Organic Fluorophore Coated Polycrystalline Ceramic LSO:Ce Scintillators for X-ray Bioimaging

Article abstract of DOI:10.1021/acs.langmuir.8b03129

The current effort demonstrates that lutetium oxyorthosilicate doped with 1-10% cerium (Lu₂SiO₅:Ce, LSO:Ce) radioluminescent particles can be coated with a single dye or multiple dyes and generate an effective energy transfer between

Full text of DOI:10.1021/acs.langmuir.8b03129

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Organic fluorophore coated polycrystalline
ceramic LSO:Ce scintillators for x-ray bioimaging
Mary K. Burdette, Yuriy P. Bandera, Eric Zhang, Artem Trofimov, Ashley
Dickey, Isabell Foulger, Joseph W. Kolis, Kelli E Cannon, Aundrea F Bartley,
Lynn E Dobrunz, Mark S Bolding, Lori McMahon, and Stephen H. Foulger
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03129 • Publication Date (Web): 05 Dec 2018
Downloaded from http://pubs.acs.org on December 16, 2018
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Organic fluorophore coated polycrystalline
ceramic LSO:Ce scintillators for x-ray
bioimaging
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Mary K. Burdette,† Yuriy P. Bandera,† Eric Zhang,†,‡ Artem Trofimov,† Ashley
Dickey,¶ Isabell Foulger,§ Joseph W. Kolis,¶ Kelli E. Cannon,k Aundrea F.
Bartley,⊥ Lynn E. Dobrunz,⊥ Mark S. Bolding,# Lori McMahon,@ and Stephen H.
Foulger∗,4,‡,§
†Department of Materials Science and Engineering, Clemson University, Clemson SC
29634, USA.
‡Center for Optical Materials Science and Engineering Technologies, Clemson University,
Anderson SC 29625, USA.
¶Department of Chemistry, Clemson University, Clemson SC 29634, USA.
§Department of Bioengineering, Clemson University, Clemson SC 29634, USA.
kDepartment of Vision Science, University of Alabama at Birmingham, Birmingham AL
35294, USA.
⊥Department of Neurobiology, Evelyn F. McKnight Brain Institute & Civitan International
Research Center, University of Alabama at Birmingham, Birmingham AL 35294, USA.
#Department of Radiology, University of Alabama at Birmingham, Birmingham AL 35294,
USA.
@Department of Cell, Developmental, and Integrative Biology, University of Alabama at
Birmingham, Birmingham AL 35294, USA.
4Department of Materials Science and Engineering, Clemson University, Anderson SC
29625, USA.
E-mail: foulger@clemson.edu
2
Phone: 01 864-656-1045. Fax: 01 864-656-1049
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Abstract
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The current effort demonstrates that lutetium oxyorthosilicate doped with 1-10%
cerium (Lu2SiO5:Ce, LSO:Ce) radioluminescent particles can be coated with a single
dye or multiple dyes and generate an effective energy transfer between the core and
dye(s) when excited via x-rays. LSO:Ce particles were surface modified with an alkyne
modified naphthalimide (6-piperidin-1-yl-2-prop-2-yn-1-yl-1H -benzo[de]isoquinoline-1,3-
(2H )-dione, AlNap) and alkyne modified rhodamine B (N -(6-diethylamino)-9-{2-[(prop-
2-yn-1-yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -ethylethanaminium, AlRhod)
derivatives to tune the x-ray excited optical luminescence from blue to green to red us-
ing Förster Resonance Energy Transfer (FRET). As x-rays penetrate tissue much more
effectively than UV/visible light, the fluorophore modified phosphors may have appli-
cations as bio-imaging agents. To that end, the phosphors were incubated with rat
cortical neurons and imaged after 24 hours. The LSO:Ce surface modified with AlNap
were able to be successfully imaged in vitro with a low-output x-ray tube. To use the
LSO:Ce fluorophore modified particles as imaging agents, they must not induce cyto-
toxicity. Neither LSO:Ce nor LSO:Ce modified with AlNap showed any cytotoxicity
toward normal human dermal fibroblast cells or mouse cortical neurons, respectively.
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Introduction
X-ray radiation is able to penetrate deeper into tissues than ultraviolet/visible light, which
makes the use of x-rays for bio-imaging and theranostic applications attractive.1 Traditional
uses of x-ray stimulation in bioimaging include x-ray projection imaging and computed
tomography.2,3 More recently x-ray radiation has been utilized in advanced x-ray fluorescence
computed tomography (XRCT),4 which utilizes positive contrast imaging agents to produce
elemental maps of the tissue, and x-ray luminescence computed tomography (XLCT) that
relies on the x-ray excitation and subsequent optical detection of the imaging agent.5–8
These imaging agents employ molecules containing high atomic number elements due to
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their ability to absorb x-rays and downconvert the energy (i.e. scintillators). Once the
scintillator is irradiated with high energy photons (such as those that come from x-ray or
γ-ray radiation), electrons are promoted from the valence band to the conduction band,
which leave behind holes in the valence band. The electron-hole pairs either travel through
the conduction band until they reach the luminescent center or they get trapped in the
conduction band by defects in the material. Trapped electron-hole pairs are omitted from
the scintillation process and do not generate any light. The electron-hole pairs that reach
the luminescent centers recombine and transfer their energy to the active center, which then
emits visible light9–13 For example, successful nanoparticle x-ray imaging agents include
gadolinium containing compounds, such as gadolinium oxide,14 gadolinium oxysulfide doped
with europium or terbium,15 sodium gadolinium tungstate,16 and alkali metal gadolinium
fluorides doped with europium.7 Additional imaging agents that have been recently utilized
for x-ray radiation induced bio-imaging include lanthanum oxysulfide doped with europium
or terbium15 and alkali metal yttrium fluorides doped with both ytterbium and erbium17 or
gadolinium.18
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While changing the phosphor to suit various emission needs is an option, many scin-
tillators are hygroscopic, meaning that they lose their scintillation properties upon being
in a water-rich environment.19–21 Therefore, these compounds are not suitable for use as
bio-imaging agents. While recent investigations into color-tuning of perovskite nanocrystals
based on counter ion type and concentration have shown promise, these materials may in-
duce toxicity as there is no shielding of these particles from their environment.22 It would be
desirable to use a bright, non-hygroscopic scintillating particle whose emission can be tuned
by attaching an organic fluorophore, or multiple fluorophores, with various emission charac-
teristics; however, these materials systems have not been studied in depth.23–25 To this end,
we demostrate that a lutetium oxyorthosilicate doped with cerium (Lu2SiO5:Ce, LSO:Ce)
particle can be coated with a single fluorophore or multiple fluorophores and generate an
effective energy transfer between the dye and core when excited through X-ray radiation.
