The synthesis of rare earth metal-doped upconversion nanoparticles coated with d-glucose or 2-deoxy-d-glucose and their evaluation for diagnosis and therapy in cancer

Article abstract of DOI:10.1039/d0nj00666a

Rare earth metal-doped upconversion nanoparticles (UCNPs) are emerging as a new class of biomedical imaging materials due to their higher energy anti-Stokes shift, high optical penetration depth and long term repetitive imaging. In the present study, upconversion nanoparticles based on NaYF4 doped with thulium (Tm) and ytterbium (Yb) were prepared via a thermolysis method using oleic acid as a capping agent and 1-octadecene as a solvent. The X-ray diffraction pattern of the synthesized nanoparticles was found to match the standard hexagonal phase. The nanoparticles were coated with silica using tetraethyl orthosilicate (TEOS) and in order to avoid agglomeration, IGEPAL CO-520 was used as the surfactant. The coatings of SiO2 over NaYF4 were confirmed by the TEM image and XRD pattern. NaYF4@SiO2 was further functionalized by the addition of (3-aminopropyl)trimethoxysilane (APTMS) followed by either d-glucose or 2-deoxy-d-glucose (2-DG). UCNPs-d-glucose and UNCPs-2DG were examined for cell viability (MCF-7 cells) by MTT assay. The cellular uptake of UCNPs in MCF-7 cells was seen in terms of emission of a blue light. Furthermore, the uptake rate of UCNPs coated with 2-deoxy-d-glucose was found to be much faster than that of UCNPs alone under d-glucose starved conditions. The functionalization of UCNPs with 2-deoxy-d-glucose (2-DG) not only increased the uptake of nanoparticles, but also blocked the glycolysis pathway resulting in the inhibition of tumor growth as 2-deoxy-d-glucose (2-DG) is mimicking the d-glucose. The results are indicative that these upconversion nanoparticles may find applications in bio-imaging, removal of tumor by precision surgery, therapy and targeted drug delivery. This journal is

Full text of DOI:10.1039/d0nj00666a

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Synthesis of rare earth metals doped upconversion nanoparticles coated
DOI: 10.1039/D0NJ00666A
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with D-glucose or 2-deoxy-D-glucose and their evaluation for diagnosis and
therapy in cancer
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K. Shitaljit Sharma,a Maikho Thoh,b Akhil K. Dubey,c Prasad P. Phadnis,a,d* Deepak
Sharma,b,d, Santosh K. Sandur,b,d and Rajesh K. Vatsaa,d*
a
Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, India,
Email: phadnisp@barc.gov.in, Tel.: +91 22 2559 2229; rkvatsa@barc.gov.in; Tel.: +91 22
2559 2438, Fax: +91 22 2550 5151
b Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai-
400 085, India
c Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
d Homi Bhabha National Institute, Anushaktinagar, Mumbai-400 094, India
Abstract
Rare earth metal-doped upconversion nanoparticles (UCNPs) are emerging as a new
class of biomedical imaging materials due to their higher energy anti-stokes shift, high optical
penetration depth and long term repetitive imaging. In the present study, upconversion
nanoparticles based on NaYF4 doped with thulium (Tm) and ytterbium (Yb) were prepared
by thermolysis method using oleic acid as a capping agent and 1-octadecene as a solvent. The
X-ray diffraction pattern of synthesized nanoparticles was found to be matching with the
standard hexagonal phase. The nanoparticles were coated with silica using tetraethyl
orthosilicate (TEOS) and in order to avoid agglomeration, IGEPAL CO -520 was used as the
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surfactant. The coatings of SiO2 over NaYF4 were confirmed by TEM imagDeOIa: 1n0d.103X9/RD0DNJ00666A
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pattern. NaYF4@SiO2 was further functionalized by addition of (3-
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aminopropyl)trimethoxysilane (APTMS) followed by either D-glucose or 2-deoxy-D-glucose
(2-DG). UCNPs-D-Glucose and UNCPs-2DG were examined for cell viability (MCF-7 cells)
by MTT assay. The cellular uptake of UCNPs in MCF-7 cells was seen in terms of emission
of blue light form. Further, the uptake rate of UCNPs coated with 2-deoxy-D-glucose was
found much faster than that of UCNPs alone under D-glucose starved conditions. The
functionalization of UCNPs with 2-deoxy-D-glucose (2-DG) not only increased the uptake of
nanoparticles, but also blocked the glycolysis pathway resulting in inhibition of tumor growth
as 2-deoxy-D-glucose (2-DG) is mimicking the D-glucose. The results are indicative that
these upconversion nanoparticles may find applications in bio-imaging, removal of tumor by
precision surgery, therapy and targeted drug delivery.
