Precise ratiometric co-loading, co-delivery and intracellular co-release of paclitaxel and curcumin by aid of their conjugation to the same gold nanorods to exert synergistic effects on MCF-7/ADR cells

Article abstract of DOI:10.1016/j.jddst.2019.101383

It is necessary for effective combinational effects that multiple chemotherapeutic agents enter and act on the same tumor cells simultaneously at a suitable ratio. Due to their individual physicochemical or biopharmaceutical properties, they cannot reach

Full text of DOI:10.1016/j.jddst.2019.101383

JournalofDrugDeliveryScienceandTechnologyxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Precise ratiometric co-loading, co-delivery and intracellular co-release of
paclitaxel and curcumin by aid of their conjugation to the same gold
nanorods to exert synergistic effects on MCF-7/ADR cells
Shaohui Xua,1, Zhu Jina,1, Zhipeng Zhangb,1, Wei Huangc,d, Yuanyuan Shena,∗
Zhongmin Wangc,d,∗∗, Shengrong Guoa,∗∗∗
,
a School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China
b School of Pharmacy, Hubei University of Science and Technology, Xianning, 437100, China
c Department of Interventional Radiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
d Department of Interventional Radiology, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong University School of Medicine, Shanghai, 200020, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
It is necessary for effective combinational effects that multiple chemotherapeutic agents enter and act on the
same tumor cells simultaneously at a suitable ratio. Due to their individual physicochemical or biopharma-
ceutical properties, they cannot reach the same cells at its predetermined ratio after co-administration. Herein,
we report a novel system with precise ratiometric co-loading, co-delivery and intracellular co-release of pacli-
taxel (PTX) and curcumin (CUR), to investigate their synergistic effects. We prepared the system by conjugating
PTX and CUR at different ratios onto the same gold nanorods (GNRs). We demonstrated that PTX and CUR could
be precisely ratiometric co-loaded to form dual-drug conjugated biotin-PEG modified GNRs (abbreviated as PTX/
CUR@BPGNRs), which could co-deliver dual drugs into tumor cells with a predetermined ratio and co-release
them intracellularly. The PTX/CUR@BPGNRs with the mass ratio of PTX and CUR at 1:1 exhibited the best
synergistic effect on multidrug resistant MCF-7/ADR cells while the free PTX and CUR mixture with the mass
ratio of at 1:0.75 displayed the strongest synergism. Cytotoxicity studies showed that PTX/CUR@BPGNRs are
superior to free drug mixture. Furthermore, PTX/CUR@BPGNRs could remarkably induce apoptosis of MCF-7/
ADR cells under near-infrared irradiation. Moreover, we found that PTX/CUR@BPGNRs significantly inhibited
P-glycoprotein (P-gp) expression in MCF-7/ADR cells.
Gold nanorods
Precise ratiometric
Synergistic effect
Co-release
Co-delivery
1. Introduction
synergistic therapeutic effects will be maximized.
The approaches to loading multiple drugs into a single nanocarrier
Multidrug resistance (MDR) is one of the main causes leading to
chemotherapy failure and the overexpression of ATP-dependent drug
efflux pumps such as permeability glycoprotein (P-gp) is considered as
the primary cause [1,2]. Combination therapy has shown superior an-
titumor efficiency to reverse MDR due to their different mechanisms of
action. As is widely recognized, synergistic effects, additive effects and
antagonistic effects may occur among different ratios of drug combi-
nations [3]. Synergistic effects will be achieved only when drug com-
binations enter tumor cells at an optimal ratio [4,5]. Therefore, it is
essential to ensure the drug combinations are at a predetermined ratio
when tumor cells are exposed to multiple drugs, therefore the
can be achieved by physical encapsulation, covalent conjugation or a
combination of both methods. To precisely control the loading ratio of
different drugs, the method of covalent conjugation is more advanta-
geous because physical encapsulation often leads to premature drug
release during blood circulation and is unable to maintain the pre-
determined drug ratio by the time drugs enter tumor cells [6,7].
With the development of nanotechnology, several nano-drug de-
livery systems (NDDS), such as liposomes and micelles, have provided
the possibility to co-deliver multiple therapeutic agents within a single
drug delivery vehicle. However, precisely controlling the loading ratio
and release characteristics of multiple drugs remain formidable
∗ Corresponding author.
∗∗ Corresponding author. Department of Interventional Radiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
∗∗∗ Corresponding author.
E-mail addresses: shenyuanyuan@sjtu.edu.cn (Y. Shen), wzm11896@rjh.com.cn (Z. Wang), srguo@sjtu.edu.cn (S. Guo).
1 These authors made equal contributions to this work.
https://doi.org/10.1016/j.jddst.2019.101383
Received 14 July 2019; Received in revised form 4 November 2019; Accepted 9 November 2019
1773-2247/©2019ElsevierB.V.Allrightsreserved.
Pleasecitethisarticleas:ShaohuiXu,etal.,JournalofDrugDeliveryScienceandTechnology,https://doi.org/10.1016/j.jddst.2019.101383

S. Xu, et al.
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Fig. 1. Schematic illustration of Precise ratiometric co-loading, co-delivery and co-release of PTX and CUR from PTX/CUR@BPGNRs.
challenges [4,8,9]. Functional gold nanomaterials, such as gold na-
norods (GNRs) and gold nanocages, have comprehensive applications in
the biomedical field, e.g. medical diagnosis, drug delivery and photo-
thermal therapy [10]. Owing to the excellent biocompatibilities, che-
mical stabilities, unique optical and photothermal properties, gold na-
norods have been widely used as platforms for cancer therapy including
chemotherapy, photothermal therapy, photodynamic therapy and
tumor imaging [11]. GNRs with a longitudinal absorption peak at ap-
proximately 808 nm wavelength are frequently used for near-infrared
(NIR) triggered drug delivery due to their strong ability to convert NIR
irradiation to local heating [12].
Herein, we developed a GNR-based dual-drug conjugation where
different drug molecules were conjugated to the same GNRs (Fig. 1).
