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The impact of doped silicon quantum dots onhuman osteoblasts
Lucie Ostrovska,ab Antonin Broz,bc Anna Fucikova,d Tereza Belinova,eHiroshi Sugimoto,f Takashi Kanno,f Minoru Fujii,f Jan Valentad
and Marie Hubalek Kalbacova*ab
Silicon (Si) nanostructures allow for the expansion of the application spectrum of this important
semiconductor material with respect to the ﬁelds of optoelectronics and photonics. At the same time,
the signiﬁcant potential of Si quantum dots (SiQDs) has been revealed in terms of their potential
application in the areas of biology and medicine due to their biocompatibility, low toxicity and natural
biodegradability, unlike currently used semiconductor quantum dots. As far as this study is concerned,
SiQDs co-doped with boron and phosphorus were used for the in vitro evaluation of their cytotoxicity inhuman osteoblasts. Two chemically identical types of SiQD diﬀering in terms of their size andphotoluminescence (PL) were studied. They both display long-lasting dispersion in methanol and even inaqueous media as well as PL which is not sensitive either to changes in the environment or surface
modiﬁcations. Our experiments revealed signiﬁcant diﬀerences between the two types of SiQD tested in

Received 3rd June 2016
Accepted 26th June 2016

regard to their behavior in a cell culture environment depending on increasing concentration (25–125
mg ml 1) and cultivation conditions (the presence or absence of proteins from the fetal bovine serum –DOI: 10.1039/c6ra14430f

a component of the cultivation medium). A detailed description of their optical parameters and the

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evaluation of zeta potential enhance the understanding of the complexities of the in vitro results obtained.

1

Introduction

Various types of nanoparticles have been studied and used in
diﬀerent elds of science including their application in the eldof bio-medicine. In general, nanoparticles (so-called quantum
dots – QD) based on a variety of materials provide a promising
tool with regard to potential as drug and gene carriers and as
imaging and diagnostic platforms.
Silicon-based nanoparticles make up one of the mostpromising platforms yet determined for medicinal application
due to their high level of biocompatibility and biodegradability
which arise from the fact that silicon (Si) is an essential traceelement in the human body. Currently, the development ofvarious types of Si-based nanoparticles is focused principally on
silica (SiO2) and pure silicon materials which are biodegradablea

Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen,
Czech Republic. E-mail: marie.kalbacova@lf1.cuni.cz

bInstitute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University
in Prague, Prague, Czech RepubliccInstitute of Physiology, Academy of Sciences of the Czech Republic, Prague-Krč, Czech
Republicd

Faculty of Mathematics and Physics, Prague, Czech RepubliceDepartment of the Cell Biology, Faculty of Science, Charles University in Prague,
Prague, Czech Republicf

Department of Electrical and Electronic Engineering, Graduate School of Engineering,
Kobe, Japan

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due to the nature of their Si–Si and Si–O bonds.1,2 In addition, it
is essential to note that most Si-based nanoparticles possess thequality of uorescence (photoluminescence) and therefore
facilitate their own imaging without any additional intervention
into their structure being required.3,4 Moreover, their dispersibility in aqueous solutions is also crucial for bio-application.5 Such properties are similar to the afore-mentioned QDs,
thus the term silicon quantum dots (SiQD) is particularly
appropriate.
This study is concerned particularly with the assessment ofcytotoxicity since this makes up the most important initial step
preceding the actual application of any of the materials studiedin terms of human medicine. Previous studies have shown thatthe cytotoxicity of Si-based nanoparticles is inuenced bya range of properties such as particle shape, size, zeta potential,
dose and chemical composition6–12 and some of these properties may play a role in the formation of so-called biomolecularcoronas. This term describes a layer of biomolecules, mainly
proteins (protein corona), originating from the biological environment surrounding nanoparticles which accord them a new
identity which diﬀers from that of bare nanoparticle in terms ofa number of characteristics especially their reaction withorganisms.13,14 Generally, the concept of the protein corona isgiven as a biological identity via which nanoparticles are presented to cells. It has been suggested that the presence, andpossibly the composition, of the protein corona provides the key

