Subcellular co-delivery of two different site-oriented payloads based on multistage targeted polymeric nanoparticles for enhanced cancer therapy

Article abstract of DOI:10.1039/c8tb02230e

The co-delivery of two or more anti-tumor agents using nanocarriers has shown great promise in cancer therapy, but more work is needed to selectively target drugs to specific subcellular organelles. To this end, our research has reported on "smart" polymeric nanoparticles that can encapsulate two different site-oriented pro-drug molecules, allowing them to reach their targeted subcellular organelles based on NIR-mediated controlled release, allowing for targeted modifications in the nucleus or the mitochondria. Specially, an all-trans retinoic acid (RA) conjugated cisplatin derivative (RA-Pt) can be delivered with high affinity to the nucleus of target cells, facilitating the binding of cisplatin to double-stranded DNA. Similarly, a synthesized derivative generated by conjugation of triphenylphosphine (TPP) and celastrol (TPP-Cet) may facilitate mitochondrial targeted drug delivery in tumor cells, inducing ROS accumulation and thereby leading to apoptosis. Relative to nanoparticles loaded with a single therapeutic agent, dual antitumor agent-loaded nanocarriers showed promising synergy, exhibiting significant tumor inhibition in vivo (81.5%), and less systemic toxicity than the free therapeutic agents alone or the drug-loaded nanoparticles without targeted ligands. These results indicated that site-oriented payloads can effectively enhance antitumor therapeutic efficiency and these studies offer a novel "multistage targeted-delivery" strategy in synergistic therapy for cancer treatment.

Full text of DOI:10.1039/c8tb02230e

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Materials Chemistry B
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Subcellular co-delivery of two different
site-oriented payloads based on multistage
targeted polymeric nanoparticles for
enhanced cancer therapy†
Cite this: J. Mater. Chem. B, 2018,
6, 6752
Chao-Qun You,ab Hong-Shuai Wu,a Zhi-Guo Gao,a Kai Sun,a Fang-Hui Chen,a
a
W. Andy Taoc and Bai-Wang Sun
*
The co-delivery of two or more anti-tumor agents using nanocarriers has shown great promise in
cancer therapy, but more work is needed to selectively target drugs to specific subcellular organelles.
To this end, our research has reported on ‘‘smart’’ polymeric nanoparticles that can encapsulate two
different site-oriented pro-drug molecules, allowing them to reach their targeted subcellular organelles
based on NIR-mediated controlled release, allowing for targeted modifications in the nucleus or the
mitochondria. Specially, an all-trans retinoic acid (RA) conjugated cisplatin derivative (RA–Pt) can be
delivered with high affinity to the nucleus of target cells, facilitating the binding of cisplatin to double-
stranded DNA. Similarly, a synthesized derivative generated by conjugation of triphenylphosphine (TPP)
and celastrol (TPP–Cet) may facilitate mitochondrial targeted drug delivery in tumor cells, inducing ROS
accumulation and thereby leading to apoptosis. Relative to nanoparticles loaded with a single thera-
peutic agent, dual antitumor agent-loaded nanocarriers showed promising synergy, exhibiting significant
tumor inhibition in vivo (81.5%), and less systemic toxicity than the free therapeutic agents alone or the
drug-loaded nanoparticles without targeted ligands. These results indicated that site-oriented payloads
can effectively enhance antitumor therapeutic efficiency and these studies offer a novel ‘‘multistage
targeted-delivery’’ strategy in synergistic therapy for cancer treatment.
Received 23rd August 2018,
Accepted 2nd October 2018
DOI: 10.1039/c8tb02230e
rsc.li/materials-b
materials (such as mesoporous silica, and graphene), liposomes,
and lipid-based nanocarriers have been developed and have shown
1. Introduction
In recent decades, various strategies have been employed for great potential in diagnostic and therapeutic applications.3
the study of cancer therapy. Nanotechnology in particular has
One very advantageous strategy has been to encapsulate
been of great interest in this field, owing to the synthesis, therapeutic agents in nanocarriers, enhancing their stability
characterization and application of novel nanomaterials and allowing for targeted delivery and controlled release. These
with useful properties. The unique advantages of these small beneficial features allow for improved therapeutic efficacy
materials allow for their potential application to nanoscale and significantly decreased toxicity due to a higher localized
biological structure modifications.1 In recent years, significant intratumoral drug concentration.4–6 Moreover, therapeutic
advances have been made in the application of nanotechnology agents such as proteins, peptides, and nucleic acids are better
to biological and medical fields.2 A variety of nanostructures preserved when encapsulated in nanocarriers, as many such
including biodegradable polymers, dendrimers, inorganic carbon molecules are susceptible to enzymatic degradation by nucleases
and proteases.7 However, not all nanocarriers show the expected
therapeutic responses when these drug carriers are targeted at
the organ-, tissue-, or cellular-level owing to targeting limitations
a School of Chemistry and Chemical Engineering, Southeast University,
Nanjing 210089, P. R. China. E-mail: chmsunbw@seu.edu.cn;
that result in drugs not localizing to the appropriate sub-
cellular compartments (such as the cytosol, mitochondria, or
nucleus).8–10 Therefore, the development of a novel nanomedicine
system that can specifically target not only tumor cells of interest,
but also specific subcellular compartments is of great interest as
such a platform could maximize the therapeutic efficiency of
nanocarrier-loaded drug molecules for the treatment of cancer.11–14
Fax: +86 25 52090614; Tel: +86 25 52090614
b College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037,
P. R. China
c Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907,
USA
† Electronic supplementary information (ESI) available: Synthesis procedure of
Pt(IV)–COOH, synthetic route for RA–Pt and TPP–Cet, 1H NMR spectra and ESI-MS
data of products and other supplementary data. See DOI: 10.1039/c8tb02230e
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Of all subcellular organelles, the nucleus is the preferred been shown to offer key advantages relative to single agent
target for delivery of most therapeutic agents, particularly in the delivery. Such synergistic treatments can more effectively kill
context of cancer because it is the site of DNA replication and is tumor cells by simultaneously blocking apoptotic signaling
vital for cell proliferation and survival. In general, nuclear pathways and inhibiting DNA replication, potentially increasing
localization sequence (NLS)-tagged carriers or drugs are the therapeutic efficacy while reducing the risk of tumor drug
most common approaches to nuclear targeting.15,16 However, resistance.37–41 To this end, we have designed and synthesized
whereas NLS-tagged peptide ligands are fragile, small chemical two novel pro-drug molecules (RA–Pt and TPP–Cet) that can
molecules are more stable and easier to modify. It has been specifically target the nuclear and mitochondrial subcellular
reported that all-trans retinoic acid (RA) can easily bind to compartments, respectively. Specially, RA–Pt has a high affinity
cellular retinoic acid-binding protein II (CRABP-II), and this for the nucleus and can facilitate the binding of cisplatin to
adduct shows excellent affinity for the nuclear RA receptor.17,18 double-stranded DNA. The TPP–Cet conjugate may facilitate
As it is already widely used in clinical settings, cisplatin is mitochondrial drug targeting in tumor cells, inducing ROS
typically the preferred therapeutic agent for many cancers even accumulation and leading to apoptosis. This delivery process
though it does not show an expected therapeutic response can be traced by ICG imaging. To maximize therapeutic efficacy
against some tumors. Moreover, cisplatin may cause systemic and significantly decrease toxicity, these functional molecules
toxicity, particularly nephrotoxicity, in patients as it can have a were trapped in folate and cRGD conjugated polymeric nano-
high concentration distribution and extended accumulation in particles, and release was controlled by ICG-mediated photo-
kidneys.19–21 Therefore, the conjugation of RA with cisplatin thermal conversion using NIR irradiation. When taken into the
may allow for the nuclear targeting and delivery of this chemo- cytoplasm by endocytosis, these nanocarriers were depolymerized
therapeutic agent in tumor cells, thereby maximizing therapeutic by the induced heat, and the encapsulated pro-drug molecules
efficacy.
were released. The therapeutic efficacy of this prepared functional
As the key site of cellular energy generation, mitochondria nano-platform was evaluated using tumor cells in vitro and further
also play a critical role in the homeostasis of various physio- validated in a breast cancer tumor model of BALB/c nude
logical functions including electron transfer and apoptosis.22,23 mice in vivo. Our results indicate that site-oriented payloads
A variety of human disorders are caused in part by mitochondrial can effectively enhance chemotherapeutic efficiency, and these
dysfunction, including diabetes, neuromuscular diseases, and studies thus offer a new strategy for multistage targeted delivery
even cancer. A major basis for these symptoms is the over- and synergistic therapy based on functional nanomedicines.
production of reactive oxygen species (ROS).24,25 Thus, a strategy
for cancer treatment based on the controlled induction of mito-
chondrial ROS and disrupted homeostasis exists. Celastrol (Cet), a
natural product with a variety of biological activities, has been
2. Materials and methods
2.1 Materials
shown to possess strong antioxidant and antitumor angiogenesis
FA–PEG2k–DSPE, PLGA3k, and cyclic (Arg-Gly-Asp-D-Phe-Lys)
potential. It has been reported that celastrol can induce ROS
peptide (cRGD) were purchased from Polymtek Biomaterial
accumulation by acting on the mitochondrial respiratory chain
Co., Ltd (Shenzhen, China). Lecithin, and triphenylphosphine
(MRC) complex I, causing decreased mitochondrial membrane
(TPP) were purchased from Sinopharm Chemical Reagent Co.,
Ltd (China). All-trans retinoic acid (RA), indocyanine green
(ICG) and celastrol (Cet) were purchased from Sigma-Aldrich
potential and cytochrome C release into cytoplasm. Importantly,
celastrol can also regulate mitochondrial signaling pathways
via activating caspase 3 and caspase 9, inducing tumor cell
apoptosis.26–28 Therefore, mitochondrial targeting of celastrol
Chemical Company (St. Louis, MO, USA). Cisplatin (Pt) was
purchased from Shandong Boyuan Pharmaceutical Co., Ltd
may be a viable therapeutic strategy in tumor cells.