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Keywords: Monodispersed, Upconversion Nanoparticles, Warburg effect, 2-deoxy-D-
glucose, Bio-imaging, 980 nm laser etc.
1. Introduction
Rare earth metal-doped upconversion nanoparticles (UCNPs) are luminescent
bioimaging agents having higher energy anti-Stokes emission formed by sequential
absorption of multiple photons through the use of ladder-like energy levels of trivalent
lanthanide ions tightly held by an appropriate inorganic host lattice.1 Generally upconversion
process consists of sequential absorption of two or more low-energy excitation photons of
NIR light, resulting in the emission of shorter wavelength (e.g., NIR, visible and UV) with
the efficiency that is much higher than that of nonlinear multi-photon absorption, under low-
cost continuous-wave (CW) diode laser excitation. Use of UCNPs in bioimaging has many
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advantageous over the existing fluorophores due to zero auto-fluorescence background which
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increases the signal-to-noise ratio.2 Properties like emission with large anti-Stokes shift as
compared to the excitation wavelength, sharp emission and high resistance to photo bleaching
of UCNPs allow ease and suitable long-term repetitive imaging.3 Some of the promising
applications of UCNPs are differentiating or visualization of diseased area at the time of
precision surgery,4 delivery or release of therapeutic entities to the treatment site5 and
activation of certain compounds like photosensitizer for photodynamic therapy.6 Utilization
of UCNPs for biological applications also included surface functionalization that decides the
behavior of UCNPs in biological systems.7 By carefully choosing the functional groups to be
functionalized on the surface of UCNPs, it can be used for diagnosis, treatment or both for
many types of human diseases.
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Cancer is the major cause of mortality around the globe and the five year survival rate
of some of the cancer is just 15%.8 The poor survival of cancer patients is due to diagnosis of
cancer in advanced stage and recurrence after certain period of time.9 In addition, the
techniques used for diagnosis of cancer are expensive and sometimes time consuming.10
Several techniques are being used in the clinic for diagnosis of cancer. One of the effective
techniques is positron emission tomography (PET) where 18F-2-deoxy-D-glusoe (FDG) is
used as a tracer.11 The technique is based on the differential uptake of glucose between tumor
and normal cells. Usually tumor cells have elevated glucose uptake than the normal cells,
known as Warburg effect. For example, FDG has been taken up preferentially by the tumor
cells giving a very bright spot of the tumor region on the image. But this technique has
several limitations due to the use of radioisotope.12
After diagnosis of cancer, proper treatment modalities are assigned for most of the
cases, either removal of the tumoral region by precision surgery is performed, along with
radiotherapy or administration of chemotherapy drugs.13 The major limitation is survival of
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resistant cells (cancer stem cells) or leaving behind few tumor cells that can lead to
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recurrence.14 To overcome this limitation, strategies which can precisely distinguish the
tumoral region should be employed during the surgery. Use of UCNPs functionalized with D-
glucose (DG) could be a good strategy as uptake of D-glucose by cancer cells is twenty-fold
higher than that of the normal cells.15 Also, the hydroxyl groups on the surface of UCNPs
functionalized with D-glucose can be used to load certain drugs by forming ester linkage or
silyl ether linkage.16
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Considering these facts, we have prepared monodispersed NaYF4 nanoparticles doped
with rare earth elements. We noted from literature, various methods have been employed for
synthesis of NaYF4 based UCNPs exhibiting versatile applications.17 Taking synthetic
guidelines from literature and optimizing the composition and reaction conditions, we have
prepared of Tm and Yb doped NaYF4 UCNPs. They were coated with silica using tetraethyl
orthosilicate
(TEOS)
and
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by
functionalization
with
(3-
aminopropyl)trimethoxysilane (APTMS) giving an amine group functionalization on the
surface. Analogues of D-Glucose and 2-deoxy-D-glucose (2-DG) were prepared with slight
modification of the protocol from the literature. These compounds were conjugated with the
amine functionalized UCNPs through amide or thiourea linkage. The presence of D-glucose
or 2-DG on the surface of the nanoparticles was confirmed by FT-IR and elemental analyses.
The cytotoxicity and uptake studies of the said D-glucose and 2-DG functionalized UCNPs
were performed in human breast cancer cells (MCF-7 cell lines).