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Furthermore, we show that the fluorophore coated LSO:Ce particles could be directly im-
aged with x-rays after incubation with rat cortical neurons. The particles as bare LSO:Ce
or single fluorophore coated LSO:Ce were shown to be non-cytotoxic to normal human der-
mal fibroblast cells and rat cortical neurons, respectively. These fluorophore coated LSO:Ce
particulates are promising candidates for applications as x-ray excited optical luminescence
imaging agents.
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Experimental Section
Reagents and solvents
All reagents were purchased from commercial suppliers, such as Aldrich or Alfa Aesar, and
were used without purification. All solvents used for reactions were distilled under nitrogen
after drying over an appropriate drying reagent. LSO:Ce phosphors (median particle size:
4 µm) were purchased from Phosphor Technologies. To decrease the size of the phosphor
particles, the particles were ball milled and a final size of ca. 800 nm as measured by a Coul-
ter N4 Plus DLS (dynamic light scattering) was obtained. All other commercial reagents
were used without further purification. All solvents were dried according to standard meth-
ods. Deionized water was obtained from a Nanopure System and exhibited a resistivity of
ca. 1018 Ohm−1cm−1. Analytical thin-layer chromatography was performed on glass plates
coated with 0.25-mm 230-400 mesh silica gel containing a fluorescent indicator. Column
chromatography was performed using silica gel (spherical neutral, particle size 63-210 µm).
Chemical characterization methods
1H NMR spectra were recorded on a JEOL ECXâĂŞ300 spectrometer. Chemical shifts for
protons are reported in parts per million downfield from tetramethylsilane and are referenced
to residual protium in the NMR solvent (CDCl3: δ 7.26 ppm).
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Syntheses
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6-(piperidin-1-yl)-2-(prop-2-yn-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (AlNap)
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6-(piperidin-1-yl)-2-(prop-2-yn-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione was synthesized
1
according to a previously reported method26 (cf. Figure 1). H NMR (CDCl3) δ 1.73 (m,
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2H), 1.89 (m, 4H, J=5.5 Hz), 2.17 (t, 1H, J=2.4 Hz), 3.24 (t, 4H, J=5.5 Hz), 4.95 (d, 2H,
J=2.4 Hz), 7.18 (d, 1H, J=8.3 Hz), 7.68 (d.d, 1H, J=7.2 Hz), 8.40 (d.d, 1H, J=8.3 Hz,
J=1.4 Hz), 8.53 (d, 1H, J=8.3 Hz), 8.61 (d.d, 1H, J=7.2 Hz, J=1.4 Hz).
O
N
O
O
O
O
O
N
O
NH2
N
H
DMSO
MeOH
N
Br
Br
AlNapBr
AlNap
Figure 1: Synthesis of 6-(piperidin-1-yl)-2-(prop-2-yn-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (Al-
Nap).
Mono(N-(6-(diethylamino)-1-(2-((prop-2-yn-1-yloxy)
carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium) monocarbonate
(AlRhod) Mono(N-(6-(diethylamino)-1-(2-((prop-2-yn-1-yloxy)carbonyl)phenyl)-3H-xanthen-
3-ylidene)-N-ethylethanaminium) monocarbonate (AlRhod) was synthesized according to a
1
previously reported method27 (cf. Figure 2). H NMR (CDCl3) δ 1.33 (t, 12H, J=7.2 Hz),
2.42 (t, 1H, J=2.4 Hz), 3.65 (m, 8H, J=7.2 Hz), 4.62 (d, 2H, J=2.4 Hz), 6.84 (d, 2H, J=2.4
Hz), 6.92 (m, 2H, J=9.3Hz, J=2.4 Hz), 7.05 (d, 2H, J=9.3 Hz), 7.34 (d, 1H, J=7.6 Hz),
7.75 (m, 1H, J=7.6 Hz), 7.85 (m, 1H, J=7.6 Hz), 8.31 (d, 1H, J=7.6 Hz).
(3-azidopropyl)trimethoxysilane (1) (3-Chloropropyl)trimethoxysilane (1 g, 5.03 mmol),
and sodium azide (0.684 g, 10.1 mmol) were mixed with dry dimethylformamide (3 ml), and
the obtained mixture was stirred at 90oC for 1h. After cooling, the mixture was diluted with
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Cl2-
Cl
N
O
N
N
O
N
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Br
O
O
+
K2CO3
DMF
O
OH
AlRhod
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Figure 2: Synthesis of mono(N-(6-(diethylamino)-1-(2-((prop-2-yn-1-yloxy)carbonyl)phenyl)-3H-xanthen-
3-ylidene)-N-ethylethanaminium) monocarbonate (AlRhod).
dry dichloromethane (10 ml) and filtered. The filtrate was collected and evaporated under
vacuum to give a 25% solution of product in DMF. This solution was used in the next step.
1H NMR (CDCl3) δ 0.68 (t, 2H, J=8.3 Hz), 1.70 (m, 2H, J=6.9 Hz, J=8.3 Hz), 3.25 (t, 2H,
J=6.9 Hz), 3.56 (s, 9H).
