A new trick (hydroxyl radical generation) of an old vitamin (B2) for near-infrared-triggered photodynamic therapy

Article abstract of DOI:10.1039/c6ra23440b

The photosensitizer has been supposed to be the key component in photodynamic therapy (PDT). Natural products and their intricate molecular frameworks are often used as starting points for drug discovery. Riboflavin (RF), also known as vitamin B₂, has a unique conjugate structure of an isoalloxazine ring, and is a potential photosensitizer for use in the PDT of cancers. In this study, a novel near-infrared (NIR) mediated nanocomposite for PDT is presented, using this old vitamin (RF) as a PDT photosensitizer, integrated with the upconversion nanotechnology. Mesoporous silica coated sodium yttrium fluoride:ytterbium/thulium (NaYF₄:Yb/Tm) nanoparticles were fabricated and used as drug carriers and photo-transducers. Chemical modification of RF was performed to obtain a photostable photosensitizer, 2′,3′,4′,5′-tetraacetylriboflavin (RTA). There is a good overlap between the fluorescence emission of NaYF₄:Yb/Tm nanoparticles and the UV-visible absorption of RTA. The RTA molecules were incorporated into the mesoporous silica shell and the fluorescent emission from the NaYF₄:Yb/Tm nanoparticles can be absorbed by RTA molecules under NIR irradiation. NIR-initiated reactive oxygen species generation was validated using electron paramagnetic resonance (EPR) spectroscopy combined with spin trapping. The results from the in vitro cell test show that this nanocomposite has good photodynamic efficacy. All the chemicals involved in this process are non-toxic, environmentally benign, and easily available. Thus, this nanocomposite has great potential in PDT applications.

Full text of DOI:10.1039/c6ra23440b

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Page 1 of 35
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A New Trick (Hydroxyl radical generation) of an Old Vitamin (B2)
for Near-infrared-triggered Photodynamic Therapy
Fang Xu*, a, Jin Lia, Tingꢀting Zhua,b, ShengꢀSong Yub, Chong Zuoa, Riꢀsheng Yaoa
and Haiꢀsheng Qiana
aDepartment of Pharmaceutical Engineering, Hefei University of Technology, Hefei,
b
230009, China; Department of Chemistry, University of Science & Technology of
China, Hefei, 230026, China.
Corresponding author:
Fang Xu, Eꢀmail: Fangxu@hfut.edu.cn; Fax: +86 551 62901771
1

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ABSTRACT:
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Photosensitizer has been supposed to the key component in photodynamic therapy
(PDT). Natural products and their intricate molecular frameworks are often used as
starting points for drug discovery. Riboflavin (RF), also known as vitamin B2, bearing
a unique conjugate structure of isoalloxazine ring, is a potential photosensitizer for
use in the PDT of cancers. In this study, we present a novel nearꢀinfrared (NIR)
mediated nanocomposite for PDT, using this old vitamin as a PDT photosensitizer,
integrated with the upconversion nanotechnology. Mesoporousꢀsilicaꢀcoated
NaYF4:Yb/Tm nanoparticles were fabricated and used as drug carriers and
photoꢀtransducers. Chemical modification of RF was performed to obtain a
photostable photosensitizer (2',3',4',5'ꢀtetraacetylriboflavin, RTA). There is a good
overlap between the fluorescence emission of NaYF4:Yb/Tm nanoparticles and the
UVꢀvisible absorption of RTA. RTA molecules were incorporated into the
mesoporous silica shell and the fluorescent emission from NaYF4:Yb/Tm
nanoparticles can be absorbed by RTA molecules under NIR irradiation. NIRꢀinitiated
reactive oxygen species generation was validated by electron paramagnetic resonance
spectroscopy combined with spin trapping. The results from inꢀvitro cell test show
good photodynamic efficacy of this nanocomposite. All ingredients involved in this
process are nontoxic, environmentally benign, and easilyꢀavailable. Thus, this
nanocomposite might has great potential in PDT applications.
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1. Introduction
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Photodynamic therapy (PDT) has emerged as a promising and noninvasive treatment
for various types of cancers, involving three key components: photosensitizer (PS,
also called PDT drug), light source and tissue oxygen.1ꢀ3 The combination of these
three components leads to the generation of different reactive oxygen species (ROS),
such as singlet oxygen (1O2) through a energyꢀtransfer process (Type II mechanism),
•ꢀ
and superoxide anion radical (O2 ) through a electronꢀtransfer process (Type I
mechanism).4ꢀ6 Subsequently, ROS cause oxidative damage to biological substrates
and ultimately result in cancer cell death.7ꢀ9 This strategy has gained wide research
interest as a powerful technique for cancer treatment, due to its builtꢀin selectivity,
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low systemic toxicity, minimal invasiveness as well as the possibility of its use in
combination with other anticancer therapies.
Among which, PS is the key agent in PDT application and is also the research
focus in the PDTꢀrelated field.10ꢀ16 In clinical practice, porphyrins and its derivatives,
such as Photofrin (a mixture of hematoporphyrin monomers, dimers, and oligomers,)
and Foscan (mꢀtetrahydroxyphenylchlorin), are the most commonlyꢀused PDT PSs.17
However, these compounds suffer from several drawbacks: (1) tedious synthesis and
purification, (2) poor water solubility, and (3) slow clearance from the body leading to
possible photosensitivity after PDT treatment.18ꢀ24 Therefore, it is still imperative to
develop efficient, highly photostable, and excellent water dispersible PSs. Features
desired for ideal PDT drugs, including: (1) high photochemical reactivity, that is, it
can effectively produce ROS under appropriate irradiation; (2) low dark toxicity; and
(3) can be excited at a wavelength in the region where tissue penetration of irradiation
is at a maximum.
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Natural products and their intricate molecular frameworks are often used as
starting points for drug discovery.11, 25ꢀ27 Riboflavin (RF), also known as vitamin B2,
is an important vitamin and widely present both inꢀvivo and natural environments.28ꢀ31
It contains a unique conjugate structure, called isoalloxazine ring, which has been
reported to function as an excellent natural PS for the generation of ROS, such as 1O2
and/or O2•ꢀ.32ꢀ34 In our previous studies, ROS generation, including highly reactive
hydroxyl radical (•OH), has been validated during the photosensitization process of
RF (Fig. 1). In which, RF play a dual role of photosensitizer and electron
mediator.35ꢀ37 All ingredients involved in this process are nontoxic, environmentally
benign, and easilyꢀavailable. Thus, this process might have broad medical
implications and inspires us to probe the possibility of this vitamin workingas a novel
PDT drug.
