Photoresponsive real time monitoring silicon quantum dots for regulated delivery of anticancer drugs

Article abstract of DOI:10.1039/c5tb02045j

Recently, photoresponsive nanoparticles have been widely used to develop drug delivery systems (DDSs) wherein light is used as an external stimulus to trigger drug release in a spatially and temporally controlled fashion. Real time monitoring DDSs are also gaining much interest due to their capability of monitoring drug release in situ. In this context we designed a new photoresponsive real time monitoring nanoparticle based on photoluminescent silicon quantum dots (SiQDs) using the o-nitrobenzyl (ONB) derivative as a phototrigger for the controlled release of anticancer drug chlorambucil (Cbl). The strong fluorescence of SiQDs was initially quenched by ONB. Upon irradiation ONB triggered the release of the drug switching on the fluorescence of SiQDs to monitor the drug release. We reported a new and simple strategy to synthesise amine functionalised silicon quantum dots and covalently conjugated phototrigger ONB with caged anticancer drug Cbl onto it. Newly designed photoresponsive theranostic ONBCbl-SiQDs performed three important functions: (i) nanocarriers for drug delivery, (ii) controlled drug release under both one photon and two-photon excitation, and (iii) photoswitchable fluorescent nanoparticles for real-time monitoring of drug release based on the photoinduced electron transfer (PET) process. In vitro biological studies revealed the efficient cellular internalisation and cancer cell destruction ability of ONBCbl-SiQDs upon photoirradiation. ONBCbl-SiQDs exhibit a successful example of combining multiple functions into a single system for drug delivery systems.

Full text of DOI:10.1039/c5tb02045j

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Photoresponsive real time monitoring silicon
quantum dots for regulated delivery of anticancer
Cite this:DOI: 10.1039/c5tb02045j
drugs†
Amrita Paul,a Avijit Jana,b S. Karthik,a Manoranjan Bera,a Yanli Zhao*b and
N. D. Pradeep Singh*a
Recently, photoresponsive nanoparticles have been widely used to develop drug delivery systems (DDSs)
wherein light is used as an external stimulus to trigger drug release in a spatially and temporally
controlled fashion. Real time monitoring DDSs are also gaining much interest due to their capability of
monitoring drug release in situ. In this context we designed a new photoresponsive real time monitoring
nanoparticle based on photoluminescent silicon quantum dots (SiQDs) using the o-nitrobenzyl (ONB)
derivative as a phototrigger for the controlled release of anticancer drug chlorambucil (Cbl). The strong
fluorescence of SiQDs was initially quenched by ONB. Upon irradiation ONB triggered the release of the
drug switching on the fluorescence of SiQDs to monitor the drug release. We reported a new and
simple strategy to synthesise amine functionalised silicon quantum dots and covalently conjugated
phototrigger ONB with caged anticancer drug Cbl onto it. Newly designed photoresponsive theranostic
ONBCbl–SiQDs performed three important functions: (i) nanocarriers for drug delivery, (ii) controlled
drug release under both one photon and two-photon excitation, and (iii) photoswitchable fluorescent
nanoparticles for real-time monitoring of drug release based on the photoinduced electron transfer
(PET) process. In vitro biological studies revealed the efficient cellular internalisation and cancer
cell destruction ability of ONBCbl–SiQDs upon photoirradiation. ONBCbl–SiQDs exhibit a successful
example of combining multiple functions into a single system for drug delivery systems.
Received 1st October 2015,
Accepted 10th December 2015
DOI: 10.1039/c5tb02045j
www.rsc.org/MaterialsB
Recently real time monitoring of drug delivery from DDSs has
been performed through Forster resonance energy transfer
Introduction
Photoresponsive nanodrug delivery systems (DDSs) combining (FRET),7 aggregation induced emission (AIE),8 or a quenching
both therapeutic and diagnostic functionalities in a single system,9 consisting of a fluorophore or nanoparticles. By any
system have captured great attention because of their key means, if we can develop a single component photoresponsive
ability to regulate drug release spatially and temporally by real time monitoring drug delivery system, it will be helpful to
externally regulated light.1 In general, photoresponsive nano- maximize control over drug release and minimize undesirable
DDSs consist of two main components, namely, biocompatible side effects.