Paclitaxel (PTX) is an anticancer drug widely used in the clinical
treatment of breast cancer that induces tubulin polymerization and
stabilizes microtubules [13]. Curcumin (CUR), a natural polyphenolic
compound, possesses pleiotropic anticancer effects such as inhibiting P-
gp expression, NF-κB signaling pathway and angiogenesis [14,15]. Sy-
nergistic effects of PTX and CUR have been demonstrated to overcome
MDR of PTX in recent studies [16,17]. The covalent bond between gold
and sulfur (namely, the Au–S bond) is a robust bond with high binding
efficiency that can provide the feasibility to precisely co-load dual-
drugs to GNRs [18]. To load drugs onto GNRs, PTX and CUR were first
reacted with lipoic acid (LA) to synthesize paclitaxel-lipoic acid ester
(PTX-LA) and curcumin-lipoic acid ester (CUR-LA), respectively. To
enhance the internalization of GNRs and facilitate effective drug de-
livery, we modified GNRs with biotin moiety conjugated polyethylene
glycol (Biotin-PEG). As reported in many studies, biotin moiety can be
specifically bound to biotin receptors, which are overexpressed in
various cancer cell lines [19–21]. Next, PTX-LA and CUR-LA at pre-
cisely controlled ratios were co-loaded onto the same biotin-PEG-
modified GNRs (abbreviated as BPGNRs) via the Au–S bonds to prepare
dual-drug loaded GNRs (abbreviated as PTX/CUR@BPGNRs). The two
drugs were precisely ratiometrically taken up by MCF-7/ADR cells via
biotin receptor-mediated endocytosis. The abundant glutathione (GSH)
in the cytoplasm promoted the dissociations of PTX-LA and CUR-LA
from GNRs through the ligand exchange reaction of Au–S [22–25].
Additionally, the high-level of esterase in cytoplasm enables ester
prodrugs to be quickly hydrolyzed to release free drugs [26–29].
Moreover, the GNRs here absorb and transform NIR irradiation to lo-
calized photothermal heating to expedite the hydrolysis of prodrugs
[30,31]. Taken together, the same Au–S bond and ester linkage will
allow PTX and CUR to be co-released from PTX/CUR@BPGNRs. The
therapeutic effects of PTX/CUR@BPGNRs on multidrug-resistant
human breast cancer MCF-7/ADR cells were evaluated and the me-
chanisms of action were investigated. We expect this combinatorial
drug delivery system would provide a new paradigm for precise control
over drug ratio in combination therapy.
2. Materials and methods
2.1. Materials
Silver nitrate (AgNO3), cetyltrimethylammonium bromide (CTAB),
sodium borohydride (NaBH4), 7-Bromo-3-hydroxy-2-naphthoic acid (7-
BrHNA), L-ascorbic acid (L-AA), 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT), curcumin (CUR), Porcine liver es-
terase (PLE) and dimethyl sulfoxide (DMSO) were purchased from
Sigma-Aldrich (USA). Chloroauric acid (HAuCl4·3H2O), hydrochloric
acid (HCl), nitric acid (HNO3), thionyl chloride (SOCl2), triethylamine
(TEA), dichloromethane (DCM), hexane (HEX), tetrahydrofuran (THF),
petroleum ether (PE), Tween 80 andethyl acetate (EA) were obtained
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from Sinopharm Chemical reagent Co., Ltd (Shanghai, China). Biotin-
poly (ethylene glycol)-lipoic acid (biotin-PEG-LA, MW = ~2000Da)
and methoxyl poly (ethylene glycol)-lipoic acid (mPEG-LA,
MW = ~2000Da) were purchased from Ponsure Biotechnology
(Shanghai, China). Paclitaxel (PTX) was purchased from Ziyun
Biotechnology Co., Ltd, (Yunnan, China). Lipoic acid (LA), 4-(di-
methylamino) pyridine (DMAP), glutathione (GSH) and N-(3-dimethy-
laminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC·HCl) were
purchased from Aladdin reagent Co., Ltd (Shanghai, China).
Acetonitrile (HPLC grade) was purchased from CINC High Purity
Solvents (Shanghai) Co.,Ltd. Trifluoroacetic acid (TFA, HPLC grade)
was bought from J&K Chemicals (Beijing). Other chemicals were ana-
lytically pure and used as received unless otherwise specified. Water
was purified by distillation, deionization, and reverse osmosis (Milli-Q
plus).
solution was filtered and the filtrate was evaporated under reduced
pressure. The obtained CUR-LA was purified by column chromato-
graphy on a silica gel with a mobile phase of PE-EA (3:2, v/v).
The chemical structure was confirmed by 1H NMR recorded on a
Varian Mercury Plus-400 NMR spectrometer (Varian, USA).
2.4. Fabrications of BPGNRs and PTX/CUR@BPGNRs
To prepare BPGNRs, mPEG-LA and biotin-PEG-LA (at the mass ratio
of 4:1) was dissolved in 2 mL of water, followed by mixing with 1 mL of
concentrated CTAB-GNRs. The mixture was stirred at room temperature
for 24 h and centrifuged at 10, 000 rpm for 10 min three times to re-
move residual reactants. The obtained precipitate was re-dispersed in
water before use. UV–Vis–NIR spectrophotometer and Zetasizer Nano
ZS/ZEN3600 were also used to characterize BPGNRs.
Breast cancer cell lines MCF-7 and MCF-7/ADR (multidrug re-
sistant) were generously donated by Shanghai Institute of Material
Medica, Chinese Academy of Sciences. Penicillin-streptomycin, RPMI-
1640 medium, fetal bovine serum (FBS), 0.25% (w/v) trypsin-0.03%
(w/v) ethylenediaminetetraacetic acid (EDTA) solution and phosphate
buffer solution (PBS) were purchased from Gibco BRL (Gaithersburg,
MD, USA).
A series of PTX/CUR@BPGNRs loading dual drugs in precisely
controlled mass ratios were prepared by adding PTX-LA and CUR-LA in
different mass ratios (PTX-LA: CUR-LA = 1:0.619, 1:0.929, 1:1.238,
1:2.477; corresponding PTX: CUR = 1:0.5, 1:0.75, 1:1, 1:2) to 40 μg/
mL BPGNRs. The mixture was stirred at room temperature for 24 h. The
resulting PTX/CUR@BPGNRs were centrifuged, washed and re-dis-
persed in water. PTX loaded BPGNRs (PTX@BPGNRs) and CUR loaded
BPGNRs (CUR@BPGNRs) were prepared by a similar method. The
mixture of PTX@BPGNRs and CUR@BPGNRs with the mass ratio of
PTX and CUR at 1:1 was prepared by mixing PTX@BPGNRs containing
20 μg/mL PTX and CUR@BPGNRs containing 20 μg/mL CUR in equal
volumes.
