Somatostatin receptor-mediated specific delivery of paclitaxel prodrugs for efficient cancer therapy

Article abstract of DOI:10.1002/jps.24438

In this study, a novel PTX prodrug, octreotide(Phe)-polyethene glycol-paclitaxel [OCT(Phe)-PEG-PTX], was successfully synthesized and used for targeted cancer therapy. A nontargeting conjugate, mPEG-PTX, was also synthesized and used as a control. Chemica

Full text of DOI:10.1002/jps.24438

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Somatostatin Receptor-Mediated Specific Delivery of Paclitaxel
Prodrugs for Efficient Cancer Therapy
MEIRONG HUO, QINNV ZHU, QU WU, TINGJIE YIN, LEI WANG, LIFANG YIN, JIANPING ZHOU
State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China
Received 9 January 2015; revised 1 March 2015; accepted 5 March 2015
Published online 27 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24438
ABSTRACT: In this study, a novel PTX prodrug, octreotide(Phe)–polyethene glycol–paclitaxel [OCT(Phe)–PEG–PTX], was successfully
synthesized and used for targeted cancer therapy. A nontargeting conjugate, mPEG–PTX, was also synthesized and used as a control.
1
Chemical structures of OCT(Phe)–PEG–PTX and mPEG–PTX were confirmed using H nuclear magnetic resonance and circular dichroism.
The drug contents in both the conjugates were 12.0% and 14.0%, respectively. Compared with the parent drug (PTX), OCT(Phe)–PEG–PTX,
and mPEG–PTX prodrugs showed a 20,000- and 30,000-fold increase in water solubility, respectively. PTX release from mPEG–PTX and
OCT(Phe)–PEG–PTX exhibited a pH-dependent profile. Moreover, compared with mPEG–PTX, OCT(Phe)–PEG–PTX exhibited significantly
stronger cytotoxicity against NCI-H446 cells (SSTR overexpression) but comparable cytotoxicity against WI-38 cells (no SSTR expression).
Results of confocal laser scanning microscopy revealed that the targeting prodrug labeled with fluorescence probe was selectively taken into
tumor cells via SSTR-mediated endocytosis. In vivo investigation of prodrugs in nude mice bearing NCI-H446 cancer xenografts confirmed
that OCT(Phe)–PEG–PTX prodrug exhibited stronger antitumor efficacy and lower systemic toxicity than mPEG–PTX and commercial Taxol.
These results suggested that OCT(Phe)–PEG–PTX is a promising anticancer drug delivery system for targeted cancer therapy. ꢀC 2015 Wiley
Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2018–2028, 2015
Keywords: prodrugs; cancer chemotherapy; targeted drug delivery; peptide delivery; polymeric drugs
1
2
INTRODUCTION
Various ligands have been investigated, including folate,
transferrin, antibodies, peptides, and aptamers.
13
14
15
16
Paclitaxel (PTX) exhibited a significant activity against various
solid tumors because of its ability to promote tubulin assembly
Somatostatin (SST) is a polypeptide that is released in the
gastrointestinal tract by delta cells and the hypothalamus. It
is a key regulatory peptide that inhibits hormones such as gas-
trin, cholecystokinin, glucagon, growth hormone, insulin, and
secretin. Cellular actions of SST are mediated by five SST
receptors (SSTR 1–5). SSTRs are G-protein-coupled receptors
that are widely distributed in various tumors, including small
cell lung cancer, neuroendocrine tumors, prostate cancer, breast
cancer, colorectal carcinoma, gastric cancer, and hepatocellu-
1
into microtubules. However, serious drawbacks hamper the
clinical application of PTX. First, PTX lacks selective cytotox-
icity against cancer cells and frequently leads to serious side
17
2
effects. Poor water solubility of PTX (0.3 :g/mL) is another
concern that significantly reduces its wider clinical application.
To enhance its water solubility, PTX is currently formulated as
a 50:50 mixture of Cremophor EL and ethanol (Taxol). How-
ever, the amount of Cremophor EL required to solubilize PTX
is considerably high (26 mL Cremophor EL for a single intra-
venous administration in an average patient), which leads to
significant side effects such as hypersensitivity, neurotoxicity,
18,19
lar carcinoma.
Clinical usefulness of SST is limited by its
very short half-life. Several synthetic SST analogs, including
octreotide (OCT), lanreotide, and vapreotide, have improved
20
metabolic stability and increased affinity to SSTRs.
