Journal of Pharmaceutical Sciences xxx (2018) 1-12

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Journal of Pharmaceutical Sciences

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Pharmaceutical Nanotechnology

Dual-Targeting Nanoparticles: Codelivery of Curcumin and

5-Fluorouracil for Synergistic Treatment of Hepatocarcinoma
Wenfeng Ni, Zhengye Li, Zengquan Liu, Yuting Ji, Lijia Wu, Sizhe Sun, Xiqi Jian,
Xiujun Gao*
Institute of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China

a r t i c l e i n f o a b s t r a c t

Article history: Chemotherapy has been the standard for cancer therapy, but the nonspecific cytotoxicity of chemo-
Received 19 July 2018 therapeutic agents and drug resistance of tumor cells has limited its efficacy. However, multidrug
Revised 2 October 2018 combination therapy and targeting therapy have resulted in enhanced anticancer effects and have
Accepted 16 October 2018
become increasingly important strategies in clinical applications. In this study, a biotin-/lactobionic acid
emodified poly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly(ethylene glycol) (BLPP) copolymer
was synthesized, and curcumin- and 5-fluorouracil-loaded nanoparticles (BLPPNPs/C þ F) were prepared
Keywords:
nanoparticles to enhance the treatment of hepatocellular carcinoma. Blank BLPPNPs were shown to have great
targeted drug delivery biocompatibility via both in vitro and in vivo studies. Good targeting of tumor cells of BLPPNPs was
cancer chemotherapy confirmed by flow cytometry, fluorescence microscopy, and biodistribution. The synergistic anticancer
polyglycolic acid
effects of BLPPNPs/C þ F were demonstrated by cytotoxicity and animal studies, while western blotting
polyethylene glycol
was used to further verify the synergistic effect of curcumin and 5-fluorouracil. The dual-targeting and
drug-loaded codelivery nanosystem demonstrated higher cellular uptake and stronger cytotoxicity for
tumor cells. Therefore, these dual-targeting NPs are a promising codelivery carrier that could be made
available for cellular targeting of anticancer drugs to achieve better intracellular delivery and synergistic
anticancer efficacy.
© 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction tumor site and increase the efficacy of chemotherapeutic drugs on

Hepatocellular carcinoma (HCC) is the sixth most common
cancer in the world and the third leading cause of cancer death.
Most hepatocellular carcinoma cases (54%) can be attributed to
HBV infection in China. Today, chemotherapy, among the 3
traditional cancer treatment strategies, is still the most effective
treatment of cancer.1 However, nonspecific distribution of chemo-
therapeutic drugs in the human body causes systemic toxicity and
side effects. Moreover, chemotherapeutic drugs have a dose-
dependent cytotoxicity against cancer cells, and drug resistance is
easily developed, leading to reduced therapeutic efficacy.2,3 To
avoid these problems, targeted nanotechnology and combined
therapy have been used in the treatment of cancer. Targeting NPs
can be used as drug carriers to promote their aggregation at the
Conflicts of interest: The authors have declared that no competing interests exist.
This article contains supplementary material available from the authors by request
or via the Internet at https://doi.org/10.1016/j.xphs.2018.10.042.
* Correspondence to: Xiujun Gao (Telephone: þ86-22-83336939).
E-mail address: gaoxiujun201007@163.com (X. Gao).
tumors, while combination therapy contributes to reducing the
high doses of drugs to alleviate side effects and, more importantly,
avoid drug resistance.4,5
The main potential mechanism proposed for nanomedicine-
based cancer therapies was associated with enhanced perme-
ability and retention (EPR) effects in tumor tissue.6 However, pre-
vious research has shown that the EPR effect plays an enormous
role in animals such as mice but has less of an effect in the human
body due to tumor heterogeneity or hypoxic areas.7,8 Active tar-
geting based on single-receptor recognition has become a potential
strategy in targeted therapy research due to the discovery of mul-
tiple specific receptors overexpressed on the cancer cells' sur-
face.4,5,7 Therefore, nanocarriers have been modified with various
targeting ligands, such as folate,9,10 hyaluronic acid,11,12 trans-
ferrin,13,14 biotin,15,16 and lactobionic acid.17,18 Unfortunately, the
outcome of current single-targeted therapy based on a saturated
process of ligand-receptor binding is suboptimal, which affects the
efficiency of administration of single-targeted nanodrugs. In addi-
tion, the complexity of the tumor microenvironment has a great
impact on therapeutic efficacy of single-targeted therapy.4,19
However, a dual ligand strategy exhibits promising potential and