2.2. Preparation of CTAB capped GNRs
CTAB capped GNRs (abbreviated as CTAB-GNRs) with a maximum
optical absorption peak at 808 nm were synthesized by the seed-
mediated approach [32]. Firstly, the seed solution was prepared by
blending 5 mL of 0.2 M CTAB solution with 5 mL of 0.5 mM HAuCl4
solution, followed by adding 0.6 mL fresh ice-cold aqueous solution of
NaBH4 (0.01 mM). Then the mixture was stirred vigorously for 2 min
and kept undisturbed in a water bath at 30 °C for 2 h before use. For
preparing the growth solution, 1.080 g CTAB and 0.0612 g 7-BrHNA
were dissolved in 30 mL warm water and the solution was kept in a
water bath at 30 °C after adding 0.96 mL AgNO3 (4 mM). After being
settled for 15 min, 30 mL HAuCl4 (1 mM) and 0.1 mL HCl (37%) were
added and stirred slowly for 15 min. Afterward, 0.3 mL L-ascorbic acid
(0.1 M) was added and the mixture was stirred vigorously for 1 min.
Finally, 96 μL of the seed solution was injected into the growth solution
and was stirred for 30 s. For GNRs growth, the resultant mixture was
kept constant in a water bath at 30 °C for 12 h. Gold nanorods were
purified by centrifugation to remove remaining CTAB (three times at
10, 000 rpm, 10 min each). The precipitates were collected and then
dispersed in water.
2.5. Characterization of CTAB-GNRs, BPGNRs and PTX/CUR@BPGNRs
UV–Vis–NIR spectrophotometer (Hitachi U-2910, Japan) and
Zetasizer Nano ZS/ZEN 3600 (Malvern Instruments, Herrenberg,
Germany) were used to characterize CTAB-GNRs, BPGNRs and PTX/
CUR@BPGNRs. Their morphologies were observed on a transmission
electron microscopy (TEM) (JEM-2100F, JEOL, Japan).
To obtain the drug loading capacities (DLCs) and encapsulation
efficiencies (EEs) of PTX@BPGNRs, CUR@BPGNRs and PTX/CUR@
BPGNRs, the supernatants during centrifugation were merged for the
determination of free PTX-LA and CUR-LA content by HPLC. DLCs and
EEs were calculated according to Eq. (1) -(4):
MWPTX
MWPTX
MWPTX−LA
DLCPTX (%) =
DLCCUR (%) =
× DLCPTX−LA
=
MWPTX−LA
WPTX−LA − wPTX−LA
×
× 100
(1)
M
2.3. Synthesis of PTX-LA and CUR-LA
MW
MW
CUR
CUR
× DLCCUR−LA
=
MW
MW
CUR−LA
CUR−LA
Synthesis of PTX-LA is schematically represented in Fig. 2A. A so-
lution of lipoic acid (103.2 mg, 0.5 mmol)in 5 mL of DCM were added to
EDC•HCl (95.9 mg, 0.5 mmol)and DMAP (61.1 mg, 0.5 mmol)followed
by paclitaxel (427 mg, 0.5 mmol)pre-dissolved in 5 mL of DCM. After
being stirred for 24 h at room temperature, the reaction mixture was
poured into 30 mL of water. The organic phase was collected, washed
with sodium bicarbonate and brine, and dried over sodium sulfate.
After removing DCM by rotary evaporation, the obtained PTX-LA was
purified by column chromatography on a silica gel with a mobile phase
of EA-HEX (3:2, v/v).
Synthesis of CUR-LA is schematically shown in Fig. 2B. To synthe-
size CUR-LA, lipoic acid was activated. Lipoyl-chloride was prepared by
adding dropwise1mL SOCl2 to 5 mL DCM solution of lipoic acid
(103 mg, 0.5 mmol) and stirred at room temperature for 1 h. DCM and
excess SOCl2 were removed by rotary evaporation and 5 mL THF was
added to prepare the lipoyl-chloride solution. Afterward, the lipoyl-
chloride solution was added dropwise to 10 mL THF solution of cur-
cumin (185 mg, 0.5 mmol)under N2 atmospheres in ice bath, followed
by injection of 0.2 mL of TEA. After stirring overnight, the reaction
W
− wCUR−LA
CUR−LA
×
× 100
(2)
(3)
(4)
M
WPTX−LA − wPTX−LA
EEPTX(%) =
EECUR (%) =
× 100
WPTX−LA
W
− wCUR−LA
CUR−LA
× 100
W
CUR−LA
where MW is the molecular weight of each reagent, W is the total
weight of the inputted reagent, w is the free drugs in the supernatants,
and M is the total weight of the resulting drug loaded BPGNRs. The
subscripts in the above equations are used to indicate which reagent the
MW, W and w belong to.
2.6. Combinational effects of PTX and CUR
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) assay
was conducted to evaluate the cytotoxicity. Firstly, the MCF-7/ADR
cells were tested for their drug resistance on PTX. In brief, MCF-7 and
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Fig. 2. Synthesis of PTX-LA(A) and CUR-LA (B).
(D)1
(D50)1
(D)2
(D50)2
MCF-7/ADR cells were seeded in 96-well plates at a density of
5000 cells/well and incubated for 24 h. Then the cells were treated with
different concentrations of free PTX. After 48 h of incubation, the
medium in each well was replaced with 200 μL of 0.5 mg/mL MTT.
After 4 h of incubation, the medium containing MTT was removed and
200 μL of DMSO was added followed by oscillation in the dark for
10 min. The absorbance was determined at 570 nm by a microplate
reader (Bio-Rad 680, USA). The 50% inhibitory concentrations (IC50) of
different samples were calculated by SPSS 21.0 software.
Next, we investigated the synergistic effects of free PTX and CUR
combinations on MCF-7/ADR cells by MTT assay. The MCF-7/ADR cells
were treated with different concentrations of free PTX, free CUR and
combinations of free PTX and CUR with different mass ratios (PTX:
CUR = 1:0.5, 1:0.75, 1:1, 1:2) for 48 h. The remaining steps were op-
erated using the same procedure as previously described.
Afterward, we explored the synergistic effects of the PTX and CUR
in PTX/CUR@BPGNRs on MCF-7/ADR cells. Similarly, MCF-7/ADR
cells were seeded in 96-well plates at a density of 5000 cells/well and
incubated for 24 h. Then the MCF-7/ADR cells were treated with dif-
ferent concentrations of PTX/CUR@BPGNR with different mass ratios
between PTX and CUR (PTX: CUR = 1:0.5, 1:0.75, 1:1, 1:2; corre-
sponding to PTX-LA: CUR-LA = 1:0.619, 1:0.929, 1:1.238, 1: 2.477) for
48 h. The remaining steps were operated using the same procedure as
previously described.
CI =
+
(5)
where (D)1 and (D)2 are the IC50 values of PTX and CUR in combination
therapy, (D
alone, respectively. Based on the method, CI < 1 suggests synergistic
effects, CI = 1 suggests additive effects, and CI > 1 suggest antag-
onistic effects.