3
nephrotoxicity, and cardiotoxicity.
Octreotide, one of the most extensively studied SST analogs,
selectively binds to SSTR2 and SSTR5. It is composed of
2
N-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-
Thr-ol (disulfide bridge Cys2-Cys7). OCT has been used clin-
ically to prevent carcinoid crisis and to visualize tumors con-
taining high density of SSTRs by using scintigraphy. For
Tc-OCT and In-
OCT, are very useful for detecting small neuroendocrine tu-
The prodrug strategy is promising to overcome these
4
,5
problems. Over the last decades, several studies have been
conducted on prodrug synthesis of PTX. Poly(ethylene glycol)
eight amino acids H
6
7
methyl ether methacrylate, cyclotriphosphazene, poly(vinyl
21
8
9
alcohol), and poly(L-glutamic acid) have been proposed as
PTX carriers. However, only a part of the prodrug can effi-
ciently accumulate in the tumor site through the enhanced
permeability and retention (EPR) effect, which decrease drug
efficiency and increase its toxicity. Further improvement
can be achieved by conjugating targeting ligands onto poly-
22
99m
1l1
example, radiolabeled OCT derivatives,
23,24
mors that cannot be detected by conventional methods.
In
addition to its successful application on radio-oncology, OCT is
further used to enhance the delivery of drugs to tumor cells
10
meric prodrugs to achieve selective delivery to tumor cells.
25,26
by modifying them on the surface of nanocarriers.
These
Receptor-targeted polymeric prodrugs have been shown to
promising results prompted us to develop OCT as a specific tar-
geting moiety for delivering a polymeric prodrug of PTX into
tumor cells via SSTR endocytosis.
improve therapeutic responses both in vitro and in vivo.11
Polyethylene glycol (PEG) modification of drugs, also called
PEGylation, is widely performed to improve the solubility and
in vivo stability of drugs. Use of PEG is widespread be-
Correspondence to: Zhou Jianping (Telephone: +86-25-8327-1102; Fax: +86-
2
5-8547-2579; E-mail: cpu zhoujp@163.com)
27
Journal of Pharmaceutical Sciences, Vol. 104, 2018–2028 (2015)
C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
cause of its unique physicochemical characteristics such as low
ꢀ
2
018
Huo et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2018–2028, 2015

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2019
polydispersity in molecular weight and solubility in aqueous acid (6:1:0.1) as eluent to give the intermediate (yield = 90.8%)
28
as well as many organic solvents. Moreover, PEGylation pre- for the next step.
vents rapid renal clearance of drugs and decreases their uptake
by mononuclear phagocyte system, thus prolonging their in vivo
Synthesis of OCT(Phe)–PEG–NH
2
2
9
half-life. In addition, passive tumor targeting of drugs can be
Octreotide was conjugated to Boc-NH-PEG-NHS using the
following procedure. Briefly, Boc-NH-PEG-NHS, OCT, and
EDC·HCl were dissolved in 5 mL acetonitrile (molar ratio of
Boc-NH-PEG-NHS:OCT = 3:1) in the presence of 5 :L triethy-
lamine. The mixture was stirred at 4°C for 12 h. Because OCT
could be PEGylated at the N-terminus (phenylalanine, Phe)
and the lysine (Lys) residue, the crude product was purified by
loading directly onto a reversed-phase high-performance liquid
chromatography (HPLC) system (LC-10AT; Shimadzu, Kyoto,
30
achieved by their PEGylation through the EPR effect.
In this study, novel polymeric prodrug system of
a
OCT(Phe)–PEG–PTX was developed for targeted tumor ther-
apy. Targeting ligand OCT helps in achieving high local con-
centrations of antitumor drugs to improve therapeutic effi-
ciency, and water-soluble PEGs improve water solubility of
parent drugs and improve the pharmacokinetic profile of PTX
while reducing its side effects. Nontargeted prodrug mPEG–
PTX was prepared as a control. The present study investigated
the aqueous solubilities of OCT(Phe)–PEG–PTX and mPEG–
PTX and in vitro release profiles of parent drugs from polymeric
drugs. Then, the cytotoxicity, cellular uptake, and intracellular
distribution of prodrugs were evaluated on human nonsmall
lung cancer cells (NCI-H446; SSTR overexpression) and hu-
man embryonic lung fibroblasts (WI-38 cells, no SSTRs expres-
sion). Finally, in vivo antitumor efficacy and systemic toxicity of
OCT(Phe)–PEG–PTX on nude mice bearing NCI-H446 cancer
2
Japan) with Delta-Pak C18 25 × 100 mm column and eluted
with an acetonitrile gradient (35%–50%, v/v) in water [0.1%
trifluoroacetic acid (TFA)] at a column temperature of 25°C.
