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Bioconjugação OH
Bioconjugação OH
Bioconjugação OH
Journal of
Materials Chemistry B
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PAPER
spectroscopy, UV-visible spectroscopy and spectrouorometry have been used to characterize the crystal
structure, the doping status, and the optical properties of the doped-dots. Folic acid (FA) was linked to
TG-capped Mn:ZnS nanocrystals to produce Mn:ZnS@TG-FA nanobioconjugates that were used for
DOI: 10.1039/c2tb00247g
targeted in vitro delivery to a human cancer cell line. Folate receptor mediated cellular uptake of FA-
www.rsc.org/MaterialsB
Introduction
Universite de Lorraine and CNRS, Laboratoire Reactions et Genie des Procedes (UPR
3349), 1 rue Grandville, BP 20451, 54001 Nancy, France. E-mail: raphael.schneider@
univ-lorraine.fr; Tel: +33 3 83 17 50 53
b
c
Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30 Street,
60-780 Poznan, Poland
d
Institut de Science des Materiaux de Mulhouse LRC 7228, 15 rue Jean Starcky, 68093
Mulhouse, France
Electronic supplementary
10.1039/c2tb00247g
information
(ESI)
available.
See
DOI:
Paper
Mn:ZnS nanocrystals with good photoluminescence quantum
yield (up to 13.2%), with pure dopant emission and without
shell introduction. To the best of our knowledge, there is no
report of such good quality Mn:ZnS d-dot preparation in
aqueous solution without shell deposition on the doped core.
Furthermore, we have chosen to decorate the surface of the dots
with a tumor-specic ligand, folic acid (FA), by taking advantage
of the functional polyhydroxyl shell of Mn:ZnS@TG nanocrystals. Folic acid was used as the targeting ligand for this
study because the a-folate receptor (FR) is observed to be upregulated in various types of human cancers, while it is only
minimally distributed in normal tissues.29,30 FA, a high anity
ligand to FR, is internalized into the cell through the receptor
mediated endocytosis even when conjugated with a wide variety
of molecules or nanoparticles.28,31,32 The uptake of FA-conjugated Mn:ZnS d-dots into T47D breast cancer cells was
conrmed by confocal imaging under biphotonic excitation. In
addition, the toxicity of the dots to human model cells was
assessed.
2.1
Materials
Characterization of d-dots
Cell cultures
In vitro cytotoxicity
Paper
of concentration. The experimental plate included wells without
cells (in order to exclude direct reaction between MTT and ddots), wells with cells treated with d-dots and wells of untreated
cells. Each treatment was performed in triplicate.
XTT assay: an XTT cell proliferation assay kit was purchased
from Cayman Chemical and used to study cell proliferation
according to the manufacturer's protocol. The assay is based on
the extracellular reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide
(XTT) by a reduced form of nicotinamide adenine dinucleotide
(NADH) produced in the mitochondria via trans-plasma
membrane electron transport and an electron mediator.
Reduction of XTT produces a water-soluble formazan which
dissolves directly into the culture medium, eliminating the
need for an additional solubilization step. Immediately before
use the Electron Mediator Solution was used to reconstitute the
entire vial of XTT Reagent and mixed well.
The experimental plate included wells without cells (in order
to exclude direct reaction between XTT and d-dots), wells with
cells treated with d-dots and wells of untreated cells. Each
treatment was performed in triplicate.
T47D human breast cancer cells were seeded in 96-well
microtiter plates at a density of 2 104 cells per well and led to
growth for 24 h in a CO2 atmosphere at 37 C. Aer this time
dierent concentrations (0.085 mM) of Mn:ZnS@TG and
Mn:ZnS@TG-FA d-dots in culture medium were added. Aer
72 h, 10 mL of the reconstituted XTT mixture to each well were
added. The microtiter plates were gently shaken and then
incubated for two hours at 37 C in a CO2 atmosphere. The
plates were gently shaken for 1 minute and absorption of each
well was measured using a microplate reader at a wavelength of
450 nm.