(Jinan, Shandong, China). cRGD–PEG5k–PCL10k was synthe-
Recently, triphenylphosphonium (TPP), a common chemical
sized according to previously reported procedures.42 All the
molecule, has been shown to have great binding affinity for the
dye kits were obtained from Beyotime Biotechnology Co., Ltd
inner mitochondrial membrane, and it has therefore been
(China). All the other chemical reagents were obtained from
suggested as an optimal candidate for mitochondria targeting
of drug conjugates or nanocarriers.29–35 The high binding
Sinopharm Chemical Reagent Co., Ltd.
affinity of TPP for the inner mitochondrial membrane allows
for the delivery of drugs or nanocarriers across the negatively
2.2 Instruments
charged mitochondrial membrane due to its highly lipophilic 1H NMR spectra were recorded on a Bruker AVANCE III HD-600
nature and positive charge.36 Conjugation of TPP with celastrol spectrometer using deuterated reagents as solvents. TEM
may therefore facilitate the mitochondrial delivery of this agent images were obtained on a JEM-2100 transmission electron
in the tumor cells, inducing ROS accumulation in a targeted microscope at an acceleration voltage of 100 kV. The UV-vis-NIR
manner. While this approach shows great promise, further absorption and fluorescence emission spectra were measured
studies are still needed as there are many regulatory barriers on a UV-2600 and Shimadzu RF5301PC spectrophotometer,
to therapeutic molecule delivery.
respectively. Hydrodynamic diameters were determined using
An important point of note is that the co-delivery of two or a Malvern Zetasizer Nano-ZS system at 25 1C. CLSM images
more anti-tumor agents simultaneously using nanocarriers has were acquired using a LSM700 laser confocal microscope.
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Flow cytometry experiments were conducted using a BD (0.77 g, 4.0 mmol), and NHS (0.35 g, 3.0 mmol) were dissolved
LSRFortessa X-20 cell analyzer.
in DCM (30 mL) under an ice-water bath and stirring for 2 h,
then TPP–NH2 (1.41 g, 3.0 mmol) was added and reacted
overnight at room temperature. Then the mixture solution
2.3 Synthesis of two site-oriented pro-drug molecules
2.3.1 Synthesis of the all-trans retinoic acid conjugate was washed with water, extracted with EA and separated by
cisplatin compound (RA–Pt). The nuclear targeting molecules column chromatography (PE : EA = 3 : 2). The obtained crude
(RA–Pt) were obtained from conjugate Pt(IV)–COOH (the syn- products were purified from ethanol and dried under vacuum
thetic procedure is shown in ESI†) and amino modified all-trans to give the desired product as a palm red solid (0.78 g, 43%).
retinoic acid (RA–NH2). At first, the all-trans retinoic acid (RA) 1H NMR (600 MHz, DMSO-d6): d (ppm) 7.84–7.70 (dt, 7H), 7.51–
was modified with amino. Briefly, RA (0.90 g, 3.0 mmol), EDCl 7.44 (dt, 5H), 7.26–7.13 (dt, 8H), 6.59 (b, 1H), 6.42–6.33 (d, 1H),
(1.15 g, 6.0 mmol), and NHS (0.52 mg, 4.5 mmol) were dissolved 5.91–5.84 (b, 1H), 2.86–2.77 (d, 4H), 2.55–1.93 (d, 13H), 1.71–
in DCM (35 mL) under an ice-water bath and stirring for 2 h, 0.54 (d, 36H). ESI-MS (m/z): 903.4 [M + H]+.
then ethylenediamine (0.36 g, 6.0 mmol) was added and reacted
overnight at room temperature. Then the mixture solution was
washed with water, extracted with DCM and separated by
column chromatography (PE : EA = 3 : 1). The desired intermediate
products were obtained after being concentrated and drying
under reduced pressure. RA–NH2 was recovered as a dark yellow
2.4 Formation of folate and cRGD modified RA–Pt and
TPP–Cet loaded nanoparticles (RA–Pt/TPP–Cet@Fc-INPs)
The designed drug carriers were prepared using a thin film
hydration method. Briefly, FA–PEG2k–DSPE (1.0 mg), cRGD–
PEG5k–PCL10k (1.0 mg), PLGA3k (10.0 mg) and lecithin (30.0 mg)
were dissolved in chloroform (40 mL) and a thin film was
formed after solvent evaporation under reduced pressure. To this
film was added 15 mL of RA–Pt aqueous solution (0.2 mg mLÀ1),
10 mL of TPP–Cet aqueous solution (0.3 mg mLÀ1), and 5 mL of
ICG aqueous solution (0.4 mg mLÀ1) and it was ultrasonically
vibrated for 8 min. The prepared nanoparticle suspension was
then centrifuged at 3000 rpm for 15 min and the supernatant
was then filtrated through a 0.22 mm cellulose acetate filter
membrane and lyophilized. The obtained solid powder was
re-dissolved in CH3OH and characterized with UV-vis spectroscopy.
1
solid (0.65 g, 63%). H NMR (600 MHz, DMSO-d6): d (ppm) 7.93
(d, 1H), 6.91 (d, 1H), 6.31–6.14 (d, 4H), 5.83 (b, 1H), 3.17–3.09
(m, 4H), 2.27 (b, 3H), 2.03–1.94 (b, 5H), 1.69–1.02 (d, 15H). ESI-MS
(m/z): 343.5 [M + H]+.
Next, the conjugation process of RA–Pt was carried out in a
similar manner with the EDCl/NHS groups as an activator.
Pt(IV)–COOH (1.07 g, 2.0 mmol), EDCl (0.77 g, 4.0 mmol), and
NHS (0.35 g, 3.0 mmol) were dissolved in DMF (30 mL) under
an ice-water bath and stirring for 2 h, then RA–NH2 (0.69 g,
2.0 mmol) was added and reacted overnight at room temperature.
The desired products were obtained after solvent evaporation
under reduced pressure and then washed with deionized water,
and ethanol in turn. The crude product was purified from
2.5 Photothermal effects and in vitro drug release
methanol and dried under vacuum to give the desired product To evaluate the photothermal conversion induced by ICG,
1
as a yellow solid (0.79 g, 46%). H NMR (600 MHz, DMSO-d6): several different concentrations of RA–Pt/TPP–Cet@Fc-INP
d (ppm) 7.99 (d, 1H), 6.93–6.88 (d, 1H), 6.31–6.15 (d, 4H), 5.81 nanoparticle suspension (0.2, 0.4, and 0.8 mg mLÀ1) were
(b, 1H), 3.29–3.16 (m, 5H), 2.91–2.54 (b, 2H), 2.47–2.20 (b, 6H), analyzed after irradiation with an 808 nm near-infrared laser
2.05–1.91 (b, 7H), 1.72–1.01 (d, 17H). ESI-MS (m/z): 858.3 [M + H]+. (1.54 W) for 5 min. Next, we investigated the thermal efficiency
2.3.2 Synthesis of the triphenylphosphine conjugate celastrol of the ICG loaded nanoparticles. The temperature variation of
compound (TPP–Cet). The mitochondrial targeting molecules the nanoparticle suspension was detected with an infrared (IR)
(TPP–Cet) were obtained from the conjugation of TPP–NH2 and thermal camera. Before the evaluation, five cycles of irradiating
celastrol with a similar amination reaction. Besides, the triphenyl- and chilling processes were tested to evaluate the photostability.