2. Experimental
2.1
Materials and methods
The chemicals and solvents required for this study, viz., paraformaldehyde, D-
glucose, 2-deoxy-D-glucose, yttrium acetate, thulium acetate, ytterbium acetate, oleic acid, 1-
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octadecene, sodium hydroxide, ammonium hydrogen fluoride, IGEPAL CO-520, (3-
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aminopropyl)trimethoxysilane (APTMS), tetraethyl orthosilicate (TEOS), HBr (33%) in
acetic acid, allyl alcohol, silver carbonate, benzoyl chloride, pyridine, zinc metal, ammonium
chloride, Dowex 500 resin, lithium bromide, potassium bromide, silver nitrate, potassium
thiocyanate and N, N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP)
were purchased from Sigma Chemical Co (St. Louis, MO, USA). Hoechst 33342 was
purchase from Molecular Probes Life Technologies (Eugene, OR, USA). Trypsin-EDTA,
fetal bovine serum, antibiotic solution and DMEM were purchased from Himedia
Laboratories (Mumbai, India). Acetic anhydride was purchased from Spectrochem (Mumbai,
India). All other chemicals used in our studies were obtained from reputed manufacturers and
were of analytical grade. All solvents were distilled and dried following the standard
protocols. The NMR spectra were recorded on the Agilent/ Varian 600 MHz NMR
spectrometer with a Unity Inova console operating at 599.88 (1H), 150.84 and (13C{1H}). The
1H and 13C{1H} NMR chemical shifts were relative to internal CHCl3 peak from CDCl3
solvent. The powder X-ray diffraction (XRD) patterns were recorded on a Pan analytical
X′pert PRO X-ray diffractometer using Cu-Kα radiation in the 2θ range of 10-70° with step
size and time of 0.02° and 1.20 s, respectively. XRD patterns were analyzed by comparing
with reported data for the end member compositions (JCPDF files). The transmission electron
microscopic (TEM) image was recorded on Libra-120 plus TEM (Carl Zeiss, Germany)
operated at 120 kV as the accelerating voltage. TEM images were analyzed by using Image J
software taking 100 nanoparticles statistically. For internalization studies, the images of
adherent cells were taken using phase contrast microscope (Olympus, Japan). The
cytotoxicity studies were performed using a flow cytometer and its results were analyzed
using FlowJo software.
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2.2 Synthesis
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2.2.1 Synthesis of monodispersed NaYF4 nanoparticles doped with rare earth metalViioewnAsrticle Online
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The synthesis of the monodispersed NaYF4 nanoparticles was done with a slight
modification from the reported literature. Briefly, yttrium acetate (1 g, 3.75 mmol), thulium
acetate (64.8 mg, 0.18 mmol), ytterbium acetate (263 mg, 0.75 mmol) were dissolved in a
solution of oleic acid (5 ml) in 1-octadecene (30 ml) contained in a three-necked flask under
an inert Ar atmosphere. Subsequently, sodium hydroxide (600 mg, 15 mmol) and ammonium
hydrogen fluoride (555 mg, 15 mmol) dissolved in methanol were added and heated to 100
°C for 1 h. After methanol was evaporated, a yellow liquid residue was obtained. The residue
was further heated to 300 °C with the heating rate of 5 °C per minute. The reaction mixture
was heated for 2 h and then cooled to the room temperature. The nanoparticles were
precipitated with acetone, dispersed in a minimum amount of hexane and then re-precipitated
with acetone. The process was repeated thrice. The obtained nanoparticles were characterized
by X-ray diffraction (XRD) analyses, transmission electron microscopy (TEM) and
photoluminescence spectrophotometry.
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2.2.2 Synthesis of NaYF4@SiO2-NH2 (UCNPs-NH2)
Cyclohexane (20 ml) and IGEPAL CO-520 (0.5 g) were added to conical flask (50
ml) and stirred for 15 min. To this solution, UCNPs (20 mg in 10 ml of cyclohexane) were
added and stirred for 20 min, followed by the addition of aq. ammonia solution (30 % 500
µl). After stirring for additional 20 min, TEOS (200 µl) was added at an interval of 12 h to 48
h. (3-aminopropyl)trimethoxysilane (APTMS) (200 µl) was added and stirred for additional
24 h. Then methanol (10 ml) was added for phase separation between cyclohexane and
methanol. The particles were collected from methanol layer by centrifugation. The particles
were characterized with TEM and FT-IR.