https://doi.org/10.1016/j.xphs.2018.10.042
0022-3549/© 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12
may lead to enhanced cell selectivity and cellular uptake in cancer
treatments. Biotin is an active tumor-targeting ligand, whose re-
ceptors are highly expressed on the surface of many cancer cells
such as breast cancer cells15 and hepatoma cells.16,20 Grafting biotin
onto the surfaces of NPs could selectively deliver antitumor drugs
to cancerous tissues or organs, thus improving drug utilization side
effects.21 The asialoglycoprotein receptor is an endocytic receptor
of a heterologous oligomer mainly expressed by hepatocytes that
recognizes glycoproteins with D-galactose (Gal) or N-acetylga-
lactosamine (GalNAc).22 Hence, modifying lactobionic acid on the
surface of NPs could be used for active targeting HCC carriers for
anticancer drugs to enhance specific targetability.17,23 Conse-
quently, biotin-mediated and lactobionic acidemediated dual-
targeted nanocarriers treatment of liver tumors could be a prom-
ising strategy.
With deep study of the mechanisms underlying the compro-
mised therapeutic efficacy of monotherapy, drug combination
therapy has been extensively exploited and is increasingly
becoming a promising strategy to combat cancer.24 One of the most
commonly used antimetabolic chemotherapy drugs for the treat-
ment of liver cancer is 5-fluorouracil (5-Fu), which works by
inhibiting thymidylate synthase and reducing the conversion of
deoxyuronic acid to deoxythymidylate, thereby inhibiting the
synthesis of DNA.25,26 However, the clinical application of 5-Fu is
limited on account of its inevitable toxicity to normal cells, which
ultimately reduces the efficacy of chemotherapy.25 To overcome
this limitation, 5-Fu had been combined with other drugs, such as
cisplatin,27 paclitaxel,28 curcumin (CUR),29 to reduce the dose of
chemotherapy drugs and enhance the cytotoxicity. CUR, a hydro-
phobic polyphenol extracted from the rhizome of turmeric, has
proven to exhibit a wide range of therapeutic effects, such as
antitumor, anti-inflammatory, and anti-angiogenic activity.30 Its
potential anticancer effects result from the induction of cancer cell
apoptosis through various apoptotic signaling pathways such as the
NF-kB,31 PI3K/Akt,32 and STAT3 pathways.33 Furthermore, there are
2 main mechanisms by which CUR may inhibit cancer cell growth.
On the one hand, it can inhibit the expression of genes such as
survivin, bcl-2, and cyclin D1, whereas on the other hand, it can
promote the expression of other genes including caspase-3, cyto-
chrome c (cyt c), bax, and p53.34-36 It is worth noting that CUR has
no cytotoxic effect on normal cells and is commonly combined with
chemotherapy drugs for the treatment of various cancers.37-39
Therefore, taking into account the different antitumor mecha-
nisms of 5-FU and CUR, the combined use of these 2 drugs has been
applied to treat various types of cancer.29,40,41
In this article, we linked 2 ligands (BIO and LAC) in an amphi-
philic triblock PEG-PLGA-PEG to prepare dual-targeting NPs
(BLPPNPs) that were loaded with CUR and 5-FU to treat liver cancer.
The synergistic anti-HCC activity of BLPPNPs coloaded with 2 drugs
was investigated both in vitro and in vivo. In this dual-targeted drug
delivery system, the targeting receptor provides a high local NP
concentration at the site of the cancer, which is vital to achieve
better therapeutic outcomes. CUR combined with 5-FU not only
enhances the antitumor outcome but also decreases the concen-
tration of 5-FU, preventing the damage of normal cells. Hence, dual-
targeting NPs and drug combination therapy will be one of the
future mainstream methods of antitumor treatment.
Materials and Methods
Materials, Cells, and Animals
Poly(lactic-co-glycolic acid) (PLGA 50:50) with a molecular
weight of about 10 kDa (Mw z 10 kDa) and poly(ethylene glycol)
(PEG) (Mw z 5 kDa and Mw z 10 kDa) were purchased from
Sigma-Aldrich Trade Co., Ltd. (Shanghai, China). CUR, 5-FU, and
fluorescein isothiocyanate (FITC) were purchased from Shanghai
Yuanye Biotechnology Co., Ltd. (Shanghai, China). 1-
hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine, 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT),
and hematoxylin and eosin (H&E) were all purchased from Tianjin
Biyou Biotechnology Co., Ltd. (Tianjin, China). Ultrafiltration tubes
(10 kDa cutoff) with different volumes were purchased from Tianjin
Lianxing Biotechnologies Co., Ltd. (Tianjin, China). Membrane bags
used for dialysis were purchased from Shanghai Green Bird Sci and
Tech. Co., Ltd. (Shanghai, China). Dulbecco's Modified Eagle's Me-
dium was acquired from Tianjin Haimingwei Trading Co., Ltd.
(Tianjin, China). The human hepatoma cells (Hep G2 cells) and the
human hepatocytes (HL-7702 cells) were acquired from Shanghai
cell bank in the Chinese Academy of Science (Shanghai, China).
Female nude BALB/c mice were purchased from National Institutes
for Food and Drug Control (Beijing, China). All animals received
humane care in strict accordance with the Regulations for the
Administration of Affairs concerning Experimental Animals (Tian-
jin, revised in June 2004), which conforms to the Guide for the Care
and Use of Laboratory Animals published by the US National In-
stitutes of Health (NIH Publication no. 85-23, revised 1996).
Synthesis and Characterization of BPP and LPP
The targeting copolymers BIO-PEG-PLGA-PEG-BIO (BPP) and
LAC-PEG-PLGA-PEG-LAC (LPP) were synthesized by a typical
esterification. The detailed synthesis route is described in
Supplementary Material S1 (SM S1). Briefly, PLGA reacted with
succinic anhydride resulting in carboxylation. Next, carboxylated
PLGA and anhydrous PEG were catalyzed by HOBt and 4-
dimethylaminopyridine to synthesize the triblock copolymer
PEG-PLGA-PEG (PPP). Finally, PPP and BIO/LAC were completely
reacted in dimethyl sulfoxide by the catalysis of DCC and HOBt to
synthesize the targeting copolymer (BPP/LPP).
The structure of copolymers (PLGA, PPP, BPP, and LPP) was
analyzed by 1H NMR (Avance III, Brooke, Brooke) as shown in Figure
S2.
Preparation and Characterization of Dual-Targeting NPs
Dual-targeting NPs were prepared by a nanoprecipitation
method,42,43 encapsulating 1 or 2 drugs (5-Fu/CUR). BPP (4 mg), LPP
(4 mg), PLGA (2 mg), and CUR (4 mg) were dissolved in 1 mL of
tetrahydrofuran. Then, the mixture solution was dropped into a 9
mL aqueous solutions of 5-FU (1.6 mg) and stirred for 1 h to prepare
the drug-encapsulated NPs. The mixture was filtered to remove any
impurities, then centrifuged at 4 C (12,000 rpm, 1 h), and the su-
pernatant was removed. The dual-targeting NPs BIO/LAC-PEG-
PLGA-PEG-BIO/LAC-NPs (BLPPNPs) solution was lyophilized and
stored at 4 C until further use. In addition to prepared BIO-PEG-
PLGA-PEG-BIO-NPs (BPPNPs), LAC-PEG-PLGA-PEG-LAC-NPs
(LPPNPs), PEG-PLPEG-NPs (PPPNPs), and PLGANPs (PLGANPs)
were synthesized following a similar protocol.
Dynamic light scattering analysis (DelsaNano; Beckman) was
used to determine the diameter and zeta potential of the drug-
loaded NPs. Transmission electron microscopy (TEM; HT7700,