phosphine (TPP) was modified with amino. Briefly, TPP (1.29 g,
The drug release investigation of RA–Pt/TPP–Cet@Fc-INPs
3.0 mmol), EDCl (1.15 g, 6.0 mmol), and NHS (0.52 g, 4.5 mmol) was performed using a dialysis method as our previous work
were dissolved in DCM (40 mL) under an ice-water bath and described.43,44 A predetermined amount of the prepared nano-
stirring for 2 h, then ethylenediamine (0.36 g, 6.0 mmol) was particles (3 mL, 1.5 mg mLÀ1) in dialysis tubing (MW, 15 000)
added and reacted overnight at room temperature. Next, the was immersed in phosphate buffered saline (PBS, PH = 7.4, 6.8
mixture solution was washed with water, extracted with DCM and 5.5) as dissolution media. The release investigation was
and separated by column chromatography (PE : EA = 1 : 1). performed with near-infrared laser (808 nm, 1.54 W, 5 min)
The desired intermediate products were obtained after being irradiation (+, À). At designed time intervals, a certain volume
concentrated and drying under reduced pressure. TPP–NH2 was (2 mL) of the release medium was taken out and the fluores-
1
recovered as a white powder (0.74 g, 54%). H NMR (600 MHz, cence emission intensity at 425 nm under an excitation
DMSO-d6): d (ppm) 7.94–7.67 (dt, 6H), 7.58–7.39 (dt, 4H), 7.31– wavelength of 280 nm was determined by the fluorescence
7.05 (dt, 6H), 3.58–3.46 (b, 1H), 3.34–3.27 (b, 1H), 2.76–2.66 spectrometer to calculate the amount of TPP–Cet, while an
(b, 1H), 2.11–1.87 (b, 1H), 1.84–1.60 (b.1H), 1.31–1.19 (b, 2H). equal volume (2 mL) of fresh PBS was added to proceed with the
ESI-MS (m/z): 471.1 [M + H]+.
release. The amount of RA–Pt presented in the dialysate was
Next, the conjugation process of TPP–Cet was carried out calculated by ICP-AES. The release experiments were tested in
in a similar way. Briefly, celastrol (0.90 g, 2.0 mmol), EDCl triplicate, and the mean value was calculated as the final result.
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2.6 Cellular uptake study
(ICG: 38.2 mg mLÀ1, 2 mL) conjugate TPP (INPs) or not (TPP-INPs).
Finally, the cells were stained with Lysotracker Green (lysosome
staining, green color) and observed with CLSM. Next, the induced
singlet oxygen was generated by TPP–Cet to further achieve
synergistic therapy and the intracellular ROS were detected by
an oxidation-sensitive fluorescent probe (DCFH-DA).50 Similarly,
MCF-7 cells was seeded in 6-well plates and further incubated for
12 h after being treated with celastrol and ICG loaded, folate and
cRGD modified nanoparticles (Cet@Fc-INPs); TPP–Cet and ICG
loaded, folate and cRGD modified nanoparticles (TPP–Cet@
Fc-INPs); and RA–Pt, TPP–Cet and ICG loaded, folate and cRGD
modified nanoparticles (RA–Pt/TPP–Cet@Fc-INPs) (celastrol:
48.3 mg mLÀ1; ICG: 38.2 mg mLÀ1, 2 mL). Then DCFH-DA
(10.0 mM) was employed to measure the ROS by celastrol. Finally,
the fluorescence intensity of ROS production in MCF-7 cells was
acquired by a flow cytometer with excitation at 488 nm.
In breast cancer, as an infinite tumor with extremely high
incidence, the expression level of avb3/avb5 in MDA-MB-231
cells is very high, while the expression level of folate receptor in
MCF-7 cells is relatively higher.45–48 Thus, we choose these two
kinds of cells to study the targeted delivery of folate and cRGD
modified drug-loaded nanoparticles we prepared.
Human breast cancer cells (MDA-MB-231 and MCF-7) were
cultured with Dulbecco’s modified Eagle medium (DMEM)
containing 10% fetal bovine serum (FBS) and 1% penicillin–
streptomycin solution under 5% CO2 atmosphere at 37 1C. In
the CLSM study, MDA-MB-231 and MCF-7 cells were severally
seeded in a confocal culture dish (2 mL medium) at a density of
8.0 Â 104 cells per dish. After being cultured under 5% CO2
atmosphere at 37 1C for 24 h, a predesigned amount of RA–Pt/
TPP–Cet@Fc-INP nanoparticle suspension (2 mL) with an
equivalent ICG concentration of 50.0 mg mLÀ1 was added into
the cell culture medium. After further incubation for 1, 3, and
12 h, the culture medium was removed, and the cells were
washed three times with PBS. Then, the cells were stained with
Hoechst 33258 and further mounted for observations with
CLSM. In addition, MCF-7 cells treated with the prepared
nanoparticle conjugate targeted ligands (Fc-INPs) or not (INPs)
were also incubated to evaluate the receptor-mediated endocytosis.
Finally, the fluorescence intensity of the cellular uptake was
calculated using a flow cytometer and the intracellular RA–Pt
was quantitated by ICP-AES.
2.9 In vitro cytotoxicity study
To evaluate the synergistic therapy of two different pro-drug
molecules, MDA-MB-231 and MCF-7 cells were seeded in
96-well plates (100 mL medium) and incubated overnight. When
the cell confluence reached around 70%, the medium was
replaced with fresh DMEM containing different formulations
of nanoparticle suspensions with various RA–Pt concentrations
(0.1, 0.5, 1.0, 5.0, 10.0 mg mLÀ1, equivalent to Pt: 0.03, 0.17,
0.30, 1.70, 3.00 mg mLÀ1) added; the concentration of TPP–Cet
is calculated as the ratio of nanoparticles to RA–Pt. After 12 h of
incubation, the medium was replaced, and the cells were
further irradiated with an 808 nm NIR laser (1.54 W, 3 s
wellÀ1).51 After being incubated for 3 h, the cells were further
irradiated by 670 nm near-infrared light for 10 minutes and
then incubated for 9 hours. After the incubation for 24 h in
total, the standard MTT assay was performed to evaluate the
cell viability.
2.7 Nuclear targeting and quantification of RA–Pt
The experimental process of nuclear targeting evaluation was
performed as described above. MCF-7 cells were seeded in
a confocal culture dish (2 mL medium) at a density of 8.0 Â
104 cells per dish. After being cultured for 24 h (at 37 1C, 5%
CO2), RA–Pt and ICG loaded nanoparticle conjugate RA ligand
(RA–Pt@RA-INPs) or not (RA–Pt@INPs) suspensions (2 mL)
with an equivalent RA–Pt concentration of 47.0 mg mLÀ1 were
added into the confocal culture dish. After further incubation
for 6 h, the waste was removed, and the cells were washed three
times with PBS. Then the cells were stained with DAPI (nucleus
staining, blue color) and further observed with CLSM. Besides,
quantification was performed with the same procedures of cell
incubation and we also added the RA–Pt/TPP–Cet@Fc-INP
group with the same RA–Pt dosage (47.0 mg mLÀ1, 2 mL). After
been incubated for 6 h, the cells were then trypsinized and
suspended at a concentration of 5 Â 106 cells per mL for 10 min
at 4 1C in 1% Triton X-100 solution (0.1 M NaCl, 1 mM EDTA,
10 mM Tris buffer, pH 7.4). The suspension was further
2.10 Animal experiments and tumor models
Female BABL/c nude mice (each 18–22 g) were purchased from
Shanghai Lingchang Biotechnology Co. LTD (Shanghai, China)
and raised under the principles of care and use of laboratory
animals. The MCF-7 tumor models were generated by subcu-
taneous injection of 100 mL cell suspension (in saline medium,
1 Â 107 cells mLÀ1) into the right flank of each mouse.
After incubation for 12 days, the mice were used for further
experiments when the tumor size had grown to B60 mm3.
All animal experiments were performed under the guidance
approved by the Laboratory Animal Ethics Committee at the
School of Medicine, Southeast University (Nanjing, China).
centrifuged (15 min, 1000 rpm), and the pellets (nuclei) were 2.11 In vivo imaging and biodistribution analysis
separated from the supernatant (cytosol).49 Finally, the collected
precipitation was washed and determined by ICP-AES.