)
50 1 and (D50 2
)
are the IC50 values of free PTX and free CUR
2.7. Cytotoxicity
To evaluate the cytotoxicities of BPGNRs and the combinations of
free PTX and free CUR (free PTX/CUR) with or without NIR irradiation,
MCF-7/ADR cells (5000 cells per well) were seeded in 96-well plates
and incubated for 24 h. Then the cells were treated with BPGNRs and
free PTX/CUR at various concentrations for 8 h. Then, the cells were
treated with or without NIR irradiation (2.5 W/cm2) for 10 min. After
further incubation for 40 h, the cell viabilities were determined by the
MTT assay.
Cytotoxicity of single drug loaded BPGNRs (PTX@BPGNRs and
CUR@BPGNRs) and the physical mixtures PTX@BPGNRs and CUR@
BPGNRs with a PTX/CUR ratio at 1:1 (the preparation was described
above, see 2.4) were also investigated by MTT assay. The resistance
reversal index (RRI) = IC50 value of free PTX/IC50 value of PTX in drug
loaded BPGNRs.
To evaluate the combinational effects of PTX and CUR, the combi-
nation index (CI) was calculated based on the Chou and Talalay method
[33]. The CI values were calculated according to Eq. (5):
2.8. Cellular uptake
Cellular uptake of the PTX/CUR@BPGNRs was measured with
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inductively coupled plasma mass spectrometry (ICP-MS, iCAP Q,
Germany). MCF-7/ADR cells (1 × 106 cells/well) were seeded in 6-well
plates and incubated for 24 h. Then the medium was replaced with
PTX/CUR@BPGNRs containing 2.5 μg/mL of PTX and CUR with 40 μg/
mL BPGNRs and the cells were incubated for 2, 4, 8, 12 and 24 h. At the
predetermined time, the cells were digested with trypsin and washed
with ice-cold PBS to remove the extracellular PTX/CUR@BPGNRs.
Subsequently, the cells were stained with 0.4% trypan blue and the
living cells were counted under inverted microscope (Olympus BX51,
Japan). The cells were digested by aqua regia (HCl/HNO3 = 3: 1, v/v)
overnight and the aqua regia was volatilized to dryness in a draught
cupboard. Finally, the residues were dissolved in 2% HNO3 and de-
termined by ICP-MS.
2.12. P-gp expression
P-gp expression of MCF-7/ADR cells was detected using a Human
permeability glycoprotein (P-gp) ELISA Kit (Beijing BaiaoLaibo
Technology, China). Firstly MCF-7/ADR cells (5 × 105 cells/well) were
seeded in 12-well plates and incubated for 24 h. Then the medium was
discarded and the cells were treated with free PTX, free PTX/CUR, PTX/
CUR@BPGNR, and PTX/CUR@BPGNR + NIR. The mass ratio between
PTX and CUR in these groups were 1:1 and the final concentration was
2.5 μg/mL. Untreated cells were also tested for control. The NIR irra-
diation for 10 min was employed after 8 h incubation with PTX/CUR@
BPGNR. After further incubation for 40 h, the cells were digested and
washed with ice-cold PBS. Then 1 × 106 cells in each group were
collected and split by freezing and thawing. The cell lysate was cen-
trifuged and the supernatant was extracted for the P-gp analysis fol-
lowing the manufacturer's protocol. The P-gp level was expressed as
percent relative to the untreated group.
2.9. In vitro drug release
0.2 mL condensed PTX/CUR@BPGNRs was placed in dialysis bags
(MWCO = 3500 Da). After sealed tightly with threads, the dialysis bags
were immersed into 10 mL of the following release media: (1) PBS (pH
7.4) with Tween 80 (0.5%, w/v); (2) PBS (pH 7.4) with Tween 80
(0.5%, w/v) under NIR irradiation of power intensity at 2.5 W/cm2 for
10 min at the predetermined time points; (3) PBS (pH 7.4) with Tween
80 (0.5%, w/v) containing 5 μM GSH; (4) PBS (pH 7.4) with Tween 80
(0.5%, w/v) containing 5 mM GSH; (5) PBS (pH 7.4) with Tween 80
(0.5%, w/v) containing 30 U/mL PLE; (6) PBS (pH 7.4) with Tween 80
(0.5%, w/v) containing 5 mM GSH and 30 U/mL PLE. Then the above
release media was placed in a shaking water bath (37 °C, 100 rpm) for
72 h. At predetermined time intervals, 0.1 mL of the release medium
was taken out for HPLC analysis and the equal volume of fresh medium
was supplemented. The 10 min NIR irradiation was exerted soon after
the fresh medium was added.
2.13. Statistical analysis
Data were expressed as the mean
standard deviation (SD).
Statistical analysis was performed using one-way ANOVA. The statis-
tical difference between the data of two groups was considered to be
significant when p < 0.05.
3. Results and discussion
3.1. Synthesis and characterization of PTX-LA and CUR-LA
In order to enable the conjugation of drugs to GNRs, lipoic acid (a
kind of carboxylic acid containing sulfur atoms) was first used to es-
terify the drugs. The similar ester bonds allow PTX-LA and CUR-LA to
be hydrolyzed to release free drugs simultaneously. As illustrated in
Fig. 2, PTX-LA was synthesized via an EDC/DMAP coupling reaction.
Then the reaction mixture was purified with column chromatographic
separation under the monitoring of thin layer chromatography (TLC).
The chemical identities of PTX-LA were confirmed by 1H NMR spec-
trum. Compared with the 1H NMR spectra of LA (Fig. 3A) and PTX (the
upper one in Fig. 3B), characteristic peaks of PTX and LA were observed
in the 1H NMR spectrum of PTX-LA (the lower one in Fig. 3B), in-
dicating the successful conjugation of LA and PTX. It is noteworthy that
the resonance at δ = 4.8 ppm (s, 1H), which is assigned to the proton of
the 2′-OH, was observed in the spectrum of PTX (the red arrow in
Fig. 3B) and not observed in the spectrum of PTX-LA. This difference
suggested that LA was conjugated at this position. Besides, we also
found that the 2′-OH was more active than 7-OH, perhaps due to the
larger steric hindrance around the 7-OH.