2.2.3. Functionalization of UCNPs with D-glucose or 2-deoxy-D-glucose
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For the functionalization of NaYF4@SiO2-NH2 with either D-glucose or D2O-ID: 1e0.o10x3y9/-DD0N-J00666A
glucose, the respective glucose derivatives viz., carboxy glucose i.e., 2-[(2,3,4,6-tetra-O-
acetyl-D-glucopyranosyl)oxy]acetic acid (Suppl. Info. compound 3) or isothiocynate glucose
i.e., 3, 4, 5-tri-O-benzoyl-2-deoxy-D-glycopyranosyl isothiocyanate (Suppl. Info. compound
8) were synthesized and characterized (Suppl. Info. Scheme S 1 and S 2). They were further
reacted as given below.
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2.2.4. Functionalization of amine functionalized UCNPs by D-glucose
To the carboxyl glucose (100 mg) dissolved in dry dimethylformamide (DMF, 5 ml)
and treated with DCC (61 mg) for 30 min. at 0 °C, amine functionalized UCNPs were added
and stirred for overnight. Catalytic amount of 4-dimethylaminopyridine (DMAP) was added.
After stirring for overnight, the UCNPs were washed with acetone and transferred to a round
bottle flask, containing aq. ammonia solution (33%, 10 ml). The UCNPs were collected by
centrifugation, washed with ethanol, dried under vacuum and characterized with FT-IR and
elemental analyses.
2.2.5. Functionalization of amine functionalized UCNPs by 2-deoxy-D-glucose
The 3, 4, 5-tri-O-benzoyl-2-deoxy-D-glycopyranosyl isothiocyanate (150 mg) was
dissolved in dry DMF (5 ml) and amine functionalized UCNPs were added. The reaction
mixture was heated at 60 °C for 8 h. The nanoparticles were collected by centrifugation and
washed with acetone for three times. The nanoparticles were dispersed in the aq. ammonia
solution (33%, 15 ml) and stirred for overnight at room temperature. The nanoparticles were
collected by centrifugation, washed with water followed by acetone and characterized by FT-
IR.
2.2.6. Labeling of nanoparticles by FITC for internalization study
The labeling of UCNPs with FITC was done first on amine functionalized UCNPs.
Using these nanoparticles, further functionalization with D-glucose or 2-deoxy-D-glucose
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moieties was performed. Briefly, the amine functionalized UCNPs (50 mg) wereDdOiIs:p10e.1r0s3e9d/D0inNJ00666A
dry dimethyl sulphoxide (DMSO, 5 ml). 100 μl of FITC in DMSO (500 μg/ml) was added
and stirred for overnight. UCNPs were collected with centrifugation, washed with water and
then transferred to dialysis tubing and placed in dialysis buffer containing 0.9 M saline. The
buffer was periodically changed after every 12 h till 48 h. The FITC functionalized UCNPs
were collected by centrifugation then washed with acetone and dried under reduced pressure.
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2.3.
Cell culture
MCF-7 cells were procured from Merck Millipore and maintained as monolayer using
DMEM media supplemented with 10% fetal bovine serum at 37 °C with 5% CO2 in a
humidified environment. For every experiment, seeding of cells was carried out one day
ahead of the experiment.
2.4.
Cytotoxicity study by MTT assay
MTT assay was performed in accordance with the standard protocol.18 Briefly, MCF-
7 cells (5000 cells per well) were seeded in a 96-well plate and treated with UCNPs
functionalized with D-glucose or 2-deoxy-D-glucose as indicated for 48 h. MTT solution (10
µl; stock solution = 5 mg/ml) was added 3 h prior to addition of DMSO. 100 µl DMSO was
added to each well and mixed using a pipette to fully dissolve the formazan crystals. The
absorbance was measured at 590 nm using a plate reader.
2.5.
Internalization study
MCF-7 cells (5 x 105 per well) were seeded in 6-well plate and treated with UCNPs
for 12 h. The cells were washed with PBS to remove the trace of UCNPs that were not
internalized. The images of the cells were captured using phase contrast microscope in the
presence and absence of 980 nm laser irradiation exposure. For flow cytometry studies,
MCF-7 cells were treated with UCNPs or UCNPs-2-DG labeled with FITC as indicated.
Cells were harvested by trypsinization, washed, fixed with 2% formaldehyde for 20 min and
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then stained with Hoechst. Cells were acquired on flow cytometer and analyzed uDsOinI:g10.F10l3o9w/DJ0oNJ00666A
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3. Results and Discussion
The NaYF4 based upconversion nanoparticles doped with Tm and Yb, have been
synthesized (vide experimental). The percentage of the dopants are 16% ytterbium and 4%
thulium of the corresponding yttrium (80%), hence these UCNPs can be as represented by
NaY0.8Yb0.16Tm0.4F4 formula (section 2.2.1.). Their synthesis was done by thermolysis
method using oleic acid as the caping agent and 1-octadecene as the solvent. The XRD
pattern of synthesized nanoparticles was found to be matching with the standard hexagonal
phase (JCPDF 28-1192; Figure 1 A). In the same XRD pattern we observed a broad peak at
2θ = 20° (Figure 1 A) which is attributable to oleic acid. The long chain organic molecules
show very broad amorphous peaks in XRD patterns. From literature it is evident that the
XRD pattern of the oleic acid coated nanoparticles exhibit this peak and after removing oleic
acid, the broad peak corresponding to oleates was not observed.20 Further, the morphology of
the synthesized UCNPs was studied using TEM and the UCNPs were found to be uniform in
shape and size. The average size of the nanoparticles is in the range of 24 1 nm with the
cuboid shape (Figure 1 B).