The nude mice were randomly divided into two groups (three
per group). Mice in group 1 as a control were injected with
200 mL of ICG loaded nanoparticles without targeted ligands
(INPs) via their tail veins. Mice in group 2 were injected with
2.8 Mitochondria targeting and ROS generation in cells
All the experimental procedures in this section for observation 200 mL of ICG loaded folate and cRGD conjugate NPs (Fc-INPs,
were consistent with those in the nuclear targeting investigation. both containing 50 mg mLÀ1 ICG) via their tail veins. Images
Specifically, the cells were incubated with ICG loaded nanoparticles and FL quantitative analysis of ICG were taken at 0, 1, 3, 6, 12,
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and 24 h after injection using the ex/in vivo imaging system 3. Results and discussion
(CRI maestro, USA) with a 704 nm excitation wavelength and a
735 nm filter to collect the FL signals of ICG. The mice were
3.1 Synthesis and characterization
To obtain the subcellular targeting effect, two novel pro-drug
molecules RA–Pt and TPP–Cet were designed and synthesized
in the present work. The synthesis route is shown in Fig. S1
(ESI†). The major amination reaction was controlled with the
EDCl/NHS group as an activator, which showed high product
yield. The chemical structures of the two pro-drug molecules
and the intermediates were confirmed by standard spectro-
scopic techniques including 1H NMR and ESI-MS, and the
results are exhibited in Fig. S2–S5 (ESI†). The chemical dis-
placement of each proton exactly appeared on the 1H NMR data
images and the molecular ion peaks in ESI-MS results were
consistent with the designed compounds, which have fully
confirmed the successful synthesis of RA–Pt and TPP–Cet.
sacrificed 24 h after injection and organs including the heart,
liver, spleen, lung, kidneys and tumor were collected for
imaging and quantitative biodistribution analysis.
2.12 In vivo antitumor efficacy studies in the breast cancer
model
MCF-7 tumor-bearing mice were divided into 5 groups to
determine the tumor growth rate. For the treatment groups
(n = 6 per group), mice bearing MCF-7 tumors were injected with
200 mL of PBS (group 1), free RA–Pt (group 2), RA–Pt/TPP–Cet and
ICG loaded nanoparticles (RA–Pt/TPP–Cet@INPs, group 3), and
RA–Pt/TPP–Cet and ICG loaded, folate and cRGD modified
nanoparticles (RA–Pt/TPP–Cet@Fc-INPs, group 4) (containing
150 mg mLÀ1 of RA–Pt solution, equivalent to Pt: 0.52 mg kgÀ1
per mouse; Cet: 0.75 mg kgÀ1 per mouse) via their tail veins and
3.2 Self-assembly behavior
group 5 was RA–Pt/TPP–Cet@Fc-INPs irradiated by an 808 nm Next, the RA–Pt and TPP–Cet dual-loaded functional nano-
NIR laser (1.54 W, 5 min) after incubation for 48 h. Each group particles (RA–Pt/TPP–Cet@Fc-INPs) were prepared and the assembly
was injected a total of five times (once every 3 days). For the instruction are shown in Scheme 1. The FA–PEG2k–DSPE, and
control group, mice were treated with the same volume of cRGD–PEG5k–PCL10k chains were synthesized and self-assembled
PBS without NIR irradiation. The tumor sizes were measured by as our previous work described.52 We have tested various propor-
an electronic digital caliper every three days after treatment and tional combinations of FA–PEG2k–DSPE, cRGD–PEG5k–PCL10k
,
calculated as follows: tumor volume (TV) = 0.5 Â (tumor length) Â lecithin, and PLGA3000 to obtain excellent overall performance
(tumor width)2. The relative tumor volumes (RTV) were normal- of drug carriers (Table S1, ESI†). The DLS results (Fig. 1B) showed
ized to their initial sizes. To further detect the effect of enhanced a pleasing particle size of 99.65 nm with a harmonious poly-
antitumor therapy in vivo, the tumors, livers and kidneys at 24 d dispersity of 0.261, while it was about 131.20 nm (Fig. 1A) before
after treatment were stained with hematoxylin and eosin.
conjugating with targeted ligands. We further verified that
2.12.1 Statistical analysis. Data are expressed as mean Æ SD. the particle size was only 79.88 nm (Fig. S6, ESI†) before
The differences among groups were determined using one-way encapsulating drug molecules. According to reports,53,54 if the
ANOVA analysis followed by Tukey’s post-test; a P value of particle size of the nanoparticles is too small or too large they
o0.05 was considered significant. Statistical significance was can easily be filtered by the kidney or swallowed by Kupffer cells
defined as *P o 0.05, **P o 0.01, and n.s. P 4 0.05.
in the liver. In addition, nanoparticles with a particle size of
Scheme 1 Self-assembly of folate and cRGD dual-targeted polymeric nanoparticles loaded with RA–Pt and TPP–Cet.
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Fig. 1 (A) Size distribution of RA–Pt and TPP–Cet loaded nanoparticles without folate and cRGD ligands (RA–Pt/TPP–Cet@INPs). (B) Size distribution of
RA–Pt and TPP–Cet loaded targeted nanoparticles (RA–Pt/TPP–Cet@Fc-INPs). (C) UV-vis-NIR spectra of ICG in methanol, free TPP–Cet, free RA–Pt,
Fc-NPs and RA–Pt/TPP–Cet@Fc-INP suspensions in phosphate buffer (pH = 7.4). (D) TEM images of RA–Pt/TPP–Cet@Fc-INPs in PBS (pH = 7.4) at 25 1C.
(E) TEM images of RA–Pt/TPP–Cet@Fc-INPs in PBS (pH = 7.4) with NIR irradiation for 3 min. (F) The drug retention profile and particle size variation under
normal conditions during four weeks (PBS or 0.9% NaCl).
about 100 nm have better drug loading capacity and they the obtained nanoparticles showed acceptable stability in blood
will not be cleared by the body during blood circulation.55,56 circulation, avoiding the revealing of encapsulated drugs over a
Thus, the present nanoparticles with a particle size of 99.65 nm period of time.
showed great potential in nanomedicines. The results further
We further use UV-vis spectroscopy to determine the RA–Pt
exhibited the z potential on the surface of NPs decreased and TPP–Cet loading amounts of the prepared nanoparticles.
from À24.93 to À49.97 mV after conjugating NPs with folate The absorption spectra of free ICG, free TPP–Cet, free RA–Pt,
and cRGD.
folate and cRGD modified hollow nanoparticles (Fc-NPs), and
The nanoparticles without targeted ligands showed poor RA–Pt/TPP–Cet@Fc-INPs are shown in Fig. 1C. The spectrum
dispersion with a polydispersity index of 0.440, which is poten- did not show obvious characteristic absorbance for RA–Pt,
tially due to the imbalance of the potential on the surface of the while the free ICG, free TPP–Cet and Fc-NPs had maximum
nanoparticles. The introduction of the targeted ligands well characteristic absorption peaks around 760, 409, and 259 nm in
regulates the balance of the nanoparticles’ surface potential in the UV spectrum, respectively. The chemical shift of protons at
the aqueous solution, resulting in a good dispersion pattern. each position of coupled amphiphilic copolymer chains was
The TEM images further exhibited the morphological charac- characterized by 1H NMR as shown in Fig. S7 and S8 (ESI†).
teristics of the present folate and cRGD modified nanoparticles The characteristic peaks of the benzene ring CH on cRGD at
under normal conditions (Fig. 1D). The individual dispersion d 7.15–8.11 ppm obviously appeared in the picture of cRGD–
form was still clearly visible though the relatively rough surface PEG5k–PCL10k at 7.16–7.89 ppm while the PEG5k–PCL10k
may be due to conjugation with targeted ligands. The depoly- showed nothing in this region, which confirmed the conjuga-
merized state of the nanoparticles when exposed to the tion of cRGD. The characteristic peaks of the folate at d
NIR laser indicated the prepared nanoparticles showed highly 4.55–11.48 ppm obviously appeared in the picture while the
controllable release (Fig. 1E).
HOOC–PEG2k–DSPE showed nothing in this region, which
To study the influence of morphological characteristics on confirmed the conjugation of folate. Therefore, the obtained
the colloidal stability of the prepared nanoparticles, we inves- results indicated that FA and cRGD had been successfully
tigated the drug release profile and particle size variation under conjugated with NPs.
normal conditions during four weeks (Fig. 1F). The results
indicated the release rate of encapsulated drugs during four
3.3 Photothermal effects and drug release
weeks was acceptable (RA–Pt, 27.66%; TPP–Cet, 27.59%) while To evaluate the impact of ICG mediated photothermal conversion
the particle size increased from 99.65 nm to 153.40 and on drug release, the photothermal conversion performance of
172.80 nm (in 0.9% NaCl and pH = 7.4 PBS, respectively). Thus, RA–Pt/TPP–Cet@Fc-INPs was investigated and the results are
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Fig. 2 (A) Heating curves of phosphate buffer (pH = 7.4), hollow nanoparticles (Fc-NPs, 0.8 mg mLÀ1), and different concentrations of RA–Pt/TPP–
Cet@Fc-INPs (0.2, 0.4, and 0.8 mg mLÀ1) suspended in phosphate buffer (pH = 7.4) under 808 nm laser irradiation at a power of 1.54 W. (B) Temperature
increments of the RA–Pt/TPP–Cet@Fc-INP suspension (0.5 mg mLÀ1) under 808 nm laser irradiation at a power of 1.54 W for five cycles (5 min of
irradiation for each cycle). NIR-dependent release of drugs (C, RA–Pt; D, TPP–Cet) from the RA–Pt/TPP–Cet@Fc-INPs at different pH (7.4, 6.8, and 5.5).
shown in Fig. 2. As the results exhibited (Fig. 2A), neither the RA–Pt (or TPP–Cet) was liberated from the NPs at pH = 7.4
PBS nor the hollow nanoparticles (Fc-NPs) showed obvious without irradiation. The drug release profile implied that the
temperature change and the inner temperature was still lower drugs can efficiently be released from the RA–Pt/TPP–Cet@
than 35 1C. Next, the induced temperature of RA–Pt/TPP–Cet@ Fc-INPs via NIR irradiation. In addition, the water solubility of
Fc-INP suspensions at various concentrations (0.2, 0.4, and RA–Pt is better than that of TPP–Cet, in this way, the release
0.8 mg mLÀ1) was recorded and the results showed a typical rate of RA–Pt is slightly faster than TPP–Cet.
concentration-dependent temperature increase. In addition,
The polymer and lecithin mixture enhanced the stability of
the fastest temperature increase was observed at the concen- the lipid bilayer structure of the prepared nanoparticles, which
tration of 0.8 mg mLÀ1, and the temperature increased by shows a compact arrangement in the normal environment.
