International Journal of Pharmaceutics 580 (2020) 119123

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International Journal of Pharmaceutics

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

Development of (G3-C12)-mediated camptothecin polymeric prodrug T

targeting to Galectin-3 receptor against androgen-independent prostate
cancer
Xia Yuana,b,1, Li Liua,1, Wei Wanga, Ya-ru Gaoa, Die Zhanga, Ting-ting Jiaa, Hai-rong Zenga,
⁎ ⁎
Gang Panc, , Yi Yuana,
a
Department of Pharmacy, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China
b
Department of Pharmacy, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China
c
Department of General Surgery, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

A R T I C LE I N FO A B S T R A C T

Keywords: The development of small molecule anticancer drugs, with low water solubility and high toxicity, into polymeric
Camptothecin prodrugs has developed into a promising strategy in clinical application. In this study, we synthesized a novel
Prostate cancer G3-C12-mediated esterase-sensitive tumor-targeting polymeric prodrug of camptothecin (CPT), P(OEGMA-co-
G3-C12 peptide CPT-co-G3-C12), and explored its anticancer activity against androgen-independent prostate cancer in vitro and
Polymeric prodrug
in vivo. Compared to free CPT, the multifunctional polymeric prodrug demonstrated improved water solubility
Tumor targeting
and stability, higher intracellular uptake, and enhanced cytotoxicity in DU145 cells in vitro. Furthermore, it
displayed an improved accumulation in the tumor and an enhanced anticancer activity in vivo. Hence, P
(OEGMA-co-CPT-co-G3-C12) could be a promising drug in the treatment of androgen-independent prostate
cancer.

1. Introduction
Prostate cancer is a common malignant tumor and the second most
common cause of cancer-related deaths in American and Western
European males (Siegel et al., 2019). The major treatment methods for
prostate cancer include surgical and hormone therapy; however, the
tumor cells ultimately become resistant to anti-androgen therapy and
continue to progress. Chemotherapy has been used in whom endocrine
therapy has failed and surgical intervention is unsuitable. Furthermore,
it has been known to increase the survival rate in prostate cancer pa-
tients (Parker et al., 2015).
Camptothecin (CPT) was first isolated from Camptotheca acuminata
by Wall and Wani in 1966 (Wall et al., 1966). CPT selectively inhibits
Topoisomerase I (Topo I), binds to Topo I-DNA complex, and then
stabilizes the complex, thereby preventing the cleaved DNA from re-
joining, inhibiting DNA replication, and leading to cell death
(Hertzberg et al., 1989; Jones et al., 2000; Young, 1985). The antic-
ancer activity of CPT has been extensively researched in prostate
cancer, with significant efficacy reported (Liu et al., 2010). However,
owing to its low solubility and high instability the clinical applications
of CPT remain limited. In addition, CPT exerts side effects including
myelosuppression and hemorrhagic cystitis due to its non-selective
accumulation in both cancer and normal cells (Martino et al., 2017).
Therefore, there is a need to develop new CPT formulations to over-
come these disadvantages.
Nanotechnology-based drug delivery is reportedly a suitable ap-
proach to increase the antitumor activity of CPT and reduce the toxic
side effects (Gigliotti et al., 2016; Lee et al., 2015; Minelli et al., 2012;
Sun et al., 2007). Polymeric prodrugs can assembles into micellar na-
noparticles in liquid, demonstrating inherent advantages such as im-
proved water solubility, increased stability, and passive targeting via
the enhanced permeability and retention (EPR) effect (Dozono et al.,
2016; Maeda et al., 2001; Matsumura and Maeda, 1986). A polymeric
prodrug can release the active pristine drug under stimulation in cancer
cells by using a responsive chemical bond to attach the drug to polymer,
releasing little drug in circulation (Zhu et al., 2014). It can effectively
solve the problem of drug burst release, which often exists in traditional
physically encapsulated nanoparticles. In recent years, thioester bonds
have been employed in polymeric prodrugs owing to their good bio-
degradability (Hu et al., 2014; Liu et al., 2008; Wang et al., 2013a;

⁎
Corresponding authors.
E-mail address: yuanyi0625@163.com (Y. Yuan).
1
The two authors contributed equally to this work.

