Synthesis and in vitro evaluation of charge reversal photoresponsive quinoline tethered mesoporous silica for targeted drug delivery

Article abstract of DOI:10.1039/c4tb01367k

We developed excellent charge reversal photoresponsive nanoparticles for targeted delivery of the anticancer drug chlorambucil. The charge reversal photoresponsive nanoparticles were constructed using two main ingredients, namely folic acid decorated mesoporous silica and quinoline chromophore. The newly synthesized quinoline chromophore performed three important roles, i.e., (i) fluorescent chromophore for cell imaging, (ii) phototrigger for regulated release of anticancer drug, and (iii) charge reversal based on its zeta potential for nuclear localization. Furthermore, folic acid decorated mesoporous silica facilitated active internalization of the drug inside the cancer cells. In vitro biological studies reveal that our photoresponsive DDS could deliver the anticancer drug chlorambucil into the tumor cells, killing the cancer cells by both one photon (≥365 nm) and two photon (675 nm) irradiation. This journal is

Full text of DOI:10.1039/c4tb01367k

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Materials Chemistry B
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PAPER
Synthesis and in vitro evaluation of charge reversal
photoresponsive quinoline tethered mesoporous
silica for targeted drug delivery†
Cite this: J. Mater. Chem. B, 2014, 2,
7971
S. Karthik,a Avijit Jana,b Biswajit Saha,d B. Krishna Kalyani,a Sudip Kumar Ghosh,d
bc
a
*
*
Yanli Zhao
and N. D. Pradeep Singh
We developed excellent charge reversal photoresponsive nanoparticles for targeted delivery of the
anticancer drug chlorambucil. The charge reversal photoresponsive nanoparticles were constructed
using two main ingredients, namely folic acid decorated mesoporous silica and quinoline chromophore.
The newly synthesized quinoline chromophore performed three important roles, i.e., (i) fluorescent
chromophore for cell imaging, (ii) phototrigger for regulated release of anticancer drug, and (iii) charge
reversal based on its zeta potential for nuclear localization. Furthermore, folic acid decorated
mesoporous silica facilitated active internalization of the drug inside the cancer cells. In vitro biological
studies reveal that our photoresponsive DDS could deliver the anticancer drug chlorambucil into the
tumor cells, killing the cancer cells by both one photon ($365 nm) and two photon (675 nm) irradiation.
Received 16th August 2014
Accepted 1st October 2014
DOI: 10.1039/c4tb01367k
www.rsc.org/MaterialsB
In general, nuclear localization peptides (NLPs) and cationic
polymers, such as polyethyleneimine (PEI), have been used to
construct nuclear targeted photoresponsive nanoparticles.7
Introduction
Photoresponsive nanoparticles have recently received consid-
erable attention for their applications especially in the area of
drug and gene delivery, because they allow precise control over
the release, including location, timing and dosage.1,2 In general,
photoresponsive nanoparticles are composed of two main
ingredients: biocompatible nanocarrier and phototrigger. A
major drawback of photoresponsive nanoparticles is their non-
specicity to cancer cells, which can lead to high toxicity to
normal cells causing undesirable side effects. To overcome this
problem, several folate-decorated photoresponsive nano-
particles have been constructed for targeted drug delivery to
cancer cells.3,4 However, these types of tumor targeted photo-
responsive nanoparticles were found to be largely retained in
cytoplasmic organelles, including lysosomes, rather than the
nucleus of cancer cells.5,6
Recently, pH dependent targeted charge reversal nanoparticles
(TCRNs) have drawn signicant attention for nuclear targeted
drug delivery, because they undergo a negative to positive charge
reversal when exposed to the extracellular acidic environment
(pH < 7) and within the acidic lysosomal environment (pH 4–5)
of cells. Because negatively charged TCRNs have some interac-
tions with blood components, they have been used extensively in
vitro. Shen8 group has prepared several TCRNs that were nega-
tively charged under neutral conditions and positively charged
at endosomal pH. They have demonstrated the application of
TCRNs for lysosomal pH responsive protein and gene delivery.