Figure 1. Characterization of NaYF4: Tm, Yb by X-ray diffraction analysis (A), TEM image
captured using Jeol TEM (B), the particle distribution (inset B) and Selected area
electron diffraction pattern (C).
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The selected area electron diffraction pattern further confirmed that theDsOyI:n10th.10e3s9i/zDe0dNJ00666A
UCNPs were crystalline (Figure 1 C). As the synthesized nanoparticles were found to be
hydrophobic due to the presence of oleic acid on the surface, the nanoparticles were
dispersible into the nonpolar solvent like chloroform or hexane. In order to use these
nanoparticles for biological applications, the particles should be dispersible in water.21 In
order to make the particles water dispersible, the nanoparticles were coated with silica using
tetraethyl orthosilicate and agglomeration was anticipated to be avoided by using IGEPAL
CO-520 as the surfactant which helps in formation of uniformly silica coated nanoparticles
and avoid the agglomeration. But some aggregation of uniformly silica coated nanoparticles
was seen in TEM image (Figure 2 B). The reason for this agglomeration is sonication process
or uneven mixing while preparing the sample for TEM. In this process, the interparticle
collisions occur under the influence of ultrasonication which leads to agglomeration which
results in changing the morphology of the particles.22 The coating of the nanoparticles with
SiO2 was confirmed by TEM (Figure 2 B). Due to the difference in the refractive index
between NaYF4 and SiO2, the formation of silica layer above NaYF4 was clearly observed.
The average size of the nanoparticles ranges between 60-65 nm (Figure 2 C). The thickness
of the silica shell is approximately 20-30 nm.
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Figure 2. Characterizations of silica coated NaYF4: Tm, Yb UCNPs by XRD (A), TEM (B)
and the particle size distribution (C).
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The XRD pattern of NaYF4@SiO2 is shown in Figure 2 A. The presence DoOfI:t1h0e.10b39r/oDa0dNJ00666A
peak centered at around 2θ = 23° which attributes to amorphous silica which also confirms
the formation of silica layer on the surface of NaYF4 which is evidenced in the earlier
research reports also.23 Further, the photoluminescence of the synthesized UCNPs was
measured with 980 nm laser excitation and the exhibited emission is shown in Figure 3 A.
The emission spectra of NaYF4: 16 % Yb, 4 % Tm consists of the blue emission bands at
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450.5 and 475 nm which correspond respectively to the G4→ 3H6 and G4→3H6 transitions
of thulium (Figure 3 A). A strong predominant NIR emission at 801.5 nm is attributed to the
1G4→3H5 transition.24
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For functionalization of the synthesized UCNPs, amine functional groups were
introduced on the surface of UCNPs by treating with (3-aminopropyl)trimethoxysilane
(APTMS). The presence of amine functional groups on the surface of the UCNPs after
treating with APTMS, was confirmed by the FT-IR spectroscopy which exhibited peaks
centered at 1619 cm-1 (N-H bend) 3360 cm-1 (broad, N-H str.) (Figure 3 B).25 The obtained
amine functionalized UCNPs were stored at 0 °C.
Figure 3. Photoluminescence spectrum of UCNPs with 980 nm laser excitation (A); its
expansion (inset A) and FT-IR spectrum of UCNPs: NaYF4: Tm, Yb@SiO2
functionalized with amine (B); its expansion (inset B)
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The synthesis of D-glucose or 2-deoxy-D-glucose compounds was done by either
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acetylation or benzoylation of the hydroxyl groups of glucose, which made the compound
hydrophobic, allowing the reactions to be performed in non-polar organic solvents.
Bromination at the acetal carbon (C-1) was done by substitution of the acetyl group. This was
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confirmed by H and C{1H} NMR spectroscopy (Suppl. Info. Figure S 1 and S 2). It was
observed that the bromo derivatives were very reactive and unstable. The synthesized bromo
derivatives were used for next reaction without any further purification.
