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Doxorubicin-loaded gold nanorods: a multifunctional chemo-photothermal nanoplatform for cancer management

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Two of the limitations associated with cancer treatment are the low efficacy and the high dose-related side effects of anticancer drugs. The purpose of the current study was to fabricate biocompatible multifunctional drug-loaded nanoscale moieties for co-therapy (chemo-photothermal therapy) with maximum efficacy and minimum side effects. Herein, we report in vitro anticancerous effects of doxorubicin (DOX) loaded on gold nanorods coated with the polyelectrolyte poly(sodium-4-styrenesulfonate) (PSS-GNRs) with and without NIR laser (808 nm, power density = 1.5 W/cm ² for 2 min) irradiation. The drug-loading capacity of PSS-GNRs was about 76% with a drug loading content of 3.2 mg DOX/mL. The cumulative DOX release significantly increased after laser exposure compared to non-irradiated samples ( p < 0.05). The zeta potential values of GNRs, PSS-GNRs and DOX-PSS-GNRs were measured as 42 ± 0.1 mV, −40 ± 0.3 mV and 39.3 ± 0.6 mV, respectively. PSS-GNRs nanocomplexes were found to be biocompatible and showed higher photothermal stability. The DOX-conjugated nanocomplexes with NIR laser irradiation appear more efficient in cell inhibition (93%) than those without laser exposure (65%) and doxorubicin alone (84%). The IC 50 values of PSS-GNRs-DOX and PSS-GNRs-DOX were measured as 7.99 and 3.12 µg/mL, respectively, with laser irradiation. Thus, a combinatorial approach based on chemotherapy and photothermal strategies appears to be a promising platform in cancer management.
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Doxorubicin-loaded gold nanorods: a multifunctional
chemo-photothermal nanoplatform for cancer management
Uzma Azeem Awan1,2, Abida Raza*2,§, Shaukat Ali3, Rida Fatima Saeed1
and Nosheen Akhtar1
Full Research Paper Open Access
Address:
1Department of Biological Sciences, National University of Medical
Sciences (NUMS), Rawalpindi, Pakistan, 2NILOP Nanomedicine
Research Laboratories, National Institute of Lasers and Optronics
College, (PIEAS), Islamabad, Pakistan and 3Medical Toxicology Lab,
Department of Zoology, Government College University Lahore,
Lahore-54000, Pakistan
Email:
Abida Raza* - abida_rao@yahoo.com
* Corresponding author
§ Tel: +92519248671-6 ext 3103, 3177; Fax: +92 51 2208051
Keywords:
chemotherapy; doxorubicin; gold nanorods; NIR laser; photothermal
therapy
Beilstein J. Nanotechnol. 2021, 12, 295–303.
https://doi.org/10.3762/bjnano.12.24
Received: 11 December 2020
Accepted: 10 March 2021
Published: 31 March 2021
Associate Editor: A. Salvati
© 2021 Awan et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Two of the limitations associated with cancer treatment are the low efficacy and the high dose-related side effects of anticancer
drugs. The purpose of the current study was to fabricate biocompatible multifunctional drug-loaded nanoscale moieties for
co-therapy (chemo-photothermal therapy) with maximum efficacy and minimum side effects. Herein, we report in vitro anti-
cancerous effects of doxorubicin (DOX) loaded on gold nanorods coated with the polyelectrolyte poly(sodium-4-styrenesulfonate)
(PSS-GNRs) with and without NIR laser (808 nm, power density = 1.5 W/cm2 for 2 min) irradiation. The drug-loading capacity of
PSS-GNRs was about 76% with a drug loading content of 3.2 mg DOX/mL. The cumulative DOX release significantly increased
after laser exposure compared to non-irradiated samples (p < 0.05). The zeta potential values of GNRs, PSS-GNRs and DOX-PSS-
GNRs were measured as 42 ± 0.1 mV, 40 ± 0.3 mV and 39.3 ± 0.6 mV, respectively. PSS-GNRs nanocomplexes were found to be
biocompatible and showed higher photothermal stability. The DOX-conjugated nanocomplexes with NIR laser irradiation appear
more efficient in cell inhibition (93%) than those without laser exposure (65%) and doxorubicin alone (84%). The IC50 values of
PSS-GNRs-DOX and PSS-GNRs-DOX were measured as 7.99 and 3.12 µg/mL, respectively, with laser irradiation. Thus, a combi-
natorial approach based on chemotherapy and photothermal strategies appears to be a promising platform in cancer management.