However, this idea begs the following questions: (1) the limited penetration
depth of the excitation wavelength of RF (<520 nm), which inflicts biological damage
and cannot take advantage of the optical window of tissue (700ꢀ1100 nm),32ꢀ39 and (2)
the poor photostability of RF under UV or visible irradiations.32ꢀ36 Nonlinear
processes (involving more than one photon) have been investigated as a potential
solution to address question (1). In this context, upconversion nanoparticles (UCNP),
a photoꢀtransducer capable of converting NIR light to UV or visible light, was
employed as an excitation method for PDT treatment.40ꢀ46 To further underline the
feasibility of RF working as a PDT drug, 2',3',4',5'ꢀtetraacetylriboflavin (RTA) was
designed and synthetized from RF through a esterification process of its ribose chain
with acetic anhydride.34, 47ꢀ49
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In this study, RTA (a photostable derivative of RF) was used as a novel PDT
agent. Mesoporousꢀsilicaꢀcoated NaYF4:Yb/Tm nanoparticles with a core/shell
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structure were fabricated and used as drug carriers and photoꢀtransducers for PDT
treatment. The emission of UCNP was used to excite the PS, as shown in Fig. 2. RTA
molecules were incorporated into the mesoporous silica shell and the fluorescent
emission from the UCNP can be absorbed by RTA molecules coated on their surfaces
under NIR irradiation. Subsequently, the excited RTA molecules interact with
surrounding groundꢀstate molecular oxygen for the generation of ROS, leading to
oxidative damage of cancer cells.
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2. Experimental section
2.1. Chemicals and reagents
Riboflavin
(RF),
2,2,6,6ꢀtetramethylꢀ4ꢀpiperidone
(4ꢀoxoꢀTEMP),
5,5ꢀdimethylꢀ1ꢀpyrrolineꢀNꢀoxide (DMPO), catalase, yttrium chloride (YCl3, 99%),
ytterbium chloride (YbCl3, 99%), thulium chloride (TmCl3, 99%), octadecene (ODE),
ammonium fluoride (NH4F), octadecyltrimethoxysilane (C18TMS), tetraethyl
orthosilicate (TEOS), oleic acid, igepal COꢀ520 and cyclohexane were purchased
from SigmaꢀAldrich Co. (USA). Ferrous sulfate heptahydrate (FeSO4.7H2O), sodium
hydroxide (NaOH), hydrochloric acid (HCl), dimethyl sulfoxide (DMSO), acetic
anhydride, HPLCꢀgrade acetic acid, and HPLCꢀgrade ammonium acetate were
purchased from Shanghai Chemical Reagent Co. (China). HPLCꢀgrade methanol was
purchased from Merck Inc. (Germany). Ultrapure (MilliQ Inc., USA) water
(resistivity of 18.2 Mꢁꢀcm) was used in the experiments. All chemicals were of
analytical grade (or HPLCꢀgrade) and used without further purification.
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RTA, a synthetic derivative of RF, was prepared through a easyꢀtoꢀoperated
esterification process of RF with acetic anhydride (Scheme 1).34, 46ꢀ48 In brief, RF (5.0
g) was suspended in acetic anhydride solution (50 mL), then added sulfuric acid (0.1
mL) to initiate the esterification reaction, and magnetic stirred at 80oC for 4 h under
argon atmosphere. The cooled solution was neutralized with 1.0 M NaHCO3 (200
mL), extracted with CH2Cl2 and washed with ultrapure water. The organic layer was
dried with anhydrous magnesium sulfate, and evaporated under reduced pressure. The
raw product was further recrystallized from MeOH/H2O (4:1; v/v) to yield pure RTA.
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Scheme. 1. Schematic representation of synthetic procedure to obtain
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2’,3’,4’,5’ꢀtetraacetylriboflavin (RTA) from riboflavin (RF).
A LTQ XL orbitrap high resolution mass spectrometry (Thermo Fisher Sci. Inc.,
USA), coupled with an electrospray ionization (ESI) source, was used for direct
injection ESIꢀMS analysis. Data acquisition was performed with Xcalibur 2.0
software (Thermo Fisher Sci. Inc., USA). The spray voltage was set to 5.0 kV,
capillary temperature to 280oC. The sheath and auxiliary gas flow rate (both nitrogen)
were set at 18 and 3 arbitrary units, respectively. Full MS scans were acquired in the
orbitrap analyzer in a positive mode with the resolution set to a value of 60000.
1HꢀNMR spectrum were collected using a Agilent 600 MHz NMR spectrometer
(Agilent Inc., USA). FTIR spectrum of RTA was recorded on a VERTEX 70 FTꢀIR
spectrometer (Bruker Inc., Germany) operated at a setting of 32 scans at a spectral
resolution of 4 cmꢀ1. Analysis data: HR-MS (ESI) m/z calculated for C25H29N4O10
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[M+H]+ 545.1883, experimental found 545.1879. H-NMR (600 MHz, DMSOꢀd6,
ppm): 10.32 (s, 1H, ArꢀNH), 7.88 (s, 1H, ArꢀH), 7.72 (s, 1H, ArꢀH), 5.46 (m, 1H,
ꢀCHOAc), 5.33 (m, 1H, ꢀCHOAc), 4.83 (m, 1H, ꢀCHOAc), 4.36ꢀ4.25 (d, 2H,
ꢀCH2OAc), 4.22ꢀ4.15 (dd, 2H, ꢀCHH), 2.50 (s, 3H, ArꢀCH3), 2.45 (s, 3H, ArꢀCH3),
2.38 (s, 3H, ꢀCOOCH3), 2.18 (s, 3H, ꢀCOOCH3), 1.98 (s, 3H, ꢀCOOCH3), 1.60 (s, 3H,
ꢀCOOCH3). FT-IR (neat KBr): ν (cm‐1) = 3200 (br), 3029(m), 1745 (m), 1668 (s),
1370 (s), 1509 (s), 1360 (s), 1231 (vs), 1171 (vs), 1035 (s), 850 (vs).
2.2. Electrochemical analysis of RTA
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests were
conducted in a 3ꢀelectrode system with a CHI 660C electrochemical workstation
(Chenhua Instrument Co., China). The system utilized glassy carbon (GC) electrode
as working electrode, KClꢀsaturated Ag/AgCl as reference electrode, platinum wire as
counter electrode. Before each test, the GC electrode (d = 3 mm) was polished with
0.3ꢀ and 0.05ꢀꢂm alumina powder in succession, and sonicated in water for 30
seconds. The electrolyte, consisted of 100 ꢂM RTA in 100 mM PBS (pH 7.0), was
deoxygenated by nitrogen prior to the electrochemical tests. All experiments were
conducted at ambient temperature of 25oC.