The bromo-derivative of acetylated glucose was reacted with allyl alcohol using silver
carbonate giving allyl glucosides following the reported method. The formation of allyl
glucoside was confirmed by 1H and 13C{1H} NMR spectroscopy (Suppl. Info. Figure S 3 and
S 4). The terminal double bond of allyl glucoside was converted to carboxylic acid with the
loss of a carbon atom, using sodium periodate and ruthenium chloride. The formation of
carboxyl glucoside was confirmed by H and 13C{1H} NMR spectroscopy (Suppl. Info.
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Figures S 5 & S 6). The carboxyl group was conjugated with the amine functional groups
present on the surface of UCNPs. Using DCC, followed by deacetylation, aq. ammonia
solution (30%). The presence of the glucose moiety on the surface of UCNPs was confirmed
by FT-IR spectroscopy. In FT-IR spectra, after treating amine functionalized UCNPs with
carboxyl glucoside, the peaks at 3320 cm-1 for N-H str. of primary amine and 2929 cm-1 and
2846 cm-1 of C-H str. is suppressed by broad peak of O-H str. centered at 3248 cm-1 (Figure 4
A).
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Figure 4. FT-IR spectra of UCNPs: NaYF4: Tm, Yb@ SiO2 were functionalized with D-glucose
(DG) (A); its expansion (inset A) and 2-deoxy-D-glucose (2-DG) (B); its expansion
(inset B), MTT assay data (C)
On the other hand, when bromo derivative of acetylated glucose was treated with zinc
metal and ammonium chloride, a glycal was formed with the elimination of acetyl group at
C-2. When the resultant glycal was treated with acetic acid, acetic anhydride and HBr under
an inert Ar atmosphere, 2-deoxy-tetra-acetylglucose was generated. This was subsequently,
treated with HBr (33%) in acetic acid to form 2-deoxy-triacetylglucopyranose bromide. This
2-deoxy-triacetylglucopyranose bromide is so reactive that it decomposes with just exposure
to normal air. To circumvent that, benzoylation of D-glucose, instead of acetylation, was
carried out using benzoyl chloride and pyridine in dried dichloromethane (DCM) and similar
procedure for glycal synthesis was performed. The 2-deoxy-tribenzoylglucopyranose
bromide obtained was found to be stable enough for recording 1H and 13C{1H} NMR (Suppl.
Info. Figure S 13 and S 14) and proceeding to the next reaction.
The 2-deoxy-tribenzoylglucopyranose bromide was reacted with freshly prepared
silver thiocyanate formed by reacting silver nitrate and potassium thiocyanate in toluene
under reflux. The 2-deoxy-tribenzoyl isothiocyanate formed (Suppl. Info. Scheme S 2) was
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confirmed by H and 13C{1H} NMR (Suppl. Info. Figure S 15 and S 16). The 2-deoxy-
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tribenzoyl-glucopyranose isothiocyanate was conjugated to the amine functionalized UCNPs
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in dimethyl formamide (DMF) by heating at 60 °C for 8 h.26 The 2-deoxy-
tribenzoylglucopyranose moiety was attached to the surface of UCNPs via thiourea linkage.
The benzoyl protection of 2-DG bound on the surface of UCNPs was removed by treating
with aqueous ammonia solution (25%) (Scheme 1). The presence of 2-deoxy-D-glucose
moieties on the surface of UCNPs was confirmed by FT-IR (Figure 4 B).
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Scheme 1. Functionalization of UCNPs with D-glucose and 2-Deoxy-D-glucose
After functionalization of UCNPs with D-glucose or 2-DG, cytotoxicity was
investigated in human mammary cancer cells (MCF-7) using different concentrations for 48
h. The UCNPs functionalized with either D-glucose or 2-DG, showed marginal loss of cell
survival as compared to control (Figure 4 C).
Cellular internalization of UCNPs was studied by microscopy and flow cytometry
using MCF-7 cells. The cells were seeded in 6-well plate and treated with or without UCNPs
for 12 h. Cells were washed with PBS and observed under microscope. The images were
captured in phase contrast mode or in the presence of 980 nm laser. As the concentration of
UCNPs increased from 29.17 µg/ml to 58.34 µg/ml the emission of UCNPs at blue region
also increased indicating the internalization of UCNPs by MCF-7 cells (Figure 5).
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Figure 5. UCNPs internalization studies: MCF-7 cells (0.2 x 106) were seeded in 6-well plate
and then treated with UCNPs for 12 h. Cells were washed with PBS and images
were taken using phase contrast microscope with or without exposure to 980 nm
laser. Scale barꢀ=ꢀ50ꢀμm.