https://doi.org/10.1016/j.ijpharm.2020.119123
Received 23 August 2019; Received in revised form 7 January 2020; Accepted 4 February 2020
Available online 05 February 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.
X. Yuan, et al.
Zhang et al., 2013a; Zou et al., 2014). The thioester bonds were easily
hydrolyzed by esterase, which is abundant in tumor cells and tumor
tissues (Shen et al., 2010; Tsukigawa et al., 2015; Zhang et al., 2013b).
The anticancer drug paclitaxel hydrogel containing a β-thioester bond
was first developed by Schoenmakers (Schoenmakers et al., 2004). In
previous studies, we designed a bufalin prodrug containing the β-
thioester bond, PEGS-BUF, which demonstrated good drug release
characteristics (Liu et al., 2016). In this context, the esterase-sensitive
β-thioester bond was employed in the polymeric prodrug, P(OEGMA-
co-CPT). In the presence of esterase, the active drug CPT is released by
hydrolysis of the β-thioester bond. Galectin-3 is overexpressed in an-
drogen-independent prostate cancer and would be a potential target site
for prostate cancer (Wang et al., 2013b). G3-C12 (sequence: ANTPC-
GPYTHDCPVKR) is a peptide that can specifically bind to the Galectin-3
receptor with high affinity (Liu and Huang, 2016; Yang et al., 2012).
Therefore, G3-C12 could be used as a potential ligand in the receptor-
mediated nano-delivery system. In this context, the tumor-targeting
moiety, G3-C12 peptide, was conjugated onto the surface of the poly-
meric prodrug.
In this work, we aimed to develop a novel multifunctional polymeric
prodrug of CPT, P(OEGMA-co-CPT-co-G3-C12). Additionally, we eval-
uated the accumulation of this prodrug in tumor tissues and the anti-
tumor effects in androgen-independent prostate cancer.
2. Materials and methods
2.1. Materials
Oligo (ethylene glycol) monomethyl ether methacrylate (OEGMA,
Mn = 300 g/mol, mean degree of polymerization: 4–5) obtained from
Aladdin was purified by passing through a neutral alumina column. The
purified OEGMA was stored at −20 °C. 2,2′-Azobis (2- methylpropio-
nitrile) (AIBN) was recrystallized from 95% ethanol prior to use. N-
Hydroxysuccinimide (NHS, 99%), 4-dimethylaminopyridine (DMAP),
and N, N′-dicyclohexylcarbodiimide (DCC) were purchased from
Sinopharm Chemical Reagent Co. Ltd. Camptothecin (CPT, 97%,
Aladdin), esterase (Sigma), G3-C12 peptide (sequence: ANTPCGPYTH-
DCPVKR, Chinese Peptide Company, Hangzhou, China), Cyanine5
amine (Cy5; Lumiprobe, Hallandale Beach, FL, USA), fetal bovine
serum (FBS, Gibco), penicillin–streptomycin (100×, Gibco), RPMI
1640 medium (Gibco), MTT (Amresco), 4% paraformaldehyde solution
(Beyotime, China), FITC Annexin V/PI apoptosis detection Kit (BD), and
Cell Cycle and Apoptosis Analysis Kit (Beyotime, China) were used as
received. Benzyl 2-methyl-2-(((propylthio)carbonothioyl)thio) pro-
panoate (BPTPA) was synthesized based on previous literature
(Development of octreotide-conjugated polymeric prodrug of bufalin for
targeted delivery to somatostatin receptor 2 overexpressing breast cancer in
vitro and in vivo).
2.2. Sample synthesis
The preparation methodology of the multifunctional polymeric
prodrug, P(OEGMA-co-CPT-co-G3-C12), is as shown in Scheme 2.
Deuterated solvents CDCl3 and DMSO‑d6 were used to characterize the
samples by Bruker AV300 1H nuclear magnetic resonance (NMR)
spectrometer (300 MHz, Fourier transform mode). The molecular
weight (Mn) and molecular weight distribution (Mw/Mn) of the samples
were determined by gel permeation chromatography (GPC, pump:
Waters 1515, detector: Waters 2414 differential refractive index de-
tector). The eluent was DMF with a flow rate of 1.0 mL/min.
Synthesis of P(OEGMA-co-BSMA). By using the copolymerization
method we synthesized P(OEGMA-co-BSMA) with β-carboxyl groups
according to previous methods available in literature (Development of
octreotide-conjugated polymeric prodrug of bufalin for targeted delivery to
somatostatin receptor 2 overexpressing breast cancer in vitro and in vivo).
Briefly, 2.70 g OEGMA (9.0 mmol), 33 mg BPTPA (0.1 mmol), 218 mg
2
International Journal of Pharmaceutics 580 (2020) 119123
Fig. 1. DMF GPC traces recorded for (a) P(OEGMA-co-BSMA), (b) P(OEGMA-co-
CPT), and (c) P(OEGMA- co-CPT-co-G3-C12).
BSMA (1.0 mmol), and 1.6 mg AIBN (0.01 mmol) were added into a
glass ampoule. After three freeze–pumpthaw cycles the ampoule was
degassed and sealed under vacuum. The polymerization was quenched
in liquid nitrogen after 5 h reaction at 70 °C. The polymer was dialyzed
against water, freeze-dried, and further treated with an excess of AIBN
(1.0 mmol, 160 mg) to remove the trithiocarbonate end-groups. P
(OEGMA-co-BSMA) was characterized by GPC and the Mn and Mw/Mn
were determined to be 22.0 kDa and 1.02, respectively (Fig. 1 and Table
S1). 1H NMR analysis revealed that the degree of polymerization,
conversion of OEGMA and BSMA were ~74, ~72% and ~89%, re-
spectively (Figure S1 and Table S1). The polymer was denoted as P
(OEGMA0.88-co-BSMA0.12)74 and abbreviated as P(OEGMA-co-BSMA) in
the following sections.
Synthesis of P(OEGMA-co-CPT). Briefly, 215 mg P(OEGMA-co-
BSMA) (88.8 μmol COOH moieties), 27 mg DCC (130 μmol), and 1 mg
DMAP was added into 15 mL anhydrous CH2Cl2. The mixture was
cooled in an ice-water bath. Next, CPT (29 mg, 83 μmol) in anhydrous
CH2Cl2 (5 mL) was added into the solution. After stirring in an ice-water
bath for 2 h, the mixture was stirred for 48 h at room temperature. The
polymer was purified by dialysis against methanol for 8 h (200 mg,
yield: 82.0%). The Mn and Mw/Mn were calculated as 23.6 kDa and
1.14, respectively, by the GPC method (Fig. 1 and Table S1). The
content of CPT in the polymer was ~ 11.3 wt%. Hence, the polymer
was denoted as P(OEGMA0.88-co-CPT0.105-co-BSMA0.015)74 and abbre-
viated as P(OEGMA-co-CPT) in the following sections.
Synthesis of P(OEGMA-co-CPT-co-G3-C12). P(OEGMA-co-CPT)
(240 mg, 11 μmol COOH moieties), DMAP (0.5 mg), and DCC (4.5 mg,
22 μmol) were added into dried CH2Cl2 (10 mL). NHS (2.5 mg,
22 μmol) and dried CH2Cl2 (5 mL) were mixed and added into the re-
action mixture drop-by-drop over 5 min. After 48 h at RT (25 °C) the
reaction mixture was filtered and washed with water and dried by
evaporation. The product was then added into DMF (5 mL).
Triethylamine (TEA, 2 μL) and G3-C12 (34 mg, 17 μmol) were added.
After stirring overnight, the final modified polymer was dialyzed
against water for 8 h (205 mg, yield: 74.8%). GPC analysis revealed that
P(OEGMA-co-CPT-co-G3-C12) possessed an Mn of 26.3 kDa and an Mw/
Mn of 1.12 (Fig. 1 and Table S1). The content of CPT in the polymer
was ~ 11.3 wt%. Hence, the final polymer was denoted as P
(OEGMA0.88-co-CPT0.105-co-G3-C120.015)74 and abbreviated as P
(OEGMA-co-CPT-co-G3-C12).
X. Yuan, et al. International Journal of Pharmaceutics 580 (2020) 119123