2.3. Synthesis of mesoporous silicaꢀcoated NaYF4:Yb/Tm nanoparticles
NaYF4:Yb/Tm nanoparticles were fabricated and used as photoꢀtransducers in
this study. NaYF4:Yb/Tm nanocrystals with uniform size distribution were
synthesized via an efficient and userꢀfriendly method.41,42 NaYF4:Yb(18%)/Tm(2%)
nanocrystals were synthesized following a protocol that was reported previously.16, 41
YCl3 (0.8 mmol), YbCl3 (0.18 mmol), and TmCl3 (0.02 mmol) were mixed with 12
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mL oleic acid and 15 mL octadecene (ODE) in a 50 mL flask. The solution was
heated to 160oC to form a homogeneous solution, and then cooled to ambient
temperature (25oC). A 10 mL methanol solution containing NaOH (2.5 mmol) and
NH4F (4 mmol) was slowly added into the flask and stirred for 30 min. The solution
was slowly heated to remove methanol, degassed at 100oC for 10 min, and then
heated to 300oC and maintained for 2.0 h under argon protection. After the solution
was cooled naturally, nanocrystals were precipitated from the solution with ethanol
and washed with ethanol/water (1:1 v/v) three times.
Porous silicaꢀcoated NaYF4:Yb/Tm nanoparticles were further achieved
successfully by one pot facile synthetic process.39 Octadecyltrimethoxysilane
(C18TMS) was used as soft template, which has been used for preparing hydrophobic
coatings and selfꢀassembled monolayers. Briefly, COꢀ520 (1.0 mL), cyclohexane (6.0
mL) and NaYF4:Yb/Tm nanocrystal solution in cyclohexane (40 mL, 0.01 M) were
mixed and stirred for 60 min. Then, 1.0 mL ammonia (28 wt%) was added into the
previous solution and sonicated for 30 min until a transparent emulsion was formed.
The mixture solution of 120 ꢂL TEOS and 50 ꢂL octadecyltrimethoxysilane
(C18TMS) was then added into the previous solution. Finally, the solution was stirred
for 48 h at a speed of 800 rpm at ambient temperature of 25oC. Silicaꢀcoated
NaYF4:Yb/Tm nanoparticles were collected by centrifugation after precipitation by
adding acetone, washed with ethanol three times and finally dried at 60oC for 10 h.
The asꢀprepared sample was calcinated at 500oC in static air with a heating rate of
1.0oC/min and a dwelling time of 2.0 h.
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Mesoporousꢀsilicaꢀcoated NaYF4:Yb/Tm nanoparticles (20 mg) were soaked in
aqueous solutions of PSs (RTA or RF, 50 mL, 10 ꢂg/mL) for 24 h at ambient
temperature (25oC). The nanoparticles were then collected by centrifugation (5000 g,
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5 min) and washed with phosphate buffer saline solution (PBS, 0.02 M, pH=7.40).
Initial and residual concentrations of PSs were analyzed using a high performance
liquid chromatography system (HPLCꢀ1100, Agilent Inc., USA) equipped with a
VWD detector and a Hypersil ODS column. A mixture of methanol and water (30:70,
v/v) was used as the isocratic mobile phase with 0.1% acetic acid in water. The flow
rate was set at 1.0 mL/min, the UV detector was set at 360 nm, and the column
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temperature was set at 30oC.
The morphology and size of the asꢀprepared samples were investigated by
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transmission electron microscopy (TEM, JEOL 3010, JEOL Co., Japan). UVꢀvisible
absorption spectra were performed using a UVꢀvisible spectrometry (UVꢀ2450,
Shimadzu Co., Japan). The spectra were collected from 200 nm to 800 nm by an
increment of 0.5 nm. All measurements were performed in standard 10 mm quartz
cuvettes. Excitation–emission matrix fluorescence spectroscopic analysis was
performed using a luminescence spectrometry (LSꢀ55, PerkinꢀElmer Co., USA)
equipped with a 980 nm excitation source. Fluorescence spectra were collected with
subsequent scanning emission spectra from 200 to 800 nm by an increment of 0.5 nm
under the 980 nmꢀexcitation.
2.4. Photostability analysis of RTA
The photostabilities of RTA and RF under both visible and NIR irradiation were
evaluated in aqueous solutions (pH=7.0). A 50ꢀW Xeꢀarc lamp (XDꢀ300, Nanjing
Yanan Co., China) equipped with an UVꢀcutoff (>400 nm) was used as visible light
source and a 980 nm NIR laser (MDLꢀ980, Changchun New industries
optoelectronics tech. Co., China) was used as NIR light source. Samples were
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irradiated, and aliquots were removed and analyzed at given time intervals (10 min).
The light dependence processes were monitored and analyzed using both high
performance liquid chromatography (HPLCꢀ1100, Agilent Inc., USA) and UVꢀvisible
spectrometry (UVꢀ2450, Shimadzu Co., Japan).
2.5. ROS detection using electron paramagnetic resonance spectroscopy
EPR spectra were obtained using a JEOL JESꢀFA200 EPR spectrometer (JEOL
Co., Japan) with a 500ꢀW Xeꢀarc lamp equipped with an UVꢀcutoff (>400 nm) as
visibleꢀlight source and a 980ꢀnm laser with an output power of 1000 mW as a
nearꢀinfrared (NIR) light source. Spectrometer with Xꢀband microwave frequency of
9.072 GHz, microwave power of 2.02 mW, spectral window of 100 G, and
modulation amplitude of 1.00 G was used at ambient temperature of 25oC.
