Biosensors and Bioelectronics 71 (2015) 300–305
Contents lists available at ScienceDirect
Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
In situ monitoring of doxorubicin release from biohybrid nanoparticles
modiﬁed with antibody and cell-penetrating peptides in breast cancer
cells using surface-enhanced Raman spectroscopy
Md. Khaled Hossain a,1, Hyeon-Yeol Cho b,1, Kyeong-Jun Kim b, Jeong-Woo Choi a,b,n
a
b
Interdisciplinary Program of Integrated Biotechnology, Sogang University, 121-742 Seoul, Republic of Korea
Department of Chemical and Biomolecular Engineering, Sogang University, 121-742 Seoul, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history:
Received 4 February 2015
Received in revised form
9 April 2015
Accepted 17 April 2015
Available online 20 April 2015
In situ monitoring of drug release in cancer cells is very important for real-time assessment of drug
release dynamics in chemotherapy. In this study, we report label-free in situ monitoring and control of
intracellular anti-cancer drug delivery process using biohybrid nanoparticles based on surface-enhanced
Raman spectroscopy (SERS) for the ﬁrst time. Each biohybrid nanoparticle consisted of gold nanoparticle,
cell-penetrating peptide (Tat peptide), and cancer-targeting antibody to increase the efﬁcacy of the anticancer drug delivery with speciﬁc targeting and increased uptake rate. The doxorubicin (Dox)-loaded
biohybrid nanoparticles were showed speciﬁc SERS spectra of Dox, speciﬁcally immobilized on the target
cell membrane and quickly penetrated into the cells when treated on the mixed cell culture condition.
The intracellular release of Dox from the biohybrid nanoparticle was continuously monitored with timedependent change of intracellular SERS signals of Dox. The releasing rate of Dox was successfully controlled with the addition of glutathione on the cells. The anti-cancer effect of intracellular released Dox
was conﬁrmed with cell viability assay. With the proposed monitoring system, speciﬁc cancer cell targeting and improved uptake of the anti-cancer drug were detected and time-dependent intracellular
release of the anti-cancer drug was monitored successfully. The proposed novel in situ monitoring system
can be used as a spectroscopic analysis tool for label-free monitoring of the time-dependent release of
various kinds of anti-cancer drugs inside cells.
& 2015 Elsevier B.V. All rights reserved.
Keywords:
In situ monitoring
Intracellular release
Cell-penetrating peptide
Surface-enhanced Raman spectroscopy
1. Introduction
In situ monitoring of drug release is very important for realtime assessment of drug release kinetics (Hu et al., 2009; Wang
et al., 2014) in cancer therapy. Several analytical techniques have
been employed to study drug release from therapeutic nanoparticles, including high-performance liquid chromatography
(HPLC) (Zagotto et al., 2001) and ﬂuorescence microscopy (Isben
et al., 2013; Nakamura et al., 2015). HPLC is the most popular
method for quantifying released drugs by reading the characteristic UV absorbance upon elution from a proper HPLC column, but
this technique lacks real-time monitoring capability and suffers
from excessive down-time and a lack of a sensitive, universal detector (Lurie et al., 1984). In some cases, to measure drug release
kinetics, dialysis devices are used to collect the released drugs
(Zhang et al., 2008; Chan et al., 2009). While these techniques are
n
Corresponding author at: Interdisciplinary Program of Integrated Biotechnology, Sogang University, 121-742 Seoul, Republic of Korea. Fax: þ82 2 3273 0331.
E-mail address: [email protected] (J.-W. Choi).
1
Equal contribution.
http://dx.doi.org/10.1016/j.bios.2015.04.053
0956-5663/& 2015 Elsevier B.V. All rights reserved.
capable of quantifying the drug release proﬁle, they usually involve complex procedures and labor-intensive sample preparation.
In the case of ﬂuorescence microscopy, additional dye is needed.
Therefore, there is still a need for an easy and suitable technique
for the effective monitoring of drug release.