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to the determination of the cytotoxicity of nanoparticles.15,16 In
vivo, the composition of the attached proteins is spatial (lunguids, bloodstream) and time dependent and, moreover, thecondition of the living organism should be considered.16 On theother hand, in vitro conditions are limited by the presence of
certain types of proteins contained within the selected supplemented serum; thus, it appears that the protein corona is not
subject to the same degree of signicant dynamic change incomposition as it is in vivo.17
Previous studies that have focused on the impact of silica
nanoparticles (SiO2 NPs) on keratinocytes revealed that theviability of cells is both dose- and size-dependent and, in
addition, that this particular type of particle causes damage tothe cellular membrane which may be of either chemical ormechanical stress origin.7,8 It has been proposed that cytotoxicity caused by SiO2 NPs may be the result of oxidative stressinduced by the production of intracellular reactive oxygen
species.11,18 However, when compared to other types of nanoparticles, e.g. ZnO particles, SiO2 NPs reveal lower cytotoxicity
levels dependent on the cell type.7,9,19 Moreover, a number of
papers has reported a signicant correlation between cytotoxicity and the dose of Si-based nanoparticles (irrespective of theactual concentration range used for a particular cellular type).
Generally, the higher the nanoparticle dose applied to the cells,
the higher is the cytotoxicity observed following a certain periodof time, an example of which is provided by studies by Bhattacharjee et al. wherein increasing concentrations from 0.1 ng
ml 1 to 100 mg ml 1 of nanoparticles were added to a cell
culture of NR8383 macrophages. The same cytotoxicity trend
was also evident with respect to keratinocytes, although theconcentrations applied were signicantly higher (25–500 mg
ml 1).8,10,11 It is possible to obtain a better understanding of thecauses of the cytotoxicity of particular nanoparticles by meansof a description of specic cell–nanoparticle interactions.
It is widely accepted that nanoparticles of diﬀerent types
enter cells by means of endocytosis, i.e. a form of active transport; however, the specic ways in which individual particles
enter certain cell types remain to be discovered. Considerable
interest has been devoted to the discovery of principal nanoparticle uptake pathways and most of the experiments conducted in this respect were performed via the selective gradualblocking of diﬀerent pathways and the subsequent determination of which was most frequently used. Clathrin- and caveolindependent endocytotic pathways have been suggested as themain routes used by silicon-based particles; however, a numberof studies also suggest the use of the otillin-dependent
pathway.12,20 It has been implied that the principal pathway isdetermined not only by particle size and shape but also by
cellular type. It is important to gain an understanding of all theinteractions which take place between cells and nanoparticles
so as to avoid any harmful eﬀects in terms of potential use in
human medicine.
This study employed two types of SiQD of diﬀerent characteristics (SiQD 1050 – 3 nm size, 750 nm peak emission and
SiQD 1100 – 4 nm size, 850 nm peak emission) and theirinuence on a biological system represented by a humanosteoblast-like cell line (SAOS-2) was compared. The main focus

63404 | RSC Adv., 2016, 6, 63403–63413

Paperof the study was (i) to determine the appropriate concentrationof both types of SiQD based on the induced cytotoxicity and (ii)to evaluate the cellular uptake of both SiQDs at 2, 6 and 24
hours by means of uorescence wide-eld and confocal
microscopy. The inuence of the presence of fetal bovine serum(FBS) in the cultivation media made up an important variable
parameter in terms of understanding the impact of the protein
corona on SiQD behavior in vitro.

2 Experimental
2.1

The fabrication of co-doped SiQDs

P and B co-doped colloidal Si QDs were synthesized by means ofa previously reported procedure.21,22 Si-rich borophosphosilicate(BPSG) lms were deposited on thin stainless steel plates via theco-sputtering of Si, SiO2, B2O3, and P2O5 using the rf-sputtering
apparatus. The lms were then peeled from the plates andcrushed to powder form in a mortar. The powder was thenannealed at diﬀerent temperatures (1050  C and 1100  C thus
obtaining SiQD 1050 and SiQD 1100 respectively) in an N2 gas
atmosphere for 30 minutes so as to cultivate SiQDs of diﬀering
size in BPSG matrices. During the growth of the SiQDs, P and B
atoms were incorporated into Si–NCs from the BPSG matrices.
2.2

The detailed optical characterization of co-doped Si-QD

Transmission electron microscopy images were obtained using
JEOL JEM-200CX, and the IR absorbance of the suspensions
measured by means of drop-casting on a gold covered silicon
substrate in an FT-IR spectrometer (Perking Elmer, Spectrum
GX). UV-VIS spectra were measured by UV-3101PC (Shimadzu).
The PL spectra (Fig. 1d) were obtained using a spectrouorometer (Fluorolog-3, Horiba Jobin-Yvon).
The photoluminescence external quantum yield (EQY) of the
SiQD suspensions was measured in specially-designed equipment based on an integrating sphere.23 Excitation wavelengths
were tuned over a broad spectral range from UV to yellow using
either a set of light-emitting diodes or a white-light emitting
laser-driven light source (LDLS, Energetiq) coupled to a 15 cm
monochromator. The absorption cross-section s was obtainedby means of the intensity-dependent PL-modulation techniqueapplied to a thin liquid layer of SiQD in methanol.24 Under 405
nm excitation and an emission between 700 and 900 nm, s wasdetermined at around 3  10 16 cm2 for both of the samples
studied.
The PL kinetics of SiQDs under high-repetition short pulses(simulating the conditions of a confocal microscope witha “white” ber laser) were tested for SiQD 1050 in a cuvetteexcited via a pulsed diode laser at 408 nm (pulse duration below
0.1 ns, 20 MHz repetition rate and 840 W cm 2 power density)(Fig. 3b).
2.3

Zeta-potential assessmentz was measured using a Malvern Zetasizer Nano ZS equipedwith MPT-2 titration unit where 0.25 M NaOH, 0.2 M HCl and0.02 HCl were used as titration agents. The SiQD suspensions
were titrated in the acid to base and base to acid direction.

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RSC AdvancesFig. 1 (a) Optical transmittance spectrum and photograph of colloidal dispersion (methanol) of SiQD 1100; (b) TEM image of SiQD 1100, inset:
high-resolution TEM image of a QD with lattice fringes corresponding to {111} plane of Si crystals; (c) IR absorption spectrum of SiQD 1100; (d) PL
spectra of SiQD 1050 and 1100 (d).