295
Introduction
Despite the enormous advances in medical research, cancer is
still the second most common cause of death worldwide from
which 9.6 million people died in 2018 [1]. Hepatocellular carci-
noma (HCC) is one of the major types of liver cancer with high
incidence of mortality [2]. Currently, there are a number of
treatment modalities, including chemotherapy, immunotherapy,
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296
targeted therapy, irradiation, and surgery [3]. Among these,
chemotherapy is the most commonly used method as most of
the HCC patients are diagnosed at advanced stages and are not
good candidates for liver transplantation or surgical resection
[4,5]. However, the use of conventional chemotherapeutic
agents in cancer treatment is limited due to several unwanted
characteristics of poor solubility, broad bioavailability range,
narrow therapeutic index, rapid elimination from systemic
circulation, unselective site of action after oral/intravenous
administration, and cytotoxic effects on normal tissues [6]. The
anticancer drug doxorubicin (DOX) is extensively used in the
management of different tumors [7] and exerts antitumor activi-
ty by interaction with DNA replication [8]. DOX-based chemo-
therapy is one of the main treatments for HCC but its efficacy is
limited by pre-existing and acquired drug resistance due to
long-term chemotherapy [9]. Also, high-dose regimens of DOX
are associated with sever cardiotoxicity and bone marrow
suppression. Different strategies were used to encapsulate the
drug to minimize its side effects; however, this decreased the
chemotherapeutic effectiveness [10]. Henceforth, new treat-
ment modalities are urgently needed to kill cancerous cells
without damaging normal cells or tissues. One approach is to
selectively remove cancer cells using the advanced drug
delivery systems. These carrier systems hold sufficient amounts
of the drug with prolonged circulation time and sustained drug
release at the tumor site [11].
Nanotechnology provides a means to overcome these hurdles as
nanocarriers, which improve the pharmacological properties of
free drugs, contribute to enhanced therapeutic efficacy in physi-
ological environment [12]. Nanocarriers as multifunctional
tumor targeting and therapeutic agents exhibit properties such
as significant absorption or scattering in the visible and near-in-
frared (NIR) regions, tunable aspect ratio, biocompatibility,
fluorescence properties, and the ease of biofunctionalization,
which makes them ideal in biomedical applications [13]. Gold-
based nanomaterials (i.e., nanospheres, nanorods, nanoshells,
and nanocages) have great potential in photothermal cancer
therapy due to plasmonic properties and the ease of biofunction-
alization. Gold nanorods (GNRs) are more preferable than other
gold nanomaterials because of their photothermal conversion
efficiency. Better nanotherapeutics can be obtained by utilizing
external stimuli, such as pH value, light, or ultrasound, to
deliver the anti-cancerous drug into tumor tissue with spatial
and temporal control [14]. Photothermal therapy (PTT) is an
emerging minimally invasive cancer therapy. It can efficiently
induce cytotoxicity by conversion of absorbed NIR light to heat.
In cancer intervention, NIR-mediated photothermal therapy is
gaining more attention due to the deep tissue penetration with
minimal absorbance by healthy tissues [15,16]. Gold nanorods
are potential delivery carriers for sustained drug release in
response to an external stimulus [13]. Additionally, the NIR
light-induced heat can improve the sensitivity of cancer cells
towards chemotherapeutic agents by increasing blood vessel
dilation and membrane permeability. These findings provide an
incentive to combine photothermal therapy and chemotherapy
for cancer treatments.
Regardless of the various beneficial properties, GNRs have lim-
itations in clinical applications due to the cytotoxicity of the
surfactant cetyltrimethylammonium bromide (CTAB), which
acts as a template in the synthesis process of GNRs [17]. Differ-
ent polymers can be used to coat GNRs to enhance their bio-
compatibility and dispersion at physiological pH values. The
positive CTAB layer on the GNR surface facilitates electro-
static adsorption of anionic compounds, such as poly(sodium
4-styrenesulfonate) (PSS), which ultimately facilitates electro-
static interaction with cationic anticancerous drugs, such as
DOX [18]. Advanced synergistic therapies, such as the combi-
nation of chemotherapy and photothermal therapy, have been
applied to enhance the overall therapeutic efficacy [19]. This
includes magnetic cores capped with gold nanorods, silica
nanorattle gold shells, and DNA-based platforms loaded with
GNRs and DOX [20-22].