nanoparticles and a small organic molecule ‘‘phototrigger’’.2
Recently, photoluminescent silicon quantum dots (SiQDs)
On the other hand real time monitoring DDSs are also attracting have emerged as promising materials in the area of cellular
utmost interest for drug delivery because they are capable of imaging. SiQDs are well explored as in vitro10–12 and in vivo13,14
monitoring the drug delivery location and the delivered amount imaging agents because of their unique optical properties, high
in situ,3–6 allowing us to determine the drug delivery kinetics. natural abundance, low toxicity, excellent surface tailorability,
size dependent tunable emission, strong fluorescence and low
photobleaching properties.15 Drug conjugation to SiQDs and
a Department of Chemistry, Indian Institute of Technology Kharagpur, 721302,
controlled release of the loaded drug in response to the pH
change have been reported recently.16–18 The aforesaid unique
features of SiQDs have inspired us to design a photoresponsive
nano-DDS based on SiQDs for the first time. Here SiQDs are
used as biocompatible fluorescent nanocarriers and o-nitrobenzyl
(ONB) as a phototrigger for the controlled release of the anticancer
drug, chlorambucil. In this study, we used the o-nitrobenzyl (ONB)
West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ernet.in
b Division of Chemistry and Biological Chemistry, School of Physical and
Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link,
637371, Singapore. E-mail: Zhaoyanli@ntu.edu.sg
† Electronic supplementary information (ESI) available: Synthetic schemes of
ONBCbl, 1H NMR and 13C NMR spectra, EDX patterns of ONBCbl–SiQDs,
quantum yield calculations, HPLC profiles and other experimental details. See
DOI: 10.1039/c5tb02045j
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fluorescence emission spectra were recorded on a Hitachi F-7000
fluorescence spectrophotometer, and FTIR spectra were recorded
on a Perkin Elmer RXI spectrometer. Transmission Electron
Microscopy (TEM) was carried out on a FEI Tecnai G220S-Twin
at 200 kV. The TEM sample was prepared by dispersing compounds
in methanol and dropping on the surface of a copper grid.
Photolysis of all the ester conjugates was carried out using a
125 W medium pressure Hg lamp supplied by SAIC (India).
Chromatographic purification was carried out with 60–120 mesh
silica gel (Merck). For reaction monitoring, precoated silica gel
60 F254 TLC sheets (Merck) were used. Reverse phase HPLC
(RP-HPLC) was carried out using mobile phase acetonitrile, at a
flow rate of 1 mL minÀ1 (detection: UV 254 nm).
Synthesis of 4-(3-bromopropoxy)5-methoxy-nitrobenzaldehyde20
1,3-Dibromopropane (3.98 g, 2.01 mL, 19.71 mmol) was added
to a solution of vanillin (1 g, 6.57 mmol) in DMF (2 mL),
containing K2CO3 (1.18 g, 8.54 mmol). The reaction was then
stirred at 80 1C for 1 h. Subsequently the reaction mixture was
cooled to room temperature, diluted with ethyl acetate and
washed many times with brine (200 mL) and the organic layer
was dried over Na2SO4. The crude material was purified by
column chromatography through silica gel using EtOAc/hexane
(1 : 2) to yield 954 mg (3.49 mmol, 53%) of the product as
a white solid. 1H NMR (CDCl3, 200 MHz): d = 9.83 (1H, s),
7.45–7.41 (1H, d, J = 8.2 Hz), 7.39 (1H, s,), 7.02–6.98 (1H, d,
J = 8.2 Hz), 4.25–4.19 (2H, t, J = 6 Hz), 3.89 (3H, s), 2.42–2.36
(2H, q, J = 6 Hz); 13C NMR (CDCl3, 50 MHz): d = 190.9 (1C), 153.8
(1C), 150.0 (1C), 130.41 (1C), 126.7 (1C), 111.9 (1C), 109.6 (1C),
66.6 (1C), 56.1 (1C), 32.1 (1C), 29.8 (1C).
Scheme 1 Schematic representation of controlled release and real time
monitoring of anticancer drug chlorambucil from ONBCbl–SiQDs.
moiety as a phototrigger for two reasons (i) phototrigger to
cage the anticancer drug chlorambucil and (ii) nitrobenzene
derivatives are well known fluorescent quenchers as they decrease
the LUMO level of the molecules, making them excellent acceptors
in the photoinduced electron transfer (PET) process inducing
fluorescence quenching. Initially the o-nitrobenzyl (ONB) moiety
with caged anticancer drug chlorambucil quenches the fluores-
cence of SiQDs due to the PET process and this state is known as
the fluorescence ‘‘off’’ state. Upon irradiation uncaging of the drug
occurred and this led to the switch on of the fluorescence of SiQDs,
considered as the fluorescence ‘‘on’’ state (Scheme 1). This
phenomenon was utilised for the real time monitoring of the
drug release. Moreover, the ONB moiety has high photochemical
quantum yield with very fast and efficient release ability upon both
one-photon (1PE) and two photon (2PE) excitation19 which could
be further useful in releasing the drug deep inside the body.
