Journal of Photochemistry and Photobiology B: Biology 136 (2014) 8190

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Targeted nanoparticles for simultaneous delivery of chemotherapeutic

and hyperthermia agents An in vitro study q
Supriya Srinivasan a, Romila Manchanda a,b, Tingjun Lei a,c, Abhignyan Nagesetti a,
Alicia Fernandez-Fernandez a,d, Anthony J. McGoron a,
a

Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, EC 2600, Miami, FL 33174, USA
Chemistry Department, Galgotias University, Greater Noida, UP, India
Cirle, 1951 NW 7th Ave, Suite 13016, Miami, FL 33136, USA
d
Physical Therapy Department, Nova Southeastern University, Fort Lauderdale, FL 33328, USA
b
c

a r t i c l e

i n f o

Article history:
Received 24 February 2014
Received in revised form 10 April 2014
Accepted 17 April 2014
Available online 29 April 2014
Keywords:
Doxorubicin
Drug delivery
Hyperthermia
Image-guided therapy
Indocyanine green
Molecular imaging
Theranostics

a b s t r a c t
The purpose of this study was to prepare targeted Poly lactide-co-glycolide (PLGA) nanoparticles with
simultaneous entrapment of indocyanine green (ICG) and doxorubicin (DOX) by surface decorating them
with tumor specic monoclonal antibodies in order to achieve simultaneous therapy and imaging. ICG
was chosen as an imaging and hyperthermia agent and DOX was used as a chemotherapeutic agent.
ICG and DOX were incorporated into PLGA nanoparticles using the oil-in-water emulsion solvent
evaporation technique. These nanoparticles were further surface decorated with antibodies against
Human Epithelial Receptor-2 (HER-2) using carbodiimide chemistry. The uptake of antibody conjugated
ICG-DOX-PLGA nanoparticles (AIDNP) was enhanced in SKOV-3 (HER-2 overexpressing cell lines)
compared to their non-conjugated counterparts (ICGDOXPLGA nanoparticles (IDNP)). The uptake of
antibody conjugated ICGDOXPLGA nanoparticles, however, was similar in MES-SA and MES-SA/Dx5
cancer cells (HER-2 negative cell lines), which were used as negative controls. The cytotoxicity results
after laser treatment (808 nm, 6.7 W/cm2) showed an enhanced toxicity in treatment of SKOV-3. The
negative controls exhibited comparable cytotoxicity with or without exposure to the laser. Thus, this
study showed that these antibody conjugated ICGDOXPLGA nanoparticles have potential for
combinatorial chemotherapy and hyperthermia.
2014 Elsevier B.V. All rights reserved.

1. Background
Cancer is the second leading cause of death next to cardiovascular diseases. With the increasing mortality rate associated with
cancer, there is an increasing need to develop new strategies for
detection and treatment. Current cancer therapies include chemotherapy, radiotherapy, immunotherapy, hormone therapy, etc.
Combination therapy is the simultaneous co-delivery of two or
more therapeutic agents or different therapies such as chemotherapy, hormone therapy, immunotherapy and radiotherapy. Coadministration of anti-cancer agents or approaches is a common
q
Sources of support for research: this work was conducted using the facilities of
the Biomedical Engineering Department at Florida International University, and
partially funded by FLDOH (Grant #08-BB-11) and the FIU Wallace H Coulter
Foundation Biomedical Engineering Young Inventor Award to R.M. Electron
microscopy was performed at the Advanced Materials Engineering Research
Institute (AMERI).
Corresponding author. Tel.: +1 305 348 1352; fax: +1 305 348 6954.
E-mail address: mcgorona@u.edu (A.J. McGoron).

http://dx.doi.org/10.1016/j.jphotobiol.2014.04.012
1011-1344/ 2014 Elsevier B.V. All rights reserved.

clinical practice for treatment as it often helps in bringing in a better clinical outcome than with single drug administration. This
helps in reducing the overall drug dose administered, thereby
reducing their dose-dependent toxicity to non-target tissues [1].
Doxorubicin (DOX) is a common anticancer drug effective
against a wide spectrum of neoplastic diseases. There are two
mechanisms of DOX mediated cytotoxicity. One of the mechanisms
is through generation of free radicals and the other is through DNA
intercalation. Free radical independent DNA intercalation of DOX
in the nucleus, however, is the most predominant mechanism of
cytotoxicity in tumor cells. Lack of tumor specicity, dose-dependent cardio toxicity and increasing resistance to DOX limits its clinical application [2].
There is a growing interest in development of combined nearinfrared based thermotherapy and chemotherapy to enhance the
therapeutic effect of chemotherapy. Cancer cells can be selectively
killed by temperatures in the 4143 C range with minimal effects
on the surrounding normal cells [3]. Tumor cell environment, such
as hypoxia, poor nutrition, and low pH are the reasons for the higher