Venkatesan et al. developed a DOX-loaded PSS-coated GNR
nanoplatform via electrostatic interaction that selectively
delivered DOX to target cells and effectively inhibited tumor
growth in MCF-7 cells [18]. The killing effect of the
DOX@PSSAuNRs was more pronounced at low concentra-
tions (0.5–2 µg/mL) and higher cytotoxicity compared to free
DOX was observed. However, no significant difference was re-
ported at a higher concentration of 5 µg/mL. 68.5% and 62.4%
of cells was killed by the DOX@PSS-Au NR conjugate and
free DOX, respectively. To achieve significant cytotoxicity with
the nanocomplex compared to free DOX, herein, we have used
the same strategy as described in an earlier report [18] to design
a multifunctional PSS-coated GNRs-based nano-platform that
facilitate chemotherapy by delivering anticancerous drug at the
site of action. DOX release with precise temporal and spatial
control is triggered under local hyperthermic conditions in-
duced by NIR laser irradiation. Heat from the GNR surface not
only promotes drug delivery into the tumor, but also increases
the drug toxicity to tumor cells by the hyperthermic effect. A
significantly higher cell death rate was achieved in the tumor
cells treated with chemo and photothermal co-therapy com-
pared to the free drug. One of the major limitations associated
with photothermal therapy is the usage of high laser powers for
long time durations. We used a NIR laser power density of
1.5 W/cm2 for 2 min, which are a lower power density and a
shorter irradiation time, respectively, compared to many previ-
ously reported studies. Liao et al. reported cell death at higher
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297
Figure 1: (a) TEM image of monodispersed GNRs. (b) Histogram showing the aspect ratio of GNRs. (c) UV–vis absorption spectrum of bare GNRs,
PSS-coated GNRs and DOX-loaded PSS-GNRs. (d) Zeta potential of CTAB-coated GNRs, PSS-GNRs and DOX-PSS-GNRs.
laser power (2.5 W/cm2) with longer exposure times [23]. Al-
though, Chen and colleagues have reported cell death at a low
laser power density of 1.8 W/cm2 but they used a high expo-
sure time and a comparatively high drug concentration
(20 µg/mL) [24]. We observed significant cell death at a lower
laser power density using a shorter exposure time and a lower
drug concentration. This will minimize thermotoxicity associat-
ed with laser exposure.
Results and Discussion
Synthesis of the DOX-loaded GNR
nanocomplex
In the present study, DOX-conjugated GNRs and hyperthermia
were employed as a treatment strategy for HCC cells. First,
GNRs were synthesized according to the well-known seed-
mediated growth method [25] with a slight modification re-
ported in our previous work [26]. The uniform GNRs were syn-
thesized with an aspect ratio of 4.3 (26 ± 2 nm in length and
6 ± 3 nm in width), by keeping pH value (pH 3) and tempera-
ture (T = 28 °C) constant.
The prepared GNR suspension has a surplus of cytotoxic
CTAB, which was removed by repetitive cycles of centrifuga-
tion and re-dispersion. A CTAB bilayer remained non-
covalently bound onto the GNRs surface to maintain the
stability of the final product. The longitudinal localized
plasmon resonance (LSPR) and the transverse plasmon reso-
nance (TSPR) of the prepared GNRs were found to be 780 and
526 nm, respectively. TEM images display mono-dispersed
rods with an aspect ratio of 4.2 (Figure 1a,b). GNRs could be
potential candidates for photothermal therapy because their
LSPR absorption band lies in the NIR region in which trans-
mitted light caused no obvious damage to healthy tissues.
Biocompatible GNRs were prepared through coating their sur-
face with PSS. The LSPR peak of the PSS-coated GNRs was
slightly redshifted to 783 nm (Figure 1c). The shift of the LSPR
peak after PSS coating is due to the side-by-side assembly of
the PSS-GNRs [27]. The surface charge of the GNRs changed
from strongly positive (+42 mV, due to CTAB presence) to
negative after PSS modification, which also confirmed the suc-
cessful surface modification as described in previous reports
[28].