Since its development, surface-enhanced Raman spectroscopy
(SERS) has been widely used for biological sensing and molecular
imaging as an ultrasensitive spectroscopic tool (Pallaoro et al.,
2010). The SERS technique has shown promise in overcoming the
problem of low sensitivity inherent in conventional Raman spectroscopy (Doering et al., 2007). In addition, Raman microscopy has
made unique contributions to intracellular activity monitoring
(Pully et al., 2010; Boyd et al., 2011; Zong et al., 2011; Zacharia
et al., 2010). Considering the advantages above, we used SERS in
this study for label-free in situ monitoring of time-dependent anticancer drug release at the single-cell level.
SERS has recently been used for label-free in situ monitoring of
anti-cancer drug release by Kang et al. (2013) and Oak et al. (2012).
In their studies, nanomaterials were used to monitor the intracellular drug release and these particles were loaded with anticancer drugs. Furthermore, light exposure (Kang et al., 2013) and
Md.K. Hossain et al. / Biosensors and Bioelectronics 71 (2015) 300–305
301
Fig. 1. Schematic diagram for (a) conjugation of the biohybrid nanoparticle, (b) time-dependent monitoring of the nanoparticle's speciﬁc targeting, cellular uptake, and drug
release, and (c) uptake of the Dox-loaded biohybrid nanoparticles by the cells and the intracellular release of Dox by GSH.
addition of glutathione (GSH) (Oak et al., 2012) were tested to
control intracellular drug release. However, techniques for targeting of speciﬁc cells and fast uptake of nanoparticles have not
been reported in the above studies. Non-targeted therapeutic nanoparticles not taken up can cause harmful effects to healthy cells.
In this study, an in situ label-free intracellular drug release
monitoring system based on biohybrid nanoparticle was proposed
for the ﬁrst time. The biohybrid nanoparticle, consist of a gold
nanoparticle (AuNP), a cell-penetrating peptide (CPP), and a breast
cancer-targeting antibody, was newly conjugated for facilitated
speciﬁc cell targeting, increased uptake, and time-dependent intracellular anti-cancer drug release using SERS (Fig. 1a). The cysteine-modiﬁed Tat peptide (Tat-C) was used as a CPP in the biohybrid nanoparticles for increased uptake by the cancer cells. In
addition, the anti-HER2 antibody was used to target the breast
cancer cells (SK-BR-3). We monitored the GSH-dependent intracellular release rate of doxorubicin (Dox) as an anti-cancer drug
and conducted a cell cytotoxicity assay to observe the effects of
intracellular anti-cancer drug release in the target cells (Fig. 1b
and c) (Fig. 1).
2.3. Cancer cell culture
The human breast cancer cell line (SK-BR-3) and neuroblastoma
cell line (SH-SY5Y) were obtained from ATCC (Manassas, VA, USA).
The cells were cultured at 37 °C in an RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1%
antibiotics (streptomycin and penicillin) in a humidiﬁed atmosphere of 95% air with 5% CO2. The cells were grown in TC-grade
petri dishes. After every 48 h of incubation, the medium was replaced with a fresh medium.
2.4. Treatment of mixed cultured cells with biohybrid nanoparticles
At ﬁrst, SK-BR-3 and SH-SY5Y cells were seeded on a glass
substrate attached with chamber at a concentration of
2 104 cells/ml media and incubated at 37 °C in a cell culture incubator. After a 48 h incubation period, the cells were treated with
the biohybrid nanoparticles and incubated at 37 °C for 2 h for
speciﬁc targeting of, and uptake by, the SK-BR-3 cells. After incubation, the biohybrid nanoparticle-containing media was removed. Cells were rinsed ﬁve times with PBS (10 mM, pH 7.4),
fresh media was added, and then the cells were incubated at 37 °C
for next 22 h for Dox-release measurements.
2. Materials and methods
2.1. Materials
2.5. Measurement of SERS on biohybrid nanoparticle-treated cells
Unless otherwise stated, all chemicals were obtained from
Sigma-Aldrich (St. Louis, MO, USA), and were of the highest purity
available.