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heat sensitivity of cancer cells over normal cells. Heat induction

also helps sensitize cancer cells to the effects of other cytotoxic
agents by increasing their membrane permeability [4]. Delivering
photothermal therapy with agents that absorb in the near-infrared
region provides the advantages of greater tissue penetration of light
(compared to visible light) and non-invasive treatment [5].
Indocyanine green (ICG) is a near-infrared absorbing dye widely
used for determining cardiac output and ophthalmic angiography
[6]. It can also be used as a hyperthermia agent for localized hyperthermia treatment [7,8]. However, its high aqueous degradability
and rapid plasma clearance limit its in vivo applications [9].
Nanomedicine, a relatively new, yet rapidly progressing area in
the eld of cancer therapy, offers great potential in combining multiple therapeutic modalities and functionalities into a single platform.
Several materials, such as inorganic materials (gold, iron etc.), carbon nanotubes and polymers have been investigated for combinatorial cancer therapeutic strategies [10]. Degradable polymeric
biomaterials (both synthetic and natural) are preferred candidates
for drug delivery. The biodegradation of polymers can be easily
manipulated for efcient and controlled drug release [11]. Poly lactide co-glycolide (PLGA) is a polymer approved by the Food and Drug
Administration for use as a drug delivery vehicle in humans. It is one
of the most successful polymers for drug delivery as its degradation
products, lactic acid and glycolic acid, are easily metabolized by the
body. Further, the ability to manipulate the copolymer ratio and
polymer weight for controlled drug release makes PLGA the most
widely investigated polymer for drug delivery [12].
The encapsulation of ICG and DOX into nanoparticles is advantageous compared to free agent administration because it can
potentially increase in vivo stability, enhance tumor uptake, and
provide more localized therapy with reduced off-target toxicity
[13,14]. Nanoparticles can be used for targeted delivery of therapeutic drugs through passive and active targeting. Passive targeting involves accumulation of drug loaded nanoparticles through
extravasation of leaky tumor vasculature (of the size of 100
780 nm) [15,16]. Passive targeting is, however, not achievable in
all cases because of variations in tumor vasculature and porosity
depending on tumor type and status [16]. Thus, the current focus
is being gradually shifted from depending on passive transport
only, to active targeting of nanoparticles, which is achieved by decorating nanoparticle surfaces with tumor-selective ligands.
A myriad of tumor-selective ligands such as monoclonal antibodies, peptides, and carbohydrates are used for targeted delivery.
Monoclonal antibodies are widely investigated for their escorting
capability due to their high specicity to tumor markers. Several
studies have used HER-2 (Human Epithelial Receptor-2) for targeting, because it is over expressed in 2025% of ovarian cancers [17].
The over expression of HER-2 on tumor surfaces has often been
correlated with angiogenesis, metastasis and resistance against
apoptosis-inducing therapeutic agents [18].
In recent work we reported the simultaneous entrapment of ICG
and DOX into PLGA nanoparticles for combined imaging and therapy [19]. The current paper presents the development of targeted
nanoparticles by surface conjugation of the nanoparticles
described earlier with monoclonal antibodies against HER-2. These
nanoparticles have been investigated for their potential to achieve
targeted combined chemotherapy, hyperthermia and imaging in
cancer cells with different drug resistance mechanisms.

2. Methods
2.1. Drugs and chemicals
Poly (DL-lactide-co-glycolide) (PLGA, L:G 50:50; MW: 40
75 KDa; glass transition temperature (Tg): 4550 C), doxorubicin