Synthesis of 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzaldehyde21
4-(2-Bromoethoxy)-3-methoxybenzaldehyde (900 mg, 3.3 mmol)
was slowly added to 20 mL of ice-cold conc. HNO3. The
temperature of the solution was slowly increased to room
temperature, and the reaction was stirred for 1 h. The reaction
mixture was then poured onto ice water and the precipitate was
filtered off and then washed with cold water. The product was
recrystallized from boiling EtOH (approximately 30 mL), giving
4-(3-bromopropoxy)-5-methoxy-2-nitrobenzaldehyde in 97%
Experimental section
Materials and method
1
yield as a yellow solid (1.02 g, 3.2 mmol). H NMR (200 MHz,
All reagents were purchased from Sigma Aldrich and used CDCl3): d = 10.42 (1H, s), 7.63 (1H, s), 7.39 (1H, s), 4.32–4.26
without further purification. Toluene was distilled using (2H, t, J = 5.8 Hz,), 3.99 (3H, s), 3.66–3.59 (2H, t, J = 6.2 Hz),
sodium and benzophenone and dichloromethane (DCM) was 2.45–2.39 (2H, q, J = 6 Hz). 13C NMR (50 MHz, CDCl3): d = 190.0
distilled using CaH2 before use. 1H NMR spectra were recorded (1C), 153.7 (1C), 151.7 (1C), 143.9 (1C), 125.9 (1C), 110.1 (1C),
on a BRUKER-AC 200 MHz spectrometer. Chemical shifts are 108.4 (1C), 67.3 (1C), 56.8 (1C), 31.9 (1C), 29.5 (1C).
reported in ppm from tetramethylsilane with the solvent resonance
Synthesis of 4-(3-bromopropoxy)-5-methoxy-2-
nitrophenyl)methanol22
as the internal standard (deuterochloroform: 7.26 ppm). Data
are reported as follows: chemical shifts, multiplicity (s = singlet,
d = doublet, t = triplet, and m = multiplet), and coupling 4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzaldehyde (1 g, 3.1 mmol)
constant (Hz). 13C NMR (50 MHz) spectra were recorded on a was dissolved in 20 mL of methanol and made ice cold. To the
BRUKER-AC 200 MHz Spectrometer with complete proton ice cold solution NaBH4 was added slowly and the resultant
decoupling. Chemical shifts are reported in ppm from tetra- solution was stirred at room temperature for 1 h. The solvent
methylsilane with the solvent resonance as the internal standard was then evaporated, and the residue was partitioned between
(deuterochloroform: 77.23 ppm). UV/vis absorption spectra were EtOAc and water. After extraction with EtOAc, organic layers
recorded on a Shimadzu UV-2450 UV/vis spectrophotometer, were washed with brine, dried over Na2SO4, filtered, and evaporated.
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Recrystallization from EtOAc gave 4-(3-bromopropoxy)-5-methoxy-2- temperature for 15 min. LiAlH4 (0.45 g, 11.8 mmol) was added to the
nitrophenyl)methanol (908 mg, 2.8 mmol) as a yellow solid and resultant solution and it was stirred for 3 h at room temperature.
the yield was 91%. 1H NMR (CDCl3, 200 MHz): d = 7.69 (1H, s), Then anhydrous methanol (20 mL) was slowly added to quench any
7.17 (1H, s), 4.92 (2H, s), 4.22–4.16 (2H, t, J = 6 Hz), 3.95 (3H, s), excess LiAlH4 after that the solvent was removed to yield silicon
3.64–3.58 (2H, t, J = 6 Hz), 2.40–2.34 (2H, q, J = 6 Hz). 13C NMR quantum dots as a white solid. Silicon quantum dots thus formed
(50 MHz, CDCl3): d = 154.5 (1C), 147.1 (1C), 139.6 (1C), had a hydrogen terminated surface. The surface of SiQDs was then
132.9 (1C), 111.2 (1C), 109.9 (1C), 67.1 (1C), 62.8 (1C), 56.5 functionalised with the amine functional group by suspending
(1C), 32.1 (1C), 29.8 (1C).
hydrogen terminated SiQDs (300 mg) and (3-aminopropyl)-
triethoxysilane (APTS) (1.4 g) in dry toluene and refluxing the
resultant mixture for 12 h. After modification of the surface,
the sample was purified in a centrifuge (7000 rpm 20 min) to yield
Synthesis of 1-(bromoethyl)-4-(3-bromopropoxy)-5-methoxy-2-
nitrobenzene23
4-(3-Bromopropoxy)-5-methoxy-2-nitrophenyl)methanol (847 mg, (787 mg) amine functionalised SiQDs as a white solid.