Absorption spectra confirmed the successful loading of DOX on
the PSS-coated GNRs (Figure 1c). The polyelectrolyte coating
allowed the GNRs to easily interact with the surrounding envi-
ronment. Consequently, the LSPR wavelength of the GNRs
perceptively responded to the refractive index change caused by
molecular adsorption. The conjugation of DOX onto the sur-
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298
Figure 3: In vitro DOX release profile from PSS-GNRs. (a) NIR-triggered DOX release at different pH values. Fluorescence intensity was measured
from 0 to 30 h. (b) Heating curves of water, PSS GNRs and PSS-GNRs-DOX (10 µg Au/mL) under NIR (808 nm) laser irradiation.
face of the PSS-GNRs resulted in a redshift of the LSPR band,
while the TSPR peak did not shift. The increased local refrac-
tive index around GNRs due to adsorption of DOX might lead
to a stronger Columbic restoring force and a redshift of the
LSPR peak [29]. The zeta potential of unrefined GNRs was
measured to be 60 ± 0.2 mV, which decreased to 42 ± 0.1 mV
after removal of excess CTAB (two rounds of centrifugation
and re-dispersion). A negative zeta potential of 30 ± 0.3 mV
was measured after successful coating of the GNR surfaces with
PSS. The positive zeta potential (40.3 ± 0.6 mV) of DOX-PSS-
GNRs, due to the positive charge of DOX, confirmed the chem-
istry changes to the GNR surfaces (Figure 1d). Our results
revealed a successful conjugation of DOX on the surface of
PSS-GNRs with a higher stability in aqueous media than in
other studies [18,30]. The percentage yield of the DOX-PSS-
GNRs was found to be 81.2 ± 0.21 wt %.
Drug loading efficiency
The loading efficacy of DOX on the PSS-GNRs was measured
systematically using a standard curve of absorption of DOX (at
490 nm) by changing the concentration of DOX while keeping
the concentration of PSS-GNRs constant (40 µg/mL). The drug
loading capacity of PSS-GNRs was about 76% with a drug
loading content of 3.2 µg DOX/mL of GNRs.
Photothermal stability of PSS-GNRs
Optical characterization of PSS-GNRs showed that the LSPR
peak of GNRs strongly depends on their aspect ratio. Hence, the
LSPR peak position is an excellent indicator for any shape
changes of GNRs. An aqueous solution of PSS-GNRs after
laser exposure for 2 min (power density = 1.5 W/cm2) remained
stable. The LSPR peak shifted by approximately 4 nm
(Figure 2). The stability of PSS-GNRs after NIR laser exposure
was sufficient for photothermal therapy.
Figure 2: PSS-GNRs before and after 808 nm laser exposure, the
LSPR peak shifted about 4 nm.
In vitro DOX release after NIR irradiation
Drug release from PSS-GNRs can be easily controlled with NIR
laser irradiation. The cumulative DOX release almost doubled
after laser exposure (1.5 W/cm2) compared to non-irradiated
samples (Figure 3). Enhanced drug release stimulated by laser
(808 nm) may be related to the heat generated by the nanomate-
rial. Almost 40% of DOX was released at pH 5 from the laser-
irradiated sample, compared to 22% from the non-irradiated
sample at the same pH after 5 h (Figure 3). DOX release was
reduced during the subsequent hour of incubation. The data
showed that 50% of conjugated DOX was released from PSS-
GNRs over a period of 30 h at pH 5. The microenvironment of
the tumor cells could facilitate enhanced drug release due to the
acidic pH value (approximately pH 5) of intracellular lyso-
somes and extracellular tissues of tumors [31].