The SERS signal was measured on the biohybrid nanoparticletreated cells through confocal Raman spectroscopy (NTEGRA
Spectra, NT-MDT) (An et al., 2014; Chae et al., 2013; Kim et al.,
2013). The distribution of biohybrid nanoparticles inside the cells
was detected with SERS map imaging. The speciﬁc Raman band of
Dox was selected to create an SERS map. Then, the intensity of the
SERS spectra was measured at ten different spots on three individual cells and the spectra were averaged to create every single
curve (El-Said et al., 2011a,b; An et al., 2011a, b; El-Said et al.,
2010). The release of Dox from the biohybrid nanoparticle surface
2.2. Formation of biohybrid nanoparticles
The Dox loaded biohybrid nanoparticles were prepared in
conjugation with AuNP, Dox, Tat-C, polyethylene glycol (PEG) and
anti-HER2 antibody (described in Supplementary material).
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Md.K. Hossain et al. / Biosensors and Bioelectronics 71 (2015) 300–305
Fig. 2. Conﬁrmation of speciﬁc cell targeting using biohybrid nanoparticles and time dependent release of Dox inside cells. (a) and (c) SERS map images of biohybrid
nanoparticles added to co-cultured SH-SY5Y and SK-BR-3 cells respectively. SERS mapping images were measured at the 1275 cm 1 Raman band. (b) and (d) Bright-ﬁeld
images of the SH-SY5Y and SK-BR-3 cells respectively. (scale bar: 20 μm (b), and 5 μm (d)). (e) SERS spectra of the SK-BR-3 cells treated with Dox loaded biohybrid
nanopartcles at different incubation times. (f) Relationship between time and release of Dox. The error bars indicate the standard deviation of ten measurements in three
individual cells.
was measured at each 4 h interval using a 785 nm near-infrared
laser with 3 mW laser power on the sample plane. During the
interval between two measurements, the cells were incubated in
the cell culture incubator.
2.6. Cytotoxicity assay
The cytotoxicity of the conjugated nanoparticles was measured
via an MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium
Md.K. Hossain et al. / Biosensors and Bioelectronics 71 (2015) 300–305
bromide) assay following the method described previously (Aroui
et al., 2009). After 48 h of seeding, the cells (5000 cells per well of a
96-well plate) were treated with the biohybrid nanoparticles and
incubated at 37 °C for 2 h for the speciﬁc targeting of, and uptake
by, the SK-BR-3 cells. After incubation, the biohybrid nanoparticlecontaining media was removed, the cells were rinsed ﬁve times
with PBS, and fresh media was added to the cells. MTT solution was
added to each well at a ﬁnal concentration of 1.2 mM and incubated
at 37 °C for 3 h. After incubation, the MTT solution-containing
media was removed and 200 μl of dimethyl sulfoxide (DMSO) was
added to each well. The optical density was measured with a universal microplate reader (EL-800, BioTek Instrument Inc.) at a wavelength of 540 nm.
3. Results and discussion
3.1. Particle conjugation and characterization
Biohybrid nanoparticles were prepared by the conjugation of
AuNPs, Tat-C, PEG, and the anti-HER2 antibody. To increase the
payload of the anti-cancer drug and to prevent the degradation of
biological elements during conjugation, Dox was immobilized to
the AuNPs ﬁrst, followed by Tat-C, PEG, and the anti-HER2 antibody. The size of the conjugated nanoparticle was characterized by
TEM imaging and DLS. The TEM image shows three 30 nm conjugated AuNP (Fig. S1a). According to the DLS results, the average
size of the bare AuNPs was 31.2 nm in diameter. After every step of
the conjugations of Dos, Tat-C, PEG and antibody the diameter of
the conjugated nanoparticles increased (Fig. S1b, S2) and, at the
end of the conjugation, the average size of the nanoparticles was
50 nm, but after GSH treatment the average size is decreased to
44 nm (Fig. S1b). The decrease in size was due to release of doxorubicin from the nanoparticle's surface after GSH treatment.