hydrochloride (MW: 579.95 Da), dimethylsulfoxide (DMSO > 99.9%

reagent grade), and polyvinyl alcohol (PVA, 8789% hydrolyzed;
1323 KDa) were purchased from SigmaAldrich (St. Louis, MO,
USA). Indocyanine green was purchased from Acros Organics.
Dichloromethane (DCM) was purchased from Burdick & Jackson
(Muskegon, MI, USA).
2.2. Synthesis of IDNPs
PLGA nanoparticles simultaneously entrapping ICG and DOX
(IDNP) were prepared using an oil-in-water emulsion solvent
evaporation technique [20]. The protocol was previously optimized
for maximum drug entrapment. Briey, 60 mg of PLGA, 1 mg of
ICG, and 1 mg of DOX were dissolved in 4 mL of methanoldichloromethane (1:3 v/v) mixture. The organic phase was emulsied
with 8 mL of PVA solution (3%, w/v) by sonication at 50 W for
1 min in an ice bath. The organic solvent was then removed under
reduced pressure at 39 C. The nanoparticle suspension was centrifuged at 14,000 rpm (16,000  g) for 30 min. The precipitate
obtained was further washed with equal volumes of distilled
water, centrifuged again, and freeze-dried.
2.3. Antibody conjugation to IDNPs to prepare AIDNPs
The IDNPs were conjugated with anti-HER-2 using a modied
protocol explained by Yang et al. [21]. Briey, the lyophilized
IDNPs were resuspended in PBS (0.1 M, pH 7.4, 1 mg/mL). 1 mL
of 2.0 mM NHS and 2.0 mM of EDC was added to the IDNPs and left
to react for 20 min. Further, the activated IDNPs were reacted with
50 lL (100 lg) of IgG antibody for 1 h. The product (AIDNP) was
recovered by centrifugation at 12,200 rpm (13,943  g), 6 C for
30 min. The pellets were washed, resuspended in PBS (0.1 M, pH
7.4), and lyophilized.
2.4. Characterization of NPs
Size and size distribution of IDNPs and AIDNPs were measured
by dynamic light scattering (DLS) using Malvern Zetasizer (Malvern Instruments, Worcestershire, United Kingdom). The measurements were taken at 25 C using a 1:30 (vol/vol) dilution of the NP
suspension in distilled water. Zeta potential of the NPs dispersed in
deionized (DI) water was measured by the same Zetasizer. The
shape and size of the nanoparticles was conrmed using SEM
(JEOL-JEM).
2.5. Dye loading into NPs
The concentrations of ICG and DOX encapsulated in the NPs
(both IDNPs and AIDNPs) were determined using a Fluorolog-3
spectrouorometer (HORIBA Jobin Yvon Inc., USA) in steady-state
mode. The NPs dissolved in DMSO were evaluated for the drugs
entrapped as previously reported by our group [20]. Briey, serial
dilutions of the supernatant were done in order to reach a linear
uorescence range. The maximum peak intensities of the supernatant solution were blank corrected and used to t to a linear model
for drug loading estimation.
2.6. Antibody conjugation efciency estimation
The amount of antibody conjugated per nanoparticle (antibody
conjugation efciency) was measured using a modied protocol
reported by Mo and Lim [22]. 1 mg of lyophilized particles (AIDNP)
was dissolved in 0.5 ml DMSO and 1 mL of 0.5% (wt./vol.) SDS/
0.05 M NaOH (salinated SDS) and incubated for 4 h. 1 mL of this
solution was mixed with an equal volume of BCA reagent for
60 min at 60 C. The absorbance of this mixture was estimated at