In addition, Shen9 and co-workers have synthesized pH
responsive charge reversal polymeric micelles decorated with
folic acid for both cellular and nuclear targeted drug delivery.
Recently, Wang et al.10 have also developed a pH responsive
charge conversional nanogel for promoted tumoral cell uptake
and doxorubicin delivery. Inspired by the nuclear targeted drug
delivery ability of TCRNs, in this work, we developed for the rst
time charge reversal photoresponsive nanoparticles for both
cellular and nuclear targeted delivery of anticancer drug.
Hence, there is a real need to design multifunctional pho-
toresponsive nanoparticles that could target not only cancer
cells but the nucleus of cancer cells. They may effectively
magnify the therapeutic potential of anticancer drugs.
aDepartment of Chemistry, Indian Institute of Technology, Kharagpur, India. E-mail:
ndpradeep@yahoo.co.in
bDepartment of Chemistry and Biological Chemistry, Nanyang Technological
The basic requirement to construct cellular and nuclear tar-
geted charge reversal photoresponsive nanoparticles is to
develop two essential ingredients: (i) chromophore that has a
combination of pH sensitive charge reversal property and pho-
totrigger ability and (ii) tumor targeted nanocarriers. For the
current study, we selected quinoline moiety as a chromophore
for three main reasons: (i) “quinoline derivatives are highly pH
sensitive”, and pKa of quinoline is 4.8.11 Hence, under slightly
University, Singapore
cSchool of Materials Science and Engineering, Nanyang Technological University,
Singapore
dDepartment of Biotechnology, Indian Institute of Technology, Kharagpur, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4tb01367k
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standard (deuterochloroform: 77.0 ppm). UV/vis absorption
spectra were recorded on a Shimadzu UV-2450 UV/vis spectro-
photometer, uorescence emission spectra were recorded on a
Hitachi F-7000 uorescence spectrophotometer, FT-IR spectra
were recorded on a Perkin Elmer RXI spectrometer. Trans-
mission electron microscopy (TEM) was measured on a FEI
Tecnai G220S-Twin at 200 kV. The TEM sample was prepared by
dispersing compounds in water and dropping on the surface of a
copper grid. Low angle powder XRD was measured by a Phillips
PW 1710 X-ray diffractometer (XRD). The surface area of the
mesoporous TP was measured by the N2 sorption experiment
using BET (Brunauer–Emmett–Teller) technique, performed at
liquid N2 temperature on Quantachrome Autosorb1 surface area
analyzer aer degassing the samples at 200 ꢀC for 4 h. The pore
size distribution curve of mesoporous TP was obtained from the
analysis of the adsorption branch of the isotherm by the BJH
(Barrett–Joyner–Halenda) method. The surface charge of the
nanoparticles was investigated through zeta potential
measurements (Zetasizer 4, Malvern Instruments, U.K.). DLS
measurements at different pH were done using a Brookhaven
90 Plus particle size analyzer. Thermal analysis was done with a
thermal analyzer (Pyris Diamond TG/DTA) with a heating rate
8 ꢀC minꢁ1 in a temperature range 50 to 1000 ꢀC. Photolysis of
the ester conjugate was carried out using a 125 W medium
pressure Hg lamp supplied by SAIC (India) and laser diode of
675 nm, 15 mW supplied by Thor Labs. Chromatographic
purication was done with 60–120 mesh silica gel (Merck). For
reaction monitoring, pre-coated silica gel 60 F254 TLC sheets
(Merck) were used. RP-HPLC was taken using mobile phase
acetonitrile, at a ow rate of 1 mL minꢁ1 (detection: UV 254 nm).
Cell imaging was done with an Olympus confocal microscope
(FV1000, Olympus) using the respective lter.
Synthesis of Qucbl. Quinoline chlorambucil conjugate
(Qucbl) was synthesized using a previously reported procedure
(ESI Scheme S1†).13
Synthesis of trimethoxysilyl tagged Qucbl. Amino propyl tri-
methoxy silane (0.5 mL) was added to an ice-cooled solution of
compound 1 (150 mg) in dry DCM (10 mL). Aer stirring for 6 h,
the solvent was removed by rotary evaporation to yield 175 mg of
trimethoxysilyl tagged Qucbl, which was used in the next step
without purication.