Table 1
Hydrodynamic diameter (< Dh > ), polydispersity index (μ2/Γ2), and zeta potentials (ξ) of copolymer micellar nanoparticles.
Samples < Dh > (nm)a PDI (μ2/Γ2)a Zeta Potential (mV)b

P(OEGMA0.88-co-BSMA0.12)74 70.3 ( ± 1.38) 0.26 −17.7 ( ± 0.15)

P(OEGMA0.88-co-CPT0.105-co-BSMA0.015)74 84.5 ( ± 2.14) 0.18 −10.3 ( ± 0.56)
P(OEGMA0.88-co-CPT0.105-co-G3-C120.015)74 78.8 ( ± 2.26) 0.25 −4.50 ( ± 0.45)

a
Determined by dynamic LLS at a copolymer concentration of 1.0 g/L and pH 7.4.
b
Determined by Malvern Zetasizer Nano ZS at a copolymer concentration of 1.0 g/L in PBS buffer (10 mM, pH 7.4).

2.3. Preparation of micellar nanoparticles

Micellar nanoparticles were obtained using the common solvent

method. Briefly, P(OEGMA-co-CPT-co-G3-C12) (10 mg) dissolved in
DMF (1 mL) was added to deionized water (9 mL) under vigorous
stirring. Next, DMF was removed by dialysis and diluted to different
concentrations for further tests.

2.4. Determination of hydrodynamic diameter (Dh) and zeta potential (ς)

ς and Dh were characterized by Nano ZS (Malvern Zetasizer) with

632 nm and a scattering angle of 173°. Prior to testing, the samples
(1.0 g/L in phosphate-buffered saline (PBS) (10 mM, pH 7.4)) were
sonicated for 30 s. Each measurement was conducted in triplicate.

Fig. 2. In vitro CPT release profiles (37 °C, 20 mM buffer solution) from
polymeric micellar solution of P(OEGMA-co-CPT-co-G3-C12) in the (a) absence
and (b) presence of 10 U esterase.
2.5. In vitro drug release tests
Micellar nanoparticles (1.0 g/L, 100 μL) were added into a dialysis
cell (molecular weight cutoff: 2.0 kDa) and immersed in PBS (3.4 mL,
pH 7.4) with and without 10 U esterase at 37 °C. The amount of CPT in
the dialysate was measured using the fluorescence intensity of CPT at
442 nm (F-4600, Hitachi, Japan).

Fig. 3. Cytotoxicity of P(OEGMA-co-BSMA) in DU145 cells at 24 h, 48 h and 72 h (A). Cytotoxicity of free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12)
in DU145 cells at 24 h (B), 48 h (C) and 72 h (D). Cell viability was evaluated by the MTT assay. Data are represented as mean ± SD (n = 6).

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X. Yuan, et al. International Journal of Pharmaceutics 580 (2020) 119123

Fig. 4. Cell apoptosis of DU145 cells after treated with free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12) for 48 h at a CPT concentration of 5.58 ng/ml
(A1-A2). Cell cycle of DU145 cells after treated with free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12) for 48 h at a CPT concentration of 1.85 ng/ml
(B1-B2). (A1) and (B1) represent flow cytometric patterns of cell apoptosis and cell cycle treated with various formulations, respectively. (A2) Quantitative analysis
of cell apoptosis by flow-cytometric analysis. aP < 0.05 vs ctrl, bP < 0.05 vs CPT, cP < 0.01 vs P(OEGMA-co-CPT) (n = 3). (B2) Quantitative analysis of cell cycle
distribution by flow-cytometric analysis. aP < 0.01 vs ctrl, bP < 0.05 vs ctrl, cP < 0.01 vs CPT, dP < 0.01 vs P(OEGMA-co-CPT) (n = 3).