2.6 mmol) was added to the stirred solution of PBr3 (716.2 mg,
Synthesis of 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyl
chlorambucil (ONBCbl) loaded silicon quantum dots
(ONBCbl–SiQDs)
0.25 mL, 2.8 mmol) in CH2Cl2 (20 mL) under a N2 atmosphere with
external cooling. The solution was stirred at room temperature for
1 h and then refluxed for 2 h. The reaction mixture was then
poured into H2O (50 mL) and was neutralized with a 2 N aqueous SiQDs (100 mg) were dispersed in dry tetrahydrofuran (THF) (3
KOH solution (100 mL). The organic phase was then separated, mL) under a N2 atmosphere and potassium tertiary butoxide
and the aqueous phase was rinsed twice with CH2Cl2 (50 mL). (75 mg) was added. After stirring for 1 h at room temperature,
The solution was then dried over Na2SO4 (5 g) and the solvent 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyl chlorambucil dis-
was removed to yield 1-(bromoethyl)-4-(3-bromopropoxy)-5- solved in dry THF was added dropwise to the mixture and the
methoxy-2-nitrobenzene (766 mg, 2 mmol, 77%) as a dirty resulting solution was stirred for 24 h at room temperature in
1
yellow solid. H NMR (CDCl3, 200 MHz): d = 7.68 (1H, s), 6.93 the dark. Finally o-nitrobenzyl chlorambucil loaded SiQDs
(1H, s), 4.85 (2H, s), 4.24–4.18 (2H, t, J = 6 Hz), 3.97 (3H, s), 3.64– (ONBCbl–SiQDs) were purified by centrifugation (7000 rpm,
3.58 (2H, t, J = 6 Hz), 2.41–2.35 (1H, q, J = 6 Hz). 13C NMR (CDCl3, 20 min), yielding 90 mg of the product.
50 MHz): d = 153.6 (1C), 148.1 (1C), 140.1 (1C), 127.7 (1C), 113.9 (1C),
Photolysis of ONBCbl–SiQDs using soft visible light irradiation
(Z410 nm)
110.0 (1C), 66.9 (1C), 56.5 (1C), 31.9 (1C), 30.1 (1C), 29.5 (1C).
Synthesis of 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyl
chlorambucil (ONBCbl)
These experiments were carried out using a previously described
method. 1 mg of ONBCbl–SiQDs was dissolved in 1 mL of
Chlorambucil (100 mg, 0.32 mmol) was dissolved in 1 mL of dry acetonitrile in quartz cuvettes. They were irradiated for 30 min
DMF, to it K2CO3 (54 mg, 0.39 mmol) was added and the resultant under UV light using a 125 W cmÀ2 medium pressure Hg vapour
solution was stirred for 20 min at room temperature. Then to the lamp using NaNO2 solution (1 M) as a UV cut-off filter. At
reaction mixture 1-(bromoethyl)-4-(3-bromopropoxy)-5-methoxy-2- regular intervals of time (5 min), 15 mL aliquots were taken
nitrobenzene (126 mg, 0.32 mmol) was added and the resultant and analyzed by RP-HPLC using mobile phase acetonitrile, at
mixture was stirred for 8 h at room temperature. Then the reaction a flow rate of 1 mL minÀ1 (detection: UV 254 nm). Peak areas
mixture was diluted with ethyl acetate and washed many times were determined by RP-HPLC, which indicated a gradual
with brine (200 mL) and the organic layer was dried over Na2SO4. increase of the released chlorambucil with time, and the average
The crude product was purified by column chromatography of three runs. Based on HPLC data for each chlorambucil
through silica gel using EtOAc/hexane (1 : 1) to yield 100 mg release, we plotted normalized [A] (HPLC peak area) versus
(0.16 mmol, 50%) of 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyl irradiation time. We observed an exponential correlation for
1
chlorambucil as a dark red liquid. H NMR (CDCl3, 200 MHz): the appearance of the released chlorambucil, which suggested a
d = 7.73 (1H, s), 7.07–7.03 (2H, d, J = 8.6 Hz), 6.99 (1H, s), 6.63–6.59 first order reaction.
(2H, s, J = 8.6 Hz), 5.47 (2H, s), 4.24–4.18 (2H, t, J = 6 Hz), 3.92 (3H,
Photolysis of ONBCbl–SiQDs using a diode laser (800 nm)
s), 3.71–3.68 (4H, t, J = 6 Hz), 3.66–3.63 (4H, t, J = 6 Hz), 3.61–3.58
(2H, t, J = 6 Hz), 2.61–2.54 (2H, t, J = 6 Hz), 2.46–2.41 (2H, t, J = 6 1 mg of ONBCbl–SiQDs was dissolved in 1 mL of acetonitrile.