2.4

Cell cultivation with SiQDs

SAOS-2 cells (DSMZ, Germany) were cultured in McCoy's 5A
medium without phenol red (HyClone) and supplemented with
10 000 U ml 1 penicillin (Sigma-Aldrich) and 10 mg ml 1 streptomycin (Sigma-Aldrich) and, in some of the experiments, with 15%
fetal bovine serum (Biosera). The cells were seeded at a density of
10 000 cells per cm2 onto a 96-well plate (Techno Plastic Products)
in triplets for the measurement of cytotoxicity (100 ml) or onto cell
imaging dishes with a 145 mm glass bottom in singlets (Eppendorf) for microscopy purposes (500 ml). The cells were cultured for
24 hours in a humidied 5% CO2 atmosphere at 37  C.
The required amount of SiQD colloid in pure methanol wasadded to an equal amount of distilled water and the resulting
mixture was subsequently incubated in a dry bath incubator(Major Science) set to 70  C until half of the liquid had evaporated. The nal SiQD colloid was ready for immediate dilution
into the appropriate cultivation media. Two types of cultivation
media were used in the experiments – McCoy's 5A medium
without phenol red (HyClone) with 10 000 U ml 1 penicillin(Sigma-Aldrich) and 10 mg ml 1 streptomycin (Sigma-Aldrich)with no serum proteins (serum-free) or supplemented with
5% fetal bovine serum (Biosera) (serum-supplemented). The
nal concentrations of SiQD in the media used for cell treatment consisted of 125 mg ml 1, 50 mg ml 1 and 25 mg ml 1.
The cells were gently rinsed with phosphate buﬀer saline(PBS) prior to the addition of the cultivation media containing
SiQDs. Subsequently, the cells were cultivated to diﬀerent time

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points depending on the type of assessment. In the case of theserum-free media, an additional volume of 50 ml (96-well plate)or 250 ml (cell imaging dish) of the medium with 5% FBS wasadded aer 6 hours of cultivation and cultivated for an additional 18 hours or 42 hours (in the case of 24 hours or 48 hoursof cultivation time respectively).
2.5

Cytotoxicity assessment

The cytotoxicity of various concentrations of SiQDs in diﬀerent
media was assessed by means of the measurement of themetabolic activity of the cells at 6, 24 and 48 hours following theaddition of SiQDs to the cells. Assessment was performed bymeans of MTS assay (Cell Titer96® AqueousOne, Promega). The
principle of this colorimetric assay consists of the reduction of
an MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) compound intoa soluble colored formazan product via mitochondrial
dehydrogenases. The assay was performed according to the
manufacturer's instructions. The cells were rinsed three times
with PBS and then incubated for 2 hours with an MTS reagent
diluted in the appropriate media. Optical density was measured
using a microplate reader (Synergy H1, BioTek) at 490 nm
subtracting the background at 655 nm. The subtraction of blankvalues was conducted for each type of medium separately. All
the results obtained were compared to the results of the control
cells cultivated in a medium supplemented with 5% FBS; theresults were expressed as percentages.

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2.6

Fluorescence wide-eld and confocal microscopyAer 2, 6 and 24 hours the cells were rinsed three times with
PBS so as to remove any un-internalized SiQDs and then xed in
4% paraformaldehyde. An Eclipse Ti–S epi-uorescence microscope (Nikon) with Mercury Arc Lamp Intensilight HGF1 andequipped with DS-Qi1Mc digital camera (Nikon) was used inorder to acquire 2D uorescence images of SiQDs at an excitation wavelength of 330–380 nm and emission wavelength of
510–590 nm. The extent of the penetration of SiQDs in the z-axisof the cells was analyzed with respect solely to the 24 hour
samples by means of a Leica TCS SP8X confocal laser scanningmicroscope (Leica Microsystems). The SiQDs were excited using
a pulse continuum white light laser (475–499 nm, 80 MHz) andemissions were collected via a hybrid detector at 700–795 nm.
The elimination of cell autouorescence from SiQD signals wasachieved by the gating of signal detection with a 5 ns delay fromthe excitation pulse while maintaining a detection width of 7 ns.
Multiple line accumulation scanning and prolonged pixel dwell
were set so as to allow for the more intense and precise imagingof the SiQDs. All the confocal 3D images were acquired bymeans of a Leica DFC365 FX monochrome digital CCD camera
and further analyzed using LAS X core soware (Leica Microsystems). The confocal images were processed using Hyugenssoware for deconvolution and maximum intensity projection.
ImageJ soware was subsequently used for contrast andsmoothness correction purposes.
2.7

Statistical analysis

All the data presented was derived from three independent
experiments performed in triplicate. The results are presented
in the form of mean values with error bars indicating standard
deviations. Data distribution was evaluated using the Shapiro–
Wilk test. The nonparametric Wilcoxon matched pairs test wasused in order to determine signicant diﬀerences between thedatasets and the control with 5% FBS and the rest of the variables. An ANOVA was used to compare diﬀering SiQD concentrations at certain time points with each other. p values of lessthan 0.05 were considered statistically signicant. Extreme
values were excluded from the analysis. Statistical analysis was
performed using STATISTICA (StatSo, Inc.) soware.