S. Srinivasan et al. / Journal of Photochemistry and Photobiology B: Biology 136 (2014) 8190

562 nm using a spectrophotometer. The instrument was calibrated

with standard bovine serum albumin protein solutions (0.540 lg/
ml) in the same proportion of DMSO and salinated SDS solution.
IDNPs were used as blank. Conjugation efciency was expressed
as weight of antibody (lg) per unit weight (mg) of nanoparticles.
2.7. In vitro studies of NPs
Human uterine sarcoma MES-SA cells, and their drug resistant
counterpart (Pgp over-expressing derivative) MES-SA/Dx5 (Dx5)
cells, human ovarian carcinoma SKOV-3 cancer cells, McCoys 5A
medium, and fetal bovine serum were purchased from American
Type Culture Collection (Manassas, VA). Formalin, 24-well tissue
culture plates and d-poly cover slips were purchased from Fisher
Scientic (Pittsburg, PA). Penicillin was purchased from Sigma
Aldrich. All cells were cultured in McCoys 5A medium supplemented with 1% penicillin and 10% fetal bovine serum, and kept
in a 37 C cell incubator with a humidied atmosphere of 5% CO2
and 95% air.
2.7.1. In vitro drug release kinetics prole in AIDNP
AIDNP (5 mg) was resuspended in 3 mL of 0.01 M PBS
(pH = 7.4). This sample was divided into three centrifuge tubes.
The tubes were shaken at 35 rpm, 37 C in an incubator. These
tubes were removed from the incubator at regular intervals and
centrifuged at 14,000 rpm (16,000  g) for 30 min. The supernatants were collected for DOX estimation and the pellet was resuspended with fresh buffer. This process was repeated for each time
point up to 48 h.
2.7.2. Subcellular localization of the NPs
MES-SA, Dx5 and SKOV-3 cells (4  104) cells/well were seeded
onto poly-D-lysine coated coverslips placed into 24-well culture
plates. Cells were incubated to reach conuence. After 24 h incubation, medium was replaced by medium containing IDNP and AIDNP
corresponding to equivalent ICG and DOX concentration of 6.2 lM
and 10 lM respectively. After 24 h incubation, the cells were
washed 3X with DPBS to remove free drugs (IDNP or AIDNP). The
cells were xed with 4% paraformaldehyde solution for 20 min.
The cells were further washed 3 with DPBS. The xed cells were
viewed under uorescence microscope (Olympus IX81, Japan) and
imaged at kex (480490 nm), kem (P515 nm) for the DOX window,
and kex (775 nm), kem (845 nm) for the ICG window. The images
were taken under a water-merged 60 objective. The uorescence
signals were acquired using a CCD camera and merged using software to give a pseudo-color (IPLab, Qimaging, Canada). The settings of the microscope were kept constant throughout the
experiment; however different exposure times were used for the
ICG and DOX windows. The uorescence comparisons between
IDNP and AIDNP were done within the same cell lines.
2.7.3. Cellular uptake experiments
The cellular uptake of AIDNP and IDNP were quantied. Briey,
2  104 cells were seeded in a 24 well tissue culture plate. After
cells have reached conuency, the cell medium was removed and
IDNP and AIDNP resuspended in growth medium (equivalent
DOX and ICG concentrations described in the previous section)
for 24 h. Cells that were not subjected to any treatment were considered as controls. After incubation, the cells were washed with
ice cold DPBS (pH 7.4) and lysed using 1 mL DMSO. The cell lysis
product was centrifuged at 14,000 rpm (16,000  g) for 10 min to
remove cell debris. Fluorescence intensities of the cell lysates were
measured using a Fluorolog-3 (Jobin Yvon Horiba) spectrouorometer at kex = 496 nm, kem = 592 nm for DOX in order to determine
the uptake of IDNP and AIDNP by the cells. A DOX calibration curve
was previously created in a mixture of ICGDOX dissolved in

83

DMSO along with untreated cells. The protein content in the

lysates was measured using a BCA assay kit. The uptake data was
obtained by normalizing DOX content to the cellular protein level
for three different experiments (three wells each for each treatment per cell line). Paired t-test was used to compare 24 h DOX
cellular uptake between the treatments corresponding to the same
equivalent concentration.
2.7.4. Cytotoxicity and hyperthermia assessment
Cell viability was measured using Sulforhodamine B (SRB) assay
(Invitrogen, Carlsbad, CA, USA). This assay is used to measure cellular protein after drug treatment [23]. In this study, AIDNP and
IDNP (equivalent to 0.1, 1 and 10 lM DOX) were incubated with
MES-SA, Dx5 and SKOV-3 cells for 24 h to evaluate their toxicity.
The detailed procedure for toxicity assessment has already been
discussed in our previous report [24]. Average absorbance value
was measured from four wells corresponding to the same treatment and cell line. The average (S.D.) was determined from three
independent experiments and plotted against DOX concentration.
Cell growth fractions were determined by normalizing the data
corresponding to treatment to control.
The methods for delivering hyperthermia and measuring its
synergistic effect with DOX have been published previously [24].
ICG at a dose of 5 lM during 3 min exposure to a 808-nm laser
at 1440 J/cm2 (6.7 W/cm2) power density increases the temperature of incubated cells from a baseline of 3743 C for selective
hyperthermia. In this study, the cytotoxicity of four different treatments (IDNP w/o laser treatment, AIDNP w/o laser treatment, IDNP
w/laser treatment and AIDNP w/laser treatment) were investigated. Cells were seeded into a 96-well plate on the rst day, and
after overnight incubation they were exposed to different treatments (each corresponding to equivalent DOX concentration of
10 lM and ICG concentration of 6.2 lM).
Cytotoxicity was measured with the SRB assay 24 h post treatment in each case. Average cell growth fractions were obtained
from four wells for each treatment (n = 3). The data were acquired
and represented in the same manner as the toxicity of the formulations discussed in the previous paragraph.
2.8. Statistical signicance
Statistical signicance was identied by one-way ANOVA (SPSS,
Chicago, IL, USA) for the difference among treatment groups at the
same DOX concentration. A p-value <0.05 was considered to be
statistically signicant.
3. Results and discussion
3.1. Characterization of NPs
3.1.1. Size, size distribution, zeta potential, drug loading and
entrapment efciency
The dynamic light scattering histograms of non-targeted ICG
and DOX loaded PLGA nanoparticles (IDNP) and anti-HER-2 targeted ICG and DOX PLGA nanoparticles (AIDNP) are shown in
Fig. 1. Fig. 2 shows a SEM image of IDNP, demonstrating a uniform
distribution of particles with spherical shape. The size and size distribution, charge, drug loading and entrapment efciency of IDNP
and AIDNP are listed in Table 1. Our results show that IDNP exhibited a size of 167 5 nm with polydispersity of 0.06 0.03 in DLS
measurements. The average size of IDNPs in the SEM image is
slightly smaller. The observed difference between the two methods
is expected because DLS measures the hydrodynamic radius of the
particles, whereas SEM is performed on a dry sample. DLS shows
an increase in size and size distribution of IDNP after antibody