2.6. Cell culture 2.8. Analysis of apoptosis

The typical human androgen-independent prostate cancer DU145
cells were kindly provided by Cell Bank, the Chinese Academy of
Sciences (Shanghai, China). The DU145 cells were cultured in RPMI
1640 containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL
streptomycin at 37 °C under an atmosphere of 5% CO2.
2.7. In vitro cytotoxicity
The cytotoxicity of the polymeric prodrug in DU145 cells was
evaluated using the MTT assay. Cells (5 × 103 cells/well) were seeded
into 96-well plates with 100 μL complete medium. After incubation
overnight, the cells were replaced with fresh medium containing free
CPT, P(OEGMA-co-CPT) or P(OEGMA-co-CPT-co-G3-C12) at different
concentrations for 24 h, 48 h or 72 h. Next, 20 μL MTT (5 mg/mL) was
added to each well and further incubated for 4 h. The supernatants were
removed and DMSO (150 μL) was added to each well. Finally, the plates
were shaken for 15 min. The absorbance was determined using a mi-
croplate reader (Thermo Fisher) at 490 nm.
The apoptosis induced by the polymeric prodrug in DU145 cells was
quantitated by FITC Annexin V/PI apoptosis detection Kit. Cells were
seeded into 6-well plates (2 × 105 cells/well). After incubation over-
night, the cells were replaced with fresh medium containing free CPT, P
(OEGMA-co-CPT), or P(OEGMA-co-CPT-co-G3-C12) at different con-
centrations for 48 h. Then cells were harvested, washed twice with cold
PBS and resuspended in binding buffer at a concentration of 1 × 106
cells/mL. Later, 100 μL of the cell suspension was transferred to a new
culture tube, double-stained with 5 µL FITC Annexin V and 5 µL pro-
pidium iodide (PI) for 15 min at RT (25 °C) in the dark. Finally, cell
apoptosis was analyzed by FACS (BD Biosciences).
2.9. Cell cycle analysis
The cell cycle distribution of the polymeric prodrug in DU145 cells
was quantitated using the Cell Cycle and Apoptosis Analysis Kit. Cells
were seeded into 6-well plates (2 × 105 cells/well). After incubation
overnight, the cells were replaced with the serum-free medium for 12 h,
and then replaced with fresh complete medium containing free CPT, P
(OEGMA-co-CPT) or P(OEGMA-co-CPT-co-G3-C12) at different

4
X. Yuan, et al.
Fig. 5. Cell migration of DU145 cells after treated with free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12) at a CPT concentration of 5 ng/ml (A1). Cell
migration was evaluated by the scratch assay. (A2) Quantitative analysis of cell migration was calculated by the migration area. aP < 0.01 vs ctrl, bP < 0.05 vs ctrl,
c
P < 0.01 vs CPT, dP < 0.01 vs P(OEGMA-co-CPT) (n = 3).
concentrations for further 48 h. Later, the cells were harvested, washed
once with cold PBS, and fixed in ice-cold 70% ethanol for 24 h. The
cells were washed once with cold PBS and then stained with PI for
30 min at 37 °C in the dark. Finally, cell cycle distribution was analyzed
by FACS (BD Biosciences).
2.10. Cell migration analysis
The cell migration induced by the polymeric prodrug in DU145 cells
was evaluated using the scratch assay. Cells were seeded into 6-well
plates (4 × 105 cells/well). After incubation overnight, a cross-shaped
wound was made on the confluent cells using a 200 μL pipette tip, then
thrice washed with PBS to remove detached cells and debris. Next, the
serum-free medium containing free CPT, P(OEGMA-co-CPT), or P
(OEGMA-co-CPT-co-G3-C12), at different concentrations, was added to
the cells. The area of wounds was observed and measured by obtaining
photographs at predetermined time.
2.11. Cellular uptake
Fluorescent dye Cy5-labeled nanoparticles, P(OEGMA-co-CPT-co-
Cy5) and P(OEGMA-co -CPT-co-Cy5-co-G3-C12), were used to explore
DU145 cellular uptake. DU145 cells were seeded into 6-well plates
(2 × 105 cells/well). After incubation overnight, the cells were re-
placed with serum-free medium containing P(OEGMA-co-CPT-co-Cy5)
([Cy5] = 5 × 10-6 mol/L) and P(OEGMA-co-CPT-co-Cy5-co-G3-C12)
([Cy5] = 5 × 10-6 mol/L) for 2 h at 37 °C. The cells were washed three
times with cold PBS and then harvested, resuspended in 400 μL cold
PBS. The cellular uptake was analyzed by FACS (BD Biosciences).
DU145 cells were seeded into 35 mm confocal dishes (1.5 × 104
cell/well). After incubation overnight, the cells were replaced with
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International Journal of Pharmaceutics 580 (2020) 119123
serum-free medium containing free CPT ([CPT] = 1 × 10-5 mol/L), P
(OEGMA-co-CPT) ([CPT] = 1 × 10-5 mol/L) or P(OEGMA-co-CPT-co-
G3-C12) ([CPT] = 1 × 10-5 mol/L) for 2 h at 37 °C. The cells were
washed three times with cold PBS and then fixed with 4% formaldehyde
for 10 min at RT (25 °C). CPT was excited at 365 nm and the fluores-
cence emissions were captured between 420 and 460 nm using a con-
focal microscope (Zeiss, Germany).
2.12. Establishment of a tumor model
The athymic nude mice (BALB/c, male, 4–6 weeks old) were used to
establish a prostate cancer xenograft model by injecting DU145 cells
(1 × 107 cells in 0.2 mL) into their left armpits. All procedures were
performed according to the Laboratory Animal-Guideline for ethical
review for animal welfare (Putuo Hospital, Shanghai University of
Traditional Chinese Medicine).
2.13. In vivo biodistribution examination
Fluorescent dye Cy5-loaded nanoparticles, P(OEGMA-co-CPT-co-
Cy5) and P(OEGMA-co- CPT-co-Cy5-co-G3-C12), were used to explore
tumor-targeting in vivo. When the tumor volume reached
to ~500 mm3, P(OEGMA-co-CPT-co-Cy5) ([Cy5] = 5 × 10-5 mol/L,
200 μL) and P(OEGMA-co-CPT-co-Cy5-co-G3-C12) ([Cy5] = 5 × 10-5
mol/L, 200 μL) were injected through the tail vein to the tumor-bearing
mice. The fluorescence images of the xenografted mice were obtained at
0.5, 4, 8, 24, 48 h after injection using the small animal fluorescence
imaging system in vivo (Berthold, Germany). Next, the mice were sa-
crificed at 48 h, the fluorescence images of the tumors and organs
(heart, liver, spleen, lung, and kidney) were obtained using the above-
mentioned method.
X. Yuan, et al. International Journal of Pharmaceutics 580 (2020) 119123