Hitachi, Tokyo, Japan) was used to observe the morphology of the
NPs. Briefly, 10 mL of NPs was placed onto a 200 mesh copper grid to
ensure a uniform distribution. The water was removed in a vacuum
drying oven, and the sample was loaded in the microscope for
observation.
 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12
Drug Encapsulation and Cumulative Release
The encapsulation efficiencies of 5-FU and CUR in NPs were
evaluated through an ultrafiltration method. CUR (4 mg) was dis-
solved in a solution of BLPPNPs (10 mg) in tetrahydrofuran (1 mL)
and dropped into a solution containing 5-FU (1.6 mg) in water
(9 mL) by a nanoprecipitation method. The NPs and the mixture
solution were separated using an ultrafiltration tube (molecular
weight cutoff: 10 kDa) and washed by phosphate-buffered saline
(PBS) 3 times for purification. Next, the eluate was combined with
the initial eluate containing 5-FU and CUR, and the resulting
mixture was analyzed with an ultraviolet (UV) spectrophotometer
(UV3310; Hitachi) at 256 nm and 207.5 nm, respectively, to calcu-
late the mass of 5-FU and CUR present. The encapsulation efficiency
of the NPs was calculated indirectly. BLPPNPs/C þ F in the ultra-
filtration tube was eluted 3 times, freeze-dried, and weighed, and
the drug loading was determined. The drug encapsulation effi-
ciency (%) and drug loading efficiency (%) were calculated based on
the following formulas44:
DEE% ¼ ðW1 =W2 Þ  100%
DLE% ¼ ðW1 =W3 Þ  100%
where W1 is the amount of drugs in NPs, W2 is the amount of added
drugs, and W3 is the amount of added NPs and drugs.
Drug release from BLPPNPs, BPPNPs, LPPNPs, PPPNPs, and
PLGANPs (10 mg) was studied by dispersing the carriers in dialysis
bags (molecular weight cutoff 14 kDa). Briefly, these NPs were
respectively redispersed in dialysis bags with 2 mL of PBS (pH 7.4)
and immersed in 50 mL PBS. Drug release was conducted at 37 C
under constant shaking at 100 rpm. At predetermined intervals, 3
mL samples of the release medium were collected and equal vol-
umes of fresh PBS were added. Then, the concentrations of released
CUR and 5-FU were measured by UV spectrophotometer at 207.5
nm and 256 nm, respectively. Finally, the cumulative release
quantities were calculated separately.
Biocompatibility of Dual-Targeted NPs
The biocompatibility of blank BLPPNPs, BPPNPs LPPNPs, PPPNPs,
and PLGANPs was studied by MTT assay in Hep G2 cells and HL-
7702 cells. Next, Hep G2 and HL-7702 were respectively cultured
overnight in 96-well plates (5  103 cells/well) (the exact proced-
ures of cell culturing are given in SM S2). Next, they were incubated
with different concentrations of blank NPs (50-800 mg/mL) for 48 h.
The viability of Hep G2 and HL-7702 cells was assessed with MTT
assays, and the detailed procedure is shown in SM S2.
To further certify the biocompatibility of blank NPs in vivo, the
acute toxicity of 5 NPs was tested in BABL/c female mice. The mice
were randomly divided into 6 groups (n ¼ 3) and injected with PBS,
BLPPNPs, BPPNPs LPPNPs, PPPNPs, and PLGANPs (200 mL/time, 200
mg/kg) via the tail vein, respectively. The mice were injected 10
times at 2 h intervals. Then, the mice were observed for 1 week, and
their general behavior, toxic symptoms, and death were recorded.
Finally, the liver, spleen, kidney, heart, and lungs of each mouse
were harvested for histological analyses after 1 week.
Cytotoxicity
First, to study the synergistic effects of 5-FU and CUR in various
combination ratios of NPs, Hep G2 cells were cultured overnight in
96-well plates (5  103 cells/well). Then cells were added to new
culture with BLPPNPs at different ratios (1/1, 1/2, 1/2.5, 1/3, 2/1, or 3/
1) of 5-FU and CUR. After 48 h of culturing, the Hep G2 cell viability
3
was assessed by MTT assays. The IC50 value was calculated by SPSS
software. The combination index (CI) indicates the degree of drug
interaction when 5-FU and CUR were loaded in NPs and was
calculated according to the following Equation 3:
D1 D
CI ¼ þ 2 (3)
Dx1 Dx2
D1 and D2 are the concentrations of drug 1 and drug 2 in the
BLPPNPs combination that inhibited x% cells, respectively. Dx1 and
Dx2 are the concentrations of drug 1 and drug 2 as a single drug in
BLPPNPs with an inhibition rate of x%. CI < 1 indicates synergism,
CI ¼ 1 indicates an additive effect, and CI > 1 indicates antago-
nism.45 To evaluate the impact of drugs in BLPPNPs/C þ F, BPPNPs/
C þ F, LPPNPs/C þ F, PPPNPs/C þ F, and PLGANPs/C þ F on Hep G2
cell proliferation, cells were cultured in a 96-well plate, then used
with different concentrations of Free/C þ F, BLPPNPs/C þ F, BPPNPs/
C þ F, LPPNPs/C þ F, PPPNPs/C þ F, and PLGANPs/C þ F at the
equivalent 5-FU and CUR concentrations for 48 h. The cytotoxicity
was measured by the MTT method, and the half-maximal inhibi-
tory concentration (IC50) was calculated by SPSS software.
(1)
NoneNP-treated cells had 100% cell viability. Finally, to assay the
effect of the same dose of NPs on the proliferation of Hep G2 cells
(2) and HL-7702 cell proliferation, 2 cells were cultured in a 96-well
plate using free CUR, free 5-FU, free/C þ F, BLPPNPs/C þ F,
BPPNPs/C þ F, LPPNPs/C þ F, PPPNPs/C þ F, and PLGANPs/C þ F at
the equivalent 5-FU (16 mg/mL) and CUR (40 mg/mL) concentrations
for 48 h, respectively. The cytotoxicity was measured by MTT assay.
Cellular Uptake of Dual-Targeting NPs
Intracellular delivery of the dual-targeting NPs was observed in
Hep G2 cells and HL-7702 cells by flow cytometry. Hep G2 cells and
HL-7702 cells were cultured overnight in 6-well plates (3  105
cells/well). FITC-labeled BLPPNPs, BPPNPs, LPPNPs, PPPNPs, and
PLGANPs (with equivalent amount of 10 mg/mL FITC) were incu-
bated for 4 h. The uptake profile of FITC NPs in both cells was
determined by flow cytometry (BD FACSVerse; Becton Dickinson).
Laser confocal microscopy (FV1000; Olympus Corporation,
Tokyo, Japan) was used to visualize NP uptake. Hep G2 and HL-7702
cells (2  104 cells/well) were seeded in a laser confocal dish (NEST,
Wuxi, China) overnight. FITC-labeled BLPPNPs, BPPNPs, LPPNPs,
PPPNPs, and PLGANPs (with equivalent amount of 10 mg/mL FITC)
were added to the dish. After 2 h, excess uningested NPs were
discarded and rinsed several times with PBS, then fixed with 4%
paraformaldehyde for 10 min. Confocal images of FITC-labeled NPs
were acquired with a confocal microscope.
Blood Circulation and Biodistribution of Dual-Targeted NPs
The CUR and 5-FU levels in blood were measured by with-
drawing 0.1 mL of blood from the tail vein of Kunming mice at
different time points after injection of BLPPNPs/C þ F, BPPNPs/C þ F,
LPPNPs/C þ F, PPPNPs/C þ F PLGANPs/C þ F, and free/C þ F (CUR 10
mg/kg 5-FU 4 mg/kg). A detailed description of the experimental
steps and the experimental results are provided in SM S4.
In vivo distribution of BLPPNPs was investigated in a Hep G2
xenograft HCC model, which was initiated by subcutaneous injec-
tion of 2  106 cells into the female BABL/c nude mice flank. To track
the biodistribution of BLPPNPs, BPPNPs, LPPNPs, PPPNPs, and
PLGANPs in vivo, RhB was used to replace drugs and was encap-