Hz), 2.41–2.35 (2H, t, J = 6 Hz), 1.98–1.91 (2H, m), 13C NMR (CDCl3, Half of the solution was kept in the dark and to the remaining
50MHz): d = 172.9 (1C), 153.9 (1C), 147.5 (1C), 144.5 (1C), 140.0 half nitrogen was passed and was irradiated using a diode laser
(1C), 130.4 (1C), 129.8 (1C), 127.4 (1C), 112.3 (1C), 110.9 (1C), 109.9 (800 nm, 500 mW cmÀ2). At regular time intervals, a small
(1C), 67.0 (1C), 63.2 (1C), 56.4 (1C), 53.6 (1C), 40.6 (1C), 34.0 (1C), aliquot (100 mL) of the suspension was taken out and analyzed
33.6 (1C), 32.0 (1C), 29.7 (1C), 26.9 (1C).
by reversed phase HPLC using mobile phase acetonitrile, at a
flow rate of 1 mL minÀ1
.
Synthesis of silicon quantum dots (SiQDs)
Cell imaging of SiQDs and ONBCbl–SiQDs using HeLa cell lines
At first SiCl4 (0.5 mL, 2.9 mmol) was dissolved in the solution
of cetyl trimethylammonium bromide (CTAB) (3.2 g) and anhy- Cell imaging studies were carried out using the HeLa cell
drous toluene (100 mL) and the solution was stirred at room line, which was maintained in minimum essential medium
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(MEM) containing 10% FBS at 37 1C and 5% CO2. To study
the cellular uptake of SiQDs and ONBCbl–SiQDs, HeLa cells
(5 Â 104 cells per well) were placed on six-well plates and
allowed to adhere for 4–8 h. The cells were then incubated with
20 mg mLÀ1 of SiQDs and ONBCbl–SiQDs in cell culture medium
at 37 1C and 5% CO2 for 6 h. Thereafter, the cells were fixed in
4% aqueous formaldehyde for 15 min and washed two times
with PBS. The image was captured using a Nikon confocal
microscope (Nikon Eclipse TE2000-E) using the respective filter.
Scheme 2 Synthesis of o-nitrobenzyl chlorambucil tagged SiQDs as a
photoresponsive DDS.
Cytotoxicity of SiQDs and ONBCbl–SiQDs using the HeLa cell
line
Cytotoxicity before photolysis. The cytotoxicity in vitro was
measured using the MTT assay on the HeLa cell line. Briefly,
cells growing in the log phase were seeded into a 96-well cell
culture plate at 1 Â 104 cells per mL. SiQDs, ONBCbl–SiQDs and
chlorambucil were added into the wells with an equal volume
of PBS in the control wells. The cells were then incubated at
37 1C in 5% CO2 for 48 h. Thereafter, fresh media containing
MTT (0.40 mg mLÀ1) were added to the 96-well plate and
incubated at 37 1C in 5% CO2 for an additional 4 h. Formazan
crystals formed were dissolved in DMSO after decanting the
media, and then absorbance was recorded at 595 nm.
Cytotoxicity after photolysis. HeLa cells in a 96-well cell
culture plate at a concentration of 1 Â 104 cells per mL were
maintained in MEM containing 10% FBS. SiQDs (0.1–50 mg mLÀ1),
ONBCbl–SiQDs (0.1–50 mg mLÀ1) and chlorambucil (0.1–50 mM)
with different concentrations were incubated at 37 1C and 5%
CO2 for 48 h. Then, the cells were irradiated for 30 min using a
125 W cmÀ2 medium-pressure Hg lamp as the irradiation source
(Z410 nm) with NaNO2 solution (1 M) as a UV cut off-filter by
keeping the cell culture plate 5 cm away from the light source.
After irradiation, the cells were incubated for another 48 h. Finally,
the cytotoxicity was measured using the MTT assay as described in
the previous section.
Surface functionalization of SiQDs and attachment of ONBCbl
to the surface of SiQDs were monitored using FTIR spectroscopy
(Fig. 1). SiQDs have a broad peak at 3100–3500 cmÀ1 which
corresponds to the stretching vibration of O–H and N–H, peaks
at 1035 cmÀ1 and 1140 cmÀ1 correspond to the stretching
vibration of Si–O and C–N, respectively, and peaks at 2930 cmÀ1
and 2860 cmÀ1 correspond to aliphatic CH2 due to the APTS
assisted surface modification of SiQDs. In the FTIR spectrum of
ONBCbl–SiQDs a new peak at 1500 cmÀ1 corresponding to the
N–O stretching frequency of NO2 appeared which indicates that
ONBCbl is successfully attached to the surface of SiQDs. We also
carried out energy dispersive X-ray (EDX pattern) and elemental
mapping of Si, C, O, N, and Cl for ONBCbl–SiQDs (Fig. 2c).