Labeling of FITC was done on amine functionalized UCNPs and these nanoparticles
were further functionalized with 2-DG moieties on the amine groups. MCF-7 cells were
grown in glucose free media for overnight and treated UCNPs-FITC or UCNPs-2-DG for 10
min, 1 h, 3 h and 6 h. Cells were washed and harvested by trypsinization. Harvested cells
were fixed with 2% paraformaldehyde and then stained with Hoechst 33342. Increase in the
FITC fluorescence was used as the criteria for internalization of UCNPs by MCF-7 cells. The
percentage of UCNPs internalization was analyzed by using FlowJo software.
Functionalization of UCNPs with 2-DG significantly increased the uptake of nanoparticles in
glucose starved MCF-7 cells at all time points (Figure 6 A and 6 B). The uptake UCNPs by
MCF-7 is a time dependent and slow process, but the uptake of UCNPs-2-DG is rapid and
nearly 53% uptake of UCNPs-2-DG is seen within 10 min incubation and 83% after 1 h.
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These results clearly demonstrated the following two advantages of using UDCONI: 1P0s.1-0239-/DD0GNJ00666A
during cancer treatment. They are taken up by tumor cells easily and UCNPs give
luminescence when exposed to 980 nm laser thereby distinguishing between the tumor and
normal cells.
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Figure 6. MCF-7 cells were cultured in D-glucose free media and treated with FITC labeled
UCNPs functionalized with or without 2-Deoxy-D-glucose (2-DG). MCF-7 cells
were stained with Hoechst 33342 and acquired on a flow cytometer. The samples
were analyzed using FlowJo software (A) and bar graph represents the percentage
of FITC positive cells (B).
Now it is well known fact that the uptake of D-glucose by cancer cells is twenty-fold
higher than that of the normal cells.15 Also literature reports, regarding comparison of D-
Glucose and 2-DG uptake clearly indicate that the 2-deoxy-D-glucose (2-DG) has ability to
compete with -glucose uptake and inhibit its metabolism in tumor cells as well as non-tumor
D
cells. Preferential and fast uptake kinetics of 2-DG leads to deprivation of nutrient and energy
resulting in suppress cancer cell growth and survival and leading to their death through
apoptotic pathway.27 The 2-deoxy-D-glucose that is functionalized with the UCNPs are
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anticipated to get released under acidic conditions upon internalization by cells. The 2-deoxy-
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D-glucose mimics D-glucose and blocks the glycolysis pathway. Finally UCNPs have
advantage over fluorophores due to zero auto fluorescence and are resistant to photo
bleaching which make them suitable for long term repetitive imaging. Hence, UCNPs-2-DG
can act as multipurpose drug in diagnosis and therapy by further increasing the ability of
UCNPs to take more load of 2-deoxy-D-glucose or tag with known chemotherapeutic agent.
At this point it is worthy to note that during the anticipated clinical applications of this
modality, the exposure of to 980 nm will not cause any harm to tissues due to its low energy
(1-4 mW/cm-2) and short duration of exposure of few seconds. Its tissue penetration is also
more than the medically used red light radiation. At present the lasers have been used in
versatile medical applications especially super facial applications, lithotripsy, for the
treatment of various types of cancer and surgery too.28 Additionally, it is now known that
water is a major component of tissue. Its absorption coefficient at 980 nm is 0.482 cm-1
(better as compared at other wavelengths of available lasers) which directly proportional to
optical thermal generation in tissue. Hence the 980 nm diode laser is capable of producing
rapid and localized heating of tissue and thereby creating ablation zones with sharp
boundaries using relatively low powers of incident laser radiation.29 It is helpful for serving
our purpose for application of this modality for precision surgery.
