concentrations ranging from 5.56-50.04nM cadmium.Cells incubated with media free of QDs were used as negative controls and cells incubated with media contain uncoated CdSe/ZnS NCs were used as positive controls.WST-1 kit reagent was added 4 h prior to completion of the incubation period.The absorption of samples was measured by microtiter reader (Tecan, Austria) at 450 nm.Experiment was performed in triplet manner.

Alkaline comet assay
According to Tice and Vasquez, the purpose of this protocol is to detect DNA damage in eukaryote cells (Tice and Vasquez, 1999).Standard microscopic slides were dipped into hot 1.0% normal melting point agarose, and the bottom of the slides was cleaned to remove excess agarose.Ten µl aliquot of control and treated HepG2 cells suspended in cold mincing solution HBSSwith 20 mM EDTA, 10% DMSO, were mixed with 75 µl of 0.5% LMA and were added to the precoated slides.After solidification, slides were placed in a cold fresh lysing solution for 24 h at 4°C in the dark.Subsequently, the slides were incubated in freshly-made alkaline buffer (pH > 13) for 20 min.
Electrophoresis was performed at 300 mA and 25 V for 20 mi, then the alkali was neutralized with 0.4 m Tris (pH 7.5), fixed in 100% cold ethanol and air dried.After the complete drying, DNA was stained with ethidium bromide (2 mg/ml).Finally, about 50 images were randomly acquired for every sample using a fluorescence microscope and analyzed for: The Tail Moment, tail Length, and % DNA in the tail using Comet Assay IV software (Perceptive Instruments, Suffolk, UK).

Confocal Laser Scanning Microscopy (CLSM)
Images for HepG2 cells were captured after the incubation with CdSe/ZnS-SiO 2 NCs using confocal laser scanning microscope (CLSM, LSM 710, Carl Zeiss, Germany) supported with Zen2009 software and equipped with CO 2 incubator.Processing was done using Zen2012 (blue edition).Specimens were excited using the 488 nm laser line of an Argon laser.Oil immersion objective 40× was used to obtain images.
To study localization of CdSe/ZnS-SiO 2 NCs in Lysosomes, CellLight® Lysosomes-RFP*BacMam 2.0 (molecular probes by life technologies, USA) was used following the same procedure in a separate Petri dishes.

Live cell imaging by confocal laser scanning Microscopy (CLSM).
Cellular internalization efficiency of the prepared NCs in HepG2 cells was estimated by live cell imaging.HepG2 cells were cultured on glass-based Petridish and exposed to CdSe/ZnS-SiO 2 NCs at concentrations of 5.56 nM.Cells were incubated in 37°C with 5% CO 2 .Time series experiments of live cells were performed for 8 h starting immediately after adding the CdSe/ZnS-SiO 2 NCs.Images were captured every 30 minutes by using 0.2% of 488 nm laser line.

Statistical analysis
Cell survival and DNA damage were expressed as the mean ± standard error (SE) and the significance of the differences, compared with control, was calculated using Graph pad prism 7.00.P < 0.05 were considered statistically significant according to Student's A nova-test.

Characterizations of CdSe QDs
The absorption spectra of a series of CdSe QDs were taken during synthesis process after different growth time ranged from 2-15 min (Figure 1).Results showed relatively sharp absorption feature near the absorption onset that corresponds to the excitonic peak.As the excitonic peak position depends on the particle size, its form and width are shaped by the pattern of size distribution.Consequently, due to quantum confinement, the increase of particle size resulted in a bathochromic (red) shift of the absorption onset (Hines and Kamat, 2014).XRD pattern showed that, the core NCs was very well matching with the cubic zinc blende structure ''JCPDS 01-088-2346''.While, core/shell had the same pattern of the core plus additional peaks of cubic ZnS with zinc blende structure ''JCPDS 00-065-0722''.In addition, core/shell-coated with silica showed the same previous peaks of core and core/shell plus additional peaks of tetragonal CdSe/ZnS-SiO 2 structure ''JCPDS 01-073-3435''.As shown in (Figure 2, a), the broadening of the XRD pattern peaks indicates the small particle (crystallite) size to the nanometric size (Iranmanesh et al., 2015).This agrees with the TEM images of the CdSe, CdSe/ZnS core/shell and CdSe/ZnS-SiO 2 NCs (Figure 2, b), which illustrate that most particles have a homogeneous spherical shape and size with an average diameter of about 2, 2.5, 35 nm, respectively.Further characterizations were performed to the three prepared samples CdSe, CdSe/ZnS and CdSe/ZnS-SiO 2 .The optical UV-Vis absorption and PL spectra of CdSe (Figure 3) showed an excitonic absorption at 532 nm and PL spectrum maximum emission around 548 nm, while the DLS analysis of the core sample showed a hydrodynamic diameter of 6.5 nm.On the other hand, the absorption spectrum of CdSe/ZnS QDs showed 7 nm bathochromic shift to give a maximum excitonic absorption at 530 nm (Figure 4).Also, PL maximum emission after the addition of ZnS shell were at 553 nm.While the hydrodynamic diameter obtained from DLS showed 7.1 nm size.Whereas absorption spectrum of CdSe/ZnS-SiO 2 NCs showed that, the SiO 2 coating causes relatively broadening of the excitonic peak which influenced by the distribution in size (Figure 5).In addition, CdSe/ZnS-SiO 2 PL emission maximum located at 577 nm, with 29 nm bathochromic shift respectively relative to the core.While CdSe/ZnS-SiO 2 sample exhibited size distribution peak around 104 nm according to DLS analysis.The polydispersity index (PDI) values of all samples were typically >0.5 that distinctly ensure particles size and shape homogeneity (Bhattacharjee, 2016).The size distribution of NCs obtained from DLS analysis was slightly larger than the equivalent size appeared in TEM images; this can be explained depending on the difference between the applied techniques of each system.Whereas DLS sizes arise from the calculated hydrodynamic diameter depending on the intensity of the scattered laser from the sample's particles (Fischer and Schmidt, 2016), the TEM sizes arise from the investigation of the sample by transmitted electron beam, that gives a more real result about the particles size and shape.The difference between the sizes obtained from both techniques reached its maximum in the case of the CdSe/ZnS-SiO 2 sample, this could be explained by the aggregation of the particles, which make a confusion for the detector that could not distinguish between scattered light from one large particle, or numerous aggregated small particles.