Synthesis of emitter series no1 LSO:Ce particles (average size 800 nm) (2 g) was mixed
with methanol (15 ml), and the obtained suspension was sonicated for 15 minutes. Then the
solution of 1 in DMF (1.4 ml) was added, and the mixture was stirred one minute. Finally,
the aqueous solution of ammonium hydroxide (29%, 1 ml) was added to the mixture and
stirred for 24 hours; then the reaction was refluxed for 2 hours. After cooling, the particles
were separated by centrifugation, washed with methanol three times, and dried under vacuum
at room temperature. The product afforded was azide modified LSO:Ce particles (cf. Figure
3a). Yield 2g.
Synthesis of emitter series no2 The synthesis of emitter series no2 was performed by
utilizing a standard 1,3 Huisgen azide-alkyne cycloaddition. Emitter series no1 (300 mg)
were dispersed in THF (2 mL) and added to the reaction vessel. Then AlNap (40 mg,
0.126 mmol) in THF (1 mL) was added to the reaction mixture. Finally, copper (II) sulfate
(62.7 mg, 0.251 mmol) in DI-water (1 mL) and sodium ascorbate (124.5 mg, 0.628 mmol)
in DI-water (1 mL) were added to the reaction vessel. Then the reaction vessel was placed
in the J-KEM mini-reactor with stirring at 28oC in the dark with a nitrogen purge. The
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reaction was allowed to proceed for 24 hrs. Upon reaction completion, the product was
washed with THF. Then the product was washed with a water/EDTA solution to remove
the copper catalyst. Finally, the product was washed six more times with THF. Washes were
performed to remove any unattached dye from the particles. All washes were performed via
centrifugation at 27,216 G for 15 min (cf. Figure 3b). Once the supernatant of emitter series
no2 showed no peaks indicative of AlNap, emitter series no2 was said to be free from any
unattached fluorophore. After the purification process, FTIR of AlNap was compared to
that of emitter series no2 to ensure attachment. The vigorous washing procedure and the
subsequent FTIR, which confirms the presence of AlNap, suggests that a successful CuCAAC
reaction does take place and that the AlNap is covalently attached to the LSO:Ce core (cf.
Supporting Information).
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Synthesis of emitter series no3 The synthesis of emitter series no3 was performed by
utilizing a standard 1,3 Huisgen azide-alkyne cycloaddition. Emitter series no1 (300 mg)
were dispersed in THF (2 mL) and added to the reaction vessel. Then AlNap (5 mg, 0.016
mmol) in THF (1 mL) was added to the reaction mixture. Finally, copper (II) sulfate (39.14
mg, 0.157 mmol) in DI-water (1 mL) and sodium ascorbate (77.63 mg, 0.392 mmol) in DI-
water (1 mL) were added to the reaction vessel. Then the reaction vessel was placed in the
J-KEM mini-reactor with stirring at 28oC in the dark with a nitrogen purge. The reaction
was allowed to proceed for 1 hour. Then AlRhod (35 mg, 0.073 mmol) in THF (2 mL)
was added to the reaction vessel. The reaction was allowed to proceed for an additional 24
hrs. Upon reaction completion, the product was washed with a 60:40 THF:water solution to
remove the copper catalyst. Finally, the product was washed seven more times with THF.
Washes were performed to remove any unattached dye from the particles. All washes were
performed via centrifugation at 27,216 G for 15 min (cf. Figure 3b). Once the supernatant
of emitter series no3 showed no peaks indicative of either AlNap or AlRhod, emitter series
no3 was said to be free from any unattached fluorophore. After the purification process,
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FTIR of AlRhod was compared to that of emitter series no3 as well as emitter series no2
to ensure attachment of both AlRhod and AlNap. The vigorous washing procedure and
the subsequent FTIR, which confirms the presence of AlNap and AlRhod, suggests that a
successful CuCAAC reaction does take place and that the fluorophores is covalently attached
to the LSO:Ce core (cf. Supporting Information).
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Optical and x-ray radioluminescence characterization methods.
Absorbance spectra were taken using a Perkin Elmer Lambda 950 spectrophotometer. Pho-
toluminescence spectra were collected using a Jobin-Yvon Fluorolog 3-222 Tau spectrometer.
X-ray radioluminescence spectra were collected by irradiating the sample with a mini X-ray
tube (Amptek Inc., MA, USA), operating at a tube voltage of 50 kV and a tube current of 79
µA. The radioluminescence was collected with a fiber bundle (Oriel) coupled to a MicroHR
(Horiba Jobin Yvon) monochromator (spectral dispersion 5.25 nm/mm with spectral reso-
lution of 0.25 nm) and a cooled CCD detector (Synapse, Horiba Jobin Yvon). The signal
was collected on a grating with 600 line mm−1 and a blaze of 500 nm. The spectra was ana-
lyzed with SynerJY (Horiba Jobin Yvon) software. The exposure time varied from 1.2-20 sec
based on sample size and relative luminescence. Spectra were not corrected for the spectra
sensitivity of the system.
Infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) spectra were obtained on a ThermoScientific
Nicolet 6700 FTIR with a diamond crystal equipped with an attenuated total reflectance
(ATR) attachment. Spectra were corrected for the ATR attachment and were baseline
corrected prior to analysis. Analyses were performed with Omnic software.
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(a)
O
O
NaN3
DMF
O
O
Si
N3
Si
Cl
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O
O
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(1)
O
O
O
O
Si
N3
Si
Lu2SiO5
+
MeOH, NH4OH, H2O
O
N3
O
Lu2SiO5
(1)
emitter series no1
(b)
O
O
Si
N
O
Lu2SiO5
N
O
N
N
click
reaction
1 dye
O
emitter series no2
N
O
O
Si
O
Si
O
N
N
N3
O
Lu2SiO5
O
O
N
N
O
click
reaction
2 dyes
N
Lu2SiO5
emitter series no1
O
O
Si
O
N
N
O
N
O
emitter series no3
N
O
N
Cl
Figure 3: (a) Synthetic scheme to yield azide modified LSO:Ce particles. (b) Subsequent CuAAC reactions
with either AlNap (1 dye) or AlNap and AlRhod (2 dyes).