1
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4ꢀoxoꢀTEMP was used to detect O2, and DMPO was utilized to measure O2 and
•OH.35, 36, 50ꢀ53
1O2 was detected with the EPR method using 4ꢀoxoꢀTEMP as a spinꢀtrapping
reagent (Reaction 1).35, 50 Solutions of RTA were prepared in ultrapure water. Under
visible irradiation, the incubation of RTA at 100 ꢂM with the spinꢀtrapping agent
4ꢀoxoꢀTEMP of 100 mM resulted in the formation of a adducts. 200 ꢂL of the freshly
prepared mixture was added to a quartz EPR tube and illuminated for 0, 1.0, 2.0, 3.0,
4.0, and 5.0 min before recording the EPR spectra. Under NIRꢀirradiation, the
incubation of RTA/UCNP (5 mg/mL) with the spinꢀtrapping agent 4ꢀoxoꢀTEMP of
100 mM resulted in the formation of the adducts of 4ꢀoxoꢀTEMP/1O2. 200 ꢂL of the
freshly prepared mixture was added to a quartz EPR tube and illuminated for 10 min
1
before recording the EPR spectra. The generation of O2 was detected as an EPR
signal, because 4ꢀoxoꢀTEMPO is formed by the reaction of 1O2 with 4ꢀoxoꢀTEMP.
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•ꢀ
DMPO was used as a spinꢀtrapping agent for the detection of O2 (Reaction 2).35,
36,50
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Solutions of RTA were prepared in DMSO. Under visible irradiation, the
incubation of 100 ꢂM RTA with 100 mM spinꢀtrapping agent DMPO resulted in the
formation of the radical adducts. 200 ꢂL of the freshly prepared mixture was added to
a quartz EPR tube and illuminated for 0, 0.5 ,1.0, 1.5, and 2.0 min before recording
the EPR spectra. Under NIRꢀirradiation, the incubation of RTA/UCNP (5 mg/mL)
with 100 mM spinꢀtrapping agent DMPO resulted in the formation of the radical
adducts. 200 ꢂL of the freshly prepared mixture was added to a quartz EPR tube and
illuminated for 10 min before recording the EPR spectra.
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DMPO was also used as a spinꢀtrapping agent for the detection of HO• (Reaction
3).36, 51, 52 Solutions were prepared in ultrapure water. Under visible irradiation, the
incubation of RTA (100 ꢂM) and FeSO4 (10 ꢂM) with 100 mM spinꢀtrapping agent
DMPO resulted in the formation of the radical adducts. Then, 200 ꢂL of the freshly
prepared mixture was added to a quartz EPR tube and illuminated for 0, 1.0, 2.0, and
3.0 min before recording the EPR spectra. Under NIRꢀirradiation, the incubation of
RTA/UCNP (5 mg/mL) and FeSO4 (2 ꢂM) with 100 mM spinꢀtrapping agent DMPO
resulted in the formation of the radical adducts. Then, 200 ꢂL of the freshly prepared
mixture was added to a quartz EPR tube and illuminated for 10 min before recording
the EPR spectra.
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2.6. Cell culture and inꢀvitro PDT test
Inꢀvitro cytotoxicity assays were further performed using the human breast
cancer cell line MDAꢀMBꢀ231 from the American Type Culture Collection (ATCC).
MDAꢀMBꢀ231cells were cultured in RPMI 1640 medium, supplemented with 10%
fetal bovine serum and 2 mM Lꢀglutamine at 37oC using a humidified 5% CO2
incubator. The MDAꢀMBꢀ231 cells were seeded onto 96ꢀwell plates at 2.0×103 cells
per well and further incubated for 24 h. The culture medium was then replaced with
100 ꢂL of freshly prepared culture medium containing either free PS or PS/UCNP at
different concentrations.
Cells were treated with free RF, RTA, RF/UCNP or RTA/UCNP composites and
incubated for 24 h and then exposed to visible or 980 nmꢀNIR irradiation. All cells
were then incubated for another 48 h at 37oC and evaluated by MTT viability assay.
For MTT assay, MDAꢀMBꢀ231 cancer cells were collected and diluted to a cell
density of 1×105/mL in complete medium, and then seeded into 96ꢀwell plates (100
ꢂL/well). After being cultured for 24 h, the nanoparticles with different concentrations
(0.625, 1.25, 2.5, and 5.0 mg/mL) with and without PS were added to the cells and the
cells were incubated for 24 h at 37oC. The exposure time of NIRꢀirradiation was
divided into several sections (2 min per section) to avoid the overꢀheating possibility.
The cells were then incubated at 37oC for 48 h. The cell viability was measured by
MTT assay (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazoliumbromide) and
expressed as a percentage of the control.
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3. Results and Discussion
3.1. Electrochemical characteristics of RTA
The known ability of isoalloxazine ring to act as an excellent PS has been
supposed to play a key role in the generation of 1O2 or O2 . In our previous study, RF
•ꢀ
has been reported to play a dual role of excellent PS and effective electron mediator in
the process of •OH generation.35, 36 Some PSs have been shown to produce different
1
kind ROS, and synergistic effects of O2 and •OH on PDT treatment has been
proposed.17 However, RF is susceptible to photodegradation, likely due to the
instability of the ribose chain. Chemical modification of the ribose chain of the
molecule is highly desired, and can improve the yield and lifetime of the triplet state
and result in a more photostable derivative.34, 47ꢀ49 RTA was synthesized through a
easyꢀtoꢀoperated esterification process of RF with acetic anhydride. The electron
shuttling capacity of RF as a redoxꢀactive molecule paly a key role in the generation
of O2•ꢀ or •OH. Thus, electrochemical characterization of RTA and RF were fristly
conducted to analysis of their oxidationꢀreduction potentials.
The sterile glassy carbon electrodes were exposed to 0.05 mM RF. CV analysis
was performed at a scan rate of 100 mV/s. The potential difference between oxidation
and reduction peaks was 58 mV and the centers of the reversible voltammetric peaks
were at ꢀ0.40V (Fig. 3A). These results show the oxidationꢀreduction process of RF is
reversible and RF could be used as a mediator in electron transfer process. To test the
redox property of RTA, CV experiments were conducted in RTA solution. Compared
to the blank electrolyte (PBS solution), RTA shows a pair of obvious reversible redox
peaks with potential difference between oxidation and reduction peaks was 49 mV,
and the centers of the reversible voltammetric peaks were at ꢀ0.37 V. The redox peaks
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of RTA were more positive than that of RF, which was consistent with DPV results
(Fig. 3B). The DPV results show that the redox potential of RTA was larger than that
of RF by 30 mV. These results indicate that RTA was more easily to be reduced.
Additionally, the peak separation of RTA was also smaller, suggesting that RTA was
more electrochemically reversible than RF. These results demonstrate that RTA was
successfully synthesized from RF and its electrochemical properties were improved
after the modification.
3.2. Characteristics of NaYF4:Yb/Tm upconversion nanoparticles
TEM analysis was used to examine the morphology of the UCNP nanoparticles.