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The conclusion of this present study will be incomplete without proposing the
pathway of observed functions exhibited by D-Glucose or 2-DG coated UCNPs viz.,
internalization and cytotoxicity. It may perhaps give directive for further research. For this
we need to go through the difference in D-Glucose metabolism in cancerous as well as
normal cells. From literature it is evident that in case of normal cells, through glycolysis and
oxidative phosphorylation, mammalian cells get ATP and precursors of amino acids,
nucleotide and lipids using D-glucose as the precursor. The transport of D-glucose into the
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cytosol through the plasma membrane is a rate-limiting step in this glucose metabolism. This
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transport of D-glucose is performed by a family of glucose transporters (GLUTs) proteins
expressed in tissues. One of the most important GLUTs is GLUT-1. But, in case of cancerous
cells different scenario is seen. The cancer cells depend preferably on glycolysis (2 ATP per
glucose) which is contrast to normal cells that metabolize glucose through oxidative
phosphorylation (36 ATP per glucose) and resort to the glycolytic pathway under anaerobic
conditions. This anaerobic glycolysis (Warburg Effect) entails the generation of lactate and is
also found to be far less efficient than mitochondrial respiration. As cancer cells rely on
glycolysis, the demand of glucose is twenty fold higher than of normal cells. This leads to the
over expression of GLUT-1 on the surface of the cancer cells. This has lead the successful
treatment of 2-DG which is an antimetabolite of D-Glucose responsible for killing cancer
cells by inhibiting glycolysis.30 Analogously, when the cancer cells were treated with 2-DG
coated UCNPs, the UCNPs interact with GLUT-1 transporters present on the surface of the
cancer cells and eventually get internalized inside the cells through clathrin-mediated
endocytosis mechanism and perform their function. Once the 2-DG is released, it inhibits the
glycolysis leading to death of cancer cell.
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4.
Conclusions
Monodispersed UCNPs based on NaYF4 doped with Yb (16%) and Tm (4%) with the
hexagonal phase were prepared which exhibited luminescence upon exposure to 980 nm
laser. To make these UCNPs, hydrophilic, uniform silica coating was performed and
subsequently functionalized with D-glucose or 2-deoxy-D-glucose. UCNPs functionalized
with 2-deoxy-D-glucose were found to have enhanced uptake by MCF-7 human breast cancer
cells and they are anticipated to inhibit growth by mimicking the glucose, thereby blocking
the glycolysis pathway, resulted in killing of MCF-7 cells. Then we labeled UCNPs coated
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with 2-deoxy-D-glucose with FITC and faster uptake (80 %) within 30 min was cDoOnI:f1i0rm.10e39d/Db0yNJ00666A
flow cytometer and internalization studies. Hence, we conclude with possible application of
2-DG coated UCNPs for targeted delivery as well as in precision surgery.
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5.
Supplementary Information
† Electronic supplementary information (ESI) is available: Some experimental details
related synthesis of D-glucose, 2-deoxy-D-glucose derivatives as well as their
characterization data by NMR (1H. 13C{1H}) spectroscopy is given in supplementary
information.
6.
Acknowledgements
Authors are thankful Dr. A. K. Tyagi, Head, Chemistry Division, BARC, Trombay,
Mumbai for the encouragement and support for this research work. Authors also
acknowledge the fruitful discussions with Dr. R. S. Ningthoujam, Chemistry Division, BARC
as well as Dr. Dibakar Goswami, Bio-Organic Division, BARC. We are thankful to Ms.
Binita Kislay Kumar and Mr. Deepak V. Kathole for technical assistance during conducting
biological experiments. KSS is grateful to University Grants Commission (UGC), New Delhi
for the award of fellowship.
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Author information
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Affiliations
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Chemistry Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India
K. Shitaljit Sharma
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Chemistry Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India & Homi
Bhabha National Institute, Anushaktinagar, Mumbai-400 094, India
Prasad P. Phadnis and Rajesh K. Vatsa
Bio-organic Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India
Akhil K. Dubey
Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai-
400 085, India
Maikho Thoh
Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai
400085, India & Homi Bhabha National Institute, Anushaktinagar, Mumbai-400 094, India
Deepak Sharma and Santosh K. Sandur
Corresponding authors
Correspondence to: Dr. Prasad P. Phadnis; Dr. Rajesh K. Vatsa
Email: phadnisp@barc.gov.in; rkvatsa@barc.gov.in
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Author Contributions
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KSS and PPP designed the work and performed synthesis of silica coated NaYF4 and
its functionalization with D-glucose and 2-deoxy-D-glucose. KSS, AKD and PPP conducted
the synthesis of D-glucose or 2-DG derivatives and their functionalization over upconversion
nanoparticles (UCNPs) to give UCNPs-D-Glucose and UNCPs-2-deoxy-D-glucose
respectively. MT, DS and KSS performed cell viability evaluation by treatment of UCNPs-D-
Glucose and UNCPs-2-deoxy-D-glucose against MCF-7 cells by MTT assay. KSS and MT
performed the biological experiments. DS, MT and KSS performed imaging and
internalization studies. PPP and KSS analyzed and concluded NMR spectral analyses. KSS,
PPP, MT and RKV wrote the manuscript. PPP and KSS analyzed and concluded NMR
spectral analyses. RKV supervised overall work.
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Ethics declarations
Conflicts of interest
“There are no conflicts to declare”.
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127x52mm (96 x 96 DPI)