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Microscope imaging
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Microscope images were acquired on a Nikon Diaphot 300 inverted microscope with camera
using OpenLab 3.1.4 software. Neuronal cell cultures were imaged using a 40x objective.
Particles were added to the cell cultures using a pipette. Bright-field images were obtained
of the cells and particles in the absence of X-ray excitation. Dark-field images of the radiolu-
minescing particles were obtained by continually exciting the particles during a four-minute
exposure with an Amptek mini-X X-ray unit (tube voltage of 40 kV and a tube current of 99
µA) placed approximately 2 mm from the cell culture in a dark room. Dark-field images of
the ambient background light were obtained the same as the radioluminescing particle im-
ages without any X-ray stimulation. Images were processed using MATLAB R2014b. First,
the ambient background image was subtracted from the radioluminesing particle image to
remove any light not derived from the radioluminesence. Next, the background-subtracted
radioluminescence image was contrast enhanced such that 1% of the data was saturated at
the high and low intensities of the image. This contrast-enhanced background-subtracted
radioluminesence image was tinted green to accentuate the radioluminescence. Finally, the
green-tinted image was overlaid on the bright-field image at 20% transparency.
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Cultured cortical neurons
Approval was obtained for all experimental protocols from the University of Alabama at
Birmingham Institutional Animal Care and Use Committee. All experiments were conducted
in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the
U.S. National Institute of Health. Primary rat cortical neuronal cultures were obtained
from embryonic day 18 rats as previously described.28 Briefly, the cell culture coverslips
were coated with poly-L-lysine (50µg/ml) overnight at 37oC and then rinsed three times
with autoclaved distilled water. Dissected cortical tissue was incubated with papain for 20
minutes at 37oC. After rinsing in Hank’s Balanced Salt Solution, a single-cell suspension of
the tissue was re-suspended in Neurobasal media (Invitrogen) by trituration through a series
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of large to small fire-polished Pasteur pipets. Primary neuronal cells passed through a 70
µM cell strainer were plated on poly-lysine coated coverslips in a 12 well plate. Cells were
grown in Neurobasal media plus B-27 and L-glutamine supplement (complete Neurobasal
media) for 15-16 days in vitro in a humidified CO2 (5%) incubator at 37oC. A 50% media
change occurred weekly.
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TUNEL assay
The cortical neurons were incubated up to 24 hours with ca. 800 nm azLSO/AlNap nanopar-
ticles (final concentration 0.2 to 0.4 mg/mL) to determine effects on cell health. The nanopar-
ticles were diluted in phosphate buffer saline (PBS) and a small volume (10 to 20 µL) was
added to the wells to prevent any changes in osmolarity. Cell death of primary cortical
cultures was assessed using the TUNEL assay technique by the NeuroTACS in situ apop-
tosis detection kit (R&D Systems). After a brief rinse with PBS, coverslips were treated
with 4% paraformaldehyde for 45 minutes. The rest of the procedure followed the recom-
mended steps as indicated by the manufacturer to detect DNA fragments using the terminal
deoxynucleotidyl transferase enzyme (TdT) and revealed through a horseradish peroxidase
system. Positive and negative controls were performed. For the positive control, coverslips
were treated with TACS-Nuclease (R&D Systems) that generates DNA breaks and showed
a pale brown staining in the majority of the cells (> 95%). No labeling was observed when
TdT was not included in the reaction. Neuronal cells were counterstained with either DAPI
(4’-6-diamidino-2-phenylindole) or Blue Counterstain (R&D Systems). Three to four cov-
erslips were used for each condition. Multiple images (6-8) were taken and analyzed from
each coverslip on an Olympus BX53 (40x objective) microscope. Visibility of neurons was
reduced at the center of the application site due to the high concentration of nanoparticles,
therefore images were taken in slightly less dense areas to improve visualize of the neurons
and determine their survival. The cells were analyzed blind to the incubation period by two
separate individuals and the results were averaged together. All TUNEL analysis statistics
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were performed using Origin software (Origin Lab Corporation, 2002) and statistical sig-
nificance was P < 0.05. Data are presented as means ± SE and sample number (n) refers
to coverslips for TUNEL assay. Statistical comparisons were made using one-way ANOVA
followed by Tukey’s posthoc analysis.
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Cultured normal human dermal fibroblast (NHDF) cells
Normal human dermal fibroblast (NHDF) cells were obtained from ATCC. The NHDF cells
were cultured in Dulbeccos modified Eagles media (DMEM) containing 2% fetal bovine serum
(FBS), 7.5 mM L-glutamine, 5 ng/mL rh FGF basic, 5 µg/mL rh Insulin, 1 µg/mL hydro-
cortisone, 50 µg/mL ascorbic acid. Cells were cultured at 37oC in a humidified atmosphere
of 95% air / 5% CO2.
MTS assay
Normal human dermal fibroblast (NHDF) cells were plated in 96 well plates at a cell density
of 7,000 cells per well. After the cells were plated for 24 hours, the cells were incubated with
ca. 800nm commercial LSO:Ce particulates in concentrations of 3.6×107, 3.6×108, or 3.6×109
particles/mL. At each concentration, the cells were incubated for 24, 48, or 72 hours. At the
conclusion of each time point, the cell viability was measured using a [3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay
R
(CellTiter 96ꢀ AQueous One Solution Cell Proliferation Assay, Promega, Fitchburg, WI)
following the manufacturer’s protocol. Briefly, the media was removed and the plate was
washed several times with phosphate buffered saline (PBS) before adding a solution con-
taining 200µL of DMEM media and 40µL of the MTS assay reagent. After 3 hours, the
absorbance was measured using a plate reader at OD=490nm.
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Thermal characterization methods
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Thermogravimetric analysis (TGA) was performed on a TA Instruments Hi-Res TGA 2950
Thermogravimetric Analyzer equipped with Universal Analysis software. All TGAs were
performed from room temperature to 1000oC at 10oC/min in a nitrogen atmosphere with a
switch to air at 700oC. Before each TGA run, the sample was purged in the furnace with
nitrogen for 15 min. Thermogravitmetric analysis was performed on particulate systems to
determine fluorophore or silane ligand coverage of the particles.