The zeta potential of the conjugated nanoparticles was measured for conﬁrmation of component conjugation at each step. The
AuNPs, HS-PEG-COOH, and anti-HER2 antibody were negatively
charged (Ock et al., 2012; Chen et al., 2013; Koopaei et al., 2011),
and Dox, Tat-C and GSH were positively charged (Yousefpour et al.,
2011; Kaplan et al., 2005; Ock et al., 2012). According to Fig. S1c,
the zeta potential of the bare AuNPs was 68.64 mV. During
conjugation of Dox, Tat-C, PEG, and the antibody to the AuNPs, the
zeta potential of the conjugated nanoparticles became either positive or negative, depending upon the components conjugated. At
the end of the conjugation, the zeta potential of the Dox-loaded
biohybrid nanoparticle was 11.22 mV, but after GSH treatment
the zeta potential was decreased to 47.75. The decrease in zeta
potential was due to release of Dox from the surfaces of AuNPs
after GSH treatment. Figs. S1d and S3 show the intensity of the
SERS spectra of different concentration of Dox loaded on the biohybrid nanoparticles. The SERS intensity of Dox increased along
with the increase in loading concentration.
The SERS spectra were measured from the conjugated nanoparticles (Fig. S4). The Dox exhibited a strong Raman band at
1275 cm 1, which was due to the (νC-O) vibrational mode of ring
A of the Dox molecule (Lee et al., 2004). After the addition of Tat-C,
PEG, and the antibody, the SERS intensity of Dox reduced slightly.
This occurs due to the scattering shielding effect caused by the
increasing thickness of the coating layer (Park et al., 2009). The
Dox-loaded biohybrid nanoparticles were stable for up to 28 days
(Fig. S5).
3.2. Speciﬁc targeting of SK-BR-3 cells
To study speciﬁc targeting, HER2-expressing cells (SK-BR-3)
(Lee et al., 2013a, b) and HER2-negative cells (SH-SY5Y) were co-
303
cultured and treated with Dox-loaded biohybrid nanoparticles. To
detect the SERS signal from the treated cells, SERS mapping images
were measured at the 1275 cm 1 Raman band. Fig. 2a and c shows
the SERS map images of the biohybrid nanoparticle–treated, cocultured SH-SY5Y and SK-BR-3 cells respectively, and Fig. 2b and d
shows the bright-ﬁeld images of both cell types. The mapping
images demonstrate that the SERS signals of the biohybrid nanoparticles were detected from SK-BR-3 cells in the immobilized
area, but only non-speciﬁc and low SERS signals in the SH-SY5Y
cell area. These results indicate that the biohybrid nanoparticles
can speciﬁcally target cells that express HER2 on the membrane.
To conﬁrm the SERS results for speciﬁc targeting of the breast
cancer cells, a ﬂuorescence microscopy experiment was also conducted. Fig. S6a shows the SERS map image and ﬂuorescence
microscopy image, while Fig. S6b shows the bright-ﬁeld images of
co-cultured cells containing SK-BR-3 and SH-SY5Y (arrow). In the
case of the SERS experiment, Dox-loaded biohybrid nanoparticles
were added to the cells, while for ﬂuorescence microscopy FITClabeled biohybrid nanoparticles were added. The mapping image
demonstrates that the SERS signals of the biohybrid nanoparticles
were detected from the SK-BR-3 cell-immobilized area only and
that the areas with the SH-SY5Y cells did not exhibit any SERS
signal. The ﬂuorescence microscopy image shows that the FITClabeled (green) conjugated nanoparticles were immobilized on the
round SK-BR-3 cells only, while the SH-SY5Y cells did not have any
conjugated nanoparticles. Hoechst 33342 dye was used to locate
the nuclei of the cells (blue). Therefore, the ﬂuorescently labeled
biohybrid nanoparticle-treatment experiment demonstrated that
the biohybrid nanoparticles are capable of speciﬁc targeting at the
bulk cell level as well.