The images clearly validate the homogenous distribution of Si,
C, O, N, and Cl elements throughout the material. The EDX
pattern further describes the presence of the corresponding
constituting elements (Fig. S1, ESI†).
The size and the shape of the NH2 functionalised silicon
quantum dots (SiQDs), and ONBCbl conjugated silicon quantum
dots (ONBCbl–SiQDs) were determined by TEM and DLS analysis.
TEM images of SiQDs and ONBCbl–SiQDs are presented in Fig. 2a
and b, respectively, they show that SiQDs and ONBCbl–SiQDs were
spherical in shape and their average diameters were B6–10 nm
and B20–30 nm, respectively. The dynamic light scattering (DLS)
study further revealed that the hydrodynamic diameters of SiQDs
and ONBCbl–SiQDs were 6.7 Æ 1.6 nm and 26.4 Æ 1 nm
(Fig. 3a and b), respectively. The size of the ONBCbl–SiQDs
was within the preferred range of nanoparticles that can be used
for drug delivery.25 ONBCbl–SiQDs were larger in size compared
Results and discussion
Synthesis and characterisation of o-nitrobenzyl-chlorambucil
conjugated silicon quantum dots (ONBCbl–SiQDs)
We presented a new synthetic approach to synthesize amine
functionalised silicon quantum dots (SiQDs) by modifying the
existing synthetic procedure.24 Commercially available silicon
tetrachloride (SiCl4) was reduced with lithium aluminum hydride
(LiAlH4) in the presence of surfactant cetyl trimethylammonium
bromide (CTAB) in dry toluene at room temperature for 3 h.
Hydrogen-terminated silicon quantum dots (Si–H) thus produced
were then functionalised with amine terminals by reacting with
(3-aminopropyl)triethoxysilane (APTS) in dry toluene at 80 1C
for 12 h thereby forming SiQDs. A photocaged anticancer
drug, 4-(3-bromopropoxy)-5-methoxy-2-nitrobenzyl chlorambucil
(ONBCbl), was synthesized separately, as shown in Scheme S1,
ESI.† Finally, ONBCbl was covalently attached on the surface
of SiQDs using potassium tert-butoxide as the base in dry THF
(Scheme 2).
Fig. 1 FTIR spectra of o-nitrobenzylchlorambucil (ONBCbl), silicon quantum
dots (SiQDs) and o-nitrobenzyl chlorambucil loaded silicon dots
(ONBCbl–SiQDs).
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exhibited strong blue emission at 430 nm, when excited at
360 nm, but the fluorescence of SiQDs got quenched upon
conjugation with ONBCbl (Fig. 4b). This phenomenon can be
explained by the Photoinduced Electron Transfer (PET) process
from SiQDs to ONBCbl which was due to the presence of the
electron withdrawing group (nitro group) in the phototrigger
(Scheme 3). Fluorescence quantum yields of SiQDs and
ONBCbl–SiQDs are 18.8% and 4.2%, respectively (quinine
sulphate quantum yield: 54%)26 as shown in Table S1, ESI.†
Hence ONBCbl–SiQDs can be utilized for cellular imaging
along with real time monitoring of drug release.
Photolysis of photocaged ONBCbl–SiQDs
Fig. 2 TEM image of (a) silicon quantum dots (SiQDs), (b) ONBCbl
conjugated silicon quantum dots (ONBCbl–SiQDs), and (c) SEM X-ray
elemental mapping of ONBCbl–SiQDs showing the presence of constituting
elements: (i) SEM image, (ii) silicon (Si), (iii) carbon (C), (iv) nitrogen (N),
(v) oxygen (O) and (vi) chlorine (Cl).
The photoinduced release of the anticancer drug, chlorambucil,
from ONBCbl–SiQDs was demonstrated by irradiating the
suspension of ONBCbl–SiQDs in acetonitrile under both visible
light (Z410 nm, Hg-vapour lamp) and two-photon excitation
using a 800 nm diode laser. The course of the photorelease was
monitored by emission spectroscopy and reverse phase HPLC.
It was observed from the emission spectra (Fig. 5a) that
the emission intensity of the suspension of ONBCbl–SiQDs in
acetonitrile steadily increased with increasing irradiation time.