The results clear show that the nanoparticles were discrete and uniform with an
average diameter of 30 and 50 nm for NaYF4:Yb/Tm (Fig. 4A) and porous
silicaꢀcoated NaYF4:Yb/Tm (Fig. 4B), respectively. Brunauer–Emmet–Teller (BET)
analysis reveal mesoporous structure of silicaꢀcoated NaYF4:Yb/Tm with an average
poreꢀsize distribution of 2.30 nm and a specific surface area of 680 m2/g.
Mesoporousꢀsilicaꢀcoated NaYF4:Yb/Tm nanoparticles (100 mg) were soaked in PS
aqueous solution (RF or RTA, 100 mL, 0.1 mM) for 24 h at ambient temperature of
25oC. The nanoparticles were then collected by centrifugation and washed with PBS
solution (0.02 M, pH=7.4). Concentrations of PS solutions (RTA or RF) were
determined using HPLC method and the amount of adsorbed into the mesoporous
silica was about 2.0 wt%.
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spectrum analysis of RTA
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The luminescent spectrum of NaYF4:Yb/Tm nanoparticles and the UVꢀvisible
absorption spectrum of the RTA and RF solutions were further analyzed. The
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luminescence spectrum of the mesoporous silicaꢀcoated NaYF4:Yb/Tm upon
NIRꢀirradiation (980 nm) is shown in Fig. 5A. The dominant emissions are located at
345, 360, 451, and 476 nm. As shown in Fig. 5B, there is a good overlap between the
fluorescence emission of NaYF4:Yb/Tm nanoparticles and the UVꢀvisible absorption
of RTA and RF solutions (main peaks located at 372 and 450 nm). It is envisaged that
the PS (RTA or RF) can be aborted and activated by the UCNP to produce ROS and
kill cancer cells when exposed to NIR irradiation.
3.4. Photostability analysis of RTA
Qualitative evaluation of the photostabilities of RTA and RF were performed
under visible or NIR (980 nm, 500 mw) irradiation. Initial and residual concentrations
of RTA and RF were analyzed using HPLC. Free RF in solution was degraded
completely after one hour of visible irradiation, while RTA was degraded only 20%
under similar conditions. Compared with visible irradiation, both free RTA and RF,
and in the form of PS/UCNP nanocomposites exhibit an appreciable photostability
under NIR irradiation, as shown in Fig. 6. These results indicate that the RTA
molecule in the form of RTA/UCNP nanocomposite has a good photostability under
NIR irradiation, and could used as a potential candidate of PS for PDT applications.
3.5. Analysis of ROS using electron paramagnetic resonance spectroscopy
ROS generation during the NIRꢀtriggered photosensitization process RTA was
analyzed using EPR spectroscopy combined with spin trapping. Solution of
RTA/UCNP (5 mg/mL) with DMPO (100 mM) was prepared in ultrapure water
(pH=3.0). 4ꢀoxoꢀTEMP was used to detect 1O2, and DMPO was utilized to measure
O2•ꢀ and •OH.51, 52 200 ꢂL of this freshly prepared mixture was added to a quartz EPR
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tube and illuminated for 5.0 min before recording the EPR spectra. The specific
1
signals of 4ꢀoxoꢀTEMP/1O2 were produced by O2 generated from photoexcited RTA
(Fig. 7A). The spectrum is composed of a triplet of lines, with a peak height ratio of
1:1:1, with parameters including hyperfine constants, aN=16.30 G and a
gꢀvalue=2.0055. These parameters exactly matched with accurate EPR spectrum
simulation of 4ꢀoxoꢀTEMP/1O2. O2•ꢀ is sensitive to the presence of proton and its
lifetime is very short in protic solvents.35, 36 Thus, it is difficult to detect O2•ꢀ in protic
•ꢀ
solvent and an aprotic solvent of DMSO was used in this study to facilitate O2
•ꢀ
detection.51,53 The hyperfine splitting constants for DMPO/O2 were aN=12.80 G;
β
γ
aH =10.41 G; aH =1.35 G; g=2.0021 (Fig. 7B). An accurate EPR spectrum simulation
•ꢀ
of DMPO/O2 has also been performed.
•ꢀ
1
On the basis of the analysis of both O2 and O2, we further examined the
feasibility of NIRꢀinitiated •OH generation of RTA solution in the presence of
dissolvedꢀiron. Solution of RTA/UCNP (5 mg/mL) and FeSO4 (5 ꢂM) with DMPO
(100 mM) was prepared in acidic water (pH=3.0). Upon exposure to NIR irradiation,
a major radical/DMPO adduct was formed and assigned to DMPO/•OH. The
hyperfine splitting constants for DMPO/•OH were aH=aN=14.96 G and a
gꢀvalue=2.0050 (Fig. 7C). Among the signals for DMPO/•OH (1:2:2:1), a weak EPR
signal was composed of a triplet of lines with a peak height ratio of 1:1:1. There is a
signal of oxidized DMPO (DMPOX) in the EPR spectra. Similar results of ROS
generation were obtained with RF/UCNP solution under NIR irradiation.
3.6. Inꢀvitro PDT activity of RTA/UCNP nanocomposites
As designed, the NIRꢀtriggered release of ROS from RTA/UCNP
nanocomposites was deemed to exhibit the spatioꢀtemporal controlled anticancer
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effects. Therefore, the therapeutic potential of RTA/UCNP composite was evaluated
inꢀvitro using the human breast cancer cell. Cells with free RTA or RF (10 ꢂg/mL)
were exposed to a 50ꢀW Xeꢀarc lamp equipped with an UVꢀcutoff (>400 nm). The
cell viability with different exposure time to visible irradiation (5, 10, 20, and 30 min)
indicated that the cell viability decreased with the exposure time under visible
irradiation, suggesting an increased amount of ROS produced by the PS (RTA or RF).
Normal incubated cells was used as control (Controlꢀ1). Cells incubated without
visible irradiation show that both RF (Controlꢀ2) and RTA (Controlꢀ3) are nontoxic
and biocompatible. Cells of controlꢀ4 were incubated under visible irradiation without
adding any PS. RTA has a much better PDT effect than RF, which can be attributed to
the improved photostability of RTA. The results of the control tests show that PS and
visible irradiation were both required.
Fig. 8A shows the effect of exposure time to NIR irradiation on the cell viability
in the presence of RTA/UCNP or RF/UCNP (5 mg/mL). Cells cultured with
PS/UCNP were exposed to a 980 nmꢀNIRꢀlaser with an output power of 500 mW.