sulated in NPs by the nanoprecipitation method. When tumors
reached about 100 mm3 in size, mice bearing Hep G2 liver tumors
were randomly divided into 4 groups and were intravenously
injected by BLPPNPs, BPPNPs, LPPNPs, and PPPNPs (with equivalent
4 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12
amount of 0.5 mg/kg RhB), respectively. The fluorescence signals of
RhB were recorded on an IVIS spectrum imaging system at 0.17, 0.5,
1, 2, 4, 8, and 24 h after injection. After 24 h, the heart, liver, spleen,
lungs, kidneys, and tumor were harvested; washed with cold sa-
line; and photographed using an IVIS spectrum imaging system
(PE).
Antitumor Activity of Dual-Targeting NPs In Vivo
Hep G2 cells (2  106) were inoculated subcutaneously in BALB/
c nude mice to establish a tumor-bearing model. When the tumor
volume reached ~50 mm3, the mice were randomly divided into 6
groups (n ¼ 3) and injected with PBS, free/C þ F, BLPPNPs/C þ F,
BPPNPs/C þ F, LPPNPs/C þ F, and PPPNPs/C þ F (CUR 10 mg/kg and
5-FU 4 mg/kg), respectively. In detail, the mice were injected once
at an interval of 2 days for a total of 4 injections through the tail
vein. The weight and volume of the tumors were recorded every 3
days. After 30 days, the mice were euthanized and the tumors were
photographed. At the same time, the kidneys, spleen, heart, and
lungs were harvested for the H&E staining assay. Tumor volume
was calculated as (Eq. 4),
Length  ðWidthÞ2
Vðmm3 Þ ¼ (4)
2
Western Blot Analysis
Western blotting was performed to detect the expression of
related apoptotic proteins including p53, Bcl-2, and cyt c. Hep G2
cells were seeded on 6-well plates (3 105 cells/well) and cultured
for 24 h. Then, CUR, 5-FU, free/C þ F, BLPPNPs/C þ F, BPPNPs/C þ F,
LPPNPs/C þ F, PPPNPs/C þ F, and PLGANPs/C þ F CUR 10 mg/kg and
5-FU 4 mg/kg were added. After 48 h, the medium was discarded 3
times by PBS (precooled at 4 C). RIPA lysis buffer containing 1%
PMSF extracted total protein was used, and the concentrations were
determined using a BCA kit. After SDS polyamide gel electropho-
resis, the eluent was then wet transferred onto PVDF membranes,
which were closed for 1.5 h with 5% dried skimmed milk or 5% BSA.
First antibodies anti-rabbit: dihydropyrimidine dehydrogenase
(DPYD), cyt c, Bcl-2, GAPDH were incubated overnight at 4 C, and
second antibodies at RT were incubated for 2 h. The membrane was
washed 3 times with TBST and the ECL was illuminated. Photo-
graphs of the protein bands were acquired with a fluorescence
imaging analyzer (GBOXiChemiXT; Syngene, Cambridge, UK).
Statistical Analysis
All data are reported as mean ± SD from at least 3 repeated
experiments. Statistical analyses were performed using a 2-sided
Student's t-test. ANOVA was used for statistical analysis. A p-
value of 0.05 or less was considered to be statistically significant.
Results and Discussion
Synthesis and Characterization of BLPP
PLGA is an FDA-approved biodegradable polymer with good
biocompatibility. It can efficiently load drugs to form NPs and has
been extensively used in drug delivery systems.9,21 PEG is
frequently used to modify the surface of nanosystems to prolong
the blood circulation time.46 Thus, the PEG-PLGA-PEG (PPP) tri-
block copolymer was synthesized via esterification, as shown in
Figure S1. Detailed analysis of the resulting copolymers is presented
in SM S1.
Preparation and Characterization of BLPPNPs
The process used to prepare BLPPNPs was summarized in
Figure 1a. The morphology of various NPs was visually observed by
transmission electron microscopy (Fig. 2a). Representative TEM
photographs show that unimolecular NPs appeared to be spheri-
cally shaped and no aggregation occurred. In addition, the dual-
targeted and single-targeted NPs had an apparent core-shell
structure. The shell of the BLPPNPs consisted of PEG-BIO/LAC seg-
ments and the core consisted of PLGA segments. Then, the particle
sizes and zeta potentials were measured via dynamic light scat-
tering. The results showed that the particle size of BLPPNPs/C þ F
was about 37.2 nm larger than that of BLPPNPs/blank, indicating
that the material effectively exhibited drug-loading capacity
(Table 1). Some studies have suggested that cells preferentially
internalize NPs with a diameter of less than 200 nm.24 Small NPs
could be quickly removed from the blood by renal clearance
(<5 nm) or rapid liver uptake (10-20 nm), whereas large NPs could
be filtered out in the spleen (>200 nm) or reidentified and cleared
by the reticular endothelial system. The average sizes of NPs in this
study were all less than 200 nm and would therefore be deemed
favorable for internalization into targeted cells.47 The zeta poten-
tials of various drug-loaded NPs were 21.3 mV, 14.2 mV, 14.2
mV, 15.2 mV, and 26.7 mV, due to the addition of a certain
percentage of PLGA. Negatively charged NPs showed greater
accumulation in cells than positively charged NPs48 and avoided
clearing by reticular endothelial system.8 Furthermore, the nega-
tive charge increased the stability of the NPs.
CUR and 5-Fu Release Patterns of Dual-Targeted NPs
To study the release patterns of 5-FU and CUR from nano-
carriers, the in vitro release profiles of drug-loaded NPs were
determined in 0.01 M PBS. Figures 2b and 2c describe the CUR and
5-FU drug release models. BLPPNPs/C þ F, BPPNPs/C þ F, LPPNPs/
C þ F, PPPNPs/C þ F, and PLGANPs/C þ F showed a highly burst
release in 8-12 h due to the diffusion of hydrophilic 5-FU on the NP
surfaces. With various NPs, after 12 h, more 5-FU was released more
than CUR because hydrophilic 5-FU was loaded on the outer layer of
the NPs while hydrophobic CUR was encapsulated inside the NPs.
The cumulative release of CUR from the 5 NPs is illustrated in
Figure 2b. About 54% of CUR was released from BLPPNPs/C þ F
within 12 h, and the cumulative release reached about 90% within
144 h. However, the amount of CUR released from PLGANPs/C þ F
within 28 h reached about 55%. There was likewise a similar phe-
nomenon in 5-FU release (Fig. 2b). 5-FU released from BLPPNPs/C þ
F presented a cumulative release of approximately 90% within
144 h, followed by the release of BPPNPs (82%), LPPNPs (81%),
PPPNPs (75%), and PLGANPs (68%). These results indicate that
encapsulating the drug in the polymer BLPPNPs allows for sus-
tainable drug release. Furthermore, in the 5-FU and CUR release
phases of NPs, those BLPPNPs had faster 5-FU and CUR cumulative
release than LPPNPs and BPPNPs. On the one hand, it was possible
that lactobionic acid molecules, which are more strongly hydro-
philic, could load more 5-FU drug. On the other hand, it may be that
the BIO molecules were partially inserted into the interior of the
NPs, forming channels that increased the CUR release. Taken
together, those results revealed that dual-targeting NPs have su-
perior drug loading and controlled release capabilities.
Biocompatibility of Dual-Targeted NPs
To further evaluate the biosafety of empty BLPPNPs in vivo and
in vitro, an MTT assay was used to investigate the in vitro cytotox-
icity and the acute toxicity was tested in mice. As shown in
 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12 5