3 Results and discussion
3.1

The optical characterization of co-doped SiQD

Phosphorus (P) and boron (B) co-doped colloidal SiQDs weresynthesized by means of a previously reported procedure.21,22
The transmittance spectrum and an image of the colloid areshown in Fig. 1a. Transmittance around the band gap of bulk Si
crystal (1100 nm) is almost 100%, which indicates the absenceof signicant light scattering by QD agglomerates. Fig. 1b showsa transmission electron microscope (TEM) image demonstrating a QD monolayer without the formation of threedimensional agglomerates. Fig. 1c shows an infrared (IR)absorption spectrum of SiQD 1100 stored in methanol for 60
days. An absorption peak at 1080 cm 1 assigned to Si–O–Si
stretching vibrations can be clearly observed while no63406 | RSC Adv., 2016, 6, 63403–63413

Paper

absorption peak is evident from C–Hx (2900 cm 1). Thissuggests that following long-term storage in methanol, thesurface of SiQDs is terminated principally by oxygen. Aeraround one month's storage in methanol, the photoluminescence (PL) spectra of SiQD 1050 and 1100 under excitation at 450 nm exhibit PL peaks of around 750 and 850 nm
respectively (Fig. 1d). The size-dependence of the PL peakwavelength of B and P co-doped Si QDs has already been studied
in detail.25
With respect to the imaging of SiQD in cell cultures by meansof uorescence microscopy (wide-eld or confocal), the presence of so-called autouorescence (AF) – i.e. the natural uorescence of cell proteins and other molecules without articial
staining must be considered. AF usually peaks in green but
extends far into the red spectral region in which it overlaps with
the PL of the tested SiQDs. This is illustrated in Fig. 2 whichshows the local PL spectra of the AF and PL of SiQD 1100 under
excitation by means of a laser at 405 nm. In general, threemeasures can be applied so as to improve the ratio of SiQD PL
versus AF:(i) The shiing of the excitation wavelength into the green
region (e.g. the Ar-ion laser line at 488 nm) since AF decreases
faster with red-shi excitation than does SiQD PL.
(ii) The selection of SiQDs with an emission peaking at longwavelengths where AF disappears but where microscope
detectors remain eﬃcient; this usually means between 700 and
800 nm.
(iii) The use of diﬀerent PL decay kinetics under pulsed
excitation. While AF decays within a few nanoseconds, SiQDs
have a lifetime in the order of tens of microseconds. Therefore,gated detection can be applied which is delayed following
pulsed excitation by around 5 ns. Fig. 3a shows the slow rate of
PL decay for both the SiQD samples and Fig. 3b illustrates thatPL decay under excitation by sub-ns pulse with high repetition
rate (20 MHz – such a degree of excitation is deliverable bya large number of currently available confocal microscopes) was
unable to follow the pulse sequence and thus quasi-constant PL
was detected – the yellow rectangle indicates the temporal
position of the detection gate (delay 5–12 ns) as used later inthis paper (such excitation and detection options are available
in a large number of the current models of confocal
microscopes).
Finally, we draw attention to the aging of the luminescence
properties of SiQDs. During long-term storage (around 1 year)
a slow change in the PL peak position and the external quantum
yield (EQY) can be observed in methanol suspensions of SiQDs,
which is further accelerated in water-based suspensions used inbio-studies. Both types of SiQD exhibit a blue-shi and the EQYchanges as illustrated in Fig. 2c roughly following a curve which
has a maximum EQY of 12% for a peak at 750 nm (similar
optimal conditions were reported by Liu et al.).26 Such optimum
conditions for uorescence imaging are characteristic of fresh
SiQD 1050 samples; however, upon aging such samples degradein terms of EQY while SiQD 1100 shis to the optimum position(a shi from 850 nm to 750 nm). In brief, it is essential thataging properties are characterized in order to be able to anticipate PL evolution and select the ideal sample.

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RSC AdvancesFig. 2 Wide-ﬁeld ﬂuorescence microscopy and spectroscopy under cw excitation at 405 nm: (a) combined ﬂuorescence image with the green
layer showing the full signal (dominated by cell autoﬂuorescence) and the orange layer showing signal above 785 nm (dominated by SiQD
luminescence). The vertical stripe shows area of the spectrometer slit introduced for spectral measurements. (b) Luminescence spectrum of cell
autoﬂuorescence (blue) and emission of SiQD cluster (red line) from area indicated by a rectangle in the panel (a). The autoﬂuorescence signal
estimated from area around the SiQD cluster was subtracted and spectral shape corrected to the sensitivity of the experimental apparatus. The
peak is around 730 nm. (c) Observed shift of PL peak and quantum yield of SiQDs with time is plotted together with data from the paper by
Sugimoto et al.20 The slow changes due to aging of SiQD suspensions in methanol are accelerated in water based media during bio-studies. The
data (green line and points) on PL QY dependence on PL peak position (size of QDs) explains well why SiQD 1100 sample is well observed in cell
cultures while luminescence of SiQD 1050 vanishes.