The flow rate was 1.0 mL/min, and UV absorbance was moni-
tored at 280 nm. Then, the separated OCT(Phe)–PEG–NH–Boc
2
was deprotected to obtain OCT(Phe)–PEG–NH in the solvent
of acetonitrile–TFA (80/20) solutions using an ice-water bath
under argon.
xenografts were compared with those of nontargeting mPEG– Synthesis of OCT(Phe)–PEG–PTX and mPEG–PTX
PTX and Taxol formulation.
Octreotide(Phe)–PEG–PTX and mPEG–PTX were synthesized
by coupling the carboxyl group of 2 -SA-PTX with the amine
ꢁ
group of OCT(Phe)–PEG–NH
2
and mPEG–NH
2
, respectively,
MATERIALS AND METHODS
Materials
ꢁ
in the presence of EDC·HCl and NHS. Briefly, 24 mg 2 -SA-
PTX was dissolved in 4 mL DMF. EDC·HCl (20 mg) and NHS
(
20 mg) were successively added to the above solutions. The
Paclitaxel was obtained from Chongqing Meilian Pharma-
ceutical Company, Ltd. (Chongqing, China). OCT (molecu-
lar weight, 1019.26 Da) was kindly provided by Shang-
hai Soho-Yiming Pharmaceuticals Company, Ltd. (Shanghai,
China). Succinic anhydride (SA) was purchased from Shang-
hai LingFeng Reagent Company, Ltd. (Shanghai, China), and
mixture was stirred at room temperature for 2 h. Then, 120 mg
OCT(Phe)–PEG–NH or 100 mg mPEG–NH dissolved in 2 mL
2
2
DMF was added, and the mixture was stirred for another 24 h.
Progress of the reaction was monitored by TLC [ethyl acetate:n-
hexane = 6:1 (v/v)]. After the reaction was complete, DI water
was added gradually, and the solution was dialyzed against DI
water for 48 h (MWCO, 3.5 kDa) followed by lyophilization.
The yield of OCT(Phe)–PEG–PTX and mPEG–PTX was 89.3%
and 92.4% with respect to the PTX, respectively. The high yields
could potentially assist in the industrialization of the conjugate
manufacturing process. The resulting products were stored at
1
-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloric
acid salt (EDC·HCl) was purchased from GL Biochem
Shanghai, China). Methoxypolyethylene glycol amine (mPEG-
NH
; molecular weight, 5000 Da) and N-hydroxysuccinimide
NHS) were purchased from Aladdin Reagent Company Ltd.
Shanghai, China). Succinimidyl carboxymethyl ester (SCM,
(
2
(
(
4
°C until further use.
Boc-NH-PEG-NHS) was obtained from Creative PEGWorks
Winston-Salem, North Caroline), and 3-(4,5-dimethylthiazol-
-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased
(
2
ꢀ
Characterization of 2 -SA-PTX, OCT(Phe)–PEG–NH
2
,
OCT(Phe)–PEG–PTX, and mPEG–PTX
from Sigma Chemical Company (St. Louis, Missouri). Cre-
mophor EL was kindly gifted by BASF Corporation (Lud-
wigshafen, Germany). All other reagents were of analytical
grade and were used without further purification. Distilled and
deionized (DI) water was used in all experiments.
ꢁ
Chemical structures of 2 -SA-PTX, OCT(Phe)–PEG–NH
2
,
OCT(Phe)–PEG–PTX, and mPEG–PTX were confirmed using a
00-MHz Avance Bruker NMR spectrometer, with OCT, mPEG,
and PTX as controls. PTX, 2 -SA-PTX, OCT(Phe)–PEG–PTX,
and mPEG–PTX were dissolved in CDCl
and OCT(Phe)–PEG–NH were dissolved in D
Circular dichroism (CD) spectra of OCT, OCT(Phe)–PEG–
NH , and OCT(Phe)–PEG–PTX were recorded in the range of
90–250 nm by using Jasco J-810 spectropolarimeter (Jasco,
Easton, Maryland) with a CD cell of 0.1-cm path length and
-nm bandwidth. Scan speed of 100 nm/min was used, and in
5
ꢁ
3
, and OCT, mPEG,
2
2
O.