Fig. 6. (A)Flow cytometry analysis of cellular uptake of (a) P(OEGMA-co-CPT-co-Cy5) and (b) P(OEGMA-co-CPT-co-Cy5-co-G3-C12) for 2 h in DU145 cells.
(B)Typical confocal microscopy fluorescence images recorded for DU145 cells after treated with free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12) for
2 h. (C1) In vivo imaging of tumor-bearing mice after iv administration with P(OEGMA-co-CPT-co-Cy5) and P(OEGMA-co-CPT-co-Cy5-co-G3-C12) for varying time.
(C2) Fluorescence images of organs, including heart, liver, lung, spleen, kidney and tumor collected at 48 h post injection of P(OEGMA-co-CPT-co-Cy5) and P
(OEGMA-co-CPT-co-Cy5-co-G3-C12). (C3) Bio-distributions of P(OEGMA-co-CPT-co-Cy5) and P(OEGMA-co-CPT-co-Cy5-co-G3-C12) in tumor issues and different
organs.

2.14. In vivo anticancer efficacy
The anticancer efficacy of the polymeric prodrug was evaluated in
xenografted mice. When the tumor volume reached to ~100 mm3, the
xenografted mice were randomized into five groups (n = 5) and treated
with normal saline (NS, negative control), P(OEGMA-co-BSMA)
(10 mg/kg), CPT (0.8 mg/kg), P(OEGMA-co-CPT) (0.8 mg CPT/kg), or
P(OEGMA-co-CPT-co-G3-C12) (0.8 mg CPT/kg) through the tail vein
every three to four days (nine-time injection). The body weight and the
tumors were measured every three to four days. On day 32, the mice
were euthanized, and the tumors were harvested for obtaining images.
3. Results and discussion
3.1. Synthesis and characterization of polymeric prodrug
A well-defined polymeric prodrug, P(OEGMA-co-CPT-co-G3-C12),
was developed by the combination of reversible addition − fragmenta-
tion chain-transfer (RAFT) polymerization and polymer post-functio-
nalization. The general steps involved in the synthesis of the well-de-
fined P(OEGMA-co-CPT-co-G3-C12) have been presented in Scheme 2.
Carboxyl-containing random copolymers, P(OEGMA-co-BSMA),
were synthesized, at first, via RAFT polymerization in the presence of
BSMA and OEGMA using benzyl-containing small molecular RAFT
agent (BPTPA), showing Mn of 22.0 kDa and Mw/Mn of 1.02 (Fig. 1a,
Table S1). The DP of P(OEGMA-co-BSMA) was calculated to be ~ 74 by
1
H NMR characterization (Figure S1). The anticancer CPT was then
anchored onto P(OEGMA-co-BSMA) by an esterification reaction be-
tween P(OEGMA-co-BSMA) and CPT with an Mn of 23.6 kDa and Mw/
Mn of 1.14 (Fig. 1b, Table S1). The appearance of characteristic 1H NMR
peaks (peaks l, m, n, o, p) ascribed to CPT in Figure S2 implied the
successful attachment of CPT onto the polymers. The modification of P
(OEGMA-co-CPT) with the tumor-targeting peptide (G3-C12) led to the
formation of the targeting polymeric prodrug of CPT, P(OEGMA-co-
CPT-co-G3-C12). This was verified by 1H NMR analysis (Figure S3). The
Mn and Mw/Mn were determined to be 26.3 kDa and 1.12, respectively,
as shown in Fig. 1c and Table S1. Fluorescence examination of P
(OEGMA-co-CPT-co-G3-C12) indicated a strong blue fluorescence
emission peak at ~ 442 nm (Figure S4) and the CPT content was cal-
culated to be ~11.3 wt% using the fluorescence method.
P(OEGMA-co-CPT-co-G3-C12) and its precursors tended to assemble
into micellar nanoparticles at neutral conditions in water. As shown in
Table 1, the Dh of P(OEGMA-co-BSMA) was determined to be ~70.3
( ± 1.38) nm. The polydispersity index (μ2/Γ2) and ς was determined to
be of 0.26 and −17.7 ( ± 0.15) mV, respectively. The negatively
charged characteristics of P(OEGMA-co-BSMA) was possibly caused by
the ionization of carboxyl groups. The BSMA moieties of P(OEGMA-co-
BSMA) are partially hydrophobic depending on the pH of the medium.
Under a strong basic condition, P(OEGMA-co-BSMA) was completely
hydrophilic and dissolved in aqueous solution as polymer chains. This
was confirmed by DLS characterization and no particles were observed
at the strong basic condition. However, at strong acidic and neutral

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X. Yuan, et al. International Journal of Pharmaceutics 580 (2020) 119123