3.3. Detecting the effect of Tat-C on nanoparticle uptake by the SKBR-3 cells
Before selecting Tat-C as a CPP, the cell penetration efﬁcacy of
four kinds of CPPs, Tat-C, Penetratin-C, pVEC-C, and Pep-1-C (Table
S1), was studied through ﬂuorescence microscopy. Among them,
Tat-C exhibited the highest cell penetration efﬁcacy (Fig. S7) and,
hence, Tat-C was selected for further experiments in this study. Of
two groups of SK-BR-3 cells, one group was treated with biohybrid
nanoparticles containing Tat-C and other group was treated with
biohybrid nanoparticles without Tat-C. After 2 h of particle treatment, the cells were washed and their gold content measured
using inductively coupled plasma atomic emission spectroscopy
(ICP-AES). According to the ICP-AES results, the uptake of Tat-Cmodiﬁed biohybrid nanoparticles was approximately 5,670 per
cell, while the uptake of the biohybrid nanoparticles not containing Tat-C was too low to detect (detection limit was 0.05 ppm)
(Table S2) (Fig. 2).
3.4. Monitoring intracellular Dox release in SK-BR-3 cells
Since the chemical structure of Dox contains an aromatic ring,
it produces enhanced Raman signals when immobilized on AuNP
surfaces. Dox can be released from biohybrid nanoparticle surfaces
by intracellular GSH. GSH is the most abundant thiol species in the
cell cytoplasm, with a concentration range of 1–10 mM, and has
been used as an in situ releasing reagent in living cells, owing to its
biochemical reducing capability (Oak et al., 2012). The thiol group
in the GSH has strong afﬁnity to AuNPs and can bind with the
AuNPs through covalent coupling. For this reason, amine containing Dox can easily be replaced from AuNP surfaces. Before
studying its effects on cells, we studied the release characteristics
of Dox from biohybrid nanoparticle surfaces in the absence of cells,
but in the presence of different concentrations of GSH using SERS.
Fig. S8 shows the GSH concentration-dependent Dox release from
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the biohybrid nanoparticle surfaces in the absence of cells. The
decrease in intensity at the 1275 cm 1 Raman band of Dox indicated the release of Dox from the surface of the biohybrid nanoparticles. Ten spectra were measured and averaged to plot each
curve. The results show that the Dox release rate was high in the
presence of 5–12 mM GSH. Time-dependent Dox release from the
biohybrid nanoparticle surface in the absence of cells but in the
presence 10 mM GSH was also studied using SERS (Fig. S9). According to the SERS results, the Dox release rate was high within
1 h of GSH treatment.
Time-dependent intracellular Dox release in live SK-BR-3 cells
was studied using SERS. Fig. 2d shows a bright-ﬁeld image of an
SK-BR-3 cell that was treated with Dox-loaded biohybrid nanoparticles. The SERS map image of the cells (Fig. 2c) shows the SERS
signal of nanoparticles taken up by the cell. The SERS mapping
image was measured at the 1275 cm 1 Raman band. The SERS
spectra were measured from ten different spots on three individual cells at 4 h intervals after 2 h of nanoparticle treatment
and the spectra were averaged to make a single curve. The SERS
results (Fig. 2e and f) show that the intracellular Dox release rate
from the biohybrid nanoparticles was high up to 12 h after nanoparticle treatment, followed by a decrease in concentration and
release rate. The intracellular Dox release rate was comparatively
slower than the Dox release rate in the absence of cells but in the
presence of 10 mM GSH (Fig. S9). To observe the effect of additional GSH on intracellular Dox release, another experiment was
conducted. GSH-OEt (5 mM) was added to the cell medium after
2 h of nanoparticle treatment and SERS spectra were measured
every 15 minutes a few minutes after GSH-OEt treatment. The
results show that after adding 5 mM of additional GSH-OEt to the
cells, the Dox release rate increased and most of the Dox was released within 1 h of GSH-OEt treatment (Fig. S10).