69.3 1C after exposure to the NIR laser for 5 min. The induced However, it depolymerized to a looser structure rapidly due to
hyperthermia can effectively accelerate drug release or even the intracellular acidic conditions. In addition, the lipid bilayer
lead to tumor ablation. In addition, the light stability of RA–Pt/ structure of the nanoparticles suffered more heavily destruction
TPP–Cet@Fc-INPs was also tested and the results showed the and further collapsed while increasing the temperature by NIR
induced temperature decreased by only 1.4 1C, thus it exhibited irradiation.
excellent photostability of the present nanoparticles (Fig. 2B).
Based on the excellent photostability of RA–Pt/TPP–Cet@
3.4 Intracellular uptake
Fc-INPs, they also could be used as a suitable photothermal The crucial factor for tumor treatment is to efficiently delivery
agent for sustained photothermal therapy of tumors.
drug molecules into the cytoplasm or nucleus. It is well known
Considering the acid condition inside the tumor cells that folate receptor (FR), a glycosylphosphatidylinositol (GPI)-
accelerates the degradation of the shell structure of drug anchored membrane glycoprotein, is frequently overexpressed
carriers, RA–Pt/TPP–Cet@Fc-INPs were suspended in deionized on the surface of a variety of tumor cells. In addition, the cyclic
water (2.0 mg mLÀ1) and then immersed in PBS with different Arg-Gly-Asp (cRGD) peptide ligand is able to specifically adhere
pH values. Furthermore, 4.70% of RA–Pt and 4.83% of TPP–Cet to avb3/avb5 integrins, which are also overexpressed on various
in the prepared nanoparticles was released in 72 h (over tumor cells and the surface of tumoral endothelial cells.57 The
99.00%) in the presence of a 1.54 W NIR laser (Fig. 2C enhanced cellular uptake of folate and cRGD modified nano-
and D). In contrast, only about 12.55% (or 11.41%) of loaded particles was confirmed by confocal laser scanning microscopy
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(CLSM). As expected, FA and cRGD modified nanoparticles folate and cRGD ligands (Fc-INPs) or not (INPs) for 6 h and the
were significantly internalized by both tumor cells (Fig. 3A), results are shown in Fig. S9 (ESI†). As expected, about 1.53-fold
and the cellular internalization of the nanoparticles showed an higher fluorescence intensity was found in Fc-INP treated
obvious time-dependent increase. MCF-7 cells treated with the MCF-7 cells than that in the cells treated with INPs. To a certain
prepared nanoparticles exhibited stronger fluorescence inten- extent, the cellular uptake amount of the nanoparticles was
sity than that in the MDA-MB-231 cells after being incubated for accelerated due to the specific interaction between folate,
12 h. The fluorescence signals mainly gathered surrounding cRGD and their receptors. According to reports, FA modifica-
the cell nuclei, indicating the prepared nanoparticles were tion can strongly accelerate the nanoparticles although it
entrapped inside the lysosome vesicles.58
insignificantly increased the total amount of nanoparticles
The flow cytometric fluorescent quantitation further confirmed entering the cells.59 Thus, our obtained results were consistent
that FA and cRGD dual modification significantly accelerated with the conclusion.
the internalization of the nanoparticles due to the conjugated
3.5 RA potentiates the nuclear transport of RA–Pt
targeted ligands. Fig. 3B showed the fluorescent quantitation of
MDA-MB-231 and MCF-7 cells at three time intervals (1 h, 3 h, and
12 h). As exhibited, about 1.11-fold higher fluorescence intensity
was found in MCF-7 cells than that detected in MDA-MB-231 cells
when incubating for 3 h, while the fluorescence intensity in both
cells showed no significant difference (FL intensity rate = 1 : 1.13)
after incubation for 12 h. We further quantitated the total amount
of RA–Pt entering the cells and the results are consistent with the
data of FL intensity cumulated in the cells (Fig. 3C).
For the study of nuclear-targeted delivery, we calculated the
total amount of RA–Pt entering the nuclear and the results are
shown in Fig. 4. Compared to the RA–Pt@INP group (without
RA targeting), the red fluorescence signals detected in the
nucleus were much stronger in the RA–Pt@RA-INP group
(Fig. 4A). On account of the uses of ICGs (one was for tracing
drugs), the accumulated amount of RA–Pt was consistent
with the fluorescence intensity. Thus, RA also promoted the
localization of nanoparticles in the nucleus. Subsequently,
we quantified the RA–Pt content in the nucleus (Fig. 4B).
Compared with the total amount of RA–Pt (13.48 mg) in MCF-7
For quantitative analysis of the cellular uptake amount of
receptor-mediated endocytosis, MCF-7 cells were incubated in
the medium with ICG loaded nanoparticles conjugated with
Fig. 3 Intracellular uptake of RA–Pt@Fc-INPs. (A) CLSM images of MDA-MB-231 and MCF-7 cells incubated with the nanoparticles for different
periods of time at an equivalent ICG concentration of 50 mg mLÀ1 (scale bar, 40 mm). (B) Flow cytometric analysis of the ICG fluorescence intensity in
MDA-MB-231 and MCF-7 cells. (C) Quantitative uptake analysis of RA–Pt in MDA-MB-231 and MCF-7 cells using the ICP-AES instrument.
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Fig. 4 (A) Internalization of the ICG-labeled targeted nanoparticles (RA–Pt@RA-INPs) or nanoparticles without RA ligands (RA–Pt@INPs) (equivalent to
47.0 mg mLÀ1 RA–Pt) by MCF-7 cells for 6 h, analyzed by CLSM (scale bar, 40 mm). (B) Quantitative measurement of the RA–Pt content in the nucleus after
incubating MCF-7 cells for 6 h with RA–Pt loaded nanoparticles (equivalent to 47.0 mg mLÀ1 RA–Pt) in the presence or absence of RA. Data are presented
as the mean Æ SD (n = 3, *P o 0.05, **P o 0.01).
cells when treating with RA–Pt@INPs, the cellular uptake amount analyze the intracellular ROS (Fig. 6). According to reports,
in the RA–Pt@RA-INP group increased to 67.73 mg in 6 h, which cytotoxic ROS can damage the mitochondria in cells, resulting
exhibited an infusive cellular uptake efficiency. The amount in cell death.60 The ROS generated in MCF-7 cells when treating
of accumulated RA–Pt in the nucleus in the RA-INP group was with celastrol and ICG loaded nanopaticles (Cet@Fc-INPs),
5.02-fold that in the INP group (P o 0.01). Likewise, the amount TPP–Cet and ICG loaded particles (TPP–Cet@Fc-INPs) and
of accumulated RA–Pt in MCF-7 cells when treating with RA–Pt/TPP–Cet@Fc-INPs, were evaluated by using a DCFH-DA
RA–Pt/TPP–Cet@Fc-INPs increased to 86.97 mg, which reaches probe. The MCF-7 cells were treated with DCFH-DA as the
92.52% of the total input. Thus, RA enabled cisplatin to deliver control group. As the results exhibited, the fluorescence inten-
from cytosol to the nucleus more efficiently and enhanced the sities of positive cells increased to 63.1% after being treated
accumulation in the nucleus.
with Cet@Fc-INPs. Moreover, the fluorescence intensities
of positive cells increased to 79.0% when incubating with
TPP–Cet@Fc-INPs, while it reached 83.2% in the RA–Pt/TPP–
Cet@Fc-INP group. Compared to single TPP–Cet loaded nano-
particles, the RA–Pt and TPP–Cet dual loaded nanoparticles
showed stronger cytotoxicity and thus lead to more ROS
accumulation. The results further highlight the synergistic
anti-cancer therapy as compared with single therapy. In addition,
the obtained results highlighted the TPP mediated mitochondrial
targeting and the subsequent ROS accumulation in
mitochondria.