Methods
Synthesis of OCT(Phe)–PEG–PTX and mPEG–PTX Prodrugs
2
1
Octreotide(Phe)–PEG–PTX and mPEG–PTX were synthesized
using a three-step procedure, according to the general scheme
presented in Scheme 1.
1
all six scans were performed per sample. The spectra were ex-
2
pressed as mean residue molar ellipticity in deg·cm /dmol. Pep-
ꢀ
Synthesis of 2 -O-Succinyl-Paclitaxel
tide concentrations were set at 100 :g/mL in 0.01 M phosphate
ꢁ
ꢁ
2
-O-Succinyl-Paclitaxel (2 -SA-PTX) was synthesized as previ- buffer (pH 7.4).
31
ously reported. The crude product was purified by silica gel
Drug concentration in OCT(Phe)–PEG–PTX and mPEG–
column chromatography with ethyl acetate–n-hexane–ethylic PTX prodrugs was determined using ultraviolet visible
DOI 10.1002/jps.24438
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020
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Scheme 1. Scheme for synthesizing OCT(Phe)–PEG–PTX and mPEG–PTX prodrugs.
Huo et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2018–2028, 2015
DOI 10.1002/jps.24438

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2021
2
spectrophotometer analysis (UV–Vis; TU-1800; Beijing Purk- OCT(Phe)–PEG–NH based on the protocol for preparing PTX-
inje General Instrument Company Limited, Beijing, China) polymeric prodrugs. CLSM was employed to examine the in-
based on a standard curve generated with known concentra- tracellular uptake and distribution of OCT(Phe)–PEG–TRITC
tions of PTX in methanol (8 = 227 nm) and by assuming that and mPEG–NH
the PTX prodrug and free drug had the same molar extinction well plates and were incubated for 24 h. Next, 2 :M each of
coefficients in water and in methanol, respectively, and that OCT(Phe)–PEG–TRITC and mPEG–NH –TRITC were added
to respective wells, and the cells were incubated for another 4
2
–TRITC. NCI-H446 cells were seeded in 6-
2
32
both followed the Beer–Lambert law.
Solubilities of OCT(Phe)–PEG–PTX and mPEG–PTX pro- h at 37°C. The nuclei of the cells were stained using a blue
8
drugs were evaluated using a visual observation method.
molecular probe Hoechst 33342 for 10 min. The cells were
washed six times with growth medium. Intracellular distri-
bution of fluorescence probe was examined using Zeiss LSM
In Vitro Release of PTX from OCT(Phe)–PEG–PTX and mPEG–PTX
Prodrugs
5
10 META confocal laser scanning microscope (Carl Zeiss,
Jena, Germany) at excitation and emission wavelengths of
55 and 572 nm, respectively. The nuclei were imaged at
In vitro release of PTX from OCT(Phe)–PEG–PTX and mPEG–
PTX were evaluated using a dialysis method. Lyophilized pro-
drugs containing 0.5 mg PTX were dissolved in 1 mL phos-
phate buffered saline (PBS) (0.1 M, pH 7.4 and 5.8, respec-
tively). The resulting solutions were placed inside a dialysis bag
MWCO, 3.5 kDa), and the entire bag was sunk in 150 mL PBS
0.1 M, pH 7.4 and 5.8, respectively) at 37°C supplemented
with 0.1% (w/v) Tween 80. An aliquot (2 mL) was drawn at pre-
determined time intervals, and fresh release medium (2 mL,
7°C) was added. The concentration of free PTX in the sam-
4
excitation and emission wavelengths of 352 and 544 nm,
respectively.
In the receptor-competitive inhibition experiment, 2 mM
OCT was added to the medium and incubated for 1 h. The cells
were incubated for another 4 h with OCT(Phe)–PEG–TRITC
to evaluate the active targeting of OCT. Nuclear staining and
determination of intracellular TRITC distribution were per-
formed as described above.