The cell viability of cancer cells clearly decreased with increasing irradiation time.
Control experiments with NIR irradiation in the absence of PS/UCNP also showed
that NIR irradiation alone did not initiate cell death. Fig. 8B showed that the viability
of cells incubated with the amount of RTA/UCNP (0.625, 1.25, 2.5, and 5 mg/mL)
exposed to NIR irradiation for 30ꢀmin was significantly lower as compared to the
nanoparticles without RTA/UCNP (Controlꢀ1) or without NIR irradiation (Controlꢀ2).
Similar results were obtained with RF/UCNP solution under NIR irradiation.
Fig. 8C shows the effect of Fe2+ on the cell viability. At a low concentration
range (0ꢀ1.0 ꢂg/mL), the cell viability decreased with an increase of Fe2+
concentration. Interestingly, the adding of Fe2+ facilitates the generation of •OH, and
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results in a increase of phototoxicity. Howerver, with Fe2+ concentration further
increased, the cell viability increased with increasing Fe2+ concentration.
4. Conclusions
In summary, a novel NIRꢀlightꢀtriggered PDT nanosystem has been developed using
an old vitamin (VB2) integrated with the upconversion nanotechnology.
Mesoporousꢀsilicaꢀcoated NaYF4:Yb/Tm nanoparticles were successfully fabricated,
which played a dual role of drug carrier and photo transducer. Chemical
modification of this old vitamin was performed through a easyꢀtoꢀoperated
esterification process to improve its photostability. This nanoplatform has some
desired features for an ideal PDT drug, including: (1) high waterꢀsolubility; (2)
excellent photochemical reactivity to produce reactive oxygen species; (3) low dark
toxicity; and (4) can be excited at NIR region where tissue penetration of irradiation is
at a maximum. The results of inꢀvitro cell experiment demonstrate that RTA/UCNP
nanocomposite of the proposed design has great potential in PDT application.
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Acknowledgements
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The authors wish to thank the National Science Foundation of China (No.21207031,
No.21471043), the China Postdoctoral Science Foundation (No. 2016M592049), and
the Key Laboratory of Urban Pollutant Conversion, Chinese Academy of Sciences
(No. KF201603) for the support of this study.
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References
1
2
3
D. E. Dolmans, D. Fukumura and R. K. Jain, Photodynamic therapy for cancer,
Nat. Rev. Cancer, 2003, 3, 380ꢀ387.
R. S. Balaban, S. Nemoto and T. Finkel, Mitochondria, oxidants, and aging, Cell,
2005, 120, 483ꢀ495.
K. Ishikawa, K. Takenaga, M. Akimoto, N. Koshikawa, A. Yamaguchi, H.
Imanishi, K. Nakada, Y. Honma and J. Hayashi, ROSꢀgenerating mitochondrial
DNA mutations can regulate tumor cell metastasis, Science, 2008, 320, 661ꢀ664.
W. M. Sharman, C. M. Allen and J. E. van Lier, Photodynamic therapeutics:
basic principles and clinical applications, Drug Discov. Today, 1999, 4, 507ꢀ517.
4
5
420
421
422
423
424
425
426
427
428
429
430
431
432
P. Mroz, A. Pawlak, M. Satti, H. Lee, T. Wharton, H. Gali, T. Sarna and M. R.
Hamblin, Functionalized fullerenes mediate photodynamic killing of cancer cells:
Type I versus Type II photochemical mechanism, Free Radical Bio. Med., 2007,
43, 711ꢀ719.
6
Y. Arenas, S. Monro, G. Shi, A. Mandel, S. McFarland and L. Lilge,
Photodynamic inactivation of Staphylococcus aureus and methicillinꢀresistant
Staphylococcus aureus with Ru(II)ꢀbased type I/type II photosensitizers,
Photodiagn. Photodyn. Ther., 2013, 10, 615ꢀ625.
7
8
M. Ochsner, Photophysical and photobiological processes in the photodynamic
therapy of tumors, J. Photochem. Photobiol. B Biol., 1997, 39, 1ꢀ18.
L. Li, G. Ishdorj and S. B. Gibson, Reactive oxygen species regulation of
autophagy in cancer: Implications for cancer treatment, Free Radical Bio. Med.,
2012, 53, 1399ꢀ1410.
19

RSC Advances
Page 20 of 35
View Article Online
DOI: 10.1039/C6RA23440B
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
9
F. Cieplik, A. Späth, J. Regensburger, A. Gollmer, L. Tabenski, K. Hiller, W.
Bäumler, T. Maisch and G. Schmalz, Photodynamic biofilm inactivation by
SAPYRꢀAn exclusive singlet oxygen photosensitizer, Free Radical Bio. Med.,
2013, 65, 477ꢀ487.
10 Y. Yamakoshi, N. Umezawa, A. Ryu, K. Arakane, N. Miyata, Y. Goda, T.
Masumizu and T. Nagano, Active oxygen species generated from photoexcited
•ꢀ
1
fullerene (C60) as potential medicines: O2 versus O2, J. Am. Chem. Soc., 2003,
125, 12803ꢀ12809.
11 A. Montazerabadi, A. Sazgarnia, M. H. BahreyniꢀToosi, A. Ahmadic, A.
ShakeriꢀZadeh and A. Aledavood, Mitoxantrone as a prospective photosensitizer
for photodynamic therapy of breast cancer, Photodiagn. Photodyn. Ther., 2012, 9,
46ꢀ51.
12 A. Nadhman, S. Nazir, M. I. Khan, S. Arooj, M. Bakhtiar, G. Shahnaz and M.
Yasinzai, PEGylated silver doped zinc oxide nanoparticles as novel
photosensitizers for photodynamic therapy against Leishmania, Free Radical Bio.
Med., 2014, 77, 230ꢀ238.
13 W. J. Yan, Q. Wang, C. H. Yuan, F. Wang, Y. Ji, F. Dai, X. L. Jin and B. Zhou,
Designing piperlongumineꢀdirected anticancer agents by an electrophilicityꢀbased
prooxidant strategy: A mechanistic investigation, Free Radical Bio. Med., 2016,
97, 109ꢀ123.
14 M. Rajendran, Quinones as photosensitiser for photodynamic therapy: ROS
generation, mechanism and detection methods, Photodiagn. Photodyn. Ther.,
20

Page 21 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA23440B
455
456
457
2016, 13, 175ꢀ187.