For the synthesis of CUR-LA, LA was first reacted with SOCl2 to
generate lipoic chloride (LA-Cl). Afterward, CUR was coupled to LA to
obtain CUR-LA where triethylamine (TEA) was used as a base. Finally,
silica gel column chromatography was used to separate and purify CUR-
LA from the reaction mixture with TLC analysis. The 1H NMR spectrum
of CUR and CUR-LA were shown in Fig. 3C and D, respectively. In 1H
NMR spectrum of CUR-LA, characteristic peaks of CUR (Ph-:
δ = 7.17–6.92, and CH3-: δ = 3.97–3.83) and LA (-S-CH-CH2-:
δ = 3.59, the peak b in Fig. 3D; -S-CH2-CH2-: δ = 3.15, the peak a in
Fig. 3D) were both observed. Moreover, the molecular masses of CUR-
LA and PTX-LA were determined to be 557.1661 [M+H]+ and
1043.3760 [M+H]+ respectively (Fig. S1), which are accorded with
2.10. Intracellular drug release
MCF-7/ADR cells (5 × 105 cells/well) were seeded in 12-well plates
and incubated for 12 h. Then the medium was replaced with fresh
medium containing free PTX/CUR or PTX/CUR@BPGNR. The mass
ratios between PTX and CUR in these two group were 1:1 and the final
concentration was 2.5 μg/mL. Then the cells were incubated for 2, 4, 8
and 12 h. At the end of incubation, cells were digested by trypsin and
washed with ice-cold PBS. Cells were freeze-thawed to lyse cells and
release the drugs. The cell lysates were centrifuged at 10, 000 rpm for
10 min to collect the supernatant. The supernatant was separated into
two parts, one was used to determine the total cell protein amount by
the BCA Protein Assay Kit (Beyotime, China), and the other was sub-
jected to HPLC analysis for the content of PTX and CUR. The in-
tracellular contents of PTX and CUR were normalized to the total
protein amount.
2.11. Cell apoptosis
The cell apoptosis was quantitative analyzed by Annexin V-FITC/PI
Apoptosis Detection Kit (Beyotime, China). MCF-7/ADR cells
(5 × 105 cells/well) were seeded in 12-well plates and incubated for
24 h. Then the cells were treated with free PTX/CUR, PTX/CUR@
BPGNR, and PTX/CUR@BPGNR + NIR. The mass ratio between PTX
and CUR in these groups were 1:1 and the final concentration was
2.5 μg/mL. Untreated cells were also tested for control. The NIR irra-
diation for 10 min was employed after 8 h incubation with PTX/CUR@
BPGNR and the cells were further incubated for 40 h. The cells were
collected after digestion and wash. 195 μL binding buffer, 5 μL Annexin-
VFITC (fluorescein isothiocyanate) and 10 μL PI (Propidium Iodide)
were added to each sample and mixed gently. Then the cell suspension
was incubated in the dark for 15 min at room temperature followed by
addition of 200 μL binding buffer. Finally, the samples were detected by
the BD LSR Fortessa flow cytometry (Becton Dickinson, USA).
the predicted molecular formulas:
55H63NO15S2 for PTX-LA. These data verified that CUR-LA and PTX-LA
had been successfully synthesized.
C29H32O7S2 for CUR-LA and
C
3.2. Characterization of CTAB-GNRs, BPGNRs and PTX/CUR@BPGNRs
Firstly, the CTAB-capped GNRs were prepared using the seed
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Fig. 3. 1H NMR spectra of LA (A), PTX and PTX-LA (B), CUR (C), CUR-LA (D).
growth method. Then they were treated with PEG-LA and Biotin-PEG-
LA to replace surface CTAB surfactants to obtain biotin-PEG modified
GNRs (BPGNRs). Finally, PTX-LA and CUR-LA were loaded onto the
BPGNRs to prepare PTX/CUR@BPGNRs via Au–S bond.
3.3. Precisely ratiometric control over dual-drug loading
For controlled loading different drugs in the same nanoparticle,
high and homogeneous encapsulation efficiency for every drug is im-
perative. PTX and CUR were loaded onto BPGNRs via the same robust
Au–S bonds with high binding efficiency. In our preliminary experi-
ments, we prepared different concentrations of single drug loaded and
dual drug loaded BPGNRs, finding a constant encapsulation efficiency
(approximately 85%) of PTX-LA and CUR-LA when drug concentrations
were no more than 40 μg/mL and BPGNR concentration was fixed at
40 μg/mL.
Based on the results, we loaded PTX-LA and CUR-LA with the mass
ratio of 1:1.238 (the corresponding mass ratio of PTX and CUR is 1:1)
onto BPGNRs to obtain the PTX/CUR@BPGNRs (1:1). As shown in
Table 1, the EEs of PTX-LA and CUR-LA in PTX/CUR@BPGNRs (1:1)
were 85.3% and 85.0%, while the DLCs were 6.49% and 7.83%, re-
spectively. The DLCs of PTX and CUR were calculated by converting the
DLSs of PTX-LA and CUR-LA with the molecular weight proportions.
The DLCs of PTX and CUR were 5.32% and 5.21%, respectively.
Therefore, the loading ratio between PTX and CUR was 1:
Fig. 4A compares UV–Vis–NIR absorption spectra of CTAB-GNRs,
BPGNRs and PTX/CUR@BPGNRs. The typical characteristics of CTAB-
GNRs solution were shown in the spectra: two surface plasma re-
sonances (SPR) absorption peaks corresponding to the longitudinal
(LSPR) at 808 nm and transverse (TSPR) at 510 nm. Compared with
original GNR stabilized with CTAB, there were negligible changes in the
absorption spectra of BPGNRs and PTX/CUR@BPGNRs, indicating the
unique optical properties of GNRs were well-maintained after the sur-
face modifications. And the resulting PTX/CUR@BPGNRs showed an
LSPR peak at 809 nm, suggesting its excellent absorption property
under NIR irradiation of 808 nm. Zeta potential measurements resulted
in values of −5.53
0.73 mV for BPGNRs and −3.97
0.52 mV for
PTX/CUR@BPGNRs in contrast with +40.37
2.48 mV for CTAB
capped GNRs (Fig. 4B), as a confirmation of successful modification of
the GNR surface.
As shown in Fig. 4C and D, the CTAB-GNRs and PTX/CUR@BPGNRs
have similar rod-shape and size. The PTX/CUR@BPGNRs had an aspect
ratio of ~3.9, which averaged 50.2 nm in length and 12.9 nm in width.
The PTX/CUR@BPGNRs were dispersed in water, PBS (pH 7.4) and cell
culture medium. After 5 days standing, the mediums were still trans-
parent without any agglomeration or precipitation (Fig. S2), indicating
a good colloidal stability of PTX/CUR@BPGNRs in the above three
mediums.
(0.976
0.05) (w/w) which is almost identical with the initial feeding
ratio of PTX and CUR (1:1). These results affirmed that PTX and CUR
were precisely ratiometric co-loaded to the same BPGNRs.