In order to prove the photothermal conversion ability of the
nanorods PSS-GNRS and PSS-GNRs-DOX (10 µg Au/mL)
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Figure 4: (a) Relative viabilities of HepG2 and 3T3 cells after being incubated with different concentrations of PSS-GNRs for 24 h. (b) A hemotoxicity
assay on PSS-GNRs shows that PSS-GNRs are hemo-compatible. No significant difference was seen in the range of 5–500 µg/mL (<20%
hemolysis). A low significant (***) of a high significant difference (****) were seen for 1000 and 2000 µg/mL compared with 100 µg/mL. Each bar shows
the mean value ± SEM of triplicates.
were exposed to NIR laser irradiation (808 nm) at a power den-
sity of 1.5 W/cm2 for 2 min. There was an increase in tempera-
ture to 52 °C and 45 °C, respectively. In contrast, no significant
change in temperature was observed when water was exposed
to the same laser irradiation (Figure 3b). This confirmed the
light–heat transformation through the GNRs. This hyper-
thermic effect mediated by GNRs may be responsible for the
laser-triggered release of DOX. These findings are consistent
with previous studies [23].
PSS-GNRs nanocomplex biocompatibility
Dose-dependent biocompatibility and cytotoxicity efficiency of
the nanocarriers were measured in vitro. The efficiency of the
GNRs in mediating cytotoxicity against HepG2 (carcinogenic)
and 3T3 (non-carcinogenic) cells was evaluated. Cells were
treated for 12 h with PSS-GNRs and analyzed using the MTT
assay. As shown in Figure 4, cells treated with PSS-GNRs had
no significant reduction in cell viability compared to control
cells. The viability remained higher than 88% at concentrations
of 500 µg/mL for HepG2 cells and 1000 µg/mL for 3T3 cells
(Figure 4a). If nanoparticles interact with red blood cells
(RBCs) in the blood stream they can cause hemolysis. Hemo-
lytic properties and interaction with RBCs are the main parame-
ters for the biocompatibility of nanocarriers [23]. Analysis of
hemoglobin released from RBCs after incubation in a suspen-
sion of PSS-GNRs showed less than 20% hemolysis at a con-
centration of 1000 μg/mL (Figure 4b). The experiments
revealed a good biocompatibility of PSS-GNRs, which was
quantified by the concentration of hemoglobin in the super-
natant of GNPs-RBCs mixture by monitoring absorbance inten-
sity at 570 nm. The absence of a marked hemotoxicity of this
sample is mainly related to the presence of the polymer. The
GNR surface had no direct contact with the RBCs because it
was completely passivated by the PSS coating [23].
Cell inhibition after NIR exposure of PSS-GNR-DOX
complexes
Drug release from PSS-GNR-DOX triggered by NIR laser irra-
diation (808 nm) at an output power density of 1.5 W/cm2 with
a beam spot size of 6 mm in diameter on HepG2 cells was
studied. DOX release from PSS-GNR-DOX was increased sig-
nificantly (p < 0.05) after 2 min of NIR irradiation (Figure 5).
HepG2 cells were treated with free DOX and DOX-PSS-GNRs,
either irradiated with NIR laser or not exposed to NIR light. A
dose-dependent cytotoxicity was observed in all study groups.
About 84% of cells were killed by free DOX and 65% by DOX-
PSS-GNRs at an equivalent DOX concentration of 10 μg/mL
(Figure 5). This showed that free DOX was more toxic than
DOX conjugated to a nanocarrier at the same drug concentra-
tion. Similar findings were reported by other studies [32,33].
The high cytotoxic effect of free DOX is due to the higher
availability of the drug to the cells after cell uptake. The
decreased cytotoxicity of DOX-PSS-GNRs is because of a
delayed drug release inside cells [23]. The PSS-GNRs
nanocomplex shows potential as biocompatible nanocarrier for
drug loading and delivery in cancer therapy.
Arunkumar et al. have reported that DOX-conjugated gold
nanorods are highly biocompatible vehicles for sustained drug
delivery, reduce cardiotoxicity in vivo, and have high
photothermal efficacy [34]. A previous report showed that
DOX-loaded tiopronin-coated gold nanoparticles (Au-TIOP-
DOX) had a better efficacy in killing cancer cells than free
DOX [35]. Similarly, a study showed an improved toxicity of
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Figure 5: (a) Percentage viabilities of HepG2 cells treated with free DOX and DOX-PSS-GNRs exposed to NIR light (1.5 W/cm2 for 2 min per treat-
ment, three treatments over 2 h). The cytotoxicity values with and without laser irradiation are significantly different with p < 0.05 in the case of DOX-
PSS-GNRs according to a two-sample student t-test. (b) Percentage viabilities of HepG2 cells treated with NIR light (1.5 W/cm2) at different points in
time.