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drugs during the conjugation process, consistent with the literature [22,25,29]. Leaching during conjugation also explains the fact
that entrapment efciency was less in AIDNP than IDNP. The
entrapment efciency of ICG was found to be less than that of
DOX, as ICG is more hydrophilic than DOX.

Fig. 1. Dynamic light scattering histogram of IDNP and AIDNP.

Fig. 2. Scanning electron microscopy image of IDNPs.

conjugation (AIDNP). Similar results were observed in several

other studies [21,22,25]. While IDNPs and AIDNPs have the potential to passively accumulate in the tumor through the leaky vasculature (of the range of 100780 nm depending on the tumor type)
[26], active targeting achieved by AIDNP can further enhance drug
accumulation inside tumor cells through enhanced internalization
[25,27]. Anti-HER-2 has an isoelectric point (pI) around 8.5 and
hence exhibits positive charge at pH 7 [28]. The positively charged
antibody thus reduced the surface charge by masking the negative
charge of the IDNP. Drug loading efciency of IDNP is signicantly
reduced after antibody conjugation (p < 0.05) due to leaching of

3.1.2. In vitro release kinetics prole

The cumulative percentage of DOX released from AIDNPs is
shown in Fig. 3. Approximately 41 2% DOX is released in 8 h,
and reaches a sustained release of 48 2% in 48 h. This release
kinetic prole is similar to that of IDNP previously reported by
our group [20], and comparable to the 39% release after 8 h that
Zheng et al. reported for their ICG and DOX loaded lipid-PLGA
nanoparticles [30]. The slow sustained release of DOX from targeted AIDNP is consistent also with literature reports of targeted
DOX-loaded PLGA nanoparticles [29]. A biphasic drug release prole is exhibited by both AIDNP and IDNP, consistent with release of
DOX from PLGA reported in the literature [21,25,29]. The two
phases of drug release pertain to an initial burst release followed
by a sustained slow release of DOX. The burst release of drug is
the release of drug adsorbed onto nanoparticle surface during formulation. The sustained release of DOX, on the other hand, is due
to drug diffusion from the polymer and degradation of the nanoparticles. High molecular weight and hydrophobicity of PLGA
resulted in the slow release of DOX. Given that the Tg of PLGA is
higher than the endpoint temperature reached by 3 min exposure
of IDNP and AIDNP to an 808 nm laser (43 C), we do not expect
an increase in DOX release after exposing the PLGA NPs to laser. In
our recent publication, we reported the preparation of DOX-IR820
loaded poly(glycerol malate co-dodecanedioate) (PGMD NPs),
which are capable of triggered thermal release of DOX upon exposure to 808 nm laser. IR820 is a cyanine dye similar to ICG, which
can also generate heat upon exposure to laser, whereas PGMD is a
novel polymer with a Tg of 42.2 C [31].
High molecular weight PLGA comprises long chains of monomers inter- linked through acid- labile ester bonds. PLGA nanoparticles sequestered in acidic cellular compartments like
lysosomes undergo degradation to release monomers. Thus, polymers with higher molecular weight undergo slower degradation
in comparison to lower molecular weight polymers. Hydrophobicity of PLGA can be easily tuned by changing the ratio of lactic acid
to glycolic acid. High hydrophobicity of the polymer results in
higher drug-polymer interactions, thereby, reducing the drug
release rate. High hydrophobicity of the polymer also results in
lower water absorption and slower degradation and drug release
[32].