3.2

Zeta potential assessment

The zeta potential (z) of water suspensions of SiQD 1050 and1100 as a function of pH was determined using 0.25 M NaOH,
0.2 M HCl and 0.02 HCl titration agents. The SiQDs had been
titrated in the acid / base and base / acid direction (Fig. 4).
The iso-electric point of both samples was determined ataround pH 2. While SiQD 1100 reacted relatively smoothly tochanges in pH, the SiQD 1050 reaction to the addition of acid/base was slow and followed by sudden jumps in z. Moreover,a two-peak distribution of z was observed for SiQD 1050 at eachpH point (Fig. 4d) indicating the presence of distinct SiQD
fractions. For a pH of 7.5 (the value relevant to biologicalexperiments) the mean value of z was around – 64 mV ( 16 mV
for the smaller peak) for SiQD 1050 and – 57 mV for SiQD 1100
(with an uncertainty of around 8 mV).
3.3 The eﬀect of diﬀerent SiQD concentrations on cell
metabolic activity in serum-supplemented and serum-freemedia
Osteoblastic cells were cultivated in a medium supplemented
with 5% fetal bovine serum (5% FBS-medium) with gradually
increasing concentrations (25, 50 and 125 mg ml 1) of two types of
SiQD which was followed by the determination of their metabolic
activity aer 6, 24 and 48 hours (Fig. 5a and b). It is apparent that
SiQD 1100 in a fully-supplemented medium had no impact oncell metabolic activity irrespective of concentration at the 6 hour
time point; however, aer 24 hours the highest concentration of
SiQD 1100 decreased cell activity signicantly and, aer 48 hours,
the medium concentration was seen to have a similar eﬀect.

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On the other hand, SiQD 1050 (lowest and medium
concentrations) surprisingly increased cell metabolic activityaer 6 hours and, aer 24 hours of incubation, the metabolic
activity of SiQD 1050-treated cells was comparable to that ofcontrols with no treatment. Only aer 48 hours did the highest
concentration of SiQD 1050 signicantly decrease cell metabolic activity.
In order to distinguish between the eﬀect of SiQDs and therole of the protein corona (originating from serum proteins),
osteoblastic cells were cultivated in a medium containing no
supplements (serum free – 0% FBS-medium). The same
concentrations of SiQDs were then added and the same tests
performed as described above (under standard conditions). It isapparent (Fig. 5c and d) that the serum-free medium only (withno SiQDs) had a negative eﬀect on cell metabolic activity at the 6hour time point; however, the decrease was not so strong as tobe considered cytotoxic.27
Those cells treated in the serum-free medium with the lowest
and medium concentrations of SiQD 1100 exhibited a metabolic
activity level similar to the control sample (cells cultivated ina 5% FBS-medium) and only in the highest SiQD concentration
did cell activity decrease signicantly to the cytotoxic level aer
6 hours (in contrast to standard conditions). Subsequently, aer
24 hours, both the medium and the highest concentrations of
SiQD 1100 were found to strongly aﬀect cell activity and, nally(aer 48 hours) all the tested concentrations of SiQD 1100 in theserum-free medium were determined as being cytotoxic.
On the other hand, SiQD 1050 did not aﬀect cell behavior atany concentration aer 6 hours; however, aer 24 hours of
incubation, all the tested concentrations were found to be

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RSC AdvancesFig. 3 (a) Kinetics of PL decay under long square pulses and (b) shorthigh-repetition rate (quasi-cw) excitation. The conditions applied in
the confocal imaging (80 MHz repetition rate, detection window of 7
ns delayed by 5 ns) is indicated by the yellow rectangle.

cytotoxic, the level of which was further enhanced following 48
hours of incubation.
3.4 The microscopy assessment of SiQD eﬀects on cellmorphology and metabolic activity in serum-supplemented
and serum-free media
Osteoblasts were treated with 50 mg ml 1 of SiQD 1050 and SiQD
1100 (medium concentration) in a 5% FBS-medium for 2, 6 and24 hours and then visualized by means of wide-eld uorescence microscopy accompanied by a phase contrast for the
more precise localization of the SiQD uorescence signal within
the cells (Fig. 6a and b). Confocal microscopy images of cells
treated with the same SiQD concentrations were acquired at the
24 hour time point only (Fig. 6c and d).
Fig. 6a shows that a very bright uorescence signal is visible
in those cells treated with SiQD 1100 as soon as aer 2 hours
and that it is even stronger at the 6 hour time point. However, it
is apparent from the phase contrast image that the signal
originates from a culture medium in which aggregates of SiQD
with proteins originating within the FBS had formed and notfrom the cells themselves. Aer 24 hours, the uorescencesignal of SiQD 1100 is apparent from inside the cells as well as