3.1
We rst studied the synthesis and the optical properties of Mndoped TG-capped ZnS nanocrystals. The synthetic conditions
were found to play a critical role in determining the PL properties of the resulting Mn:ZnS@TG d-dots. First, the reaction
temperature for the synthesis inuences the PL QY of the d-dots
markedly. The experimental results showed that to obtain
nanocrystals with good optical properties, the optimal reaction
temperature was 100 C. If the temperature was too low (60 or 80
C), the PL intensity (normalized to the same absorption at the
excitation wavelength) of the Mn2+ emission at ca. 585 nm of the
resulting d-dots was signicantly lower than that obtained at
100 C. Too high temperature (130 or 150 C), obtained by
heating the reaction mixture in a sealed Teon tube, led to the
formation of poorly water-dispersible and large-sized nanocrystals exhibiting only a surface defect emission located at ca.
450 nm. In our case, this situation probably originates from
partial decomposition of TG at high temperature leading to
dangling bond states on the d-dots surface due to incomplete
surface passivation by the ligand.36,37
Initial experiments performed with 4% Mn2+ relative to Zn2+
and a ca. 0.19 M Zn2+ solution showed that good quality d-dots
Paper
were obtained when using Na2S in short supply (by ca. 0.9 fold)
relative to Zn2+. A decrease or an increase of the Zn2+/S2 ratio
yielded Mn-doped nanocrystals with poor PL eciency
(increase of the blue emission located at ca. 450 nm at the
expense of the orange emission originating from the 4T1 / 6A1
transition within the 3d shell of the Mn2+ ion). Aer injection of
Na2S, a strong orange emission evolved over a matter of hours,
reached its maximum intensity aer ca. 20 h of heating at
100 C, and remained stable if the heating time was
increased (Fig. 1a).
As shown in Fig. 1b, the Zn2+ precursor also has an inuence
on the optical properties of the Mn:ZnS@TG d-dots. The highest
PL QY (13.2%) was obtained with Zn(OAc)2. The use of ZnSO4 or
Zn(NO3)2 yielded d-dots with lower PL QYs, 11 and 2.5%,
respectively. It should also be pointed out that the purity and
the crystallinity of the d-dots prepared from Zn(OAc)2 were
higher than those prepared from ZnSO4 or Zn(NO3)2 as shown
by X-ray diraction (XRD) studies (data not shown). Finally, it
can be observed from Fig. 1b that when varying Zn2+ precursors
from Zn(OAc)2 to ZnSO4 and Zn(NO3)2, 4T1 to 6A1 luminescence
of Mn2+ ions shis from 585 to 590 and 593 nm, respectively.
Due to the limited degree of doping in Mn:ZnS@TG nanocrystals, MnMn interactions should be weak and therefore PL
emission shis should not be dependent on the Mn2+ concentration.38,39 It is more likely that the variations of the PL emission wavelengths observed in Fig. 1b are related to the
distribution of Mn2+ ions in the dots. Indeed, for the nucleation
or growth doping strategy, lattice diusion on Mn2+ dopants
plays an important role in PL intensity and PL emission wavelengths.40 This diusion into the host layer generates isolated
dopant centers within the ZnS host lattice, which could yield
highly emissive d-dots and seems to increase under our
synthetic conditions using Zn(OAc)2 compared to ZnSO4 and
Zn(NO3)2.
An investigation of the inuence of the Mn2+ concentration
on the PL eciency of Mn:ZnS@TG dots was also carried out.
Indeed, when the concentration of Mn2+ dopant ions exceeds a
certain threshold, the non-radiative energy transfers between
neighboring Mn2+ dopant ions can reduce and even annihilate
the PL.4143 A blank sample prepared without Mn2+ showed only
the ZnS-related trap emission at ca. 450 nm. As soon as Mn2+ is
incorporated in the ZnS nanocrystals, the intensity of the blue
emission decreased and Mn2+ emission at 585 nm comes up,
thus indicating that the energy transfers between the ZnS host
and the Mn2+ dopant is eective. With increasing the Mn/Zn
ratio from 2 to 4%, the PL QY gradually increased from 5.2 to
13.2% (Fig. 1c). Interestingly, a slight blue shi of the Mn2+
emission from 595 to 590 nm was observed during these variations of the Mn/Zn ratio. This blue shi may be ascribed to the
decrease of the symmetry of the crystal eld around the Mn2+
center in the ZnS host.18,40 Finally, above a Mn/Zn ratio of 4%, a
decrease of the PL intensity was observed probably due to the
formation of pairs of Mn2+ dopant ions.