3.1.3. Antibody conjugation efciency

The antibody conjugation efciency of AIDNP was measured as
described in the methods Section 2.6. The results obtained showed
that AIDNP exhibited 9.0 0.7 lg antibodies per milligram of NPs.
These results are consistent with the literature [25,33].

Table 1
Mean size, polydispersity index (PDI), zeta potential, percent drug loading, entrapment efciency, and antibody conjugation efciency for IDNPs and AIDNPs (n = 3).
Formulation

Size
(nm)

Polydispersity
(PDI)

IDNP

167 5

0.06 0.03

AIDNP

210 7

0.02 0.01

Loading% (wt.
of drug/wt. of
nanoparticles)

Entrapment efciency%
(wt. of entrapped drug/
initial wt. of drug used)

Antibody conjugation efciency

(lg protein per mg nanoparticles)

11.3 1.6

1.79 0.02% ICG

2.30 0.01% DOX

45.2 1.4% ICG

70.3 3.4% DOX

N/A

1.0 0.5

1.39 0.01% ICG

1.90 0.02% DOX

32.6 2.3% ICG

61.5 2.8% DOX

9.0 0.7

Zeta
potential
(mV)

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85

3.2. Cell culture studies

Fig. 3. DOX release kinetic prole of AIDNPs.

3.2.1. Subcellular localization studies

Fluorescence microscopy images of the sub-cellular localization
and uptake of IDNP and AIDNP in SKOV-3, MES-SA and Dx5 cell
lines, respectively are shown in Figs. 46. The image exposure
times were kept constant for each drug in the respective cell line
for comparison purposes. IDNP and AIDNP corresponding to equivalent ICG and DOX concentrations described in the previous section were used.
AIDNP exhibited higher uorescence (of both DOX and ICG) in
comparison to their non-targeted counterpart IDNP in SKOV-3
cells. AIDNP and IDNP exhibited similar uptake in both MES-SA
and MES-SA/Dx5 cells. SKOV-3 cells were chosen as the positive
control cells which exhibited an over expression of HER-2 receptors (approximately 105 receptor/cell) [34]. Targeted therapy is
achieved using receptors that have high copy number on cancer
cells with respect to normal cells (105106 receptors/cancer cell
which is 100 fold higher than that in normal cell) [35]. High internalization rate of the receptor along with high specicity of the

Fig. 4. Subcellular localization of DOX and ICG in SKOV-3 cells; the exposure times for DOX and ICG channels were 500 ms and 2000 ms respectively. a. DOX uorescence of
IDNP, b ICG uorescence of IDNP, c merged picture of a and b, d. DOX uorescence of AIDNPs, e. ICG uorescence of AIDNPs and f. merged picture of d and e.

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Fig. 5. Subcellular localization of DOX and ICG in MES-SA cells; the exposure times for DOX and ICG channel were 500 ms and 2000 ms respectively. a. DOX uorescence of
IDNP, b. ICG uorescence of IDNP, c. merged picture of a and b, d. DOX uorescence of AIDNPs, e. ICG uorescence of AIDNPs and f. merged picture of d and e.

antibody to the receptor explains the higher uptake of AIDNP compared to IDNP in SKOV3 cells [36]. MES-SA and Dx5 cells, used as
negative control cells, do not over express the receptor.
IDNP and AIDNP exhibited similar intracellular localization in
all three cell lines, MES-SA, Dx5 and SKOV-3. PLGA nanoparticles
are internalized through endocytosis [19,25]. The intracellular
release of DOX from PLGA nanoparticles is due to hydrolysis of
ester bonds in the PLGA polymer and the increased solubility of
DOX in mildly acidic intercellular components such as lysosomes
[37]. DOX uorescence in the cytoplasm, therefore, might be associated with both the internalized drug-entrapped nanoparticles as
well as drug released from the nanoparticles. DOX uorescence in
the nucleus must be from drug released from the nanoparticles
because nanoparticles cannot enter the nuclear pores due to size
limitations [19].
3.2.2. Cellular uptake experiments
The 24 h uptake of IDNP and AIDNP in MES-SA, Dx5 and SKOV-3
cells was quantied by measuring the DOX uorescence in cells
normalized to cellular protein content. Fig. 7 shows that AIDNP sig-