3.5. Cytotoxicity assay
To study the cytotoxicity of the biohybrid nanoparticles, an
MTT assay was conducted with different time condition. Fig. 3a
shows that the cytotoxic effect depends on the concentration of
Dox under different treatment conditions over the 24 h incubation
period and, in contrast with the Dox-containing treatment condition, bare AuNPs (0 μM Dox) did not cause any cytotoxicity. The
Dox-conjugated AuNP treatment caused higher cytotoxic effects
under every tested condition and had a role as a carrier of Dox into
cells. Furthermore, in AuNP-based Dox delivery, Tat-C-modiﬁed
AuNPs exhibited higher cytotoxic effects than non-modiﬁed
AuNPs, based on their increased uptake. Fig. 3b shows the timedependent cytotoxicity of Dox-loaded biohybrid nanoparticles.
After 2 h of treatment with biohybrid nanoparticles, cell viabilities
were measured up to 24 h every 4 h. The results show that Dox
was released continuously under intracellular conditions, as
shown in Fig. 2e, and induced an anti-cancer effect in the cells.
Eight hours after the particle treatment, 9.53% of cells died and cell
viability decreased continuously until 24 h (39.48%). The Dox
worked as a DNA intercalator when it was released from the
biohybrid nanoparticles and that is the reason the Dox-mediated
cytotoxicity was not obtained directly. However, the biohybrid
nanoparticles did not cause any signiﬁcant cell mortality. Therefore, the biohybrid nanoparticles can be used as carrier for anticancer drugs and as a monitoring probe for drug release (Fig. 3).
4. Conclusion
In this study, we proposed label-free in situ monitoring and
control of intracellular anti-cancer drug delivery process using
biohybrid nanoparticles based on surface-enhanced Raman
Fig. 3. Results of a cytotoxicity assay with the SK-BR-3 cell line. (a) Level of Doxdependent cytotoxicity under different treatment conditions. (b) Cytotoxicity of
time-dependent intracellular Dox release from the biohybrid nanoparticle surfaces.
The error bars indicate the standard deviation of ﬁve independent measurements.
spectroscopy (SERS) for the ﬁrst time. The biohybrid nanoparticles
were successfully conjugated with AuNP, Tat-C, PEG and anti-HER2
antibody and enhanced Raman signal of Dox when Dox was loaded. The HER2-positive cancer cell (SK-BR-3) was speciﬁcally targeted with biohybrid nanoparticles and showed the SERS signal of
Dox from entire cell. Due to the addition of Tat-C to the biohybrid
nanoparticles, an average of 5670 nanoparticles were taken up by
each cell within 2 h of treatment, but uptake was very low to in
the case of nanoparticles not containing Tat-C. Time-dependent
intracellular Dox release from biohybrid nanoparticles was monitored using SERS. According to the SERS results, 90.23% of the Dox
was released by the action of intracellular GSH with 24 h of particle treatment and releasing time of Dox can be reduced until 2 h
with addition of GSH. Cell mortality was linearly increasing until
60.86% at 24 h with Dox-loaded biohybrid nanoparticle-treated
cells, but there was no anti-cancer effect in unloaded biohybrid
nanoparticle-treated cells. Thus, by using the biohybrid nanoparticles, we successfully monitored in situ Dox release inside SKBR-3 cells. The proposed biohybrid nanoparticles suitable for application of aromatic anti-cancer drugs but SERS signal of other
kinds of drugs may not be enough to distinguish from other signals. However, our newly proposed system can be applied as a
Md.K. Hossain et al. / Biosensors and Bioelectronics 71 (2015) 300–305
spectroscopic biosensor for label-free, in situ monitoring of the
time-dependent release of other anti-cancer drugs in cells.
Acknowledgements
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government
(MSIP) (No. 2014R1A2A1A10051725).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2015.04.053.
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