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Scanning Electron Microscopy
A Hitachi 4800 field emission scanning electron microscope (FE-SEM) was used to obtain
size distribution of azLSO:Ce. The samples were platinum coated at 15 mA for one minute
under argon conditions at 200 mtorr. Images of secondary electrons were taken with an
accelerating voltage of 15 kV. Image analysis was performed using ImageJ software and
dimensions were calibrated according to the scale bar of each picture.
Results and Discussion
In the current effort, ca. 800 nm commercial lutetium oxyorthosilicate doped with cerium
(LSO:Ce) phosphors containing 1-10 at% cerium, as specified by the manufacturer, were sur-
face modified through organometallic chemistry with a silane linker (cf. Figure 4a, emitter
series no1) to enable subsequent copper (I) catalyzed azide/alkyne cycloaddition (CuAAC)
reactions. Through CuAAC reactions between emitter series no1 and 6-piperidin-1-yl-2-prop-
2-yn-1-yl-1H -benzo[de]isoquinoline-1,3-(2H )-dione (cf. Figure 4b, emitter series no2) and N -
(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -ethylethanaminium
(cf. Figure 4c, emitter series no3), the x-ray radioluminescence of LSO:Ce could be trans-
formed from its normal blue emission (cf. Figure 4d) to have elements of green (cf. Figure 4e,
emitter series no2) and red (cf. Figure 4f, emitter series no3) in the x-ray radioluminescence
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wavelength (nm)
501 447
620
551
407
376
347
(d)emitter series no1
1
0.8
0.6
0.4
0.2
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(a)
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LSO:Ce
emitter series no1
1.8
2
2.2
2.4
2.6
2.8
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energy (eV)
wavelength (nm)
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(e) emitter series n5o201
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(b)
LSO:Ce
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emitter series no2
O
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Si
N
wavelength (nm)
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N
O
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551
407
376
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1 (f) emitter series n5o301
N
O
Cl
0.8
(c)
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2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
energy (eV)
LSO:Ce
emitter series no3
Figure 4: (a) Lutetium oxyorthosilicate doped with cerium (LSO:Ce) particles of median size ca.
800 nm surface modified with 3-azidopropyl)(trimethoxy)silane (emitter series no1), which emit blue
when excited with x-rays. (b) Emitter series no1 surface modified with 6-piperidin-1-yl-2-prop-2-
yn-1-yl-1H -benzo[de]isoquinoline-1,3-(2H )-dione (AlNap) with a CuAAC transformation (emitter series
no2); emitter series no2 emits green upon x-ray irradiation. (c) Emitter series no1 surface modified
with AlNap and N -(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -
ethylethanaminium (AlRhod) with multiple CuAAC reactions; emitter series no3 emits elements of red,
green, and blue upon X-ray irradiation. X-ray radioluminescence spectra of (d) emitter series no1, (e) emit-
ter series no2, and (f) emitter series no3. X-ray radioluminescence spectra obtained with an AmpTek Mini-X
equipped with a silver target operating at 50 kV and 79µA.
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spectra through Förster resonance energy transfer (FRET).
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Photophysics of fluorophores
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6-piperidin-1-yl-2-prop-2-yn-1-yl-1H -benzo[de]isoquinoline-1,3-(2H )-dione (AlNap) has an
absorbance at 400 nm.29,30 The emission profile of AlNap exhibits a maximum emission
at ca. 515 nm.26,31,32 The maximum absorbance of N -(6-diethylamino)-9-{2-[(prop-2-yn-1-
yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -ethylethanaminium (AlRhod) was ca. 550
nm with an aggregation peak at 515 nm, while the maximum emission for AlRhod was ca.
585 nm when excited at 550 nm (cf. Figure 5).33–35 No significant emission was detected
for the naphthalimide and rhodamine B derivatives when irradiated with x-rays (data not
shown). It is obvious that the emission of LSO:Ce overlaps well with AlNap, and the emission
of AlNap overlaps well with the absorbance of AlRhod.
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1.1
emission AlNap absorbance AlRhod
emission LSO:Ce
absorbance AlNap
1
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0
emission AlRhod
300
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wavelength (nm)
Figure 5: (a) Absorbance of 6-piperidin-1-yl-2-prop-2-yn-1-yl-1H -benzo[de]isoquinoline-1,3-(2H )-dione
(AlNap), optical emission of LSO:Ce (light green), optical emission of AlNap (dark green), absorbance of N -
(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -ethylethanaminium
(AlRhod) (pink), and optical emission of AlRhod (purple). Shaded are the areas of emission/absorbance
overlap. Excitation of LSO:Ce, AlNap, and AlRhod at 357 nm, 400 nm, and 550 nm, respectively. All
concentrations were 5 µg/mL with the exception of LSO, which was measured in its solid form.
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Surface modification of LSO:Ce
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Polycrystalline ceramic LSO:Ce contains lutetium, silicon, oxygen, and a small percentage
of doped cerium atoms that occupy lutetium sites. The LSO:Ce particles are modified by at-
taching an organometallic linker with an azide group on one end ((3-azidopropyl)(trimethoxy)silane)
to the particle via a base catalyzed hydrolysis reaction in the presence of ammonium hy-
droxide in a methanol solution. Upon a basic aqueous environment the methoxy groups
on the end of the linker hydrolyze to alcohol groups, which readily react with the surface
of the LSO:Ce particle by forming Si-O-Si bonds (cf. Figure 4a, emitter series no1).36,37 At
the completion of the reaction, the product was put through a vigorous washing (through
centrifugation) purification process with a good solvent for the silane linker to remove any
unattached linker. While surfaces of larger particles are less energetic, longer reaction times
do result in modification; Fourier transform infrared spectroscopy (FTIR) confirmed attach-
ment of the azide linker as indicated by the peak at 2100 cm−1, which is related to the
stretching of the N=N (cf. Supporting Information). In this way, azide modified LSO:Ce
(azLSO:Ce) particles are able to undergo a variety of CuAAC reactions, where a variety of
fluorescent tags, proteins, and targeting moieties could be covalently attached to the parti-
cles to enhance their utility in bioimaging or other therapies.38 Therefore, non-hygroscopic
LSO:Ce can easily be surface modified via facile reactions to exhibit emissions that cover the
visible spectrum.