nicantly enhanced DOX uptake in SKOV-3 cells in comparison to

IDNP. There is an approximate twofold increase in the uptake of
AIDNP in SKOV-3 in comparison to IDNP. This can be attributed
to the HER-2 receptor mediated endocytosis in SKOV3 cells. AIDNP
and IDNP exhibited similar uptake in the negative control cell lines
MES-SA and Dx5. These results are consistent with our previous
report and other researchers reports on targeted drug delivery system based on PLGA nanoparticles [22,25]. The zeta potential
results of IDNP showed that they are negatively charged
( 11 mV). Harush-Frenkel et al. reported that negatively charged
PLGA nanoparticles are taken up by cancer cells through adsorptive
endocytosis [38]. Receptor mediated endocytosis, however, is more
efcient and specic in comparison to adsorptive endocytosis [39].
The involvement of efcient receptor mediated endocytosis contributes to the higher uptake of AIDNP than IDNP in SKOV-3
(HER-2 positive cell line).
3.2.3. Cytotoxicity and effect of hyperthermia experiments
PLGA is a widely used polymer approved by the FDA for various
applications [12]. Our previous reports have suggested PLGA

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87

Fig. 6. Subcellular localization of DOX and ICG in Dx5 cells; the exposure times for DOX and ICG channels were 500 ms and 2000 ms respectively. a. DOX uorescence of IDNP,
b. ICG uorescence of IDNP, c. merged picture of a and b, d. DOX uorescence of AIDNPs, e. ICG uorescence of AIDNPs and f. merged picture of d and e.

Fig. 7. 24 h intracellular DOX uptake data in SKOV-3, MES-SA, and Dx5 cells, n = 3
experiments, 3 wells per treatment. P < 0.05 (by ANOVA) between free drug and
NPs formulation for each cell line, indicating signicant differences due to loading
of DOX into PLGA NPs.

Fig. 8. SKOV-3, MES-SA and Dx5 cell growth for different drug formulations
(without laser hyperthermia), n = 3 experiments, 4 wells per treatment.

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Fig. 9. Cytotoxicity of nanoparticles when excited by NIR laser. n = 3 experiments, 4

wells per treatment. The concentration of IDNP and AIDNP was around 0.25 mg/mL,
which contains 10 lM DOX and 6.2 lM ICG. P < 0.05 (by ANOVA) between lasertreated and non-treated cells of nanoparticles, indicating signicant cytotoxicity
difference due to hyperthermia.

nanoparticles do not exert toxic effects to the tested cancer cell

lines up to 1 mg/mL (which is much higher than the concentration
used for toxicity and uptake experiments) [40]. This is consistent
with the safety prole of PLGA nanoparticles reported by Liu and
Lin (1.02 0.37 mg/mL) [41].
A dose-dependent increase in cytotoxicity was observed in both
IDNP and AIDNP in all three cell lines (Fig. 8). DOX (10 lM) concentration was chosen due to its clinical relevance [42]. Our previous
reports have shown synergistic cancer cell killing using 10 lM DOX
and 5 lM ICG based chemo-laser phototherapy [24]. In the present
study, setting the DOX concentration to 10 lM corresponds to an
ICG concentration of 6.2 lM ICG in our nanoparticle formulation.
This concentration of ICG is non-toxic, and it provides a thermal
dose similar to prior reports [24,43] and within the practical constraints posed by the desire to optimize the concentration of both
agents. AIDNP (without laser treatment) enhanced the cytotoxic
effect in SKOV-3 cells over IDNP due to enhanced uptake by receptor mediated endocytosis. However, the toxic effects of the AIDNP
did not reach statistical signicance over their non-targeted
counterparts.
These results are consistent with our previous report of comparable toxicity of antibody conjugated DOX loaded PLGA nanoparticles (ADNP) and DOX loaded PLGA nanoparticles (DNP) [25].
Chittasupho et al. also reported similar results, wherein c-LABL
peptide conjugated DOX loaded nanoparticles exhibited comparable toxicity to the non-conjugated nanoparticles [29]. This comparable toxicity observed between AIDNP and IDNP (without laser
treatment) can be due to the aggressive nature of SKOV-3 cell lines
[44]. SKOV-3 cells lack the ability to activate caspase-3. This along
with the presence of defective apoptotic protease factor-1 makes
these cells resistant to DOX [45]. Therefore, enhanced uptake of
DOX through AIDNP (in the absence of laser treatment) does not
signicantly enhance their toxicity in SKOV-3 cells. SKOV-3 cells
have also been reported to be inherently thermotolerant, which
could be attributed to their p53 mediated drug resistance
[46,47]. Our previous work suggested that combined laser hyperthermia and chemotherapy delivered through PLGA nanoparticles
(IDNP) has the ability to signicantly reduce the cell viability of
such aggressive cell line [19]. Additionally, AIDNP signicantly