3.6 Mitochondria targeting and ROS generation in cells
Similarly, we further evaluated the role of TPP, a mitochondria-
targeting molecule. A commonly used mitochondrial dye,
Mito-Tracker Green (MTG), was employed to label mitochon-
dria for the imaging observation. As exhibited in Fig. 5A, a
much stronger fluorescence signal was found in MCF-7 cells in
the TPP-INP group than that in the INP group for 6 h. The flow
cytometric fluorescent quantitation further confirmed that the
TPP modification significantly accelerated the internalization
of the nanoparticles due to the great binding affinity to
the inner mitochondrial membranes (Fig. 5B). Fig. 5C
3.7 Cytotoxicity in vitro
demonstrated the mitochondria-targeted delivery at higher The cytotoxicity is the primary consideration when designing a
resolution. The obtained results confirmed that the TPP novel nano-therapeutic agent. Before the experiment, the IC50
mediated mitochondria-targeting could effectively enhance the (mmol LÀ1) values for MDA-MB-231, and MCF-7 cell lines were
accumulation in mitochondria.
investigated and the results are shown in Table S2 (ESI†).
To calculate the amount of singlet oxygen produced by As expected, both pro-drug molecules (RA–Pt and TPP–Cet)
celastrol, the flow cytometer was employed to quantitative showed enhanced toxicity to selected cell lines when compared
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Fig. 5 (A) Internalization of the ICG-labeled targeted nanoparticles (TPP-INPs) or nanoparticles without TPP ligands (INPs) (equivalent to 50.0 mg mLÀ1
ICG) by MCF-7 cells for 6 h, analyzed by CLSM (scale bar, 40 mm). (B) Flow cytometric analyses of MCF-7 cells after 6 h of incubation with ICG-loaded
nanoparticles (equivalent to 50 mg mLÀ1 ICG) in the presence or absence of TPP. Data are presented as the mean Æ SD (n = 3, *P o 0.05, **P o 0.01).
(C) Internalization of the ICG-labeled targeted nanoparticles (TPP-INPs) (equivalent to 50.0 mg mLÀ1 ICG) by MCF-7 cells for 6 h, analyzed by CLSM
(scale bar, 20 mm).
TPP–Cet dual loaded nanoparticles without targeted ligands
(RA–Pt/TPP–Cet@INPs) or even RA–Pt/TPP–Cet@Fc-INPs without
NIR irradiation at a RA–Pt concentration below 1.0 mg mLÀ1
.
However, the cell viability of the RA–Pt/TPP–Cet@Fc-INPs with
added NIR irradiation group decreased obviously, and that
treated with blank Fc-NPs showed little difference as the
RA–Pt concentration reached 10.0 mg mLÀ1. Moreover, the
obtained results showed that the cell viability of RA–Pt/TPP–
Cet@INPs (10.0 mg mLÀ1) reached 28.86%, which was 25.32%
lower than that in the free RA–Pt group (54.18%), showing
the expected synergistic effect. To compare the cell-killing
performance of RA–Pt/TPP–Cet@Fc-INPs, the MCF-7 cells,
which overexpress the folate receptor, were further evaluated
and the results are shown in Fig. 7B.
As shown in Fig. 7B, the low concentration (0.1 mg mLÀ1
)
of RA–Pt did not show a remarkable effect on the viability of
MCF-7 cells as compared with the free RA–Pt group, RA–Pt/
TPP–Cet@INP group or RA–Pt/TPP–Cet@Fc-INP without NIR
irradiation group. However, after irradiation with the NIR laser
(808 nm, 1.54 W), the cell viability of RA–Pt/TPP–Cet@Fc-INPs
(1.0 mg mLÀ1) reduced from 56.43% to 38.73%, and the cell
viability of RA–Pt/TPP–Cet@Fc-INPs (5.0 mg mLÀ1) further
reduced to 22.41% after being exposed to the NIR laser.
Besides, simultaneous incubation with RA–Pt and TPP–Cet
(RA–Pt/TPP–Cet@INPs, 10.0 mg mLÀ1, 26.53%) had much
Fig. 6 The fluorescence intensities of DCFH-DA in MCF-7 cells after
being treated with Cet@Fc-INPs, TPP–Cet@Fc-INPs and RA–Pt/TPP–
Cet@Fc-INPs (48.0 mg mLÀ1) for 12 h; the medium was used as the control
group.
with single molecules without modification. Then the cytotoxi- better inhibitory effects on cells than that in the single drug
city of RA–Pt/TPP–Cet@Fc-INPs was evaluated in MDA-MB-231 group (free RA–Pt, 10.0 mg mLÀ1, 48.80%), further confirming
and MCF-7 cells using standard MTT assay. As shown in Fig. 7A, the expected synergistic effect. Specifically, the inhibition
the MDA-MB-231 cells treated with RA–Pt/TPP–Cet@Fc-INPs and rate of MCF-7 cells incubated with RA–Pt/TPP–Cet@Fc-INPs
further treated with NIR irradiation did not show a significant (10.0 mg mLÀ1) with added NIR irradiation for 5 min reached
difference in cell viability when compared with the control group, 96.97%, which showed the great potential as a new type of
hollow nanoparticles (blank Fc-NPs), free RA–Pt, RA–Pt and NIR-responsive drug release for cancer therapy.
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Fig. 7 Quantitative evaluation of cell viability for MDA-MB-231 cells (A) and MCF-7 cells (B) treated with PBS, blank Fc-INPs, free RA–Pt, RA–Pt/TPP–
Cet@INPs, and RA–Pt/TPP–Cet@Fc-INPs with NIR irradiation (+, À) for 24 h. Error bars were based on the standard error of the mean (n = 3). N.S.:
no significance; *P o 0.05; ** P o 0.01 based on Student’s t test.
Due to the dual targeting effect of folate and cRGD, the drug different antitumor agents can effectively enhance the thera-
carriers can be internalized in large quantities by tumor cells. peutic efficiency and our study highlighted the synergistic anti-
The obtained results indicated that the co-delivery of two cancer therapy. In addition, according to reports,61,62 the range
Fig. 8 (A) In vivo fluorescence imaging of subcutaneous MCF-7 breast tumor-bearing nude mice after intravenous injection of nanoparticles. (B) Images
of dissected organs of mice bearing subcutaneous MCF-7 breast tumors sacrificed 24 h after intravenous injection of nanoparticles. (C) Flow cytometric
analysis of the ICG fluorescence intensity in MCF-7 breast tumors incubated with INPs or Fc-INPs for 24 h. Data are presented as the mean Æ SD
(n = 3, *p o 0.05, **p o 0.01).
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of temperatures within which the drug-delivery system is
Fluorescence imaging in vivo was further performed to
activated, should be keep between 37 and 42 1C, because tempera- investigate the nanoparticle distribution in various organs.
tures beyond this range will cause protein denaturation or function The distributions of INPs or Fc-INPs in major organs and
disruption. In the current study, the prepared blank nanoparticles tumors 24 h post-injection are shown in Fig. 8B. The fluores-
did not cause significant cytostatic effects under NIR irradiation, cence signals were easily observed in the tumor site of the mice
indicating that the prepared nanoparticles did not induce high treated with Fc-INPs, while much weaker signals were collected
internal temperatures under current NIR irradiation conditions. In in the INP group. Moreover, the fluorescence signals of the
this way, the designed nanopharmaceutical system does not harm Fc-INP group in the five organs (heart, liver, spleen, lung, and
normal cells and shows great potential for application.
kidney) were hardly observed except those in the liver and
kidney. In addition, the fluorescence signals accumulated in
the liver were much weaker when treating with Fc-INPs. Since
the prepared nanoparticles conjugate with folate and cRGD
3.8 Drug delivery and in vivo imaging
On the basis of the in vitro cytotoxicity results, we further ligands, which have greatly enhanced the specificity of tumor
applied the RA–Pt/TPP–Cet@Fc-INPs to mice for ICG imaging cells. Therefore, the fluorescence signals in the tumor were
in vivo. The fluorescence signals were recorded at different time particularly significant in the Fc-INP group. The in vivo fluores-
intervals (Fig. 8A).
cence signals of the tumors 24 h post-injection of the INP or
As shown in Fig. 8A, the fluorescence signals of the tumor Fc-INP solutions were quantified and the results indicated
site in the Fc-INP group were obviously stronger at any time the obvious difference (Fig. 8C). Thus, the prepared Fc-INPs
post-injection ranging from 1 h to 24 h. Moreover, the fluores- showed enhanced accumulation in the tumor site due to the
cence signals of the INP group were majorly distributed in the conjugation of folate and cRGD.