DOX-loaded DNA-wrapped gold nanoparticles in drug-resis-
tant cancer cells [36]. Our results are opposite to this study, it
might be due to the higher sensitivity of HepG2 cells to DOX,
which could induce more toxicity of free drug compared to
conjugated one. The cytotoxic efficiency of the DOX-loaded
PAA-PEG-GNRs was found to be similar to free DOX and im-
proved with an increase in their concentrations [36]. In a
previous study, cell viability was significantly decreased down
to 57% using GNR-DOX-cRGD, whereas free DOX demon-
strated the highest level of cytotoxicity (41% of control) in
U87MG cells [35]. We found that DOX-PSS-GNRs complexes
killed more cancer cells (93%) after NIR laser irradiation
(Figure 5). The higher cytotoxicity of the complex is due to the
enhanced drug release upon NIR laser irradiation. The IC50
value of PSS-GNRs-DOX was 7.99 ± 0.0032 µg/mL. For PSS-
GNRs-DOX with laser irradiation it was 3.12 ± 0.0906 µg/mL.
The IC50 values of free DOX and DOX with laser exposure
were 3.999 ± 0.04211 and 4.41 ± 0.0037 µg/mL, respectively.
Previously, Au-HNS-EGFR-DOX were reported to have a sig-
nificant antiproliferative activity against lung cancer cells when
irradiated with NIR laser (125 mW/cm2, 25 s), in contrast to
non-irradiated cells [37]. Free DOX showed no significant in-
fluence on viability, neither with nor without laser irradiation.
This indicates that increased cell death upon NIR laser irradia-
tion might be attributed to the presence of the gold nanocarrier.
Without laser treatment low drug release from the nanocom-
plex was observed. Laser-triggered DOX release was measured
using the same laser treatment at different time intervals (2, 3,
and 4 h) in which drug release was improved in a time-depend-
ent manner. Less than 10% of DOX was released within 4 h
from PSS-GNR-DOX without NIR irradiation under the same
experimental conditions (Figure 5). Drug release from the
nanocomplex (PSS-GNR-DOX) might be easily turned “on”
and “off” by NIR laser exposure. The NIR laser irradiation
causes a melting of PSS that would lead to decreased stability
and an enhanced drug diffusion coefficient. No drastic change
in temperature of the solution was observed after NIR irradia-
tion. Hong et al. developed a system to estimate the
photothermal conversion efficacy of GNRs for different irradia-
tion laser powers and reported that exposure with 40 W/cm2 for
30 min generated heat on PEGylated GNRs necessary for
photothermal ablation of MDA-MB-231 [38]. To minimize the
thermotoxicity associated with laser exposure, in the current
study, we used a low laser power density of 1.5 W/cm2, a
shorter time of NIR irradiation (2 min), and a DOX concentra-
tion of 10 µg/mL. Under these conditions, we observed signifi-
cant cell death (93%). Contrary to this, about 73% cell death at
a higher laser power (2.5 W/cm2) with a longer exposure time
of 5 min is reported by Liao et al. [23]. Similarly, in other study
74% cell death was reported using a low laser power density of
1.8 W/cm2 but with long exposure time and high drug concen-
tration (20 µg/mL) [24]. Thus, we achieved a higher cell death
rate at shorter exposure time and lower drug concentration.
Conclusion
Multifunctional, biocompatible, and thermostable PSS-GNRs
could be easily prepared by simple wet chemistry. A polymer
was electrostatically conjugated, which facilitates the loading of
DOX and its phototriggered release inside cancer cells in acidic
environment. A comparatively good photothermal transfer
ability has been achieved at a very low power density of
1.5 W/cm2 of NIR laser irradiation, as evidenced by the rapid
temperature increase on the nanocarrier surface under 808 nm
laser exposure for 2 min. A high cytotoxicity was observed with
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DOX-PSS-GNRs after NIR laser irradiation, in contrast to DOX
alone. The GNRs could proficiently produce hyperthermia by
converting NIR light into heat and kill heat-sensitive cancer
cells with minimal side effects on the surrounding healthy cells
due to the low power density of the laser and the shorter time of
exposure. At the same time, the DOX release stimulated by the
temperature rise could inhibit the proliferation of residual
cancer cells.