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Paperfrom the culture medium itself. The confocal image (Fig. 6c)taken at the same time conrms the localization of these SiQDs
inside cells in a vesicular form with no apparent changes in cellmorphology. In addition, cell metabolic activity tests (Fig. 8a)
indicated that these SiQDs had no negative impact on the cells
at the same time point; only aer 48 hours of incubation did the
SiQD 1100 cause a signicant decrease in cell metabolic activity
but not to such an extent as to include cytotoxic eﬀects.27
In the case of SiQD 1050 an even stronger uorescence signalof SiQD and proteins in the cultivation medium was detectedaer 6 hours but only a very weak signal could be detected in thecells aer 24 hours (Fig. 6b). The image of cells treated with
SiQD 1050 for 24 hours presented was intentionally selected so
as to show the cells in the region not totally covered with SiQDprotein aggregates; notwithstanding, most of the sample area
was found to be covered with these foggy aggregates. Thisobservation was conrmed by means of confocal imaging(Fig. 6d) which revealed a weak uorescence signal distributed
diﬀusely within the cells (autouorescence) and a concentrated
signal issuing from the culture medium in which SiQD 1050
aggregates and FBS proteins were formed. Indeed, this corresponds well with the data presented in Fig. 8b according towhich no reduction in metabolic activity (compared to theuntreated control in the 5% FBS-medium) was detected ineither evaluation using the same concentration of SiQD 1050.
Subsequently, the same experiments were performed withusing the serum-free medium (0% FBS-medium). Fig. 7a showsthat a uorescence signal is visible in those cells treated with
SiQD 1100 as soon as aer 2 hours of incubation and it is evenstronger at the 6 hour point at which all the cells exhibita uorescence signal. Aer 24 hours, SiQD and protein aggregates were visible in the medium. The proteins originated fromthe FBS added to the culture medium aer 6 hours due to thecells being unable to survive (i.e. to avoid signicant behavioral
changes) any longer without the addition of FBS. It was expectedthat all the SiQDs would already have entered the cells by thistime point and that they would no longer be present in themedium. However, microscope images demonstrate that aer 6hours a certain amount of SiQD 1100 was still present in themedium available to react with the FBS proteins. Nevertheless,
SiQD 1100 were present on a massive scale inside the cells at 24hours (Fig. 7a) and had a negative impact on cell morphology;
moreover, this led to a signicant decrease in cell metabolic
activity (Fig. 8a). The confocal image (Fig. 7c) conrms thepresence of high quantities of SiQD 1100 inside the cells in thediﬀused form which is in contrast to the localization of SiQD
1100 in those cells cultivated in the 5% FBS-medium (Fig. 6c).
Results concerning metabolic activity indicate that the cells
were dying at a rapid rate at the 24 hour point and had died atthe 48 hour time point.
Conversely, SiQD 1050 were practically invisible at all time
points in those cells cultivated in the serum-free medium; a very
faint signal was detectable at the 2 hour time point which disappeared over time (Fig. 7b). The wide-eld microscopy data
was conrmed by the confocal images in which the uorescencesignal was very faint and originated from cell autouorescence.
However, the results presented in Fig. 8b suggest that

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RSC AdvancesFig. 4 Zeta-potential evaluation: (a) titration curves of SiQD 1100 and (b) SiQD 1050 (each point is average of three measurements). Small graphs
show an example of single measurement of zeta potential at pH 7.5 in (c) SiQD 1100 and (d) SiQD 1050.

a reduction in metabolic activity occurred aer 24 hours of
incubation (also apparent in the form of changed cellmorphology in Fig. 7b) and that at the 48 hour point the cells
had already died.

This report presents the rst study performed on the impact
of novel Si-based nanoparticles on a biological environment
consisting of a human osteoblast cell culture. The most uniqueproperty of the B and P co-doped SiQDs used consists of their

Metabolic activity of osteoblasts cultivated in a cultivation medium supplemented with 5% FBS (a, b) and in serum free medium (c, d) for
increasing concentration of SiQD 1100 (a, c) and SiQD 1050 (b, d). Relative values are expressed as a percentage of control sample in thecultivation medium with 5% FBS (dashed line). The star symbol (*) highlights a signiﬁcant diﬀerence from the control in cultivation medium with
5% FBS (Wilcoxon matched-pairs test, p < 0.05). Groups marked with diﬀerent letters express signiﬁcant inter-group diﬀerences within time
points (ANOVA, LSD post hoc test, p < 0.05).

Fig. 5This journal is © The Royal Society of Chemistry 2016

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Fig. 6 Wide-ﬁeld ﬂuorescence and phase contrast microscope images of osteoblasts treated with (a) SiQD 1100 and (b) SiQD 1050 in 5% FBSsupplemented medium in diﬀerent time points (the scale bar is 30 mm) and confocal images after 24 h of incubation with (c) SiQD 1100 (imaged
volume 75.0  75.0  7.6 mm) and (d) SiQD 1050 (imaged volume 112.6  112.6  9.5 mm).