(
(
3
In Vivo Antitumor Efficacy Test. BALB/c nude mice (age, 7
weeks; weight, 20–25 g) were used for pharmacodynamic exper-
iments. A subcutaneous tumor was established by inoculating
ples was determined using HPLC (LC-2010 system; Shimadzu)
TM
equipped with a LIChrospher
5
C18 column (particle size,
2
:m; 250 × 4.6 mm ). The mobile phase consisted of acetoni-
6
5
× 10 NCI-H446 cells into the armpit region of these mice.
trile/35 mM ammonium acetate [55:45 (v/v), pH 5.5] and had a
flow rate of 1.0 mL/min. The detection wavelength was 227 nm,
and sample injection volume was 20 :L.
3
When the tumors grew to approximately 100 mm , the mice
were randomly divided into four groups to receive different in-
jections as follows: (1) normal saline (control group, n = 5); (2)
Taxol at 10 mg PTX/kg (n = 5); (3) OCT(Phe)–PEG–PTX at
In Vitro Cell Studies
Cell Culture
1
(
0 mg PTX/kg (n = 5); and (4) mPEG–PTX at 10 mg PTX/kg
n = 5). Each sample was injected once every 5 days for 15
NCI-H446 cells (SSTR overexpression) and WI-38 cells (no days. Body weights of mice were recorded, and tumor volumes
2
SSTR expression) were obtained from Origin Biosciences Inc. were calculated using the following equation: a × b
/2, where
a was the largest diameter and b was the smallest diameter.
Tumors were also removed from the sacrificed mice after 20
days of observation and were weighed. Percentage of tumor
(
Nanjing, China) and cultured in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum and 0.1% peni-
cillin/streptomycin at 37°C in 5% CO
atmosphere. All culture
2
media were replaced every 3 days. Cells in their growth phase weight inhibition (%TWI) was calculated using the following
formula: TWI (%) = (W
C
−
W
D
)/W
C
× 100, where W
C D
and W
were used for performing the experiments.
were the mean tumor weights of control and treated groups,
respectively.
Cytotoxicity Assay
To further evaluate the antitumor effect of Taxol and PTX-
polymeric prodrugs on the tumor bearing animals, the tumors
were fixed in 10% formalin. Formalin-fixed tumors were em-
bedded in paraffin blocks to prepare hematoxylin and eosin
In vitro cytotoxicity of OCT(Phe)–PEG–PTX and mPEG–PTX
prodrugs was evaluated with NCI-H446 and WI-38 cells by us-
ing the MTT assay. Both the cells were seeded in 96-well plates
3
at a density of 5 × 10 cells/well and were incubated for 24 h at
(
H&E)-stained tumor sections. These sections were visualized
3
7°C. Next, the cells were incubated with 100 :L OCT(Phe)–
under an optical microscope (Olympus, Tokyo, Japan).
Blood samples were collected after 20 day of observa-
tion. Biological markers of liver and kidney function, that is,
alanine transaminase (ALT), aspartate transaminase (AST),
alkaline phosphatase (ALP), blood urea nitrogen (BUN),
and serum creatinine (SCr), were measured using blood
samples.
PEG–PTX, mPEG–PTX, and Taxol (at equivalent PTX concen-
tration of 0.02, 0.08, 0.16, 0.32, 0.64, and 1.28 :g/mL) diluted
in OCT-deficient RPMI 1640 medium at 37°C for 72 h. Taxol,
which is a commercial formulation, was prepared by dissolv-
ing 12 mg PTX in 1.0 mL ethanol and 1.0 mL of Cremophor
33
EL, followed by sonication for 30 min. Then, the MTT assay
34
was conducted according to previously reported procedures.
Toxicity of PTX formulations was expressed as inhibitory con-
centration at which 50% of cell growth is inhibited (IC50).
Statistical Analysis
Intracellular Uptake and Distribution of Fuorescence-Labeled Statistical analysis was performed using Student’s t-test for two
Prodrugs. To evaluate the intracellular uptake and distri- groups and one-way analysis of variance for multiple groups.