liver but less in the tumor during 1 h after injection, while the
fluorescence signals of the Fc-INP group homogeneously distri-
3.9 In vivo combinational therapeutic efficacy
bute in the whole body. We have further noticed that the We further applied the RA–Pt/TPP–Cet@Fc-INPs to mice for
fluorescence signals in the tumor (Fc-INP group) were gradually enhanced antitumor therapy in vivo. According to the results of
strengthened with the time increasing (0, 1, 3, 6, 12 and 24 h) in vivo FL imaging of ICG, synergistic therapy was performed at
and reached a maximum in 24 h after Fc-INP solution injection the time point of 24 h post-injection.
while these signals in the INP group completely disappeared in
As shown in Fig. 9, the control, or free RA–Pt group, showed
24 h after injection. Overall, the Fc-INP group showed much rapid tumor growth, indicating that the tumor growth was
stronger fluorescence in the tumor site than that of the INP affected by the biocompatible carrier. The tumor suppression
group, confirming the specific targeting of Fc-INPs in vivo.
of RA–Pt/TPP–Cet@INPs or the RA–Pt/TPP–Cet@Fc-INP group
Fig. 9 Representative photographs of mice bearing MCF-7 breast tumors treated with PBS solution (group 1), free RA–Pt (group 2), RA–Pt/TPP–
Cet@INPs (group 3), RA–Pt/TPP–Cet@Fc-INPs (group 4), and RA–Pt/TPP–Cet@Fc-INPs + NIR (group 5).
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Fig. 10 In vivo antitumor activity on solid tumors using female BABL/c nude mice based MCF-7 breast tumor models. (A) Scheme of combined therapy
of live MCF-7 breast tumor-bearing nude mice treated with RA–Pt/TPP–Cet@Fc-INP suspensions under 808 nm laser irradiation. (B) Relative tumor
volume growth curves in different groups of tumor-bearing mice after various treatments (were normalized to their initial sizes). (C) Tumor weight
growth curves in different groups of tumor-bearing mice after various treatments. Asterisk indicates p o 0.01. Error bars represent the standard deviation
of six mice per group. (D) The tumor weight in different groups of tumor-bearing mice after incubation for 28 days. Data are presented as the mean Æ SD
(n = 6, *p o 0.05, **p o 0.01 vs. PBS group).
without NIR irradiation did not show a remarkable difference,
while the RA–Pt/TPP–Cet@Fc-INP group with NIR irrradiation
caused significant inhibition after 24 days (Fig. 9). Additionally,
during the 24 days of treatment, the tumor volume increased
significantly in the PBS group (Fig. 10B), and the RA–Pt/TPP–
Cet@Fc-INP group exhibited an 81.5% tumor inhibition rate
after 24 days (Fig. 10D). During the 3 days of treatment, no
significant body weight change was observed in all groups
(Fig. 10C), showing that RA–Pt/TPP–Cet@Fc-INPs without NIR
irradiation had no significant side effects on the treated mice
in vivo. During the treatment period, the body weight of mice in
group 1 (the control group) increased by 3.9 g, while that in
groups 2, 3, and 4 also increased by 3.5 g, 2.6 g, and 2.1 g,
respectively. However, the body weight of the RA–Pt/TPP–
Cet@Fc-INP group with NIR (group 5) only increased by 1.9 g,
considering that the tumor volume of the mice decreased from
group 1 to group 5 in turn, indicating that the mice in group 5
were increased in normal body weight and the prepared nano-
particles showed a good anti-tumor effect. Therefore, the
obtained results highlighted the NIR-responsive drug release
and great potential in application for tumor inhibition.
Next, the tumors and normal organs were analyzed by H&E
staining to further investigate the potential toxicity of RA–Pt/
Fig. 11 H&E stained images of major organs from mice treated with PBS solution
TPP–Cet@Fc-INPs. The PBS group (group 1) clearly showed
tumor necrosis and destroyed blood vessels, while the free Cet@Fc-INPs (group 4), and RA–Pt/TPP–Cet@Fc-INPs + NIR (group 5).
(group 1), free RA–Pt (group 2), RA–Pt/TPP–Cet@INPs (group 3), RA–Pt/TPP–
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5 J. Kreuter, V. E. Petrov, D. A. Kharkevich and R. N. Alyautdin,
J. Controlled Release, 1997, 49, 81–87.
6 F. Alexis, E. Pridgen, L. K. Molnar and O. C. Farokhzad, Mol.
Pharmaceutics, 2008, 5, 505–515.
7 N. Toub, C. Malvy, E. Fattal and P. Couvreur, Biomed.
Pharmacother., 2006, 60, 607–620.
8 M. Zeira, P. F. Tosi, Y. Mouneimne, J. Lazarte, L. Sneed,
D. J. Volsky and C. Nicolau, Proc. Natl. Acad. Sci. U. S. A.,
1991, 88, 4409–4413.
9 S.-I. Hamano, S. Yamashita, M. Tanaka, S. Yoshinari,
M. Minamitani and Y. Eto, J. Pediatr., 2006, 148, 485–488.
10 H. Padh and N. M. Sakhrani, Drug Des., Dev. Ther., 2013, 7,
585–599.
RA–Pt group (group 2) induced moderate levels of tumor
necrosis (Fig. 11). By contrast, no significant tumor destruction
was observed in the other three control groups. The results
indicated that compared with the control group (group 1 and 2),
the morphologies of the tumor cells in nanoparticle-administered
groups (group 3, 4 and 5) were better, and the blood vessels and
other functions were restored to different degrees.
The H&E images of major detoxification organs (liver and
kidney) from MCF-7 tumor-bearing mice showed that there was
no obvious organ damage or inflammation lesions induced
by the applied synergistic therapy in all groups (Fig. 11). These
obtained results clearly demonstrated that the RA–Pt/TPP–
Cet@Fc-INPs have high biocompatibility and specificity, and
can be considered as a nanotheranostic agent for imaging-
guided synergistic therapy of cancer.
11 A. R. Maity and D. Stepensky, Int. J. Pharm., 2015, 496, 268–274.
¨
12 L. Rajendran, H.-J. Knolker and K. Simons, Nat. Rev. Drug
Discovery, 2010, 9, 29–42.
13 S. Balaz, Chem. Rev., 2009, 109, 1793–1899.
14 G. G. D’Souza and V. Weissig, Expert Opin. Drug Delivery,
2009, 6, 1135–1148.
15 V. Escriou, M. Carriere, D. Scherman and P. Wils, Adv. Drug
Delivery Rev., 2003, 55, 295–306.
16 J. Zhong, L. Li, X. Zhu, S. Guan, Q. Yang, Z. Zhou, Z. Zhang
and Y. Huang, Biomaterials, 2015, 65, 43–55.
17 R. J. Sessler and N. Noy, Mol. Cell, 2005, 18, 343–353.
18 A. Majumdar, A. D. Petrescu, Y. Xiong and N. Noy, J. Biol.
Chem., 2011, 286, 42749–42757.
19 A. A. Argyriou, P. Polychronopoulos, G. Iconomou, E. Chroni,
P. Haralabos and H. P. Kalofonos, Cancer Treat. Rev., 2008, 34,
368–377.
4. Conclusion
In summary, we successfully developed a biocompatible, folate
and cRGD dual-targeted nanotheranostic agent for synergistic
cancer therapy. The prepared RA–Pt/TPP–Cet@Fc-INPs showed
low cytotoxicity, a high capacity for RA–Pt and TPP–Cet loading,
and the ability to target MCF-7 cells or to induce ROS. The RA
and TPP mediated subcellular targeting, and ICG imaging-
guided synergistic therapy using RA–Pt/TPP–Cet@Fc-INPs
induced significant tumor cell death in vitro, and efficiently
inhibited the MCF-7 tumor growth in vivo. These results
demonstrate the potential theranostic efficacy of RA–Pt/TPP–
Cet@Fc-INPs for the synergistic treatment of solid tumors, thus
offering a new approach for multistage targeted-delivery and
synergistic therapy based on functional nanomedicines.
20 S. R. McWhinney, R. M. Goldberg and H. L. McLeod, Mol.
Cancer Ther., 2009, 8, 10–16.
21 G. Daugaard and U. Abildgaard, Cancer Chemother. Pharmacol.,
1989, 25, 1–9.
22 S. Kotecha, Biochim. Biophys. Acta, Mol. Basis Dis., 2010,
1802, 649–658.
Conflicts of interest
23 Y. Yamada and H. Harashima, Adv. Drug Delivery Rev., 2008,
60, 1439–1462.
There are no conflicts to declare.
24 P. I. Moreira, M. S. Santos and C. R. Oliveira, Antioxid. Redox
Signaling, 2007, 9, 1621–1630.