Figure 1. (a) Preparation of drug-loaded nanoparticles BLPPNPs. (b) After injection into the bloodstream, BLPPNPs were taken up by cells via receptor-mediated endocytosis. (c) 5-
FU and CUR were released from the nanoparticles and regulated the expression of relative proteins to increase the cellular apoptosis.

Figures 3a and 3b, 48 h after incubation with blank BLPPNPs,
BPPNPs, LPPNPs, PPPNPs, and PLGANPs, the average cell viability of
Hep G2 cells and HL-7702 cells was higher than 90% (<200 mg/mL),
whereas when the concentration reached 800 mg/mL, the survival
rate was still around 85%. No obvious toxicity was observed when
the concentration of BLPPNPs was increased. According to the test
standard of USP, the cytotoxicity of blank BLPPNPs was zero or
grade 1.49 More noteworthy, the targeted NPs had a slightly higher
proliferation than nontargeted NPs because the surface-targeting
molecules BIO and LAC are small molecules that are used within
cells to increase cell viability. Experimental results indicated that
NPs could be used as a safe drug carrier.
To provide insights into the biological compatibility and the
potential applications of these NPs in vivo, the acute toxicity of 5
NPs was tested in mice. Blank BLPPNPs, BPPNPs, LPPNPs, PPPNPs,
and PLGANPs were injected into mice via the tail vein. The control
mice received an intravenous injection of PBS. It was worth noting
that none of the mice died after being treated with 5 NPs for 10
doses of 200 mg/kg, which was the highest concentration of NPs
prepared. Each mouse was injected 10 times a day to reach 2000
mg/kg. Obviously, the gram equivalent of 2000 mg/kg in an average
adult man (65 kg) would translate into a 130 g dose of NPs, which
made it safe for use.49 Mice tissue sections were shown in SM
Figure S3, and the cell morphology of the main organs did not
change significantly compared with the control group, suggesting
that intravenous injection of targeted NPs did not lead to cell injury.
Upon further comparison, no obvious organ injury or inflammation
changes were observed. These results confirm that, as drug nano-
carriers, the blank BLPPNPs, BPPNPs, LPPNPs, PPPNPs, and PLGANPs
exhibited good biosafety and biocompatibility in vitro and in vivo.
These NPs exhibited biocompatibility similar to that of the lacto-
bionic acid-poly(ethylene oxide)-3-polylysine-g-PLGA copol-
ymer.49 Hence, BLPPNPs could be a more promising candidate for
drug delivery systems.
Cytotoxicity of Dual-Targeted NPs
To confirm the synergistic effect of 5-FU and CUR in NPs, the
combination index (CI 50% cells viability inhibition) was calculated
to quantify the synergistic effect of the 2 anticancer drugs. Based on
the results in Table 2, with the proportion of CUR and 5-FU
increasing, the CI value firstly increased and then decreased. The
lowest CI value of CUR/5-FU was obtained at a 2.5/1 feed ratio,
indicating that the 2 drugs manifested an increased synergistic
effect against Hep G2 cells. Therefore, the optimal feed ratio of NPs
encapsulated drugs is 2.5:1 (CUR: 5-FU).
6 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12