bution of PTX-polymeric prodrugs in NCI-H446 cell lines, All the results are expressed as mean ± SD, unless otherwise
a fluorescence probe 6-tetramethylrhodamine isothiocyanate noted. The p values less than 0.05 were considered statistically
(
TRITC) was chemically coupled with mPEG-NH
2
and significant.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 1. (a) HPLC analysis of OCT (1) before separation (2) and after separation (3) of the reaction mixture and blank solvent of deprotected
1
Boc (4) and OCT(Phe)–PEG-NH2 (5). (b) The H-NMR spectra of OCT in D2O (1), Boc-NH-PEG-NHS in D2O (2), OCT(Phe)–PEG–NH2 in D2O
ꢁ
(
(
3), PTX in CDCl3 (4), 2 -SA-PTX in CDCl3 (5), OCT(Phe)–PEG–PTX in D2O (6), and CDCl3 (7), mPEG-NH2 in D2O (8), and mPEG–PTX in D2O
9), and CDCl3 (10). (c) CD spectra of OCT. HAC (1), OCT(Phe)–PEG–NH2 (2), and OCT(Phe)–PEG–PTX (3).
Huo et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2018–2028, 2015
DOI 10.1002/jps.24438

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2023
RESULTS AND DISCUSSION
Synthesis and Characterization of OCT(Phe)–PEG–PTX and
mPEG–PTX Prodrugs
In an attempt to prepare the targeting polymeric prodrug
OCT(Phe)–PEG–PTX, the ligand OCT was first conjugated
to the carboxyl group of PEG by EDC reaction. The PEGy-
lated reaction was preferred at two sites containing a primary
amine: the N-terminus (Phe) and the lysine (Lys) residue. Sev-
eral studies have shown that N-terminus modifications of OCT
effectively retain the binding affinity to SSTR-overexpressing
35,36
tumor cells.
Therefore, it was necessary to separate the by-
products. As shown in Figure 1a, the developed HPLC method
could separate OCT(Lys)–PEG–NH–Boc from unmodified OCT
and OCT(Phe)–PEG–NH–Boc. Because of ionic polarity differ-
ence, the OCT was washed out, followed by OCT(Phe)–PEG–
NH–Boc and OCT(Lys)–PEG–NH–Boc (Fig. 1a2). The HPLC
chromatogram of OCT(Phe)–PEG–NH–Boc product is shown in
Figure 1a3. After deprotection of Boc, the polarity of OCT(Phe)–
Figure 2. In vitro PTX release profiles of (a) mPEG–PTX and
OCT(Phe)–PEG–PTX in neutral (pH 7.4) and acidic conditions (pH 5.8)
at 37°C. Each point is represented in mean ± SD (n = 6).
SA-PTX (Fig. 1b5), the peaks at 2.5–2.8 and 7.5–8.0 ppm be-
longed to the typical protons of mPEG and benzene ring of
PEG–NH
2
increased, resulting in the forward movement of the
ꢁ
chromatographic peak (Fig. 1a5). Next, 2 -SA-PTX was pre-
ꢁ
1
ꢁ
2
-SA-PTX, respectively, in H-NMR spectra of mPEG–2 -SA-
ꢁ
pared by esterification of 2 -hydroxyl group by using SA in the
PTX (Figs. 1b9 and 1b10). The results indicated that mPEG
presence of dry pyridine. After 3 h of reaction, no free PTX
was found by TLC, indicating that the reaction was complete.
The product was collected as a white powder, with a yield of
ꢁ
was successfully conjugated to 2 -SA-PTX.
Drug Concentration and Solubility of PTX in OCT(Phe)–PEG–PTX
and mPEG–PTX
8
9% and purity of 98%, respectively. Finally, by using EDC and
ꢁ
NHS as reaction promoters, 2 -SA-PTX was conjugated to the
Concentrations of PTX in the polymeric prodrugs were quan-
tified using UV absorbance (8 = 227 nm) by assuming that
the prodrugs and free drug had the same molar extinction co-
efficients in water and methanol, respectively, and that both
followed the Beer–Lambert law. The calculated weight of PTX
in OCT(Phe)–PEG–PTX and mPEG–PTX was 12.0% (w/w) and
free amines of OCT(Phe)–PEG–NH
age to produce OCT(Phe)–PEG–PTX.