Synthesis of quinoline chlorambucil loaded on MSNs Q1
(Qucbl-MSNs). Trimethoxysilyl tagged Qucbl was dissolved in
dry toluene (10 mL) containing 100 mg of mesoporous silica
nanoparticles and the mixture was reuxed for 20 h at 80 ꢀC to
afford compound Q1(Qucbl-MSN). The course of loading was
followed by UV-vis absorption spectra for a regular time interval.
Finally the materials were recovered by centrifugation, washed
twice with toluene and dried under vacuum. We calculated
Qucbl loaded on mesoporous silica nanoparticle using the
equation below:
Scheme 1 Schematic illustration of the proposed tumor targeted as
well as nuclear targeted delivery of anticancer drug.
acidic conditions, it can be easily protonated to reveal a positive
charge, which can be exploited for charge reversal property, (ii)
“quinoline-based derivatives are well known phototriggers”.12
They have been well demonstrated for the controlled release of
physiologically active messengers through both one-photon
excitation (1 PE) and 2 PE (near-IR light) that is the optimal
wavelength for tissue penetration, and (iii) “quinoline deriva-
tives are well known pH sensitive uorophores”, and they have
been exploited for cellular imaging application. Furthermore, we
also chose folic acid decorated mesoporous silica as the nano-
carriers because of their biocompatibility, enhanced cellular
uptake and high drug loading ability. The design of the targeted
drug delivery process was schematically shown in Scheme 1.
Experimental section
Materials and methods
All reagents were purchased from Sigma Aldrich and used
without further purication. Acetonitrile and dichloromethane
were distilled from CaH2 before use. 1H NMR spectra were
recorded on a BRUKER-AC 200 MHz spectrometer. Chemical
shis are reported in ppm from tetramethylsilane with the
solvent resonance as the internal standard (deuterochloroform:
7.26 ppm). Data are reported as follows: chemical shis, multi-
plicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet),
coupling constant (Hz). 13C NMR (50 MHz) spectra were recor-
ded on a BRUKER-AC 200 MHz spectrometer with complete
proton decoupling. Chemical shis are reported in ppm from
tetramethylsilane with the solvent resonance as the internal
ðInitial conc: of Qucbl ꢁ final conc: of Qucbl in reaction mediumÞ ꢂ M: wt of Qucbl
Quinoline chlorambucil loaded on MSNs ¼
7972 | J. Mater. Chem. B, 2014, 2, 7971–7977
Amount of mesoporous silica taken
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Synthesis of folate loaded Qucbl-MSNs Q2 (Qucbl-Fol-
MSNs).13 Folic acid was attached to Qucbl-MSNs. In a round
bottom ask, folic acid (50 mg) and APTS (0.2 mL) were mixed
in DMSO (3 mL). N-Hydroxysuccinimide (30 mg) and 1-(3-
Journal of Materials Chemistry B
15 min and washed two times with PBS. Imaging was done with
a Nikon confocal microscope (Nikon Eclipse TE2000-E) using
the respective lter.
Time dependent internalization studies of Q2 at pH 7.4. We
followed the same procedure except the Qucbl-Fol-MSN nano-
particles were dispersed in PBS of pH 4.8.
Nuclear co-localization studies using Qucbl-Fol-MSNs and a
nuclear staining dye propidium iodide. Cells, grown and plated
as described above, were incubated for 4 h at 37 ꢀC with 1 mL of
MEM containing 20 mM of Qucbl-Fol-MSNs. Thereaer, cells
were washed 3 times with 10 mM PBS and xed with 2% para-
formaldehyde for 15 min at room temperature. Aer xation
cells were washed 3 times with 10 mM PBS permeabilized with
0.01% Triton X 100, a non-ionic surfactant. The cells were
counterstained with 10 mg mLꢁ1 propidium iodide (PI) and
0.5 mg mLꢁ1 RNASE at room temperature in the dark for 30 min.
Aer gentle washing in 10 mM PBS for 3 times, the cells were
viewed under a confocal microscope.