3.4. Combinational effects of PTX and CUR
The IC50 values of PTX against MCF-7 cells and MCF-7/ADR cells
were calculated to investigate the levels of MDR. The results showed
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Fig. 4. Characterizations of GNR-based systems. UV–Vis–NIR absorption spectra (A) and Zeta potential (B) of CTAB-GNRs, BPGNRs and PTX/CUR@BPGNRs. TEM
images of CTAB-GNRs (C) and PTX/CUR@BPGNRs (D).
Table 1
Table 2
Drug loading capacities (DLCs) and encapsulation efficiencies (EE) of PTX-LA,
CUR-LA, PTX and CUR for the PTX/CUR@BPGNRs (1:1).
IC50 and CI Values of different treatments in MCF-7/ADR cells after 48 h in-
cubation.
Drug
DLCs (%)
EEs (%)
Treatment
IC50 (μg/mL)
CI
PTX-LA
CUR-LA
PTX
6.49
7.84
5.32
5.21
0.21
85.3%
85.0%
1.53
1.86
PTX
CUR
0.16
0.27
0.32
Free PTX
Free CUR
20.17
–
–
–
–
34.16
6.12
7.35
9.73
14.62
1.37
1.77
1.84
2.82
CUR
Free PTX: CUR (1:0.5)
Free PTX: CUR (1:0.75)
Free PTX: CUR (1:1)
Free PTX: CUR (1:2)
PTX/CUR@BPGNRs (1:0.5)
PTX/CUR@BPGNRs (1:0.75)
PTX/CUR@BPGNRs (1:1)
PTX/CUR@BPGNRs (1:2)
12.24
9.80
9.73
7.31
2.74
2.36
1.84
1.41
0.786
0.701
0.767
0.790
0.176
0.169
0.145
0.152
that IC50 value of MCF-7/ADR cells was relatively high (20.17 μg/mL),
which was 150-fold higher than the parent MCF-7 cells
(IC50 = 0.134 μg/mL). Therefore, the MCF-7/ADR cells were highly
drug-resistant to PTX and could be used for the following experiments.
The combinational effects of drug combinations based on free form
of PTX and CUR with different mass ratios were investigated. IC50 va-
lues of free PTX, free CUR and combinations of free PTX and CUR (free
PTX/CUR) with the mass ratio of 1:0.5, 1:0.75, 1:1 and 1:2 against
MCF-7/ADR cells and the CIs were displayed in Table 2. The CI values
of all the tested combinations of free drugs were all less than 1, sug-
gesting the combination of PTX and CUR could bring out notable sy-
nergistic effects. More importantly, the results showed that free PTX/
CUR with a mass ratio of 1:0.75 exhibited the strongest synergism
among the four series of combinations of free PTX and CUR.
The ratio in the bracket presents the mass ratio between PTX and CUR.
discrepancy could be that the ways of drugs entered into MCF-7/ADR
cells are different. The free drugs enter cells through diffusion, which is
related to physicochemical properties of drugs. Free PTX and CUR may
lead to different uptake behaviors, as a result, mass ratio of drugs inside
cells may be inconsistent with the initial mass ratio. As for the PTX/
CUR@BPGNRs, which has been modified with biotin, can bind with the
overexpressing biotin receptor on tumor cell membrane [19–21]. The
binding could result in receptor mediated endocytosis which internalize
the PTX/CUR@BPGNR as a whole and maintain the loading mass ratio.
The CI values of PTX/CUR@BPGNRs with different mass ratios
between PTX and CUR were also shown in Table 2. Interestingly, for
PTX/CUR@BPGNRs, we found that the mass ratio of 1:1 resulted in the
strongest synergistic effects among four tested mass ratios, which was
different from that of free PTX/CUR. The main reason for the
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Fig. 5. A: Cell viabilities of MCF-7/ADR
cells incubated with different concentrations
of BPGNRs with and without NIR irradiation
(*: p < 0.05). B: Cell viabilities of MCF-7/
ADR cells incubated with combinations of
free PTX and CUR (1:1) at different con-
centrations with and without NIR irradia-
tion. C: Quantification of gold nanorods per
million cells determined by ICP-MS after
incubation for 2 h, 4 h, 8 h 12 h and 24 h.
The NIR irradiation was performed with the
laser power intensity at 2.5 W/cm2 for
10 min. (For interpretation of the references
to colour in this figure legend, the reader is
referred to the Web version of this article.)
3.5. Cytotoxicity
Table 3
IC50 values and resistance reversal indexes (RRI) of different treatments against
MCF-7/ADR cells.
In the view of the best synergist effect, we selected the PTX/CUR@
BPGNRs (1:1) which load PTX and CUR with a mass ratio of 1:1 for the
following research. To clarify its cytotoxicity, we evaluated the cyto-
toxicities of BPGNRs, free PTX and free CUR with or without NIR ir-
radiation firstly. As can be seen from Fig. 5A, negligible cytotoxicity
was observed in a certain concentration range (1–120 μg/mL), with
over 80% cell viability when BPGNRs concentration reached 120 μg/mL
after 48 h of incubation, indicating that BPGNRs possessed good bio-
compatibility for MCF-7/ADR cells. Fig. 5A also showed that NIR ir-
radiation had an ignorable impact on cell viability when BPGNRs
concentration was below 40 μg/mL, while the temperature reached
about 42 °C (shown in Fig. S3), implying that the photothermal heating
alone generated by the NIR radiation with 40 μg/mL of BPGNRs did not
exceed cell killing threshold. However, cell viabilities decreased to
below 75% where BPGNR concentration was 120 μg/mL under NIR
irradiation, suggesting that the cytotoxicity of BPGNRs under NIR ir-
radiation became obvious in high concentrations with the increasing
temperature. Moreover, no obvious difference was found in the cell
viabilities of MCF-7/ADR cells after incubation with free PTX and CUR
with or without NIR irradiation (Fig. 5B). Finally, we continued the
experiments with a BPGNRs concentration of 40 μg/mL.