Acknowledgements
25 T. Finkel, Nat. Rev. Mol. Cell Biol., 2006, 6, 971.
26 G. Chen, X. Zhang, M. Zhao, Y. Wang, X. Cheng, D. Wang,
Y. J. Xu, Z. Y. Du and X. D. Yu, BMC Cancer, 2011, 11, 170.
27 J. H. Lee, Y. S. Won, K. H. Park, M. K. Lee, H. Tachibana,
K. Yamada and K. I. Seo, Apoptosis, 2012, 17, 1275–1286.
28 H. Mou, Y. Zheng, P. Zhao, H. Bao, W. Fang and N. Xu,
Toxicol. In Vitro, 2011, 25, 1027–1032.
We are highly grateful to the International S&T Cooperation
Program of China (No. 2015DFG42240), the financial support
from the National Natural Science Foundation of China (Grant
No. 21628101 and 21371031), The Technological Program
Foundation of Jiangsu Province (No. BY2015070-09), and the
Priority Academic Program Development (PAPD) of Jiangsu
Higher Education Institutions.
29 H. Padh and N. M. Sakhrani, Drug Des., Dev. Ther., 2013, 7,
585–599.
30 G. G. D’Souza, M. A. Wagle, V. Saxena and A. Shah, Biochim.
Biophys. Acta, Bioenerg., 2011, 1807, 689–696.
References
1 N. A. Ochekpe, P. O. Olorunfemi and N. C. Ngwuluka, Trop. 31 J. Zhou, W.-Y. Zhao, X. Ma, R.-J. Ju, X.-Y. Li, N. Li, M.-G. Sun,
J. Pharm. Res., 2009, 8, 377–390.
J.-F. Shi, C.-X. Zhang and W.-L. Lu, Biomaterials, 2013, 34,
3626–3638.
32 D. Y. Cho, H. Cho, K. Kwon, M. Yu, E. Lee, K. M. Huh,
D. H. Lee and H. C. Kang, Adv. Funct. Mater., 2015, 25,
5479–5491.
2 S. E. Leucuta, Curr. Clin. Pharmacol., 2010, 5, 257–280.
3 S. Singh, J. Nanosci. Nanotechnol., 2010, 10, 7906–7918.
4 R. S. Raghuvanshi, Y. K. Katare, K. Lalwani, M. M. Ali,
O. Singh and A. K. Panda, Int. J. Pharm., 2002, 245, 109–121.
This journal is ©The Royal Society of Chemistry 2018
J. Mater. Chem. B, 2018, 6, 6752--6766 | 6765

View Article Online
Journal of Materials Chemistry B
Paper
33 L. F. Yousif, K. M. Stewart, K. L. Horton and S. O. Kelley, 47 A. Shibata, T. Nagaya, T. Imai, H. Funahashi, A. Nakao and
ChemBioChem, 2009, 10, 2081–2088. H. Seo, Breast Cancer Res. Treat., 2002, 73, 237–243.
34 K. L. Horton, K. M. Stewart, S. B. Fonseca, Q. Guo and 48 J. Renukuntla, S. Shah, S. H. S. Boddu, A. D. Vadlapudi,
S. O. Kelley, Chem. Biol., 2008, 15, 375–382.
35 Z. Khatun, Y. S. Choi, Y. G. Kim, K. Yoon, M. Nurunnabi,
R. K. Vadlapatla, D. Pal and A. K. Mitra, Drug Dev. Ther.,
2015, 6, 52–61.
L. Li, E. Lee, H. C. Kang and K. M. Huh, Biomacromolecules, 49 Y. Y. Zhang, C. Teh, M. H. Li, C. Y. Ang, S. Y. Tan, Q. Y. Qu,
2017, 18, 1074–1085. V. Korzh and Y. L. Zhao, Chem. Mater., 2016, 28, 7039–7050.
36 E. Ju, Z. Li, Z. Liu, J. Ren and X. Qu, ACS Appl. Mater. 50 H. Ranji-Burachaloo, F. Karimi, K. Xie, Q. Fu, P. A. Gurr,
Interfaces, 2014, 6, 4364–4370.
37 P. A. Ascierto and F. M. Marincola, J. Transl. Med., 2011,
9, 115.
38 C. Zhu, S. Jung, S. Luo, F. Meng, X. Zhu, T. G. Park and
Z. Zhong, Biomaterials, 2010, 31, 2408–2416.
D. E. Dunstan and G. G. Qiao, ACS Appl. Mater. Interfaces,
2017, 9, 33599–33608.
51 C. Q. You, H. S. Wu, M. X. Wang, S. L. Wang, T. Y. Shi,
Y. H. Luo, B. W. Sun, X. Y. Zhang and J. Zhu, Nanotechnology,
2017, 28, 165102.
39 S. H. Lee, J. K. Ryu, K. Y. Lee, S. M. Woo, J. K. Park, J. W. Yoo, 52 C. Q. You, H. S. Wu, M. X. Wang, Z. G. Gao, X. Y. Zhang and
Y. T. Kim and Y. B. Yoon, Cancer Lett., 2008, 259, 39–49. B. W. Sun, Nanotechnology, 2018, 29, 015601.
40 D. W. Ahn, J. K. Seo, S. H. Lee, J. H. Hwang, J. K. Lee, 53 H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer,
J. K. Ryu, Y. T. Kim and Y. B. Yoon, Pancreas, 2012, 41,
1048–1057.
B. I. Ipe, M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol.,
2007, 25, 1165–1170.
41 B. Yao, Q. M. He, L. Tian, F. Xiao, Y. Jiang, R. Zhang, G. Li, 54 M. Dellian, F. Yuan, V. S. Trubetskoy, V. P. Torchilin and
L. Zhang, J. M. Hou, L. Wang, X. C. Cheng, Y. J. Wen, B. Kan, R. K. Jain, Br. J. Cancer, 2000, 82, 1513–1518.
J. Li, X. Zhao, B. Hu, Q. Zhou, L. Zhang and Y. Q. Wei, Hum. 55 C. Fang, B. Shi, Y. Y. Pei, M. H. Hong, J. Wu and H. Z. Chen,
Gene Ther., 2005, 16, 1075–1086. Eur. J. Pharm. Sci., 2006, 27, 27–36.
42 C. Q. You, H. S. Wu, M. X. Wang, Y. W. Zhang, J. W. Wang, 56 D. C. Perrault, C. Walkey, T. Jennings, H. C. Fischer and
Y. H. Luo, L. H. Zhai, B. W. Sun, X. Y. Zhang and J. Zhu,
Chem. – Eur. J., 2017, 23, 5352–5360.
43 C. Q. You, H. S. Wu, M. X. Wang, Z. G. Gao, B. W. Sun and
X. Y. Zhang, Mater. Sci. Eng., C, 2018, 92, 453–462.
44 C. Q. You, M. X. Wang, H. S. Wu, P. J. An, M. M. Pan,
W. C. W. Chan, Nano Lett., 2009, 9, 1909–1915.
57 F. Danhier, A. L. Breton and V. Preat, Mol. Pharmaceutics,
2012, 9, 2961–2973.
58 C. Q. You, J. Yu, Y. Sun, Y. H. Luo, X. Y. Zhang, J. Zhu and
B. W. Sun, New J. Chem., 2017, 41, 773–785.
Y. H. Luo and B. W. Sun, Mater. Sci. Eng., C, 2017, 80, 59 Q. Q. Yang, L. Wu, L. Li, Z. Zhou and Y. Huang, Nanoscale,
362–370. 2017, 9, 1547.
45 Y. M. Zhang, F. Huang, C. H. Ren, L. J. Yang, J. F. Liu, 60 Y. L. Chang, S. T. Yang, J. H. Liu, E. Dong, Y. W. Wang,
Z. Cheng, L. P. Chu and J. J. Liu, ACS Appl. Mater. Interfaces,
2017, 9, 13016–13028.
A. Cao, Y. F. Liu and H. F. Wang, Toxicol. Lett., 2011, 200,
201–210.
46 S. T. Mayne, H. A. Risch, R. Dubrow, W.-H. Chow, M. D. 61 M. H. Xiong, Y. Bao, X. Z. Yang, Y. C. Wang, B. Sun and
Gammon, T. L. Vaughan, D. C. Farrow, J. B. Schoenberg, J. Wang, J. Am. Chem. Soc., 2012, 134, 4355–4362.
J. L. Stanford, H. Ahsan, A. B. West, H. Rotterdam, W. J. Blot 62 M. Tanihara, Y. Suzuki, Y. Nishimura, K. Suzuki,
and J. F. Fraumeni Jr, Cancer Epidemiol., Biomarkers Prev.,
2001, 10, 1055–1062.
Y. Kakimaru and Y. Fukunisi, J. Pharm. Sci., 1999, 88,
510–514.
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This journal is ©The Royal Society of Chemistry 2018