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(50 mg) were then added into the mixture and stirred for 2 h. To
the reaction mixture, 50 mg of Q1 DMSO suspension was added
and the mixture was stirred for 20 h at room temperature. The
MSN have approximately 0.5 wt% of folic acid graed on the
surface of Q1. Finally, folate decorated quinoline chlorambucil
tagged MSN Q2 (Qucbl-Fol-MSN) was recovered by centrifuga-
tion, washed twice with toluene and dried under vacuum.
Hydrolytic stability of Q2. To check the stability of Q2 in the
cell culture medium, we dispersed 1 mg mLꢁ1 Q2 with 10% fetal
ꢀ
bovine serum and incubated at 37 C in the dark for 72 h. The
tubes were kept in an ultrasonic bath for 10 min to make the
ꢀ
solutions homogeneous and stored at 37 C in dark condition
for 96 h. Then all the solutions were centrifuged (5000 rpm) for
10 min and the supernatant solutions were analyzed by reverse
phase HPLC to examine the percentage of drug depleted.
Photolysis of Q2 using so UV irradiation ($365 nm) and 675
nm laser diode
Photolysis of Q2 using so UV irradiation ($365 nm). A
suspension of 5 mg/5 mL of the Q2 was prepared in acetonitrile.
Half of the suspension was kept in dark and to the remaining
half nitrogen was passed and irradiated using a 125 W medium
pressure Hg lamp as the light source (l $ 365 nm) and 1 M
CuSO4 solution in 0.1 N H2SO4; the transmittance for the above
lter ¼ 365 to 500 nm. At regular time intervals, a small aliquot
(100 mL) of the suspension was taken out and centrifuged (5000
rpm) for 10 min; the obtained transparent solution was
analyzed by reverse phase HPLC using mobile phase acetoni-
Cell imaging and cytotoxicity of MSNs, and Q2 on HeLa cell
line
Qucbl-Fol-MSNs for cell imaging studies using HeLa cell
line. Cell imaging studies were carried out using the HeLa cell
line, which was maintained in minimum essential medium
ꢀ
(MEM) containing 10% fetal bovine serum (FBS) at 37 C and
5% CO2. To study the cellular uptake of Q2, HeLa cells (5 ꢂ 104
cells per well) were plated on 12 well plates and allowed to
adhere for 4–8 hours. Cells were then incubated with 50 mg of
Q2 separately in cell culture medium for 4 h at 37 ꢀC in 5% CO2.
Thereaer, cells were xed in paraformaldehyde for 15 min and
washed two times with PBS. Imaging was done with an Olympus
confocal microscope (FV1000, Olympus) using the respective
lter.
trile at a ow rate of 1 mL minꢁ1
.
Photolysis of Q2 using red laser. 1 mg of Q2 was dissolved in
1 ml acetonitrile. Half of the solution was kept in dark and to
the remaining half nitrogen was passed and irradiated using a
675 nm laser diode (15 mW cmꢁ2). At regular time intervals, a
small aliquot (100 mL) of the suspension was taken out and
centrifuged (5000 rpm) for 10 min; the obtained transparent
solution was analyzed by reverse phase HPLC using mobile
Lyso tracking experiment. Cell imaging studies were carried
out using the HeLa cell line, which was maintained in Dulbec-
co's Modied Eagle Medium (DMEM) containing 10% fetal
ꢀ
bovine serum (FBS) at 37 C in 5% CO2. To study the intracel-
phase acetonitrile at a ow rate of 1 mL minꢁ1
.
lular localization of Q2 nanoparticles, briey HeLa cells (5 ꢂ 104
cells per well) were plated on coverslips in 6 well plates and
allowed to adhere for 4–8 hours. Cells were then incubated with
50 mg of Q2 in PBS for 6 h at 37 ꢀC in 5% CO2. Then, the medium
Cytotoxicity of Q1 and Q2 on HeLa cell line
Cytotoxicity before photolysis. The cytotoxicity in vitro was
was discarded and the cells were washed two times with PBS measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
followed by the addition of LysoTracker Red DND-99 (2 mL, 50 diphenyltetrazolium bromide, a yellow tetrazole) assay on HeLa
nM) in culture medium and incubated at 37 ꢀC in 5% CO2 for 1 cell line. Briey, cells growing in log phase were seeded into 96-
h. Thereaer, cells were xed in paraformaldehyde for 15 min well cell-culture plate at 1 ꢂ 104 cells per mL. Different
and washed two times with PBS. Imaging was done with a Nikon concentrations of Q1, Q2 and chlorambucil were added in the
confocal microscope (Nikon Eclipse TE2000-E) using the wells with an equal volume of PBS inꢀthe control wells. The cells
respective lter.