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X-ray radioluminescence of fluorophore modified LSO:Ce
Upon x-ray irradiation, the cerium dopant in the LSO crystal structure causes LSO:Ce to
emit in the blue portion of the visible spectrum. Before any modifications were performed,
x-ray radioluminescence was obtained for the commercial LSO:Ce particles as a control. X-
ray radioluminescence spectrum of the as received, commercial LSO:Ce particles (size 2-4
µm) were deconvoluted into three peaks at 396 nm (3.13 eV), 428 nm (2.90 eV), and 498 nm
(2.49 eV) (cf. Figure 6). Ce3+ resides in two distinct crystallographic sites with the first site
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(Ce1 site) being the 6-oxygen-coordinated site and the second site (Ce2 site) being the 7-
oxygen-coordinated site, where the Ce1 site is the dominant site in x-ray radioluminescence.39
Particles of 2-4 µm are too large to be successful bioimaging agents; therefore, the as received
particles were ball milled in ethanol to achieve a median particle size of 800 nm, which is a
more viable size for bioimaging agents (cf. Figure 7). Three SEM micrographs at varying
magnifications are presented to show detail of the particles post-ball milling. The ball milled
ca. 800 nm LSO:Ce particles were used in all further processes.
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In emitter series no1, the deconvolution of the x-ray radioluminescence spectrum revealed
2 peaks at 397 nm (3.12 eV) and 428 nm (2.90 eV) resulting from the Ce1 site. In each
crystallographic site there are two 4f ground states (2F5/2 (emission at higher energy) and
2F7/2 (emission at lower energy)) due to spin orbit coupling.40–42 There is a shoulder at ca. 498
nm (2.48 eV) resulting from the combination of both 4f states in the Ce2 site (cf. Figure 8a),
which agrees well with the energy profile for commercially available LSO:Ce polycrystalline
ceramics. The slight difference in the shape of the energy profile and shifted deconvoluted
peaks, when compared to commercial LSO:Ce, can be due to the surface modification of
emitter series no1 since the emission of Ce3+ is greatly influenced by its environment due to
the unshielded 4f → 5d transition.43 While the Ce3+ on the inside of the particle would not
be influenced by the silane linker on the surface, the Ce3+ close to the surface of the particle
may feel the effects of the ligand, leading to a slight shift in the energy profile. Additionally,
the overall size of emitter series no1 is smaller when compared to commercial LSO:Ce, which
can influence the energy profile44,45 though clearly none of these modifications had a great
influence on the x-ray radioluminescent emission characteristics of LSO:Ce. The emission of
emitter series no1 could be tuned via judicious choice of Förster Resonance Energy Transfer
(FRET) pairs. By utilizing LSO:Ce as the x-ray excited “pump" source for other non-xray
active fluorophores, the emission of the particles could be tuned to range the entire visible
spectrum on the basis of a blue emitter.
6-piperidin-1-yl-2-prop-2-yn-1-yl-1H -benzo[de]isoquinoline-1,3-(2H )-dione (AlNap), when
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wavelength (nm)
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443
376
428 nm
2.90 eV
Ce1
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396 nm
3.13 eV
Ce1
498 nm
2.49 eV
Ce2
1.8
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2.2
2.4
2.6
2.8
energy (eV)
3
3.2
3.4
3.6
Figure 6: Deconvolution of the x-ray radioluminescence spectrum of commercial, as received LSO:Ce
particles (2-4 µm). Deconvolutions revealed peaks at 498 nm (pink, attributed to the Ce2 site), 428 nm, and
396 nm (green and purple, respectively, both attributed to the Ce1 site). X-ray radioluminescence spectra
obtained with an AmpTek Mini-X equipped with a silver target operating at 50 kV and 79µA.
(a)
(b)
(c)
3 µm
4 µm
5 µm
Figure 7: Scanning electron micrographs (SEM) of LSO:Ce particles (median size 800 nm) at a magnifi-
cation of (a) 9,000 (scale bar 3.00 µm), (b) 12,000 (scale bar 4.00 µm), and (c) 15,000 (scale bar 5.00 µm).
Images were taken at 15 kV and 10 µA.
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covalently attached to the surface of emitter series no1, forms a FRET pair with LSO:Ce
(cf. Figure 4b, emitter series no2), causing an x-ray emission shift from blue (dominate peak
at ca. 420 nm) to green (dominant peak at ca. 525 nm) (cf. Figure 4e). It is clear from
the deconvoluted radioluminescence spectrum of emitter series no2 that a significant portion
of the energy from LSO:Ce has been transferred to AlNap as indicated by the appearance
of the peaks at ca. 525 nm (2.36 eV) and 534 nm (2.32 eV), which are attributed to the
monomeric and J-aggregate peaks, respectively, of AlNap as these peaks coincide well with
the peaks observed in the photoluminescence spectrum of AlNap.26,31,32 The Förster distance
(i.e. the distance at which FRET is 50% efficient) between LSO:Ce and AlNap is ca. 2.98
nm when 0.30 is used as the quantum efficiency of LSO:Ce;46 it should be noted that energy
contributing to FRET is only considered from the surface of the LSO:Ce core. With this in
mind, modeling AlNap as a sphere of diameter 1.4 nm, which is the fully extended length of
the dye, 0.405 nm3 are directly in contact with the surface of the LSO:Ce particle, which is
indicates FRET is a possibility. Furthermore, the volume of the sphere representative of the
dye is 1.44 nm3, and, if we look at Förster distance as a volume, which is 9 nm3, the Förster
“volume" is 6.27 times larger than the volume of the fluorophore. While the existence of
photon emission and photon re-absorbance cannot be completely obviated, there is strong
evidence to suggest that FRET is a viable energy transfer mechanism. This type of behavior
is expected for all systems reported.