Figure 2. (a) Transmission electron microscopy of BLPPPNPs/C þ F, BPPPNPs/C þ F, LPPPNPs/C þ F, PPPNPs/C þ F, and PLGANPs/C þ F, scale bar ¼ 1 mm and 200 nm, respectively. (b)
In vitro release profiles of CUR and (c) in vitro release profiles of 5-FU at 144 h. Results are expressed as the mean ± SD (n ¼ 3).

As shown in Figure 3c, the antitumor effects of all loaded dual-
drug NPs were clearly concentration dependent. BLPPNPs/C þ F
exhibited greater cytotoxicity than other NPs, indicating that dual-
targeting could increase NP uptake. The IC50 values of various NPs
toward Hep G2 cells at 48 h are shown in Table 3. The results
showed that the IC50 values were in the order free/C þ F >
PLGANPs/C þ F > PPPNPs/C þ F > LPPNPs/C þ F > BPPNPs/C þ F >
BLPPNPs/C þ F. The IC50 values of the 2 free combined drugs 5-FU
and CUR were 53.265 mg/mL and 70.163 mg/mL, respectively, in
Hep G2 cells. The IC50 of 5-FU and CUR in BLPPNPs/C þ F was
8.933 mg/mL and 22.333 mg/mL, which are approximately 6-fold
and 3-fold lower than that of the free dual drugs, respectively
(p < 0.05). More notably, the IC50 of 5-FU in BLPPNPs/C þ F was
clearly lower than in BLPPNPs/5-FU (p < 0.05). This shows that the
drug utilization was significantly improved, while the declining 5-
FU concentration avoided systemic toxicity. The results showed
that the dual-targeted and loaded drugs of NPs had a strong ability
to kill cancer cells.
Figure 3d shows the toxicity of the 5 NPs and free dual drugs
simultaneously on Hep G2 and HL-7702 cells at the same concen-
tration of action (5-FU 16 mg/mL CUR 40 mg/mL). Although free/C þ
F had strong antitumor toxicity, it was more toxic to normal

Table.1
Characteristic Properties of Blank NPs and Drug-Loaded NPs

Nanoparticles Average Size (nm) Zeta Potential (mV) Encapsulation Efficiency (%) Drug Loading (%)

BLPPPNPs/blank 150.2 ± 0.493 15.5 ± 0.687 / /

BLPPPNPs/C þ F 187.4 ± 2.456 21.3 ± 0.666 54.1 ± 1.6 23.4 ± 0.7
LPPPNPs/C þ F 156.5 ± 4.424 14.2 ± 0.265 52.7 ± 1.5 22.1 ± 1.3
BPPPNPs/C þ F 150.7 ± 1.473 14.2 ± 0.458 49.8 ± 2.4 18.7 ± 0.8
PPPNPs/C þ F 128.5 ± 1.682 15.2 ± 0.351 45.8 ± 1.8 17.4 ± 2.1
PLGANPs/C þ F 110.7 ± 0.862 26.7 ± 0.850 44.7 ± 1.9 15.6 ± 3.3
 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12 7

Figure 3. Proliferation of (a) Hep G2 cells and (b) HL-7702 cells after incubation with different concentrations BLPPNPs, BPPNPs, LPPNPs, PPPNPs, and PLGANPs for 48 h. Cell
viability of (c) Hep G2 cells after incubation with various NPs for 48 h and (d) of Hep G2 cells and HL-7702 after incubation with various NPs for 48 h. Data are given as the mean ±
SD (n ¼ 5) and ANOVA was used for statistical analysis.

hepatocytes than tumor cells. However, the toxicity of BLPPPNPs/
C þ F to HL-7702 was still lower than that of free dual drugs due to
the efficient targeting of the NPs. It was shown that targeted NPs
could recognize surface receptors of liver cancer cells and specif-
ically target these cells, thus reducing the toxic and side effects of 5-
FU drugs on normal cells.
Tumor Cellular Uptake Study of Dual-Targeting NPs
Uptake of NPs in Hep G2 cells and HL-7702 cells was observed
by FCM analysis (Fig. 4). Cellular uptake of the dual-targeted NPs
was quantitatively analyzed. As shown in Figure 4a, the intracel-
lular uptake of BLPPNPs exhibited time dependence. Figure 4b
shows the mean fluorescence intensity. At 0.5 h after the addition
of NPs, the mean fluorescence intensity of nontargeted PLGANPs
was 24, and the fluorescence intensity of BLPPNPs was 102.2, which
was about 4 times higher (p < 0.05). More importantly, the
difference gradually increased with time. After 4 h, the fluorescence
intensity of BLPPNPs was about 5.5 times higher than that of
PLGANPs and about 1.7 times higher than that of BPPPNPs (p <
0.05). This shows that BIO and LAC-modified NPs entered Hep G2
cells more efficiently. Figure 4c shows the median fluorescence
intensity which followed a similar trend as the mean fluorescence
intensity.
The intracellular localization and cellular uptake were visual-
ized using a fluorescence microscope. Blue fluorescence represents
DAPI-stained nuclei, and green fluorescence represents FITC-
labeled NPs. Compared with BPPNPs/FITC, LPPNPs/FITC, PPPNPs/
FITC, and PLGANPs/FITC, cells treated with BLPPNPs/FITC exhibited
much stronger green fluorescence after 4 h of incubation, which
was almost all within the cells and a little in the nucleus (Fig. 5a).
The green fluorescence of PLGANPs was the darkest. These results
show the effectiveness of BLPPNPs for targeted delivery of CUR and
5-FU into cells or tissues of interest. When Hep G2 cells were
pretreated with free BIO and LAC (free/B þ L), the intracellular