2
through amide bond link-
ꢁ
Chemical structures of the synthesized 2 -SA-PTX,
OCT(Phe)–PEG–NH
firmed using H-NMR. The H-NMR spectra of 2 -SA-PTX
are shown in Figure 1b5. Compared with the spectra of PTX
2
, and OCT(Phe)–PEG–PTX were con-
1
1
ꢁ
1
4.0% (w/w), respectively, which was consistent with the the-
(
Fig. 1b4), the characteristic peaks of SA appeared at 2.5–2.8
ꢁ
oretical weight of PTX in OCT(Phe)–PEG–PTX [12.2% (w/w)]
and mPEG–PTX (14.3%).
ppm (-O-CO-CH
2
-CH
2
-), and the chemical shift of 2 -hydroxyl
group of PTX changed from 4.8 to 6.0 ppm because of succiny-
1
Solubility of PTX in PTX-polymeric prodrugs was de-
lation. The H-NMR analysis confirmed the successful intro-
8
termined using a direct observation method. The solubil-
duction of SA into PTX. Succinylation took place preferentially
ꢁ
1
ity of OCT(Phe)–PEG–PTX and mPEG–PTX prodrugs was
at the 2 -hydroxyl group position of PTX. The H-NMR spectra
of OCT(Phe)–PEG–NH are shown in Figure 1b3. Compared
with the spectra of OCT (Fig. 1b1) and Boc-NH-PEG-NHS
Fig. 1b2), the peaks at 7.0–7.5 ppm and 3.52–3.64 ppm be-
5
.96 mg/mL (PTX equivalent) and 9.38 mg/mL (PTX equiva-
2
lent), respectively, which was 20,000- and 30,000-fold higher
than that of the parent drug (PTX, 0.3 :g/mL). The results
showed that PEG modification could significantly improve the
solubility of PTX.
(
longed to the typical protons of OCT and PEG, respectively, indi-
cating that PEG was successfully coupled with the N-terminal
amine of OCT. Furthermore, successful conjugation of 2 -SA-
ꢁ
In Vitro Release of PTX from OCT(Phe)–PEG–PTX and mPEG–PTX
PTX to the amine group of OCT(Phe)–PEG–NH
2
was confirmed
ꢁ
by the characteristic peaks of 2 -SA-PTX at 2.5–2.8 ppm (SA)
and 7.1–7.6 ppm (benzene ring), with some overlap with OCT
To investigate the stability of ester bond, we measured free
PTX released from the two prodrugs after incubating the prod-
ucts in PBS at pH 7.4 and 5.8, respectively, at 37°C. Free PTX
was measured using HPLC, as described above. The cumula-
tive release rate curves are depicted in Figure 2. All the pro-
drugs exhibited a slow release profile, and the amount of PTX
released from the prodrugs increased as the pH of the PBS
increased. After 168 h of incubation in PBS at pH 7.4, PTX
released from OCT(Phe)–PEG–PTX and mPEG–PTX prodrugs
was approximately 92.63% and 92.03%, respectively. However,
only approximately 45.84% and 57.54% of PTX was released
from OCT(Phe)–PEG–PTX and mPEG–PTX prodrugs, respec-
tively, after 168 h of incubation in PBS at pH 5.8. However,
there are many contradictory results reported in literature.
Some studies showed quicker free drug release from prodrugs
1
in the H-NMR spectra of OCT(Phe)–PEG–PTX (Figs. 1b3 and
1
b5–1b7).
Structural conformation of OCT in OCT(Phe)–PEG–NH
and OCT(Phe)–PEG–PTX was further evaluated using CD,
with OCT as the control. As shown in Figure 1c, the CD spectra
of intact OCT, OCT(Phe)–PEG–NH , and OCT(Phe)–PEG–PTX
2
were nearly superimposable in the range of 190–250 nm, sug-
2
ꢁ
gesting that PEGylation and conjugation of 2 -SA-PTX had no
significant effect on the secondary structure of OCT.
37
As a nontargeting control, mPEG–PTX was also synthe-
ꢁ
sized by conjugating 2 -SA-PTX with mPEG–NH
2
. The chemi-
1
cal structure of mPEG–PTX was also confirmed using H-NMR.
Compared with the spectra of mPEG–NH2 (Fig. 1b8) and 2 -
ꢁ
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38
at pH 5.5 than pH 7.4, whereas competing studies observed a
31
contrary phenomena. These prodrugs were all synthesized by
ꢁ
conjugating different macromolecules to the 2 position of PTX
31
through a spacer succinyl group. Cavallaro et al. also pointed
out that the free PTX degraded both at pH 5.5 and 7.4 by chemi-
cal pathways. Therefore, the PTX release and degradation took
place simultaneously. They also studied the in vitro release of
ꢁ
free PTX for 2 -SA-PTX and poly(hydroxyethylaspartamide)–
ꢁ
ꢁ
2
-SA-PTX (PHEA–2 -SA-PTX) conjugates at pH 5.5 and 7.4,
respectively. A different release profile was observed. In the
ꢁ
case of 2 -SA-PTX, more released PTX was determined at pH
ꢁ
5
.5 than pH 7.4. However, in the case of PHEA–2 -SA-PTX,
more free PTX was determined at pH 7.4 than 5.5. Therefore,
both the pH and the stability of free PTX and its derivatives
at different pH environments can affect the free PTX we deter-
mined in the release media.