enhanced toxicity over IDNP in SKOV-3 upon laser exposure. AIDNP upon laser exposure enhanced the toxicity three times in comparison to the same treatment with IDNP in SKOV-3 cells (Fig. 9).
The negative controls (MES-SA and Dx5) exhibited comparable
cytotoxicity after exposure to laser. Thus, targeted PLGA nanoparticles with simultaneously entrapped ICG and DOX could selectively enhance cell killing in SKOV-3 cells and hold promise for
killing such aggressive cells overexpressing the receptor.
IDNP and AIDNP exhibited similar toxicity in the negative control cells; MES-SA and Dx5. These results are consistent with our
expectations as these cells do not overexpress HER-2 receptors. It
was observed that 3 min laser hyperthermia exposure to IDNP
and AIDNP could not provide an advantage over the free drug
forms (ICG and DOX) in both MES-SA and Dx5 cells. MES-SA cells
are DOX sensitive and also show inherent sensitivity to environmental stressors [4749]. Thus, IDNP or AIDNP after laser treatment could not provide a competitive cell killing advantage over
their free drug forms in MES-SA and Dx5 cells.
The results obtained in Dx5 cells are consistent with our previous report where IDNP with 3 min laser exposure could not
enhance cell killing. Extended laser exposure of IDNP to 5 min,
however, could signicantly increase toxicity to Dx5 cells [19].
Zheng et al. also conducted a study by incorporating ICG and
DOX into lipid-PLGA (IDLPNP) nanoparticles to test their effect
on the drug sensitive and drug resistant cancer cells lines MCF-7
and MCF-7/Adr. The latter cell line receives its name because it is
resistant to Adriamycin (DOX). The authors reported that combined photo-chemotherapy through IDLPNP (with equivalent drug
concentrations of 5.5 lg/mL ICG, 6.0 lg/mL DOX) upon exposure to
NIR laser with irradiance of 1.6 W/cm2 irradiance for 5 min
resulted in only 5% cell growth inhibition of the MCF-7/Adr cells
72 h after laser exposure [30].
Multi-drug resistance in cancer cells is exhibited through over
expression of Multi-drug resistance proteins (MRP1). The level of
expression of these proteins (or transcripts for expression of this
protein; i.e., mRNA) can be correlated to their degree of drug resistance [50]. MCF-7/Adr cells are an adriamycin resistant variant of
MCF-7 cells exhibiting 6000 multi-resistant protein 1(MRP1)
m-RNA copies. Dx5 (the DOX resistant variant of MES-SA cells)
exhibited four times more MRP1 mRNA copies than MCF-7/Adr
cells [51]. Dx5 cells are more resistant to DOX than are MCF-7/Adr
cells due to higher expression levels of MRP1. Therefore, Dx5 cells
are expected to be more resistant to simultaneous exposure to ICG
and DOX loaded nanoparticles and NIR laser than are MCF-7/Adr
cells.
Kampinga reported that combinatorial heat and drug administration causes damage to the multi-drug resistant proteins;
thereby sensitizing the effect of drugs [52]. Protein denaturation
due to hyperthermia treatment is highly dependent on the thermal
dose provided. At least 5% protein denaturation is required to bring
about detectable cell toxicity [53]. The above reason suggests the
requirement of a high thermal dose to bring about appreciable protein denaturation and hence higher cell killing. Franke et al. [54]
reported additional mechanisms of cytotoxicity through combined
chemotherapeutic drug and heat administration in multi-drug
resistant cell lines. They reported a reduced expression of MRP1
in cell membranes of multidrug resistant cells upon combinatorial
drug and heat administration. They demonstrated that hyperthermia causes altered translocation of MRP1 to membrane through
increased ROS production at the protein level [54]. Our group also
found Pgp inhibition in multi-drug resistant cells after application
of rapid rate laser-mediated ICG hyperthermia in comparison to
incubator hyperthermia [43]. Therefore, there can be a complex
interplay of different mechanisms which mediate toxicity during
photo-chemotherapy treatment in multi-drug resistant cell lines.

S. Srinivasan et al. / Journal of Photochemistry and Photobiology B: Biology 136 (2014) 8190

All the mechanisms, however, depend on the thermal dose provided during combined chemo-phototherapy.
4. Conclusions
AIDNP can specically target cancer cells (SKOV-3) overexpressing the receptor corresponding to the conjugated ligand
(anti- HER-2 monoclonal antibody). AIDNPs also show great promise for combinatorial treatment employing both chemotherapy and
hyperthermia. This combination hence serves as a potential candidate for targeted hyperthermia and chemotherapy treatment
in vivo.
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