After 30 min of photoirradiation, it was found that the fluores-
cence intensity of ONBCbl–SiQD suspension in acetonitrile
to free SiQDs, which clearly indicated that ONBCbl was
anchored on the surface of SiQDs.
The total amount of ONBCbl loaded on the surface of SiQDs
was determined by UV-Vis absorption spectra (Fig. S2a–c, ESI†);
it was calculated to be about 22 mg mgÀ1 of SiQDs.
Photophysical properties of ONBCbl–SiQDs and SiQDs
Furthermore, the photophysical properties of ONBCbl–SiQDs
were studied. The UV-Vis spectrum of ONBCbl–SiQDs dispersed
in acetonitrile showed a broad absorbance band from 300 nm
to 475 nm (Fig. 4a). It can be noted that free SiQDs do not have
such a broad absorption band. SiQDs dispersed in acetonitrile
Fig. 4 (a) UV-Vis absorption spectrum of SiQDs, ONBCbl–SiQDs, and
ONBCbl and (b) emission spectrum of SiQDs and ONBCbl–SiQDs in
acetonitrile.
Fig. 3 Particle size distribution graph of (a) SiQDs and (b) ONBCbl–SiQDs
as revealed by DLS.
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Scheme 3 Proposed mechanism for the photorelease of anticancer drug
chlorambucil and fluorescence ‘‘turn on’’ ability of ONBCbl–SiQDs.
changed from faint (Fig. 5b) to bright blue (Fig. 5c) which is the
characteristic emission of SiQDs. This phenomenon
of photoinduced fluorescence switch ‘‘off’’–‘‘on’’ of ONBCbl–
SiQDs can be exploited for real time monitoring of the drug
release. By correlating the increase in fluorescence intensity
with the percentage of the drug released, it was found that after
30 min of irradiation about 74% of the drug was released as
shown in Fig. 5d.
On the basis of literature reports,27,28 we proposed a mechanism
for the photorelease of anticancer drug chlorambucil from
ONBCbl–SiQDs as shown in Scheme 3. Upon irradiation photo-
trigger conjugated chlorambucil (ONBCbl) undergoes photolysis
to release the anticancer drug chlorambucil thereby producing
nitrosobenzaldehyde-SiQDs as the photoproduct (Scheme 3).
The electron transfer process from SiQDs was stopped in the
presence of the nitrosobenzaldehyde group and so SiQDs regained
their inherent fluorescence after photolysis.
Fig. 5 (a) Emission spectra of the ONBCbl–SiQDs at different time intervals
of photoirradiation (0–30 min, Z410 nm), (b) fluorescence before photolysis
at 0 min, (c) fluorescence after 30 min of irradiation, (d) percentage of
chlorambucil released as a function of fluorescence intensity change, and
(e) the partial progress for the release of chlorambucil under bright and dark
conditions. ‘‘ON’’ indicates the beginning of light irradiation and ‘‘OFF’’
indicates the end of light irradiation.
From the HPLC profile (Fig. S3, ESI†) it was also observed
that after 30 min of irradiation (l Z 410 nm, 125 W cmÀ2) 74%
of the loaded anticancer drug chlorambucil was effectively
released, whereas 12% of the drug was released using a diode
laser (800 nm, 500 mW cmÀ2) (Fig. 6), this clearly indicates that
the percentage of drug release is highly dependent on the
external light intensity. We also studied controlled photolytic
release of the anticancer drug from ONBCbl–SiQDs by monitoring
the release processes while exposing it to light and dark conditions
alternately. Fig. 5e (inset) shows that the drug was released from
ONBCbl–SiQDs only under irradiation.
Cellular uptake and intracellular trafficking studies of
ONBCbl–SiQDs and SiQDs
Fig. 6 HPLC profiles of the percentage of loaded chlorambucil released
from ONBCbl–SiQDs under one photon (Z410 nm) and two photon
(800 nm) excitation.
To establish that ONBCbl–SiQDs can be used as a versatile
photoresponsive nano-DDS, real-time cellular uptake of SiQDs
and ONBCbl–SiQDs was investigated by cellular imaging using
HeLa cells. Cells were incubated with 20 mg mLÀ1 of SiQDs and
ONBCbl–SiQDs in cell culture medium for 6 h and imaging fluorescence (Fig. 7ib). This revealed that the SiQDs were
was done using a confocal microscope using appropriate readily internalized by the cells. On the other hand faint blue
filters. After incubation with SiQDs, cells emitted bright blue fluorescence was observed from the HeLa cells after incubation
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with ONBCbl–SiQDs (Fig. 7iib) indicating that ONBCbl–SiQDs
were also readily internalized by the cells.