Then the cytotoxicities of PTX@BPGNRs, CUR@BPGNRs, PTX@
BPGNRs + CUR@BPGNRs (the mass ratio between the corresponding
PTX and CUR was 1:1) and PTX/CUR@BPGNRs (1:1) against MCF-7/
ADR cells were investigated. As shown in Table 3, the IC50 values of
PTX and CUR in PTX@BPGNRs and CUR@BPGNRs against MCF-7/ADR
cells were 5.65 and 12.98 μg/mL, respectively, both of which were
much lower than that of their free forms, demonstrating enhanced cy-
totoxicities of the drugs loaded in BPGNRs as nanoformulations. Since
we have proved that BPGNRs were not cytotoxic towards MCF-7/ADR
cells (See Fig. 5A), the cytotoxicity of PTX/CUR@BPGNRs (1:1) could
Treatment
IC50 (μg/mL)
RRI
Free PTX
PTX@BPGNR
CUR@BPGNR
PTX@BPGNR + CUR@BPGNR (1:1)
PTX/CUR@BPGNR (1:1)
20.17
5.65
12.98
1.96
–
3.6
–
10.29
10.96
1.84
be attributed to the released drugs from the conjugates. In particular,
the IC50 value of PTX/CUR@BPGNRs (1:1) was 1.84 μg/mL and the RRI
of PTX/CUR@BPGNRs (1:1) was 10.96, indicating its strongest lethal
effect on MCF-7/ADR cells.
3.6. Cellular uptake
According to the reports, the cellular uptake characteristics of na-
nomaterials could be affected by their physicochemical properties and
is significant for their biological applications [34–36]. Herein, ICP-MS
was used to investigate the cellular uptake of PTX/CUR@BPGNRs. As
shown in Fig. 5C, the gold content per million cells reached a plateau
after 8 h of incubation, indicating that the endocytosis of PTX/CUR@
BPGNRs (1:1) by MCF-7/ADR cells nearly reached equilibrium. Based
on the results, we imposed NIR irradiation on MCF-7/ADR cells after
8 h of incubation with PTX/CUR@BPGNRs.
3.7. In vitro drug release
It was widely reported that the extracellular and intracellular mi-
croenvironment was quite different, which could be taken advantage
for nanocarriers to selectively release inside cells to reduce premature
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Fig. 6. Drug release profiles of PTX/CUR and PTX-LA/CUR-LA from the PTX/CUR@BPGNRs in following release media: PBS (pH 7.4) with Tween 80 (0.5%, w/v)
(A); PBS(pH 7.4)with Tween 80 (0.5%, w/v) with NIR irradiation (B); PBS (pH 7.4) with Tween 80 (0.5%, w/v) containing 5 μM GSH (C); PBS (pH 7.4) with Tween
80 (0.5%, w/v) containing 5 mM GSH (D); PBS (pH 7.4) with Tween 80 (0.5%, w/v) containing 30 U/mL PLE (E); PBS (pH 7.4) with Tween 80 (0.5%, w/v)
containing 5 mM GSH and 30 U/mL PLE with NIR irradiation (F). In all groups, the concentrations of free drugs and the drugs in PTX/CUR@BPGNRs were 2.5 μg/mL
and the NIR was performed at laser power of 2.5 W/cm2 for 10 min **: p < 0.01, ***: p < 0.001 compared with the corresponding amount of PTX, CUR, PTX-LA
and CUR-LA released in PBS (pH 7.4) respectively.
drug leakage [24–27,37]. To reach this goal, besides the utilization of
the biotin moiety as a targeting ligand, PTX/CUR@BPGNRs (1:1) were
also designed with responses to the following stimuli: GSH, esterase and
NIR photothermal effects. PTX-LA and CUR-LA were conjugated to
BPGNRs via Au–S bonds [18]. Au–S bonds are relatively stable under
low GSH concentration of extracellular environment but can be de-
tached from the surface of GNRs under high GSH concentration in the
cytoplasm of tumor cells [22–25]. The abundant intracellular esterase
also enabled the specific release of PTX and CUR from PTX-LA and
CUR-LA inside MCF-7/ADR cells [28,29]. Additionally, when PTX/
CUR@BPGNRs were internalized by tumor cells, external NIR irradia-
tion also contributed to the release of PTX and CUR by promoting the
hydrolysis of ester bonds. The detached PTX-LA, CUR-LA and released
PTX and CUR in different release media were measured by HPLC to
characterize the in vitro release profiles.
The PTX/CUR@BPGNRs were first incubated in PBS at pH 7.4
containing 0.5% Tween. As shown in Fig. 6A, both free drugs (free PTX
and CUR) and prodrugs (PTX-LA and CUR-LA) exhibited slow release
9

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profiles in PBS at pH 7.4 (the cumulative release percentages of PTX,
BPGNRs group (no difference between the PTX amount and the CUR
amount) at different time points, which was practically identical to the
loading ratio between PTX and CUR in PTX/CUR@BPGNRs. It sug-
gested that PTX/CUR@BPGNRs (1:1) co-delivered and co-released PTX
and CUR at a predetermined ratio in MCF-7/ADR cells. We also ob-
served that greater amount of PTX and CUR was delivered into MCF-7/
ADR cells in PTX/CUR@BPGNRs group compared with those in free
PTX/CUR group, showing a remarkable advantage of PTX/CUR@
BPGNRs in efficient delivery of PTX and CUR.
CUR, PTX-LA and CUR-LA were 3.88
0.35%, 4.13
0.48%,
3.62 0.39% and 3.65 0.25% at 72 h), indicating that PTX/CUR@
BPGNRs were relatively stable at physiological conditions. Never-
theless, the release rates of free PTX and CUR were significantly in-
creased once the NIR irradiation (2.5 W/cm2 for 10 min) was given at
predetermined time points (Fig. 6B). In detail, approximately 20% of
free PTX and CUR were detected in the release media after 72 h of in-
cubation. This could be due to the local heating generated by BPGNRs
under NIR irradiation, which facilitated hydrolysis of the ester bonds of
the prodrugs (PTX-LA, CUR-LA) conjugated to BPGNRs.
3.9. Cell apoptosis
Next, we investigated the drug release profiles in PBS solutions
containing different GSH concentrations (5 μM and 5 mM) to mimic
extracellular and intracellular reduction environments, respectively. As
shown in Fig. 6C and D, there was no obvious difference in the release
profiles of free drugs (PTX and CUR) between the groups containing
5 μM and 5 mM GSH concentrations (the cumulative release percen-
tages were no more than 6.5%). The amounts of released prodrugs were
To quantify the apoptotic effects induced by PTX/CUR@BPGNRs,
MCF-7/ADR cells were stained with Annexin V-FITC/PI Apoptosis
Assay kit and analyzed by flow cytometry. The total apoptosis rate is
the sum of late apoptosis percentage (Q2, upper right) and early
apoptosis percentage (Q3, lower right). As shown in Fig. 7B and C, free
PTX/CUR (1:1) induced 25.3
BPGNRs (1:1) induced higher apoptosis rate (50.8
1.3% cell apoptosis. And PTX/CUR@
3.2%) than that
found to increase little (PTX-LA: 6.87
7.12
0.35%, CUR-LA:
0.25%) in the release media containing 5 μM GSH at 72 h,
of free PTX/CUR (1:1), which is in accordance with the results of the
above presented MTT assay. For the PTX/CUR@BPGNRs group with an
additional NIR irradiation, the percentage of apoptotic cells increased
suggesting good stability of PTX/CUR@BPGNRs in extracellular re-
duction environment (Fig. 6C). By contrast, the amounts of detected
prodrugs reached up to 38.54
3.05% (for PTX-LA) and
to 67.2
4.3%, while the percentage of necrotic cells was about 0.1%,
38.93 2.27% (for CUR-LA) after 72 h of incubation with a high
suggesting the promotion of cell apoptosis induced by the accelerated
release of drugs rather than direct killing of the cells by photothemal
effect.
concentration of GSH (Fig. 6D). It is reported that GSH can act as a
reducing agent to exchange the therapeutic prodrugs from the surface
of the gold. These data showed that the drugs were liberated from PTX/
CUR@BPGNRs in the form of prodrugs by the GSH concentration-de-
pendent thiol group exchange reaction.