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The deconvolution of emitter series no2 revealed only one LSO:Ce peak at 413 nm (3.00
eV); this is likely a combination of the peaks at 397 nm (3.12 eV) and 428 nm (2.90 eV) found
in the spectrum of emitter series no1 as 413 nm (3.00 eV) is the average of these two peaks
(cf. Figure 8b) as these two peaks could not be individually deconvoluted reliably. Addition-
ally, the band relating to the Ce2 site could not be deconvoluted as it overlaps heavily with
the strong AlNap bands; efforts to introduce additional peaks in the deconvolution of the
spectrum in Figure 8b does not provide changes in the fitting curve, as the fitting software
attempted to minimize the influence of those peaks. This can indicate that some of the
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peaks present in Figure 8a could merge together or be suppressed in Figure 8b due to the
introduced energy transfers caused by AlNap. Based on the ratio of the integral of the AlNap
component of the x-ray radioluminescence spectrum to the total integral of the spectrum the
dye component is responsible for 78% of the total spectrum suggesting that energy transfer
is efficient. In emitter series no3 (cf. Figure 4c), LSO:Ce and AlNap remain FRET pairs with
one another, but AlNap then transfers its energy to N -(6-diethylamino)-9-{2-[(prop-2-yn-1-
yloxy)carbonyl]phenyl}-3H -xanthen-3-ylidene)-N -ethylethanaminium (AlRhod) through an
additional FRET, resulting in a shift from a blue emission to a blue emission with a promi-
nent green and red component (cf. Figure 4f). The Förster distance between AlNap and
AlRhod is ca. 3.62 nm. When comparing the integral of the AlRhod component of the x-ray
radioluminescence spectrum to the total spectrum, the AlRhod component composes 10.5%
of the total x-ray radioluminescence spectrum.
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The deconvolution of the radioluminescence spectra of emitter series no3 revealed an
AlRhod peak at 633 nm (1.96 eV), which coincides well with the photoluminescence J-
aggregate peak of rhodamine B. There is also a peak at 541 nm (2.29 eV), which is likely a
combination of a small monomeric AlRhod peak, which should be at ca. 585 nm (2.12 eV)
with a major contribution from AlNap as this peak is slightly red shifted from the expected
AlNap peak at ca. 525 nm (2.36 eV). Clearly, the energy transfers that takes place with
emitter series no3 is not as good as emitter series no2 as two LSO:Ce bands could easily be
deconvoluted; however, these bands are red shifted from their expected peaks at 397 nm (3.12
eV) and 428 nm (2.90 eV) which suggests that the peak from the Ce2 site is embedded in the
two LSO:Ce peaks observed in Figure 8c. To maintain the fit, the peak attributed to the Ce2
site could not be deconvoluted separately from the presented LSO:Ce peaks. Both AlNap
and AlRhod are stable against x-ray irradiation as the fluorophores were exposed to several
cycles of x-ray irradiation with photoluminescence of each fluorophore being obtained after
each radiation cycle. The photoluminescence spectra of AlNap and AlRhod did not change
at any time after exposure to x-ray radiation (data not shown), indicating that the molecular
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structure of the fluorophores remained intact as photoluminescence of the molecules would
change upon bond destruction.
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It is clear from the radioluminescence spectra of emitter series no2 and no3 that x-ray ra-
diation can induce FRET between an x-ray active pump source and non-x-ray active fluo-
rophores. Therefore, these particles have potential as imaging agents for deep tissue appli-
cations. In order to evaluate their imaging ability, emitter series no2 was incubated with
cultured single-cell layer rat cortical neurons and imaged (cf. Figure 9). Upon being in a
water-rich, biological environment, emitter series no2, which was a mix of a non-active pre-
cursor compound and ca. 10% AlNap, was imaged under x-ray irradiation using a standard
photomicrography microscope. However, even with AlNap in low yield, emitter series no2
was still able to be imaged under physiological conditions. This is a good indication that the
particles are not degraded in an aqueous environment.47,48 Futhermore, control studies of
neurons incubated with particles but without x-ray irradiation showed only a black picture
(data not shown). While the particles were able to be successfully imaged, we needed to
ensure that the particles did not induce any cytotoxicity. Because emitter series no2 was
imaged with rat cortical neurons, the terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) toxicity assay was also performed with rat cortical neurons. Primary
cortical neurons are particularly sensitive to slight alterations in their culturing conditions,
which can lead to their cell death.49 Cell death involves atrophy of the cell body, condensation
of the nuclear chromatin, DNA fragmentation, and neurite degeneration. A TUNEL assay
was used to determine the number of neurons that had undergone cell death for each condi-
tion (cf. Figure 10a). The presence of 1.01 ×1011 particles/mL or 2.02 ×1011 particles/mL
of emitter series no2 did not decrease the number of viable cells in culture at the various
time points observed (cf. Figure 10b). Additionally, there was no change in the neuronal
density between the various conditions (cf. Figure 10c). While emitter series no2 did not
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wavelength (nm)
376
347
620
551
501
447
407
1
0.8
0.6
0.4
0.2
0
(a)
428 nm
2.90 eV
500 nm
2.48 eV
397 nm
3.12 eV
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1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
energy (eV)
wavelength (nm)
620
447
407
376
551
501
347
1
0.8
0.6
0.4
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0
(b)
525 nm
2.36 eV
AlNap
413 nm
3.00 eV
LSO:Ce
534 nm
2.32 eV
AlNap
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
energy (eV)
wavelength (nm)
447
501
620
551
407
376
416 nm
2.98 eV
LSO:Ce
347
484 nm
2.56 eV
LSO:Ce
1
0.8
0.6
0.4
0.2
0
(c)
632 nm
1.96 eV
AlRhod
541 nm
2.29 eV
AlNap
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
energy (eV)
Figure 8: The deconvolution of the radioluminescence spectrum of (a) emitter series no1 revealed 3
peaks at 397 nm (3.12 eV), 428 nm (2.90 eV), and 500 nm (2.48 eV), which corresponds to Ce3+ 2F5/2 and
2F7/2 in the first crystallographic site and a combination of the 4f states in the second crystallographic site,
respectively; (b) Emitter series no2 showed AlNap an aggregate peak at 534 nm (2.32 eV) and a monomeric
peak at 525 nm (2.36 eV) as well as an LSO:Ce peak at 413 nm (3.00 eV), which is a combination of the 397
nm (3.12 eV) and 428 nm (2.90 eV) peak observed in emitter series no1. (c) Emitter series no3 exhibited
an aggregate AlRhod peak at 633 nm (1.96 eV), a peak at 541 nm (2.29 eV) which is a combination of the
monomeric AlRhod peak and AlNap peaks, and two LSO:Ce peaks at 484 nm (2.56 eV) and 416 nm (2.98
eV), which are slightly blue shifted from the expected peaks. Total fit for the deconvolutions is presented
in black with the actual data presented in blue. All x-ray radioluminescence spectra were obtained with an
AmpTek Mini-X equipped with a silver target operating at 50 kV and 79µA.