ability to form stable colloidal suspensions in the absence of
surfactants and organic passivation. Moreover, these SiQDs
required no protective shell layer and were of a very small size
(3–4 nm in diameter) in contrast with other semiconductor
quantum dots with a core–shell structure and which aresubstantially larger than 10 nm (not taking into accountpossible surface passivation) in order that they exhibit a stableemission in a similar spectral region on the border of red andinfrared (700–850 nm).28 We selected two types of SiQDs withsizes of 3 and 4 nm (SiQD 1050 and 1100 respectively) the PL
emission peaks of which are situated in the afore-mentioned
spectral range which is optimal in terms of uorescencemicroscopy. Somewhat surprisingly, our experiments revealed
signicant diﬀerences with respect to the interaction of the two
types of SiQD with cell cultures which could not be ascribed
simply to the 1 nm size diﬀerence – at the cellular level sucha small diﬀerence does not inuence particle cytotoxicity andonly a slight change in cellular uptake is able to occur.29–31
Zeta-potential (z) is related to colloidal suspension stability.
The critical value of z below which a suspension is unstable(and at which agglomeration can take place) is around 30
mV.32 Generally, z is result of the net electrical charge contained
within the region bounded by the slipping plane. This charge isstrongly dependent on ions present in the solution and therefore changes with pH value. The pH value, for which the net
electrical charge is null, is called isoelectric point. Around thispoint the nanoparticles aggregate rapidly. With concern tobiology, the surface potential of a particle is important in termsof the formation of protein corona – nanoparticles with
diﬀerent potentials will bind to diﬀerent proteins or to diﬀerent
active sites on individual proteins which, in turn, may signicantly inuence the overall toxicity of nanomaterials. An
example of the varying degree of toxicity of Si nanoparticles
depending on the value of z and the particle covering is reported
in;10,11 however, this eﬀect has also been observed with respect

63410 | RSC Adv., 2016, 6, 63403–63413

to other types of nanoparticles.33,34 Our experiments resulted in
diﬀerent values of z for Si QD 1050 and 1100; therefore wesuspect the formation of a diﬀering protein corona under thesame biological conditions.
Both of the tested SiQDs caused harm to the cells ina diﬀerent manner – SiQD 1100 exhibited a measurable harmful
eﬀect at the highest applied concentration level (125 mg ml 1) assoon as aer 24 hours of incubation in the fully-supplemented
medium, while the negative eﬀect of SiQD 1050 was notapparent until aer 48 hours of incubation (Fig. 5). It is conjectured that this diﬀerence may be linked to the observed
formation of dense clusters of SiQD 1050 with serum proteins(see the uorescence images in Fig. 6). These clusters act asreaction centers and continue to grow thus forming hugeaggregates which cover the outside membranes of the cells andprevent SiQD 1050 from entering the cells. When subjected touorescence microscopy, protein/SiQD 1050 aggregates arevisible as fog-like structures which grow over time (Fig. 6b andd). Despite this eﬀect, however, a number of uncovered locations were detected aer 48 hours thus providing coarse information on the incorporation of SiQD 1050 into the cells(Fig. 6b). This phenomenon was also witnessed with respect to
SiQD 1100 but to a signicantly lesser extent which, nevertheless, still allowed for the visualization of the cells and theirincorporation of SiQD 1100. Notably, literature describes thisaggregation eﬀect with respect to other types of nanoparticles.35,36 Therefore, it is safe to conclude that the presenceof FBS proteins in the cultivation medium strongly inuencespecic SiQD behavior and thus their availability and absorption by cells. It may be speculated from the uorescence images(Fig. 6c) that the SiQD 1100 visible in cell cytoplasm are localized in vesicles and thus potentially enter cells by means of
endocytosis as has been previously described.37–40
When SiQDs were incubated with cells in the serum-free
medium, SiQD 1100 exhibited a cytotoxic eﬀect at the highest

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RSC AdvancesFig. 7 Wide-ﬁeld ﬂuorescence and phase contrast microscope images of osteoblasts treated with (a) SiQD 1100 and (b) SiQD 1050 in the serumfree medium in diﬀerent time points (the scale bar is 30 mm) and confocal images after 24 h of incubation with (c) SiQD 1100 (imaged volume 85.0
 85.0  11.6 mm) and (d) SiQD 1050 (imaged volume 102.0  102.0  9.6 mm). Note – after 6 h of cell incubation in serum-free medium, FBS
was added due to the cell survival purposes.

Fig. 8 Metabolic activity of osteoblasts treated with 50 mg ml 1 of (a)SiQD 1100 and (b) SiQD 1050 for diﬀerent time points.

applied concentration level (125 mg ml 1) as soon as aer 6hours of incubation (Fig. 5c). Therefore, it was concluded thatbare SiQDs (without a protein corona) are able to cross cell
membranes more easily and eﬃciently than treated SiQDs. This
statement was subsequently proven by means of uorescenceimaging (Fig. 7a) which revealed the presence of SiQD 1100 in
the cell cytoplasm but, surprisingly, that it was localized
diﬀusely rather than in the form of whole spots as in the case ofThis journal is © The Royal Society of Chemistry 2016