were then incubated for 72 h at 37 C in 5% CO2. Thereaer,
Time dependent internalization studies of Q2 at pH 7.4. fresh media containing 0.40 mg mLꢁ1 MTT were added to the 95
Following the above procedure HeLa cells (5 ꢂ 104 cells per well plates and incubated for 4 h at 37 ꢀC in 5% CO2. Formazan
well) were plated on coverslips in 6-well cell culture plates and crystals thus formed were dissolved in DMSO aer decanting
allowed to adhere for 4–8 hours. Cells were then incubated with the earlier media and absorbance recorded at 595 nm.
ꢀ
50 mg of Q2 in PBS for different time intervals at 37 C in 5%
Cytotoxicity aer photolysis. HeLa cells maintained in
CO2. Thereaer, cells were xed in paraformaldehyde for minimum essential medium (in 96-well cell-culture plate at
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concentration of 1 ꢂ 104 cells per mL) containing 10% fetal
The amount of the quinoline chlorambucil loaded on MSNs
bovine serum (FBS) and different concentrations of Q1, Q2 and is calculated to be about ꢃ273 mg mgꢁ1, based on UV-Vis
chlorambucil were incubated for 4 h at 37 ꢀC in 5% CO2. Then absorption spectra (Fig. 1). The UV-Vis absorption and uores-
the cells were irradiated (keeping the cell-culture plate 5 cm cence spectra of Q2 are presented in Fig. S8.† Similar to quin-
apart from the light source) using a 125 W medium pressure Hg oline chromophore, Q2 also showed broad absorbance from 300
lamp as irradiation source ($365 nm) and 1 M CuSO4 solution to 365 nm and emission maxima at 460 nm. Hence, Q2 can be
as UV cut-off lter. Aer irradiation the cells were again incu- explored like quinoline chromophore for simultaneous cell
bated for 72 h. Then cytotoxicity was measured using the MTT imaging and release of the anticancer drug by both one photon
assay as described in the earlier section.
(365 nm) and two photon (675 nm) excitation.
A key pH dependent charge reversal property of Q1 and Q2
was determined by measuring their zeta potentials at different
acidities (Fig. 2a). The MSNs revealed a zeta potential of about
ꢁ19 mV in acidic pH ranges of 6.5–3, indicating that they always
remain negatively charged due to the presence of Si–OH group
on their surface. On the other hand, Q1 and Q2 showed zeta
potentials of ꢁ5 mV and ꢁ1 mV at pH 6.5, respectively. But, in
the pH ranges of 5–4.5, both Q1 and Q2 became positively
charged, and gradually, their zeta potential reached about +1.65
mV and +3.67 mV, respectively. At pH 3, the zeta potentials of
Q1 and Q2 were found to be about +9.5 mV and +11.6 mV,
respectively.
Results and discussion
We synthesized mesoporous silica nanoparticles (MSNs) using a
previously reported procedure.13a Transmission electron
microscopy (TEM), powder X-ray diffraction and nitrogen
sorption isotherm analysis reveals that MSNs are of ꢃ50–66 nm
particle size, and have a honeycomb like porous structure with a
2.9 nm average pore diameter and a surface area of 302.69 m2
gꢁ1 (ESI, Fig. S1–S3†). Subsequently, quinoline chlorambucil
conjugate (Qucbl) was synthesized using a previously reported
procedure13b (Scheme S1†). Finally, Qucbl and folic acid were
covalently anchored on the surface of MSNs with silane
coupling agent in a stepwise manner as depicted in Scheme 2.