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(a)
(c)
(b)
(d)
Figure 9: Microscopy images of neurons with emitter series no2 in culture. Neurons and particles imaged
in (a) brightfield, (b) under X-ray irradiation with background subtraction in darkfield, (c) under X-ray
irradiation with contrast enhancement under X-ray irradiation with background subtraction, and (d) under
X-ray irradiation with contrast enhancement under X-ray irradiation with background subtraction pseudo-
colored green. All scale bars are 50 µm. X-ray irradiation performed with an AmpTek Mini-X equipped
with a silver target operating at 40 kV and 99µA. Blue arrows indicate the soma of the neurons, while red
arrows indicate emitter series no2.
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exhibit any cytotoxicity, it needed to be ensured that unmodified LSO:Ce particles did not
induce cytotoxicity. Therefore, LSO:Ce particles were incubated with normal human dermal
fibroblasts for 24 hr, 48 hr, and 72 hr at particle concentrations of 3.6×107, 3.6×108, or
3.6×108 particles/mL. At all concentrations and at all time points, there was no observable
cytotoxicity as confirmed via a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium (MTS) assay statistically analyzed with the Tukey’s test (cf.
Figure 10d). As the particles do not induce cytotoxicity, these particles are potential viable
bio-imaging agents.
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24 hr Incubation with
(a)
(d)
No Nanoparticles
1.1
1
Nanoparticles
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
(b)
(c)
125
1250
100
75
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0
1000
750
500
250
0
3.6x107
particle concentration (particles/mL)
3.6x108
3.6x109
control
-
0
+
0
+
4
+
+
-
0
+
0
+
4
+
+
Nanoparticles
Nanoparticles
Incubation
14 24
Incubation
14 24
(Hours)
(Hours)
Figure 10: Cultured cortical neurons were incubated for up to 24 hours with or without emitter series no2.
The cells were then fixed, analyzed for TUNEL reactivity, and counterstained blue to visualize the neurons.
(a) Images of cortical neurons with and without emitter series no2 after processing with the TUNEL assay.
The black arrows indicate examples of healthy neurons. Dead neurons were identified as cells that showed
TUNEL reactivity through the brown staining of the condensed chromatin. Scale bar 25 µm. (b) Group
data shows no change in neuronal survival at any of the incubations time points (0, 4, 14, and 24 hours)
(ANOVA, F4,108= 1.2, P =0.31, n=3,3,3,3,4). Six to eight images from each coverslip were analyzed. (c) The
overall neuronal density was unchanged. (ANOVA, F4,108= 1.0, P =0.39, n=3,3,3,3,4) (d) MTS cytotoxicity
assay of normal human dermal fibroblast (NHDF) cell incubated without particles (control) or with 3.6×107,
3.6×108, or 3.6×108 particles/mL for 24 hrs (blue), 48 hrs (green), and 72 hrs (pink). No cytotoxicity was
observed for any of the concentrations at any time point as analyzed by the Tukey’s test.
It was noticed that acute application of nanoparticles resulted in the nanoparticles quickly
surrounding the cells. For applications involving proteins localized on the cellular membrane
extracellularly, particle uptake by the cells can be undesirable.50,51 To assess the potential
for neurons to engulf the imaging probes, neurons were incubated for 24 hours with the
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nanoparticles and then z-stack images were taken. Figure 11 shows that even after 24 hours
the nanoparticles are still sitting on the cell surface. Therefore, these particulate systems
are suitable to extracellular bioimaging.
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+4 m
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Figure 11: Cultured cortical neurons were incubated for 24 hours with nanoparticles. Each row is a z-stack
image separated by 2 µm. The white arrows indicate neurons with nanoparticles (small dark structures)
sitting on top of the neurons. The nanoparticles come into focus on a slightly different plane than the
neuronal cell bodies. All scale bars are 10 µm.
Summary and Conclusions
As x-ray bio-imaging and theranostics continue to become more clinically viable options, the
need for better, more versatile imaging agents will grow. Herein, we created a particulate
system based in LSO:Ce cores that have been surface modified with green emitting (AlNap)
and red emitting (AlRhod) to generate systems that are capable of emitting at a variety of
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colors when the core is excited with x-ray radiation. These particles were successfully imaged
in vitro with rat cortical neurons. The particulate systems were tested for cytotoxicity with
multiple assays and were never shown to be cytotoxic at high particle concentrations for up
to 72 hours. Additionally, due to the ease of surface functionalization of the particles, the
particles could be functionalized to target various extracellular targets for robust bioimaging.
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Acknowledgements
The authors wish to thank Dr. Jeremy Day (University of Alabama Birmingham) for pro-
viding the neuronal cultures. In addition, the authors wish the thank Nancy Gallus (Day
group) for preparing the neurons. The authors thank the Gregg-Graniteville Foundation and
the National Science Foundation (OIA-1632881) for financial support.
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