15 C. S. Lee, W. Park, Y. U. Jo and K. Na, A chargeꢀswitchable, fourꢀarmed
polymeric photosensitizer for photodynamic cancer therapy, Chem. Commun.,
458
459
2014, 50, 4354ꢀ4357.
16 F. Xu, L. Ding, W. Tao, X. Z. Yang, H. S. Qian and R. S. Yao,
460
461
462
Mesoporousꢀsilicaꢀcoated upconversion nanoparticles loaded with vitamin B12 for
nearꢀinfraredꢀlight mediated photodynamic therapy, Materials Lett., 2016, 167,
205ꢀ208.
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
17 J. M. Dąbrowski, L. G. Arnaut, M. M. Pereira, K. Urbańska, S. Simões, G.
Stochel and L. Cortes, Combined effects of singlet oxygen and hydroxyl radical
in photodynamic therapy with photostable bacteriochlorins: Evidence from
intracellular fluorescence and increased photodynamic efficacy in vitro, Free
Radical Bio. Med., 2012, 52: 1188ꢀ1200.
18 X. Chen, L. Hui, D. A. Foster and C. M. Drain, Efficient synthesis and
photodynamic activity of porphyrinꢀsaccharide conjugates: Targeting and
incapacitating cancer cells, Biochemistry, 2004, 43, 10918ꢀ10929.
19 I. Lanzo, N. Russo and E. Sicilia, Firstꢀprinciple timeꢀdependent study of
magnesiumꢀcontaining porphyrinꢀlike compounds potentially useful for their
application in photodynamic therapy, J. Phys. Chem. B, 2008, 112, 4123ꢀ4130.
20 J. F. Lovell, M. W. Chan, Q. C. Qi, J. Chen and G. Zheng, Porphyrin FRET
acceptors for apoptosis induction and monitoring, J. Am. Chem. Soc., 2011, 133,
18580ꢀ18582.
21 X. C. Zhu, W. T. Lu, Y. Z. Zhang, A. Reed, B. Newton, Z. Fan, H. T. Yu, P. C.
21

RSC Advances
Page 22 of 35
View Article Online
DOI: 10.1039/C6RA23440B
478
479
480
481
482
483
484
485
486
487
488
489
490
491
Ray and R. M. Gao, Imidazoleꢀmodified porphyrin as a pHꢀresponsive sensitizer
for cancer photodynamic therapy, Chem. Commun., 2011, 47, 10311ꢀ10313.
22 T. C. Lei, G. F. Glazner, M. Duffy, L. Scherrer, S. Pendyala, B. H. Li, X. L.
Wang, H. W. Wang and Z. Huang, Optical properties of hematoporphyrin
monomethyl ether (HMME), a PDT photosensitizer, Photodiagn. Photodyn. Ther.,
2012, 9, 232ꢀ242.
23 R. Dosselli, C. Tampieri, R. RuizꢀGonzález, S. De Munari, X. Ragàs, D.
SánchezꢀGarcía, M. Agut, S. Nonell, E. Reddi and M. Gobbo, Synthesis,
characterization, and photoinduced antibacterial activity of porphyrinꢀtype
photosensitizers conjugated to the antimicrobial peptide apidaecin 1b, J. Med.
Chem., 2013, 56, 1052ꢀ1063.
24 E. Alves, M. A. F. Faustino, M. G. P. M. S. Neves, Â. Cunha, H. Nadais and A.
Almeida, Potential applications of porphyrins in photodynamic inactivation
beyond the medical scope, J. Photochem. Photobiol., C: Photochem. Rev., 2015,
492
493
22, 34ꢀ57.
25 T. Rodrigues, D. Reker, P. Schneider and G. Schneider, Counting on natural
494
products for drug design, Nat. Chem., 2016, 8, 531ꢀ541; 2016.
495
496
497
498
499
26 T. A. Shell and D. S. Lawrence, A new trick (hydroxyl radical generation) for an
old Vitamin (B12), J. Am. Chem. Soc., 2011, 133, 2148ꢀ2150.
27 T. A. Shell, J. R. Shell, Z. L. Rodgers and D. S. Lawrence, Tunable visible and
nearꢀIR photoactivation of lightꢀresponsive compounds by using fluorophores as
lightꢀcapturing antennas, Angew. Chem. Int. Ed., 2014, 53, 875ꢀ878.
22

Page 23 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA23440B
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
28 H. Canstein, J. Ogawa, S. Shimizu and J. R.Lloyd, Secretion of flavins by
Shewanella species and their role in extracellular electron transfer, Appl. Environ.
Microb., 2008, 7, 615ꢀ623.
29 E. Marsili, D. B. Baron, I. D. Shikhare, D. Coursolle, J. A. Gralnick and D. R.
Bond, Shewanella secretes flavins that mediate extracellular electron transfer,
Proc. Natl. Acad. Sci. USA, 2008, 105, 3968ꢀ3973.
30 S. SanudoꢀWilhelmy, L. S. Cutter, R. Durazo, E. A. Smail, L. GomezꢀConsarnau,
E. A. Webb, M. G. Prokopenko, W. M. Berelson and . Karl, ultiple Bꢀvitamin
depletion in large areas of the coastal ocean, Proc. Natl. Acad. Sci. USA, 2009,
109,14041ꢀ14045.
31 S. Babanova, O. Bretschger, J. Roy, A. Cheung, K. Artyushkova and P.
Atanassov, Innovative statistical interpretation of Shewanella oneidensis
microbial fuel cells data Phys. Chem. Chem. Phys., 2014,16, 8956ꢀ8969.
32 Y. Barbieri, W. A. Massad, D. J. Diaz, J. Sanz, F. AmatꢀGuerri and N. A. Garcia,
hotodegradation of bisphenol A and related compounds under naturalꢀlike
conditions in the presence of riboflavin: Kinetics, mechanism and photoproducts,
Chemosphere, 2008, 73, 564ꢀ571.
33 E. Haggi, S. Bertolotti and N. A. Garcia, Modelling the environmental
degradation of water contaminants. Kinetics and mechanism of the
riboflavinꢀsensitisedꢀphoto oxidation of phenolic compounds, Chemosphere,
2004, 5, 1501ꢀ1507.
34 C. K. Remucal and K. McNeill, Photosensitized amino acid degradation in the
23

RSC Advances
Page 24 of 35
View Article Online
DOI: 10.1039/C6RA23440B
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
presence of riboflavin and its derivatives, Environ. Sci. Technol., 2011, 45,
5230ꢀ5237.