Biological evaluation of NCs effect
Based on the results of the cytotoxicity studies performed by applying different concentrations of CdSe/ZnS-SiO 2 (Figure 6 a), it could be confirmed that the prepared NCs possess good biocompatibility up to 40 nM (no significant reduction in HepG2 cells growth relative to control).While, with increasing the concentration of NCs to 50 nM, it showed low toxicity.Five nM of CdSe/ZnS at ODE was used as a negative control, which show 100% lethal effect.
The statistical significant increased (P < 0.05) in DNA damage as assessed by tail length, percentage of the DNA in tail and tail moment, indicated that the administration of CdSe/ZnS-SiO 2 at 5.5 and 16.7 µM doesn't induce significant DNA damage when compared with control group of HepG2 cells (cells without any NCs, Figures 6, c, d).While, all other higher concentrations showed a statistically significant increase of all DNA damage parameters in comparison with the HepG2 cells control group (P < 0.05).According to Tsoi's findings, we can attribute the DNA damage to the cadmium core of QDs that could generate free radical species in the surrounding media (Tsoi et al., 2013).
On the other hand, The CLSM micrographs (Figures 7 a, b) confirmed the internalization of QDs in the cytoplasm including mitochondria and lysosomes.However, it showed no internalization inside the nucleus.These results agree with the finding of prior works (Deerinck, 2009;Xiao et al., 2010).Cellular localization of CdSe/ZnS-SiO 2 QDs in HepG2 cells was tested using CLSM.By imaging HepG2 cells pre-incubated over night with Mitochondria-RFP/lysosomes-RFP and 30 min with DAPI, after seconds of adding CdSe/ZnS-SiO 2 NCs (Figures 7 c, d).
Live cell imaging of the prepared NCs in HepG2 cells showed that, cellular uptake of the prepared NCs was very fast as CdSe/ZnS-SiO 2 NCs was inside the cells from the first minute of the experiment time (Figure 8).Also, signal from CdSe/ZnS-SiO 2 NCs showed no decay or photobleaching, what ensure its stability inside the cell up to eight hours.

CONCLUSION
These results confirmed the effective role of silicon as surface coating of CdSe/ZnS NCs.Rather than the role of CdSe/ ZnS-SiO 2 in labeling and imaging of cancer cells due to their good fluorescence emission inside the cytoplasm, non-toxicity at low concentrations and their fast uptake that could be due to the silica shell that help in the internalization of the NCs throw the cells membrane.However, staining cell's nucleus still a challenge as it needs further surface modification.

CONFLICT OF INTERESTS
There are no conflicts of interest.

Fig. 1 :
Fig. 1: The absorption spectra of the CdSe nanocrystals.Monitoring the change in the absorption as a function of growth time UV-Vis absorption spectra were recorded in the range from 400 to 700 nm to CdSe QDs aliquots during synthesis process at different growth time (from 2 to 15 minutes).

Fig. 3 :
Fig. 3: PL spectra and size distribution of CdSe core.(a) UV-Vis absorption and PL spectra and size distribution of the as-prepared CdSe, (b) Show particle size distribution of CdSe.

Fig. 4 :
Fig. 4: PL spectra and size distribution of CdSe/ZnS core/shell.(a) UV-Vis absorption and PL spectra and size distribution of the as-prepared CdSe/ZnS, (b) particle size distribution of CdSe/ZnS.

Fig. 6 :
Fig. 6: Biological evaluation of core/shell-coated NCs.(a) survival % relative to controls for HepG2 cell line.After exposure to different concentrations of CdSe/ZnS-SiO 2 NCs.Error bars represent standard error from three different experiments.(b) DNA damage represented as tail length (µm), (c) % DNA in tail and (d) tail moment in different treatment groups in HepG2 cells.X axis represents QDs concentration by µM.Results are expressed as mean ± SE, significant difference with negative control using T test, bars denoted by the same letter(s) are not statistically different at P < 0.05 according to Student's A nova-test.

Fig. 8 :
Fig. 8: Confocal Laser Scanning microscopy images of live HepG2 cell.HepG 2 cell line incubated with CdSe/ZnS-SiO 2 by and continuous imaging was acquired by time lapse mode for 8 hours.