Fig. 7. In vivo antitumor activity in

DU145-bearing nude mice treated
with various formulations. (A1)
Tumor growth curves evaluated by
changes in tumor volume. aP < 0.01
vs NS, bP < 0.01 vs CPT, cP < 0.05
vs P(OEGMA-co-CPT) (n = 5). (A2)
The enlarged profiles of free CPT, P
(OEGMA-co-CPT) and P(OEGMA-co-
CPT-co-G3-C12) on the growth of tu-
mors. aP < 0.01 vs CPT, bP < 0.05
vs P(OEGMA-co-CPT) (n = 5). (B)
Alteration in body weight of DU145-
bearing nude mice after administra-
tion. aP < 0.01 vs NS, bP < 0.05 vs
P(OEGMA-co-CPT) (n = 5). (C) The
images of excised tumor at the end of
the test after 32 days observation. (D)
Tumor inhibitory rate calculated with
excised tumor volume. aP < 0.01 vs
CPT, bP < 0.05 vs P(OEGMA-co-CPT)
(n = 5).

conditions, BSMA moieties were unionized or only partially ionized.
The unionized BSMA moieties showed hydrophobic characteristics.
Hence, P(OEGMA-co-BSMA) micellar assemblies in water (pH 7.4) were
observed. However, it should be mentioned that the aggregation of P
(OEGMA-co-BSMA) can only be observed at a much higher concentra-
tion (greater than0.5 g/L) than P(OEGMA-co-CPT), aggregating into
nanoparticles even at a concentration < 0.05 g/L.
Typical micellar nanoparticles from P(OEGAM-co-CPT) showed Dh
of ~ 84.5 ( ± 2.14) nm, μ2/Γ2 of 0.18 and ς of −10.3 ( ± 0.56) mV
(Table 1). The attachment of G3-C12 onto P(OEGAM-co-CPT) showed a
slightly decreased size with Dh of ~ 78.8 ( ± 2.26) nm, μ2/Γ2 of 0.25.
The ς decreased to −4.50 ( ± 0.45) mV, exhibiting near-neutral char-
acteristics (Table 1).
3.2. In vitro drug release
In vitro CPT release from P(OEGMA-co-CPT-co-G3-C12) was in-
vestigated. As shown in Fig. 2, in the absence of esterase in PBS, the
release of CPT from P(OEGMA-co-CPT-co-G3-C12) was relatively very
slow and only ~ 20% cumulative CPT was released over 24 h. This
design could effectively reduce premature drug release during the cir-
culation process. However, with the addition of esterase which is
abundant in vivo, a dramatic CPT release was observed. As shown in
Fig. 2b, in the first 4 h, CPT was released from the nanoparticles almost
linearly, with a cumulative ~ 60% CPT released, exhibiting pseudo-first
order kinetics. After 4 h, CPT release decreased gradually and ~77%
CPT release was obtained at 24 h.
3.3. In vitro cytotoxicity
Cytotoxicity of the obtained polymeric prodrug in the human an-
drogen-independent prostate cancer DU145 cells, at different con-
centrations, was evaluated using the MTT assay. Firstly, the cytotoxicity
of the blank P(OEGMA-co-BSMA) polymer was tested. As illustrated in
Fig. 3 A, it exhibited good biocompatibility even when treated with
100 μg/mL for 72 h, and cell viability exceeded 90%. As shown in
Fig. 3B–D, free CPT, P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-
C12) demonstrated a dose-dependent and time-dependent cytotoxicity
toward the tumor cells. It was observed that the cell viability of free
CPT at ~5.56 ng/mL for 48 h was ~ 63%. However, at the same CPT
dosage, the cell viability with P(OEGMA-co-CPT-co-G3-C12) decreased
significantly to ~40%. When the culture time was extended to 72 h, the
cell viability with P(OEGMA-co-CPT-co-G3-C12) further declined
to ~ 21%. The higher anticancer efficacy could be explained as follows.
One possible reason was that when CPT was synthesized into a poly-
meric prodrug, the properties of the polymer prodrug, such as increased
water solubility, can promoted the entry of CPT into cells. Additionally,
the employment of the esterase-sensitive β-thioester bond can effec-
tively release the drug when esterase is present in cells. Moreover, at-
tachment of the G3-C12 peptide to the polymer might play an im-
portant role in improving cytotoxicity via actively targeting the
Galectin-3 receptor. We examined its mechanism using the following
experiments.
3.4. Apoptosis assay
The effects of the polymeric prodrug on apoptosis in the DU145 cells