Table 2
IC50 Values of Different Drug Ratio in Dual Drugs NPs and CI Value in Hep G2
Table 3
Nanoparticles IC50 (mg/mL) CI IC50 Values of the Loading Dual Drugs NPs and Free Drugs in Hep G2

CUR 5-FU System IC50/5-FU (mg/mL) IC50/CUR (mg/mL)

BLPPPNPs/5-FU / 33.317 / Free C þ F 53.265 70.163

BLPPPNPs/CUR 58.102 / / BLPPPNPs/C þ F 8.933 22.333
BLPPPNPs/C þ F (1:3) 6.946 47.583 1.0274 BPPPNPs/C þ F 12.554 31.385
BLPPPNPs/C þ F (1:2) 7.384 46.332 1.0191 LPPPNPs/C þ F 24.286 60.715
BLPPPNPs/C þ F (1:1) 10.285 43.547 1.0582 PPPNPs/C þ F 29.286 74.667
BLPPPNPs/C þ F (2:1) 9.506 35.350 0.8937 PLGANPs/C þ F 39.024 97.560
BLPPPNPs/C þ F (2.5:1) 8.933 22.628 0.6576 BLPPPNPs/5-FU 33.317 /
BLPPPNPs/C þ F (3:1) 10.698 23.956 0.7334 BLPPPNPs/CUR / 58.102

IC50, half-maximal inhibitory concentration. IC50, half-maximal inhibitory concentration.

8 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12
Figure 4. Cellular uptake experiments. (a) Flow cytometry results of BLPPNPs at 0.5, 1, 2, and 4 h, (b) mean fluorescence intensity, and (c) median fluorescence intensity. Flow
cytometry results for various NPs in (d) Hep G2 cells and (e) HL-7702 cells at 4 h. (f) Mean and median fluorescence intensity in Hep G2 cells and HL-7702 cells, respectively. Data
are given as mean ± SD (n ¼ 3), and ANOVA was used for statistical analysis.
fluorescence of cells incubated with BLPPNPs significantly
decreased. This study suggests that free BIO and LAC molecules
prevent the cellular uptake of the BLPPNPs by competitive binding
to the biotin and lactobionic acid receptors on the cell surface.17,18 In
addition, Hep G2 cells had stronger uptake of NPs than HL-7702
cells, as shown in Figure 5b, suggesting that Hep G2 cells’ higher
expression of receptors sped up the uptake of the dual-targeted
NPs. These findings suggested the biotin-/lactobionic acid-
receptor-mediated endocytic uptake of the NPs was more specific,
leading to greater cellular uptake.
BLPPNPs showed a considerable ability to improve the uptake of
Hep G2 cells in vitro. We next investigated their targeting proper-
ties in vivo using a Hep G2-induced liver cancer mouse model.
In Vivo Biodistribution and Targeting Effect
To further research the targetability of BLPPNPs in vivo, the
biodistribution of RhB-loaded BLPPNPs, BPPNPs, LPPNPs, and
PPPNPs intravenously injected into the Hep G2 tumor-implanted
nude mice was monitored by an IVIS spectrum imaging system.
Figure 5c shows that during the whole period of observation, the
fluorescence of 5 NPs at the tumor site remained strong. The
fluorescence signal of BLPPNPs/RhB appeared stronger than other
NPs at 1 h after injection, especially PPPNPs/RhB. Even 8 h after
injection, the BLPPNPs/RhB fluorescence signal still remained
stronger, confirming the accumulation of BLPPNPs/RhB in the tu-
mor, which could be attributed to the EPR effect and targeting ef-
fect. As we all know, the NPs with suitable sizes are trapped due to
the EPR effect in the tumor. Undeniably, targeting NPs contributed
to delivering the drug to tumor cells via receptor-specific ligands.
The cellular uptake behaviors of BLPPNPs/RhB against Hep G2 cells
and in vivo optical imaging results indicate that BLPPNPs/RhB could
target liver tumor cells both actively and passively.
After 24 h, tumors and normal tissues were isolated from mice
and visualized. As shown in Figure 5d, the RhB signal in BLPPNPs (2)
not only reduced NP accumulation and aggregation in normal tis-
sue but also significantly enhanced accumulation in the tumor
mass within 24 h in vivo model of Hep G2, a feat that is in stark
contrast to the tumor accumulation of the nontargeting NPs
(PPPNPs/RhB, 5). The probable explanation for this behavior is that
the surface modification of the dual-targeting NPs influenced their
biodistribution in mice. Enhanced targetability allowed the NPs to
accumulate specifically at the tumor site. Accordingly, it was
confirmed that BLPPNPs/RhB had high tumor targetability as a
result of the ligands modifying the NP surfaces.
Anticancer Effects of Dual-Targeting NPs
To further verify its in vivo anticancer efficacy, Hep G2
tumorebearing BALB/c mice models were used to investigate the
in vivo antitumor effects of different drug-loaded NPs. Tumor vol-
ume and body weight were registered during the experiment. As
shown in Figure 6a, the average tumor volume rapidly expanded
from 50 mm3 to 1800 mm3 in the PBS group over 30 days. The
inhibitory effect of nontargeted NPs (PPPNPs) was not sufficient,
where the tumors showed a significant rapid regeneration after the
 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12 9

Figure 5. (a) Confocal laser scanning microscopy images of Hep G2 cells after incubation with BLPPNPs/FITC, BPPNPs/FITC, LPPNPs/FITC, PPPNPs/FITC, and PLGANPs/FITC for 4 h. (b)
Hep G2 cells and HL-7702 cells after incubation with BLPPNPs/FITC for 4 h. (Nuclei were dyed blue with DAPI; NPs were labeled with FITC, green fluorescence). (c) In vivo fluo-
rescence imaging was acquired at 0, 0.5, 1, 4, 8, and 24 h after injection for PBS, BLPPNPs/FITC, BPPNPs/FITC, LPPNPs/FITC, and PPPNPs/FITC. (d) Fluorescence imaging of the tumor
and normal organs of the Hep G2 tumorebearing nude mice after the mice were euthanized at 24 h after injection. BLPPNPs/FITC (2), BPPNPs (3)/FITC, PPPNPs (4)/FITC, and PPPNPs/
FITC (5). Data are given as mean ± SD (n ¼ 3).