3.10. P-gp expression
It is generally accepted that esterase concentration inside tumor
One of the primary causes of MDR in MCF-7/ADR cells is the ele-
vated expression of P-gp, a drug efflux pump which pumps anti-tumor
drugs out of cells, leading to the reduction of intracellular drug con-
centration below effective dose. CUR was reported to down-regulate P-
gp expression, thus could decrease the efflux of intracellular anti-tumor
drugs [38]. P-gp ELISA Kit was used to determine P-gp expression after
different treatments and the P-gp expression of untreated MCF-7/ADR
cells was used as control. As we can see from Fig. 7D, free PTX exhibited
cells is 2–3 orders of magnitude higher than that in extracellular fluid26
.
Therefore, PLE was used to study the drug release in the simulated
intracellular environment of tumor cells. As depicted in Fig. 6E, the
amounts of released free PTX and CUR at 72 h were 30.67
2.35%
and 34.32 2.89%, respectively. However, the cumulative percentage
of released prodrugs (PTX-LA and CUR-LA) did not exceed 4% at 72 h.
The results indicated that PTX/CUR@BPGNRs could be enzymatically
hydrolyzed to release the free drugs through quick degradation of the
ester linkage in the presence of a high concentration of esterase.
Most importantly, when PTX/CUR@BPGNRs were incubated with
the release media 5 mM GSH and 30 U/mL PLE (simulated the in-
tracellular environment of tumor cells) under intermittent NIR irra-
the highest P-gp expression (116.4
7.8%), indicating that free PTX
could up-regulate P-gp expression in MCF-7/ADR cells [39]. Compared
with free PTX/CUR (84.2
CUR@BPGNRs (60.4
5.4%), the P-gp expression levels of PTX/
10.3%) further declined. It was found that the
P-gp expression of MCF-7/ADR cells treated with PTX/CUR@BPGNRs
with NIR irradiation was the lowest (48.2 7.5%) among all the
diation, we found that 49.89
1.78% of PTX and 51.65
2.37% of
CUR were released from PTX/CUR@BPGNRs at 72 h (Fig. 6F). The
hydrolysis of ester bonds in PTX-LA and CUR-LA caused by NIR and
groups, due to the promoted intracellular delivery and release of CUR.
esterase result in the decrease of PTX-LA (10.61
1.89%) and CUR-LA
4. Conclusion
(10.42 1.98%) in this media compared with that in media with
5 mM GSH alone. It is important that the released amount of PTX and
CUR were almost same which in accordance with their loading ratio
(1:1), suggesting the achievement of precise ratiometric co-release.
In this work, we presented a novel approach to precisely ratiometric
co-loading PTX and CUR at the optimum ratio by conjugating them
onto the same GNRs to combat MDR. By esterifying PTX and CUR with
LA, PTX-LA and CUR-LA were conjugated to the same gold nanorod
with approximately 85% encapsulation efficiency, PTX/CUR@BPGNRs
were successfully prepared. The drug loading capacities of PTX and
CUR in PTX/CUR@BPGNRs were 5.32 wt % and 5.21 wt %, which were
in accordance with the predetermined mass ratio of PTX and CUR at
approximately 1:1. In vitro and intracellular drug release profiles re-
vealed that PTX and CUR were controllably and synchronously released
inside the tumor cells. The PTX/CUR@BPGNRs with the mass ratio of
PTX and CUR at 1:1 exhibited superior cytotoxicity and greater synergy
on MCF-7/ADR cells than the free PTX and CUR mixture. In addition,
PTX/CUR@BPGNRs had enhanced inhibition of P-gp expression on
MCF-7/ADR cells especially with NIR irradiation than the free PTX and
CUR mixture. Overall, our study offers a solution to the problem of
loading different drugs into the same drug-delivery platform with a
precisely controlled ratio because this drug-carrier conjugation ap-
proach can be generalized to various drugs containing hydroxyl groups.
3.8. Intracellular drug release
MCF-7/ADR cells were incubated with combination of free PTX/
CUR (1:1) and PTX/CUR@BPGNRs (1:1) for 2, 4, 8 and 12 h with
corresponding PTX and CUR concentration of 2.5 μg/mL to study the
intracellular release behavior of PTX and CUR from PTX/CUR@
BPGNRs. As shown in Fig. 7A, the internalized amounts of PTX and CUR
in the free PTX/CUR group and PTX/CUR@BPGNRs group both in-
creased gradually during incubation. In free PTX/CUR group, the in-
tracellular amount of PTX was less and increased much slower than that
of CUR, indicating that PTX and CUR did not enter MCF-7/ADR cells
simultaneously. This significant difference could be due to the more
hydrophobic property of free CUR compared with free PTX, which was
more conducive to the diffusion into cells. However, the mass ratio of
intracellular released PTX and CUR was approximate 1:1 in PTX/CUR@
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Fig. 7. Intracellular amounts of PTX and CUR (A), cell apoptosis analysis (B), cell apoptosis percentage (C), and relative P-gp expression level (D) of the MCF-7/ADR
after treatments with none drug (untreated), free PTX, free PTX/CUR, PTX/CUR@BPGNRs and PTX/CUR@BPGNRs with NIR irradiation. In all groups, the drug
concentrations of free drugs and the drugs in PTX/CUR@BPGNRs were both 2.5 μg/mL and the NIR was performed at laser power of 2.5 W/cm2 for 10 min *:
p < 0.05, **: p < 0.01, ***: p < 0.001.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgements
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numbers YG2014ZD01, YG2016MS28, YG2016MS17), Science and
Technology Development Fund of Shanghai Pudong New Area (grant
number PKJ2017-Y04), Shanghai Municipal Science and Technology
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