Quinoline-chlorambucil loaded mesoporous silica Q1 (Qucbl-
MSNs) and folic acid decorated quinoline-chlorambucil loaded
mesoporous silica Q2 (Qucbl-Fol-MSNs) were characterized by
IR spectra, solid state UV and thermogravimetric analysis (TGA)
(Fig. S4–S6†).
The physicochemical properties of Q2 such as morphology,
size, and zeta potential were studied because they have inu-
ence on cellular uptake. DLS (Fig. S7†) studies reveal that the
average particle size of MSNs, Q1 and Q2 were 85.26 ꢄ 1.82 nm,
134.82 ꢄ 1.54 nm and 143.49 ꢄ 1.52 nm, respectively. The
increase in particle size of Q2 compared to free MSNs and Q1
implies that mesoporous silica was decorated by both folic acid
and Qucbl conjugate. TEM observation shows that MSNs, Q1
and Q2 were well dispersed and spherical in shape. The size of
the Q2 is well within the preferred range of the nanoparticles
useful for effective drug delivery.
To evaluate the proton-binding behaviour of Q2, we recorded
the emission spectra of model compound 2 in Na2HPO4–citrate
buffer at different pH values ranging from 7.0 to 2.6 (Fig. 2b).
We noted the uorescence maxima of compound 2 in neutral
pH is around 380 nm, which was red shied to 450 nm at lower
pH. Interestingly, we also observed an isoemissive point in
emission spectra at around 410 nm, indicating the presence of
two distinct species in equilibrium. This is because, at lower
pH, protonation is favoured and hence protonated 2 is the
predominant species. Further proton-binding behavior of
1
compound 2 was also supported by H NMR spectroscopy. We
recorded 1H NMR spectrum of compound 2 and its protonated
form (10 mM HCl). As shown in Fig. 1c, on protonation, the
quinolinic protons H3 and H4 displayed signicant downeld
shis to 8.99 and 8.25 ppm respectively, suggesting that the
protonation occurred at the quinolinic site.
On the other hand, we explored the pH dependent charge
reversal properties of Q2 in vitro by carrying out cellular inter-
nalization studies at two different pH, 7.4 and 4.8.8,9 The time
dependent confocal laser scanning microscopy (CLSM) imaging
studies showed a signicant difference in cellular internaliza-
tion of Q2 at pH 7.4 and 4.8. At pH 4.8, Q2 were more effectively
Scheme 2 Synthesis of quinoline-chlorambucil loaded mesoporous Fig. 1 Quinoline chlorambucil loaded on MSNs. (a) The course of
silica (Qucbl-MSNs) and quinoline chlorambucil and folic acid deco- loading was followed by UV-vis absorption spectra; (b) calibration
rated mesoporous silica (Qucbl-Fol-MSNs).
curve for the concentration of chlorambucil on the surface of MSNs.
7974 | J. Mater. Chem. B, 2014, 2, 7971–7977
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Journal of Materials Chemistry B
Furthermore, the above mentioned studies also showed that
Q2 was largely internalized in HeLa cells compared to Q1. The
intracellular distribution of Q2 was further evaluated by CLSM.
A LysoTracker Red dye DND-99 was used to stain the acidic
organelles in HeLa cells. We found that Q2 were dominantly
localized in LysoTracker-labeled acidic organelles aer 6 h of
incubation (Fig. 4a–d). Those nanoparticles that were trapped
inside endosomes/lysosomes are pink in color. Nevertheless,
some of the Q2 appeared to be able to escape from the endo-
some, and was distributed in the cytoplasm. The nitrogen of the
quinoline unit on the nanoparticles was protonated at acidic
endosomal pH, which could disrupt the endosome and
promote the escape of the nanoparticles from the endosome
into the cytoplasm. The behaviour can be attributed to the
“proton-sponge” or “endosome buffering” effect.13a
We also investigated the nuclear localization ability of Q2.