35 F. Xu, X. N. Song, G. P. Sheng, H. W. Luo, W. W. Li, R. S. Yao and H. Q. Yu,
Sunlightꢀmediated degradation of methyl orange sensitized by riboflavin: Roles
of reactive oxygen species, Sep. Purif. Technol., 2015, 142, 18ꢀ24.
36 F. Xu, X. N. Song, G. P. Sheng, H. W. Luo, W. W. Li, R. S. Yao and H. Q. Yu,
Vitamin B2ꢀinitiated hydroxyl radical generation under visible light in the
presence of dissolved iron, ACS Sustainable Chem. Eng., 2015, 3, 1756ꢀ1763.
37 Y. Suzuki, Y. Kitatsuji, T. Ohnuki and S. Tsujimura, Flavin mononucleotide
mediated electron pathway for microbial U(VI) reduction, Phys. Chem. Chem.
Phys., 2010,12, 10081ꢀ10087.
38 E. Kosarieh, S. S. Khavas, A. Rahimi, N. Chiniforush and N. Gutknecht,
The comparison of penetration depth of two different photosensitizers in root anal
s with and without smear layer: An in vitro study, Photodiagn. Photodyn. Ther.,
2016, 13,10ꢀ14.
39 S. Kishimoto, M. Bernardo, K. Saito, S. Koyasu, J. B. Mitchell, P. L. Choyke and
M. C. Krishn, Evaluation of oxygen dependence on in vitro and in vivo
cytotoxicity of photoimmunotherapy using IRꢀ700ꢀantibody conjugates, Free
Radical Bio. Med., 2015, 85, 24ꢀ32.
40 D. Gao, R. R. Agayan, H. Xu, M. A. Philbert and R. Kopelman, Nanoparticles for
twoꢀphoton photodynamic therapy in living cells, Nano Lett., 2006, 6, 2383ꢀ2386.
41 H. S. Qian, H. C. Guo, P. C. L. Ho, R. Mahendran and Y. Zhang,
24

Page 25 of 35
RSC Advances
View Article Online
DOI: 10.1039/C6RA23440B
544
Mesoporousꢀsilicaꢀcoated upꢀconversion fluorescent nanoparticles for
545
546
photodynamic therapy, Small, 2009, 5, 2285ꢀ2290.
42 B. B. Ding, H.Y. Peng, H. S. Qian, L. Zheng and S. H. Yu, Unique upconversion
547
548
coreꢀshell nanoparticles with tunable fluorescence synthesized by a sequential
growth process, Adv. Mater. Interfaces, 2016, 3, 1500649.
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
43 P. Zhang, W. Steelant, M. Kumar and M. Scholfield, Versatile photosensitizers
for photodynamic therapy at infrared excitation, J. Am. Chem. Soc., 2007, 129,
4526ꢀ4527.
44 A. V. Kachynski, A. Pliss, A. N. Kuzmin, T. Y. Ohulchanskyy, A. Baev, J. Qu
and P. N. Prasad, Photodynamic therapy by in situ nonlinear photon conversion,
Nat. Photon., 2014, 8, 455ꢀ461.
45 G. F. Wang, Q. Peng and Y. D. Li, Lanthanideꢀdoped nanocrystals: synthesis,
opticalꢀmagnetic properties, and applications, Acc. Chem. Res., 2011, 44,
322ꢀ332.
46 L. Z. Zhao, J. J. Peng, Q. Huang, C. Y. Li, M. Chen,Y. Sun, Q. N. Lin, L. Y. Zhu
and F. Y. Li, Nearꢀinfrared photoregulated drug release in living tumor tissue via
yolkꢀshell upconversion nanocages, Adv. Funct. Mater., 2014, 24, 363ꢀ371.
47 E. C. Smith and D. E. Metzler, Photochemical degradation of riboflavin, J. Am.
Chem. Soc., 1963, 85, 3285ꢀ3287.
48 P. F. Heelis, The photophysical and photochemical properties of flavins
(Isoalloxazines), Chem. Soc. Rev., 1982, 11,15ꢀ39.
49 R. A. Larson, P. L. Stackhouse and T. O. Crowley, Riboflavin tetraacetate: A
25

RSC Advances
Page 26 of 35
View Article Online
DOI: 10.1039/C6RA23440B
566
567
568
569
570
571
572
potentially useful photosensitizing agent for the treatment of contaminated waters,
Environ. Sci. Technol., 1992, 26, 1792ꢀ1798.
50 S. S. Kumar, T. P. A. Devasagayam, B. Bhushan and N. C. Verma, Scavenging of
reactive oxygen species by chlorophyllin: an ESR study, Free Radic. Res., 2001,
35, 563ꢀ574.
51 P. Pieta, A. Petr, W. Kutner and L. Dunsch, In situ ESR spectroscopic evidence
•ꢀ
of the spinꢀtrapped superoxide radical, O2 , electrochemically generated in
573
574
DMSO at room temperature, Electrochim. Acta., 2008, 53, 3412ꢀ3415.
52 J. L. Roberts, M. M. Morrison and D. T. Sawyer, Baseꢀinduced generation of
575
576
577
578
579
superoxide ion and hydroxyl radical from hydrogen peroxide, J. Am. Chem. Soc.,
1978, 100, 329ꢀ330.
53 M. J. Burkitt and R. P. Mason, Direct evidence for in vivo hydroxylꢀradical
generation in experimental iron overload: an ESR spinꢀtrapping investigation,
Proc. Natl. Acad. Sci. USA., 1991, 88, 8440ꢀ8444.
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Fig. 1. Schematic illustration of RFꢀinitiated ROS generation under visible irradiation
(RF: Riboflavin; ISC: Intersystem Crossing).
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591
Fig. 2. Schematic illustration of the PS/UCNP nanoplatform for PDT and Proposed
NIRꢀtriggered ROS generation leading to oxidative damage of cancer cells, RTA
(2’,3’,4’,5’ꢀtetraacetylriboflavin, a chemical derivative from RF) was used as PDT
agent.
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DOI: 10.1039/C6RA23440B
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Fig. 3. (A) Cyclic voltammetry (CV) analysis of RTA (blue), RF (red) and 0.1 M PBS
(pH 7.0, black) between ꢀ0.8 and 0.2 V vs. Ag/AgCl at a scan rate of 100 mV/s; (B)
Differential pulse voltammetry (DPV) analysis RTA (blue), RF (red) and 0.1 M PBS
(pH 7.0, black) between ꢀ0.8 and 0.2 V vs. Ag/AgCl at a scan rate of 100 mV/s.
29