7
X. Yuan, et al.
Scheme 1. Schematic illustration for the receptor-medicated endocytosis and release of active pristine drug (camptothecin, CPT) of tumor-targeting micellar na-
noparticles assembled from targeting peptide G3-C12-modified responsive polymer-CPT conjugate.
were quantified by FACS using FITC Annexin V/PI apoptosis detection
kit. As shown in Fig. 4 A1 and A2, untreated cells demonstrated neg-
ligible apoptotic and necrotic cells. However, it was observed that free
CPT resulted in an improvement in apoptosis and necrosis, with an
apoptosis rate of ~17.95% and necrosis rate of ~3.36% at 5.58 ng/ml.
As expected, P(OEGMA-co-CPT-co-G3-C12) further increased apoptosis
and necrosis, demonstrating an apoptosis rate of ~30.81% and necrosis
rate of ~19.46%. The data was consistent with the cytotoxicity results,
and P(OEGMA-co-CPT-co-G3-C12) was more effective in inducing
apoptosis compared to free CPT.
3.5. Cell cycle analysis
The cell cycle distribution of the polymeric prodrug in DU145 cells
was quantified by FACS using the Cell Cycle and Apoptosis Analysis Kit.
As illustrated in Figure.4 B1 and B2, most cells were distributed in the
G1 phase in the control group. However, the proportion of cells in the S
phase significantly increased from ~29% to ~43% after treatment with
1.85 ng/mL free CPT. Subsequently, the proportion of cells in the G1
phase significantly decreased from ~61% to ~42%. As expected, the
proportion of cells in the S phase further increased to ~65% and the G1
phase proportion decreased to ~19% in the DU145 cells after treatment
with P(OEGMA-co-CPT-co-G3-C12). Furthermore, P(OEGMA-co-CPT)
and P(OEGMA-co-CPT-co-G3-C12) caused a similar cell cycle arrest in
comparison to the free CPT. This implied that, when CPT was synthe-
sized into the polymeric prodrug, the cycle arrest at the S phase was not
altered, and P(OEGMA-co-CPT-co-G3-C12) was more effective in indu-
cing cell cycle arrest compared to free CPT.
8
International Journal of Pharmaceutics 580 (2020) 119123
3.6. Cell migration analysis
Cumulative reports have demonstrated that CPT could inhibit tumor
migration (Li et al., 2018). We used the scratch assay to explore the
anti-migration ability of free CPT, P(OEGMA-co-CPT), and P(OEGMA-
co-CPT-co-G3-C12). As shown in Fig. 5, the cells migrated into the
scratch and were visible after 24 h. CPT exhibited dramatical migration
inhibition compared to the control. As expected, a further decrease in
the migration ability was observed in DU145 cells after treatment with
P(OEGMA-co-CPT-co-G3-C12), indicating that P(OEGMA-co-CPT-co-G3-
C12) was more effective in inhibiting migration compared to free CPT.
3.7. In vitro cellular uptake
Improving drug selectivity into tumor cells is an advantage of
tumor-targeted polymeric prodrugs. As shown above, P(OEGMA-co-
CPT-co-G3-C12) revealed significantly improved cytotoxicity, which
might be partly due to the G3-C12 peptide specifically bounding to the
Galectin-3 receptor. We used FACS and confocal microscopy to evaluate
the selective cellular uptake of the polymeric prodrug in DU145 cells.
The selective cellular uptake was quantitatively evaluated on Cy5-
labeled nontargeting and targeting prodrugs P(OEGMA-co-CPT-co-Cy5)
and P(OEGMA-co-CPT-co-Cy5-co-G3-C12) using FACS. Fig. 6A presents
the Cy5 value of cells treated with P(OEGMA-co-CPT-co-Cy5), which
was significantly higher compared to the control. This implied that the
nanostructure can helped P(OEGMA-co-CPT-co-Cy5) to enter the cells.
As expected, the uptake of the targeting prodrugs P(OEGMA-co-CPT-co-
Cy5-co-G3-C12) was furthered enhanced.
Furthermore, the intracellular distribution of free CPT, P(OEGMA-
co-CPT), and P(OEGMA-co-CPT-co-G3-C12) was investigated using
X. Yuan, et al.
Scheme 2. Synthetic routes employed for the fabrication of P(OEGMA-co-CPT-co-G3-C12) covalently attached with anticancer drug, CPT, and tumor targeting
peptide, G3-C12.
confocal microscopy. As illustrated in Fig. 6B, weak fluorescence spots
of the CPT group were distributed in DU145 cells. As expected, the
fluorescence intensity of the P(OEGMA-co-CPT) group was significantly
strengthened compared to free CPT molecules. Notably, the underling
mechanism might be considerably complicated. Our preliminary as-
sumptions are as follows. The CPT content in P(OEGMA-co-CPT) was
calculated as ~11.3%, implying that there were ~7.8 CPT molecules
per polymer chain. In addition, it has been well-known that one poly-
meric micelle contains several hundreds and sometimes thousands of
polymer chains. Hence, it was reasonable to assume that with the in-
ternalization of one single nanoparticle more than 1,000 CPT moieties
entered the cells simultaneously. This could explain the stronger
fluorescence of P(OEGMA-co-CPT)-stained cells compared to free CPT
molecules. However, in combination with the in vitro anticancer results
in Fig. 3, the anticancer performance of P(OEGMA-co-CPT) failed to
improve compared to free CPT and even a little worse, although more
CPT molecules entered into cells from P(OEGMA-co-CPT). This in-
dicated that in addition to the internalization of drug molecules, other
factors may play important roles. For example, compared to P(OEGMA-
co-CPT), free CPT possibly enters cells in a slower manner due to its
special physicochemical characteristics; by considerably extending the
incubation time, free CPT molecules could enter cells and exert an anti-
cancer effect. We tried to extend the incubation time from the current
2 h to 24 h and even longer to examine our assumptions. Unfortunately,
after incubating the tumor cells with either free CPT or P(OEGMA-co-
CPT) for a longer time, considerably more dead cells were observed,
and the fluorescence results observed using confocal microscopy were
no longer accurate and reliable.
Interestingly, the fluorescence intensity of the P(OEGMA-co-CPT-co-
G3-C12) was further increased. As mentioned above, the main differ-