Previous literature studies indicated that nuclear localization of
TCRNs was observed aer longer time of incubation (20–24
h).15,16 Hence we incubated HeLa cells with Q2 for 24 h. To
distinguish, nuclei were stained with PI, showing red uores-
cence in the images. Fig. 4g showed strong uorescence corre-
sponding to Q2, which was quite evenly distributed throughout
the cytoplasm and nucleus, indicating that the drug might have
entered into the nucleus. In general, nanoparticles larger than
70 nm in diameter were considered to be quite large to enter
into cell nuclei. Our newly synthesised nanoparticles Q2 have an
average particle size of 143.49 nm. Hence, we presume that our
photocage compound Qucbl would have been leached out from
silica cores of Q2 due to the breakage of secondary silica coating
in acidic medium, and then, freely diffused throughout the
whole intracellular area, resulting in the accumulation of Qucbl
Fig. 2 (a) The zeta potential of MSNs, Qucbl-MSNs (Q1) and Qucbl-
Fol-MSNs (Q2) at different pH. (b) pH responsive fluorescence spectra
of compound 2. (c) 1H NMR spectra of compound 2 in 10 mM HCl (10
mL) in MeOH-D4 (inset of (c): corresponding emission images from
protonated and non-protonated 2 under UV light of 366 nm).
internalized by the cell membrane (Fig. S9†) than that at pH 7.4.
The above mentioned studies clearly indicate that Q2 is indeed
charge-reversal nanoparticles. Thus, Q2 should be negatively
charged at physiological pH and be suitable for in vivo appli-
cations. Once localized in solid tumors/lysosomes, Q2 will
undergo negative to positive charge reversal and thus be more
readily internalized by the cells. Further, the effectiveness of the
targeting group folic acid on the Q2 in binding folate receptors
and promoting the cellular uptake was evaluated using HeLa
cells and normal cells-L929, because it is well known that folate
receptors are over-expressed in HeLa cells compared to normal
cells.14 The time dependent CLSM imaging studies (0–6 h)
revealed that Q2 was internalized to a greater extent in HeLa
cells compared to normal cells-L929 (Fig. 3).
Fig. 4 Confocal fluorescence images of HeLa cells: (a and e) bright
field images of cells that were incubated with 50 mg Qucbl-Fol-MSNs
(Q2) for 6 h, (b and g) showing the uptake of Qucbl-Fol-MSNs (Q2) (lex
¼ 365 nm), (c) emission from LysoTracker Red DND-99 (25 nM, lex
¼
535 nm), (d) fluorescent and bright field overlay image of (b) and (c)
showing both Qucbl-Fol-MSNs (Q2) and DND-99 were located at the
lysosome. The blue fluorescence is from Qucbl-Fol-MSNs (Q2) and
the red fluorescence is from red dye DND-99 used to stain the lyso-
some, (f) showing the uptake of PI (propidium iodide) (25 nM, lex ¼ 535
nm), (h) fluorescent and bright field overlay image of (f) and (g) showing
Fig. 3 Time dependent CLSM images of HeLa cells and normal cell that both Qucbl-Fol-MSNs (Q2) and PI were located at the cell nuclei.
(L929) incubated with of Qucbl-Fol-MSNs (Q2) and Qucbl-MSNs (Q1) The blue fluorescence is from Qucbl-Fol-MSNs (Q2) and the red
(without folic acid) for 6 h. The blue fluorescence is from the Qucbl fluorescence is from PI used to stain the nuclei (scale bar ¼ 20 mm (a–
conjugate of the MSNs. Scale bar: 20 mm.
d) and 30 mm (e–h)).
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in the nucleus due to its intercalation with double stranded
DNA.17
Aer successful demonstration of cellular internalization
and distribution of Q2, we studied photoinduced anticancer
drug release behaviour of Q2. The time courses of the anti-
cancer drug release by Q2 under photolysis at both $365 nm
(Hg vapour lamp) and two-photon 675 nm diode laser were
monitored by HPLC. The HPLC prole indicates (Fig. 5a) that,
aer 45 min of irradiation, 65% of loaded anticancer drug
(chlorambucil) was effectively released by using UV light (l $
365 nm, 120 mW cmꢁ2), whereas 15% of the drug was released
using the diode laser (675 nm, 15 mW cmꢁ2) (Fig. S10†), sug-
gesting that external light intensity could regulate the drug
release. Furthermore, we demonstrated precise control over the
photolytic release of loaded anticancer drug by monitoring the
release of chlorambucil aer periods of exposure to light and
dark conditions (Fig. 5b), which clearly showed that the drug
release proceeded only under illumination. In addition, the
photochemical quantum yield of Q2 was measured to be 0.29,
which resembles a previous report.12
Fig. 6 (a–b) Cell viability test of (i) MSNs, (ii) chlorambucil, and (iii)
Qucbl-Fol-MSNs (Q2) and in HeLa cell line: (a) before irradiation and
(b) after irradiation. Values are presented as mean ꢄ SD.