The nanomaterial complex described here will have the capacity
for cost-effective upscaling due to ease of synthesis and surface
modification, and the tunable drug loading ability. Chemo-
photothermal treatment based on nanocomplex systems is an
efficient approach for reducing the high dose-related side
effects in cancer management.
Experimental
Materials
CTAB (99.9%), hydrogen tetrachloroaurate(III) trihydrate
(HAuCl4·3H2O 99%), ʟ-ascorbic acid (C6H8O6, 99%), sodium
borohydride (NaBH4, 98%), silver nitrate (AgNO3, 99%),
doxorubicin, (98%) poly(sodium 4-styrenesulfonate) (PSS;
Mw = 70,000) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) were purchased from Sigma-Aldrich.
Deionized (DI) water, having a resistance of 18 MΩ·cm, was
used throughout the experiments.
Gold nanorod synthesis
GNRs were synthesized through seed-mediated growth [25]
with a slight modification [26]. Gold seed particles were synthe-
sized by adding 250 μL of 10 mM HAuCl4·3H2O to 10 mL of
0.1 M CTAB under continuous stirring. 600 μL of freshly pre-
pared, ice-cold NaBH4 solution (10 mM) was added followed
by 30 min of continuous stirring. For the GNR growth solution,
50 mL of 0.1 M CTAB was added to 2.5 mL of 10 mM
HAuCl4·3H2O. To the stirring solution 400 μL HCl (1 M),
500 µL AgNO3 (10 mM), and 400 μL ʟ-ascorbic acid (10 mM)
was added. Finally, 200 μL of seed solution was added to the
growth solution. GNRs were purified by centrifugation
(14,000g for 20 min) after 24 h of incubation. Then, the
collected pellet was re-dispersed in deionized water.
PSS coating of GNRs
A reported method by Venkatesan et al. was used, with a slight
modification, for the PSS coating of GNRs [18]. Prepared
GNRs (2 mL, 40 µg/mL) were centrifuged at 12,000g for
10 min and the pellet was re-dispersed in 2 mL of deionized
water. GNR solution was added drop-wise to 2 mL of PSS
(2 mg/mL in 8 mM NaCl). For maximum adsorption, the
solution was kept under stirring at room temperature for
2 h. Excess polymer (supernatant fraction) was removed by
centrifugation (12,000g for 10 min). The PSS-stabilized
GNRs were re-suspended in 2 mL deionized water and stored at
4 °C.
Doxorubicin-loaded PSS-GNRs
The anticancer drug DOX was loaded onto the surface of PSS-
GNRs by a previously reported simple stirring method with
slight modifications [30]. PSS-GNRs (40 µg/mL, 2 mL) were
added to an aqueous solution of DOX at a final concentration of
10 µg/mL and were stirred overnight in the dark at room tem-
perature. Excess drug was removed by centrifugation at 12,000g
for 10 min and the pellet was re-dispersed in 2 mL deionized
water. UV–vis spectra of DOX-loaded GNRs were scanned at a
wavelength range of 400–1100 nm. The surface charge distribu-
tion of DOX-loaded PSS-GNRs, at a different level, was deter-
mined by using a zeta potential analyzer (Zetasizer Nano ZS90
DLS system Malvern Instruments Ltd., England).
Percentage yield
The nanoparticles were collected and weighed accurately. The
percentage (%) yield was then calculated using the formula
given below [39]:
Drug loading efficiency (DLE)
In order to calculate the drug loading efficiency, a known quan-
tity of DOX was mixed with an aqueous PSS-GNRs solution
(40 mg/mL) to get final drug concentrations of 5, 10, 15, 20, 25,
50, 100, 200, and 300 mg/mL. Then the suspension was stirred
overnight in the dark at 20 °C. The suspension was then
centrifuged at 12,000g for 10 min in order to precipitate the
DOX-PSS-GNRs nanoconjugate and then dialyzed against pure
water to remove unbound DOX by a previously described
method [30]. The quantity of loaded DOX was measured at
485 nm. Drug loading efficiency (DLE) was calculated using
the formula given below:
Photothermal stability of PSS-GNRs
The photothermal stability of PSS-GNRs was measured using a
previously described method [23]. Briefly, the aqueous solution
of PSS-GNRs was irradiated with NIR laser (power density =
1.5 W/cm2) for 2 min and analyzed by UV–vis spectroscopy.
In vitro drug release by NIR exposure
NIR-triggered drug release from PSS-GNRs was measured in
10 mM phosphate-buffered saline (PBS, pH 5.6 at 37 °C). A
Beilstein J. Nanotechnol. 2021, 12, 295–303.