standard cultivation conditions with the presence of FBS. It
would appear that the way in which SiQD 1100 enter cells varies
under diﬀerent conditions (with or without a protein corona),a fact that might be studied in greater detail in the future. SiQD
1050 required a longer incubation time (24 hours) before they
entered the cells under serum-free conditions; importantly,
however, the eﬀect was signicantly stronger – all the concentrations of SiQD 1050 tested were found to be cytotoxic aer 24hours (Fig. 5d). Both SiQDs in the serum-free medium were seen
to harm the cells earlier and at a lower concentration thanunder fully-supplemented medium conditions. It has alreadybeen mentioned that nanoparticles lower their surface freeenergy via strong non-specic interaction with cell membranes.
However, the amount of free energy is lowered by the presenceof a protein corona and both the degree of adhesion to themembrane and cell uptake are reduced.41,42 Thus the observed
eﬀect of proteins bound to SiQDs inducing a delay in the onsetof cytotoxic eﬀects is not surprising and is in agreement withprevious observations.43,44
SiQD 1100 were detected within the cells under both test
conditions – both with and without proteins originating from
FBS; however, it was found that without proteins they enter thecells more rapidly, are distributed diﬀusely and have a stronger
negative eﬀect on metabolic activity. On the other hand, SiQD
1050 were not detected in the cells by means of uorescencemicroscopy under either of the test conditions; however, thecytotoxic eﬀect thereof was determined (Fig. 8b) which may
indicate their entry into the cells. Moreover, the cytotoxic eﬀect
was found to be stronger under non-FBS protein presence
conditions. This may imply the easier cell entry of SiQD 1050
without a protein corona. However, they were not observable via
uorescence microscopy images. We suspect, as PL aging
studies suggest (Fig. 2c), that the PL of these SiQDs degrades toa signicant extent in aqueous media (i.e. in the natural biological experiment environment). Moreover, it should be noted

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RSC Advancesthat SiQD 1050 exhibit three times lower PL EQY and a slightly
lower absorption cross-section, which renders them morediﬃcult to observe by means of uorescence microscopy(Fig. 6b and d).
The formation and structure of the protein corona is crucial
in terms of the fate of all nanoparticles located in livingorganisms as has been demonstrated by a large number ofprevious studies.13,35,36,45 Bare nanoparticles are unable tosurvive within a biological system since they are immediately
covered by a layer of proteins from the uid which forms thehard protein corona and which continues to grow until theyattain a stable state, which is oen a long-term process asobserved in the case of SiQD 1050. Experiments conducted prior
to in vivo application should always mimic the environment of
the organism as precisely as possible. In vitro dose-dependent
tests of nanoparticles should be performed using biologicaluids only before forming conclusions on the cytotoxicity of thematerial. As was apparent especially in the case of SiQD 1050,
the presence of bare SiQDs or protein-coated SiQDs in the cell
culture makes a huge diﬀerence in terms of cytotoxicity and theimmense aggregation of SiQD 1050 with proteins leads to fatal
consequences for organisms.

4 Conclusion
We attempted to demonstrate to what extent novel co-doped
SiQDs react upon being introduced into a natural biologicalenvironment consisting of human osteoblasts and a cell culture
medium with or without the addition of fetal bovine serum. A
series of experiments was conducted at various concentrationlevels of two types of SiQD (1100 and 1050), an evaluation wasmade of their cytotoxicity and their localization within the cell
culture was assessed by means of uorescence wide-eld andconfocal microscopy.
The detailed luminescence characterization of the SiQDs in
colloidal suspensions at diﬀerent times following fabrication as
well as of the SiQDs inside cell cultures and water-based media
enabled the research team to uncover the continuous changes
which took place due to the aging of the SiQDs (a shi in the PL
peak to shorter wavelengths and related EQY changes, Fig. 2). A
detailed knowledge of PL aging allowed us to select the
optimum type of SiQD (i.e. SiQD 1100) for uorescence imaging
purposes in cells with an optimal PL peak of around 750 nm andan ensemble EQY of 12%.
Zeta potential measurement indicated that the tested SiQDs
diﬀer from each other not only in terms of their size and PL
properties but also with respect to zeta potential values.
Consequently, the formation of a protein corona diﬀers on thesurface of SiQDs, which aﬀects the reaction with the biologicalcomponents of the cell culture medium and overall cytotoxicity.
SiQD 1050 in particular were almost completely entrapped
within growing aggregates of proteins which hindered theiraccess into the cells which, in turn, led to the cytotoxicity of
SiQD 1050 appearing to be relatively low. However, the realextent of cytotoxicity was revealed under serum-free conditions
indicating that a high amount of SiQD 1050 enter cells despite
the fact that they become undetectable by uorescence

63412 | RSC Adv., 2016, 6, 63403–63413

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microscopes (due to a PL shi to shorter wavelengths withstrong cell autouorescence and the degradation of PL yield).
On the other hand, SiQD 1100 exhibited a low level of interaction with proteins which enabled cell incorporation even in theserum-supplemented medium. Thus, these particles are able toenter cells in the bare state as well as with the addition ofa protein corona although in each case the pathways mostprobably diﬀer.
The results provide important ndings concerning the invitro toxicity of novel co-doped SiQDs and enhance our understanding of the complexity of processes acting between SiQDs
and biological environments.

Acknowledgements
This study was supported by the project LH14246 Kontakt II of
Ministry of Education, Youth and Sports of the Czech Republic
and by the Visegrad Group (V4)-Japan Joint Research Programon Advanced Materials (project NaMSeN) and by the project of
National Sustainability Program I No. LO1503. This study was
also supported by Charles University in Prague, First Faculty of
Medicine (project PRVOUKP24/LF1/3) and Faculty of Medicine
in Pilsen (SVV 260 279/2016).

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