concentration of chlorambucil to that of Q2 (Fig. 6b), Q2
showed much lower cytotoxicity. But upon irradiation, Q2
showed an enhanced cytotoxicity to cancer cells in comparison
to chlorambucil because of the efficient photorelease of chlor-
ambucil inside the cancerous cells.
Aer successful demonstration of photoinduced anticancer
drug release by Q2, we evaluated the cytotoxicity of chlor-
ambucil, Q2, and MSNs in vitro using the MTT assay in HeLa cell
line. It was observed that cell viability remains above 90% at 50
mg mLꢁ1 of Q2 and MSNs. However, cells treated with chlor-
ambucil showed increasing cytotoxicity with increasing drug
concentration (Fig. 6a). The above mentioned studies indicated
that Q2 and MSNs were relatively nontoxic to the cells. For the
light exposure experiment, cells incubated with chlorambucil,
Q2, and MSNs were irradiated for 30 min under UV light ($365
nm). Cell viability of 27.8% was observed with free chlorambucil
at the concentration of 50 mg mLꢁ1. For the same concentration
of Q2, the cell viability of 23% was noted, which can be due to
the efficient photorelease of anticancer drug chlorambucil
inside the cancerous cell. Further, 73% cell viability was
observed at the concentration of 50 mg mLꢁ1 for two photon
irradiation (Fig. S11†). On the other hand, cell viability was
found to be largely unaffected by drug-free MSNs, indicating the
cytotoxicity was likely caused by the released drug chlorambucil
upon light irradiation on Q2. In comparison with the same
Conclusion
We have developed pH dependent charge reversal photo-
responsive nanoparticles for in vitro targeted drug delivery. The
TCRNs, i.e., Qucbl-Fol-MSNs, were negatively charged in neutral
solution and quickly transformed into positively charged at pH
6 and highly positively charged at pH 5.0–4.5. The charge
reversal has enhanced the cellular uptake of the photo-
responsive TCRNs and greater accumulation of drug in the
nucleus and cytoplasm. Even though the size of the nano-
particle was much larger than that of nuclear pores, the released
Qucbl from the silica cores was able to freely diffuse and
accumulated inside the nucleus, giving much improved cyto-
toxicity. Photoregulated drug release ability of Qucbl-Fol-MSNs
has been established by means of periodic exposure to light and
dark conditions. Strong uorescence of Qucbl-Fol-MSNs has
been explored for the in vitro cellular imaging application and
precise drug release inside the cancer cells upon irradiation.
Thus, we expect that the above mentioned study may be a
promising starting point for the utilization of charge reversal
photoresponsive nanoparticles in construction of nuclear tar-
geted drug delivery systems.
Acknowledgements
DST-SERB for nancial support. DST-FIST for 400 MHz NMR
and CLSM. S. Karthik is thankful to IIT KGP, B. Krishna Kalyani
to Inspire Fellowship and Biswajit Saha to DBT for their
fellowship. Yanli Zhao thanks nancial support from the Sin-
gapore
National
Research
Foundation
Fellowship
(NRF2009NRFRF001-015).
Fig. 5 (a) Time course for the photorelease of chlorambucil from
Qucbl-Fol-MSNs (Q2) under soft UV irradiation 1 PE ($365 nm, 120
mW cmꢁ2) and 2 PE (675 nm, 15 mW cmꢁ2). (b) Progress of release of
chlorambucil under bright and dark conditions. “ON” indicates the
beginning of light irradiation and “OFF” indicates the ending of light
irradiation.
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