Real-time monitoring of drug release
To demonstrate the real time monitoring ability of the ONBCbl–
SiQDs, in vitro time dependent photocontrolled drug release
studies on HeLa cells were performed. Fig. 7ia and b clearly
show that before irradiation the ONBCbl–SiQDs in the HeLa
cells were faintly fluorescent but upon gradual irradiation fluores-
cence the intensity increased and finally showed characteristic
fluorescence of free SiQDs (Fig. 7iva and b) after 30 min of
irradiation, indicating photolysis of ONBCbl–SiQDs and thereby
release of the anticancer drug chlorambucil.
Anticancer efficacy of ONBCbl–SiQDs before and after
photolysis
After successful demonstration of the pronounced internalization
and accumulation of SiQDs and ONBCbl–SiQDs in the HeLa cells,
the in vitro cytotoxicity of SiQDs, chlorambucil and ONBCbl–SiQDs
was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay on the HeLa cell line. Cyto-
toxicity was expressed as the percentage of cell viability relative to
the untreated control cells. The percentage of cell viability was
plotted versus concentration of SiQDs, chlorambucil and ONBCbl–
SiQDs. It was observed that cell viability remained above 80% at
different concentrations of SiQDs and ONBCbl–SiQDs in the
absence of light (Fig. 8a), whereas an increase in cytotoxicity was
observed with the increase in the amount of chlorambucil (Fig. 8a).
Photoinduced release of chlorambucil by ONBCbl–SiQDs within
the HeLa cell line and its subsequent effect on the cell viability
were investigated. For that, cells were incubated with different
concentrations of ONBCbl–SiQDs for 48 h and were irradiated with
visible light (Z410 nm) for 30 min resulting in the release of the
anticancer drug chlorambucil to induce cytotoxicity to HeLa cells,
as confirmed by the MTT assay (Fig. 8b). On the other hand,
no significant cell death was observed when the cells were
irradiated in the presence of SiQDs (Fig. 8b), clearly indicating
that the cytotoxicity caused was only because of chlorambucil
Fig. 8 (a and b) Cell viability of HeLa cells incubated with SiQDs,
ONBCbl–SiQDs, and chlorambucil (Cbl), (a) before irradiation and, (b) after
30 min of irradiation. The values are presented as mean Æ SD.
which was released upon photoirradiation from ONBCbl–SiQDs.
ONBCbl–SiQDs showed much lower cytotoxicity in the dark
compared to chlorambucil (Fig. 8a). But upon irradiation
ONBCbl–SiQDs showed enhanced cytotoxicity to the cancer cells
in comparison to free chlorambucil (Fig. 8b), because of the
efficient photorelease of chlorambucil inside the cell. Thus,
ONBCbl–SiQDs could be considered as an efficient biocompatible
photoresponsive nano-DDS for the photoinduced release of the
anticancer drug in vitro for a sustained period of time.
Conclusions
We have developed a new photoresponsive real time monitoring
nano-drug delivery system based on fluorescent SiQDs (ONBCbl–
SiQDs) for the regulated delivery of anticancer drugs. We presented
a new synthetic approach for synthesizing ONBCbl–SiQDs.
ONBCbl–SiQDs exhibited the photoinduced fluorescence ‘‘off’’–
‘‘on’’ phenomenon upon drug uncaging which was explored for
real time monitoring of the drug release. Photoregulated drug
delivery by ONBCbl–SiQDs upon one- and two-photon excitation
(1PE and 2PE) was established by periodic exposure to light
and dark conditions. Furthermore cellular internalisation of
ONBCbl–SiQDs and photoinduced enhancement of cytotoxicity
with respect to the concentration of ONBCbl–SiQDs inside the
HeLa cell were demonstrated. The low cytotoxicity and unique
Fig. 7 Confocal bright field and fluorescence images of HeLa cells
incubated with ONBCbl–SiQDs (20 mg mLÀ1) at different irradiation times
(0–30 min, time interval = 15 min): (ia–iva) bright field and (ib–ivb)
corresponding fluorescence images. Scale bar = 20 mm.
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Journal of Materials Chemistry B
11 C. Tu, X. Ma, P. Pantazis, S. M. Kauzlarich and A. Y. Louie,
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optical properties of SiQDs opened a new area of designing
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dots. In future we wish to design photoresponsive, multifunctional
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Acknowledgements
We thank DST-SERB for their financial support and DST-FIST
for 400 MHz NMR. A. Paul, S. Karthik and M. Bera are thankful
to IIT KGP for the fellowship.
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