ence between P(OEGMA-co-CPT) and P(OEGMA-co-CPT-co-G3-C12)
9
International Journal of Pharmaceutics 580 (2020) 119123
was the modification of the targeting peptide G3-C12, which demon-
strating that the G3-C12 peptide could improve cellular uptake and the
Galectin-3 receptor on the cell surface of DU145 cells played an im-
portant role in the cellular internalization of targeting prodrugs.
3.8. In vivo biodistribution examination
As observed in the above in vitro assays, attachment of the G3-C12
peptide to the polymeric prodrug increased drug internalization in the
tumor cells. Here, Cy5-labeled non-targeting prodrugs P(OEGMA-co-
CPT-co-Cy5) and targeting prodrugs P(OEGMA-co-CPT-co-Cy5-co-G3-
C12) were injected through the tail vein into the xenografted mice to
examine the in vivo biodistribution. As shown in Figure.6 C1, the real-
time distribution of prodrugs was studied at predetermined time points.
After 0.5 h post-injection, the fluorescence signals of both P(OEGMA-
CPT-co-co-Cy5) and P(OEGMA-co-CPT-co-Cy5-co-G3-C12) were ob-
served all through the body. At 4 h post-injection, the fluorescence
intensity increased in the tumor area. With time, the fluorescence in-
tensity of P(OEGMA-co-CPT-co-Cy5-co-G3-C12) at the tumor site was
significantly higher than that of P(OEGMA-co-CPT-co-Cy5). As ex-
pected, the fluorescence signals of the P(OEGMA-co-Cy5-co- G3-C12)
group showed a longer residence time, and the fluorescence signals
were retained in the tumor even at 48 h post-injection. Moreover, the
quantitative analysis of the fluorescence intensity of the isolated tumors
and organs (including heart, liver, spleen, lung and kidney) at 48 h
post-injection was conducted. As illustrated in Figure.6 C2 and C3, the
fluorescence intensity of both P(OEGMA-co-CPT-co-Cy5) and P
(OEGMA-co-CPT-co-Cy5-co-G3-C12) in the heart, liver, lung, and spleen
was significantly lower than that in the tumors. For the P(OEGMA-co-
CPT-co-Cy5) group, the fluorescence intensity in the tumor and kidney
was similar, about 25%. Surprisingly, the fluorescence intensity of the P
X. Yuan, et al.
(OEGMA-co-CPT-co-Cy5-co-G3-C12) group in the tumor was ~ 80%,
while the quantity in the kidney was relatively less. This data implied
that the accumulation and tumor targeting of the polymeric prodrug
could be attributed to the EPR and G3-C12 peptide of the nanoformu-
lation, which improved the ability of the polymeric prodrug to target
the tumor.
3.9. In vivo anticancer efficacy
The anticancer efficacy of the polymeric prodrug in vivo was eval-
uated in DU145 xenografted mice. As shown in Fig. 7 A1, A2, C, and D,
the blank polymer, P(OEGMA-co-BSMA), demonstrated no effect on the
tumor compared to normal saline, and the tumor volume increased
dramatically. The free CPT demonstrated a good efficacy on tumor
growth suppression (P < 0.01). Furthermore, the tumor volume of the
P(OEGMA-co-CPT) group was more effectively inhibited than that of
the free CPT group (P < 0.01). As expected, the P(OEGMA-co-CPT-co-
G3-C12) group further inhibited tumor growth compared to the P
(OEGMA-co-CPT) (P < 0.05). These effects could be attributed to the
advantages of nanomedicine, such as improved water solubility and
stability, prolonged circulation time, and increased accumulation in
tumor tissues due to EPR effects compared to small molecular drugs.
Furthermore, these effects could be attributed to the modification of the
targeting peptide G3-C12 on the polymeric prodrug. The safety of CPT
nanomedicines was evaluated by measuring the changes in body weight
at predetermined time points. As illustrated in Fig. 7B, the body weight
did not obviously decrease in the normal saline and P(OEGMA-co-
BSMA) treated group, implying that the blank P(OEGMA-co-BSMA)
polymer had good biocompatibility. However, a remarkable loss in
body weight was observed after treatment with free CPT. Compared to
the free CPT treatment, the loss in body weight with the P(OEGMA-co-
CPT) and P(OEGMA-co-CPT-co-G3-C12) groups was significantly de-
creased. These results indicated that P(OEGMA-co-CPT-co-G3-C12)
improved antitumor activity and demonstrated minimal toxicity com-
pared to free CPT.
4. Conclusion
In this study, we successfully synthesized a new G3-C12-mediated
multifunctional polymeric prodrug of CPT, by linking CPT via an es-
terase-responsive β-thioester bond and Galectin-3 receptors targeted
peptide, G3-C12 (Scheme 1). This prodrug revealed better water solu-
bility and stability, higher intracellular uptake, and enhanced cyto-
toxicity in DU145 cells in comparison to free CPT in vitro. Notably, it
demonstrated prolonged circulation time and improved accumulation
in tumor tissues. Moreover, it demonstrated superior anticancer per-
formance and less toxicity in vivo, owing to the ERP effect and G3-C12-
mediated tumor targeting. In conclusion, this research indicated that P
(OEGMA-co-CPT-co-G3-C12) could be a promising polymeric prodrug
in the treatment of androgen-independent prostate cancer.
CRediT authorship contribution statement
Xia Yuan: Conceptualization, Methodology, Data curation, Writing
- original draft, Funding acquisition. Li Liu: Conceptualization,
Methodology, Data curation, Writing - original draft. Wei Wang:
Methodology, Validation. Ya-ru Gao: Investigation. Die Zhang:
Investigation. Ting-ting Jia: Investigation. Hai-rong Zeng:
Investigation. Gang Pan: Supervision. Yi : . Yuan: Writing - review &
editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
10
International Journal of Pharmaceutics 580 (2020) 119123
Acknowledgements
This work was supported by Shanghai Key Clinical Pharmacy
Specialist Construction Project (district); and the Budgetary Project of
Shanghai University of Traditional Chinese Medicine [No. 18LK064]. In
addition, the authors wish to thank Tao Liu for his supporting.
Disclosure
The authors report no conflicts of interest in this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2020.119123.
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