Fig. 1d shows absorption, PL excitation, and PL emission of
Mn:ZnS@TG d-dots. A strong 585 nm ion PL band with a fullwidth at half-maximum of ca. 60 nm and a large Stokes shi of
ca. 270 nm arising from 4T1 / 6A1 transition from Mn2+ in the
This journal is The Royal Society of Chemistry 2013
Fig. 2
Paper
Fig. 3 Absorption (black), PL excitation (red), and PL emission spectra of FAconjuagted Mn:ZnS d-dots.
Fig. 4
Paper
Fig. 5 TEM images of (a) Mn:ZnS@TG and (b) Mn:ZnS@TG-FA d-dots; (c) and (d) are the corresponding size distributions, and (e) powder X-ray diraction patterns of
the nanocrystals.
Fig. 6 Confocal uorescence of T47D cells treated with (a) JC1, (b) Hoechst 33342, and (c) Mn:ZnS@TG-FA nanocrystals, (d) is the overlay of uorescence images. Scale
bars 25 mm. Confocal microscopy images (b) and (c) were obtained with laser excitation at 800 nm, while image (a) was recorded after excitation at 488 nm.
Paper
Mn:ZnS@TG d-dots aer biphotonic excitation. Fig. 6a and b
show the Hoechst and JC1 uorescence in the cells. The same
eld visualized showed internalized orange emission from FAconjugated d-dots (Fig. 6c). When the uorescence pattern of
the FA-conjugated dots was merged with the staining pattern of
Hoechst and JC1, it was evident that Mn:ZnS@TG-FA d-dots
appear to accumulate in the cytoplasm within the cells, thus
suggesting endocytotic uptake (Fig. 6d). To prove that
Mn:ZnS@TG-FA dots are specically targeted through the FA
FRa interaction, two control experiments were conducted. First,
cells were uncubated with unconjugated dots (see ESI, Fig. S9).
The second control experiment involved saturating the cells
with free FA for 2 hours in order to block the available FR on the
cell surface, followed by treatment with Mn:ZnS@TG-FA dots
(see ESI, Fig. S10). In both cases, minimal signal from the cells
was observed, thus conrming that the uptake of the dots
occurred predominantly via the FAFR interaction.
3.4
Fig. 7 Eect of d-dots concentration on the viability of T47D cells evaluated via
MTT assay. (a) Mn:ZnS@TG nanocrystals and (b) Mn:ZnS@TG-FA nanocrystals.
d-dots were 3.9 0.5 and 4.2 0.7 nm, respectively (Fig. 5ad).
The corresponding selected area electron diraction patterns
(insets of Fig. 5a and b) can be indexed to a zinc blende cubic
phase, this is in accordance with the results of powder X-ray
diraction (XRD) patterns. XRD spectra recorded on
Mn:ZnS@TG and on Mn:ZnS@TG-FA powders recorded aer
centrifugation and drying are shown in Fig. 5e. The diraction
peaks of both samples perfectly match the (111), (220), and
(311) crystalline planes of the cubic ZnS phase with very
broadened peaks indicating the formation of very small nanocrystals (JCPDS no. 05-0566). No characteristic peaks of any
foreign phase or manganese impurities were detected. In
addition, we can see that doping Mn2+ into the host ZnS
nanocrystals does not bring about a phase transformation on
the crystal structure.
3.3
Evaluation of cytotoxicity
Fig. 8 Eect of d-dots concentration on the viability of T47D cells evaluated via
XTT assay. (a) Mn:ZnS@TG nanocrystals and (b) Mn:ZnS@TG-FA nanocrystals.
Paper
modication of Mn:ZnS dots was found to increase the uptake
of these nanocrystals by cells that overexpressed the a-folate
receptor. As seen in Fig. 7b, FA-capped dots are more toxic than
the uncapped dots at all dosages probably due to higher intracellular concentrations of these nanocrystals (by ca. 45 fold).
IC50 value determined for Mn:ZnS@TG dots was higher than
5000 mM, while that of Mn:ZnS@TG-FA d-dots was lower.
XTT
(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assays were also conducted to measure
the mitochondria activity and quantify the cell death aer
exposure of T47D cells to Mn:ZnS dots (Fig. 8). As previously
observed in the MTT assays, the toxicity was increased for the
FA-modied dots (Fig. 8b), which correlated with the aforementioned enhanced cellular uptake.
Finally, MTT and XTT assays of cell viability studies show
that Mn:ZnS d-dots demonstrate low cytotoxicity and can be
used at a high concentration for imaging or other biomedical
applications.
Conclusion
Paper
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