302
continuous-wave 808 nm NIR laser (Ti-Sapphire, Spectra
Physics CA 95054, USA) was used. DOX-PSS-GNRs
(40 µg/mL, 2 mL) were dispersed in 10 mL of PBS followed
by NIR laser irradiation at an output power of 1.5 W/cm2 for
2 min and 800 μL of the solution was taken out for analysis.
Exposed media was centrifuged at 12,000g for 10 min. The
amount of DOX released from PSS-GNRs in the supernatant
was determined by fluorescence measurements (Biotek synergy
H4 multi-mode plate reader) following the method reported in
[23].
In vitro cytotoxicity assays
The in vitro cytotoxicity of PSS-GNRs was measured using
3T3 and HepG2 cells. Cells were seeded in 96-well plates
(4 × 103 cells per well) in 100 μL DMEM supplemented with
10% FBS and 1% pen–strep. After 24 h of incubation, cells
were exposed to different concentrations of PSS-coated GNRs
and were allowed to incubate at 37 °C for additional 24 h.
Viability was measured by the MTT assay [40].
Hemolysis assay
All human blood samples in this study were from healthy
volunteers and used with Institutional Review Board (IRB)
bioethics approval. The hemolysis assay was carried out accord-
ing to the protocol from National Cancer Institute (NCI). Whole
blood (5 mL) from two healthy human donors was drawn
directly into K2-EDTA-coated tubes to prevent coagulation.
Blood collection was performed by a trained phlebotomist in
order to minimize the risk to the donor. A written informed
consent was obtained from each donor prior to the blood drawn.
To the 5 mL of blood 15 mL of sterilized phosphate buffer
saline (PBS) was added and, after slow agitation, tubes were
centrifuged at 500g for 10 min. Supernatant containing plasma
was aspirated and the buffy coat was washed thrice and diluted
with normal saline to a 50% packed cell volume (hematocrit)
adjusted at pH 7.4 and stored at 4 °C. Different concentrations
of PSS-GNRs (100 μL each) were incubated with 100 μL of
RBCs suspension at 37 °C in CO2 incubator for 4 h. 0.2%
Triton X-100 was used as positive control and PBS was taken
as negative control [41]. After incubation, 50 μL of 2.5%
glutaraldehyde was added to the sample in order to stop the
process of hemolysis and centrifuged at 1000g for 10 min.
Hemoglobin release was monitored at 562 nm using a micro-
plate reader (Platos R496, Austria) by transferring supernatant
to a 96-well plate. Percentage hemolysis was calculated using
the following formula:
Cell inhibition after photothermal treatment
Combination therapy was performed by the method described in
a previous study with modifications [28]. The HepG2 cells were
seeded into 96-well plates (5 × 103 per well) and incubated for
24 h before the adding the different concentrations of PSS-
GNRs, free DOX, and PSS-GNRs-DOX conjugate. The treated
cells were incubated for 12 h for proper uptake before laser irra-
diation. After that, cells were illuminated with a 808 nm NIR
laser (power density = 1.5 W/cm2 for 2 min) with a beam spot
of 6 mm in diameter and incubated at 37 °C for 24 h. The MTT
assay was performed to measure cell inhibition.
Funding
The authors are thankful to Higher Education Commission,
Pakistan (SRGP (NO.21-2219/SRGP/R&D/HEC/2018)) for
financial support. We are grateful to Pakistan Science Founda-
tion Islamabad, Pakistan, for financial assistance (PSF/Res/C-
NILOP/Med (330)).
ORCID® iDs
Uzma Azeem Awan - https://orcid.org/0000-0001-9919-5722
Abida Raza - https://orcid.org/0000-0002-4414-1070
Shaukat Ali - https://orcid.org/0000-0003-2481-1978
Preprint
A non-peer-reviewed version of this article has been previously published
as a preprint: https://doi.org/10.3762/bxiv.2020.98.v1
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