treatment stage and reached about 1200 mm3. Attractively, the
rapid growth of the tumor was inhibited by continuous treatment
with BLPPNPs/C þ F, resulting in tumors approximately 8 times
smaller than the tumor volume observed in the PBS group (p <
0.001). The high antitumor efficacy was attributed to the syner-
gistic effect of 5-FU and CUR. At the same time, the double-targeted
NPs achieved the preferential accumulation of CUR and 5-FU in the
tumors and enhanced the cytotoxicity. These research results are in
agreement with those of the above in vitro assays. In addition, nude
mice lost body weight in the free drug combination group due to
systemic toxicity caused by 5-FU (Fig. 6b). Compared with the
control group, no significant loss of body weight was observed in
the BLPPNPs/C þ F mice. This indicates that the drug-loading NPs
avoided the systemic toxicity of the chemotherapeutic drug 5-FU.
BLPPNPs/C þ F had higher tumor targeting capabilities, which also
lead to a decrease in drug toxicity in normal tissues such as liver,
kidney, spleen, heart, and lung tissues (Fig. S5). According to the
H&E staining (40), no significant apoptosis or necrosis was found
in the tumor tissue of mice treated with PBS or free/C þ F, and the
nucleus was intact (with yellow arrows). On the contrary, mice
treated with BLPPNPs/C þ F showed significantly more tumor
apoptosis or necrosis (marked by black arrows) than those treated
with BPPNPs/C þ F, LPPNPs/C þ F, PPPNPs/C þ F (Fig. 6d). The results
showed that the coloaded dual-targeting NPs had increased anti-
tumor effectiveness, leading to more cells killed at the cellular level.
Synergistic Anticancer Mechanism of Dual Drug
To explore the mechanism of combination therapy with CUR
and 5-FU, western blotting was performed to detect the expression
of related proteins including DPYD, p53, Bcl-2, and cyt c. DPYD is
the rate-limiting enzyme in 5-FU catabolism, and DPYD gene
10 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12

Figure 6. In vivo antitumor efficacy of BLPPNPs. (a) Tumor growth curve after different treatments (n ¼ 3). The arrows indicate the time points of treatment. (b) Average body
weight changes in different groups (n ¼ 3). (c) Surgically removed tumor and normal tissues from nude mice on day 30. (d) Hematoxylin-eosin staining assay tumor. Yellow arrows
indicate tumor cells, black arrows indicate normal tissues. Scale bar ¼ 75 mm. Data are given as mean ± SD (n ¼ 3), and statistical analyses were performed using a two-sided
Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

expression is one of the major determinants of the efficacy of 5-
FU.25,50 This result suggests that BLPPNPs/C þ F significantly de-
creases the DPYD expression compared to BLPPNPs/5-FU and the
control group. However, the DPYD expression with BLPPNPs/CUR
was nearly equal to that of the control group in Hep G2 cells. The
best explanation is that CUR could enhance the cytotoxicity of 5-FU
by downregulating DPYD expression. Previous experiments have
shown that p53 could inhibit the expression of DPYD.51 Next, we
further examine the expression of the p53 protein in Hep G2 cells.52
As shown in Figure 7a, the p53 expression was significantly higher
in the dual-targeting combination treatment group than in the
BLPPNPs/5-FU, BLPPNPs/CUR, and control groups in Hep G2. More
importantly, p53 protein expression was higher in BLPPNPs/CUR
groups than in BLPPNPs/5-FU groups. This suggests that CUR could
promote p53 protein expression, in line with previous studies.35 It
has been also reported that CUR could inhibit the expression of Bcl-
2 which could increase the release of cyt c from mitochondria, thus
preventing apoptosis.53,54 As shown in Figure 7a, it was clear that
the Bcl-2 protein expression of BLPPNPs/C þ F was lower than that
of the BLPPNPs/5-FU, BLPPNPs/CUR, and control groups, while the
expression of cyt c was higher. This probably indicated that CUR
could inhibit the expression of Bcl-2 and increases the release of cyt
c to promote the apoptosis of liver tumor cells. Therefore, 5-FU
combined with CUR increases tumor lethality by enhancing
cytotoxicity.
Western blotting also further confirmed the efficacy of the
dual-targeted NPs anticancer efficacy (Fig. 7b). The Bcl-2 and
DPYD protein expressions of BLPPNPs/C þ F were lower than
those of the free/C þ F and control groups (Fig. 7b), whereas the
cyt c and p53 expressions showed different degrees of increase.
This showed that effective targeting enhances the anticancer ef-
fect of drugs.

Figure 7. (a) Effect of drug in NPs on protein expression of DPYD, P53, Bcl-2, and Cyt C in Hep G2 cells at 48 h. (b) Effect of dual-targeted NPs on protein expression.
 W. Ni et al. / Journal of Pharmaceutical Sciences xxx (2018) 1-12
Conclusions
To summarize, we successfully prepared dual-targeting drug
loading NPs and used them for tumor targeting and combined
chemotherapy. 5-FU and CUR encapsulated in NPs had controlled-
release and sustained-release properties. Dual-targeted NPs had
higher uptake efficiency and higher cytotoxicity than single-
targeted or nontargeted NPs. In addition, antitumor studies
in vivo demonstrated that NPs would be more effective than
chemotherapeutic combination therapy in nude mice with Hep G2
tumors. Dual-targeted NPs reduced the toxic side effects of
chemotherapeutic agents while effectively inhibiting tumor growth
at the same time. Furthermore, these synergistic effects depend on
CUR to enhance the anticancer effect by increasing the cytotoxicity
of 5-FU. CUR also could promote the apoptosis of hepatoma cells by
decreasing the expression of Bcl-2 and increasing the release of cyt
c. This research shows that the dual-targeting drug-loaded NPs
with synergistic effect may be used for HCC targeting of anticancer
drugs to achieve better chemotherapy.
Acknowledgments
This study was supported by the Natural Science Foundation of
China (grant no. 81272495) and a grant from the Natural Science
Foundation of Tianjin (grant no. 16JC2DJC32200).
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