In Vitro Cytotoxicity Studies
To evaluate the selectivity of OCT(Phe)–PEG–PTX, cytotoxi-
cities of mPEG–PTX and OCT(Phe)–PEG–PTX against NCI-
H446 (SSTR overexpression) and WI-38 cells (no SSTR expres-
sion) were compared. Figure 3 shows the IC50 values of Taxol,
mPEG–PTX, and OCT(Phe)–PEG–PTX prodrugs against NCI-
H446 and WI-38 cells. After 72 h of incubation, mPEG–PTX had
significantly higher IC50 values against NCI-H446 cells than
Taxol (p < 0.05; Fig. 3a). This may be because free PTX is read-
ily diffused into the cytosol by passive diffusion. In contrast, a
common polymeric prodrug such as mPEG–PTX has to be inter-
nalized by endocytosis, after which the chemically conjugated
drug is progressively released via ester bond hydrolysis by both
chemical and enzymatic pathways; this usually delays the cy-
tosolic delivery of the drug and decreases the efficacy of can-
cer cell inhibition.3 However, when the polymeric prodrug was
9
modified with OCT, the IC50 values significantly decreased. The
IC50 value of OCT(Phe)–PEG–PTX prodrug was 2.6-fold lower
than that of the mPEG–PTX prodrug. The enhanced cytotoxic- Figure 3. Cytotoxicities of Taxol, mPEG–PTX, and OCT(Phe)–PEG–
ity of OCT(Phe)–PEG–PTX might be attributed to its selective PTX against NCI-H446 (a) and WI-38 cells (b) and their IC50 values.
binding to the SSTRs expressed on the surface of NCI-H446 Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, and
*
**p < 0.001.
cells.
To further prove that the enhanced cellular uptake was be-
cause of receptor-mediated endocytosis of the polymeric pro-
drug, SSTR-deficient WI-38 cells were used for the cytotoxicity
experiment. Figure 3b shows that the enhanced cytotoxicity of
OCT(Phe)–PEG–PTX prodrug was observed only against NCI-
H446 cells and not against WI-38 cells. On the basis of these
cytotoxicity results, it can be concluded that OCT-modified poly-
meric prodrugs could increase the selectivity for killing tumor
cells.
cytoplasm facilitates the elicitation of its pharmacological re-
sponses. These results were in line with those obtained in the
cytotoxicity assay. Interestingly, fluorescence intensities of the
nuclei of cells incubated with the OCT(Phe)–PEG–TRITC were
much greater than those of nuclei of cells incubated with the
mPEG–TRITC. This might be because SSTR-mediated endocy-
tosis could transport higher proportions of the active polymeric
prodrug within the cytoplasm, thus increasing the probability
of fluorescence probes to enter the nucleus. This result sug-
gested that a novel polymeric prodrug might be obtained by
Intracellular Uptake and Distribution of Polymeric Prodrugs
Labeled with Fluorescence Probe
conjugating DNA-toxin to OCT(Phe)–PEG–NH
Competition experiment showed that in the presence of the
2
.
Intracellular uptake and distribution of the polymeric prodrug
labeled with TRITC in NCI-H446 cells were comparatively es- blocking ligand, fluorescence intensity of OCT–PEG–TRITC
timated using CLSM (Fig. 4). In the absence of OCT in the incu- reduced dramatically and became equivalent to that of the
bation medium, the intracellular fluorescence intensity of the mPEG–TRITC (Fig. 4c). Overall, the results of CLSM directly
OCT(Phe)–PEG–TRITC increased significantly (Fig. 4b) com- indicated that OCT-modified polymeric prodrug was taken up
pared with that of the mPEG–TRITC (Fig. 4a). This result in- by SSTR-mediated endocytosis, which was consistent with the
dicated that receptor-mediated endocytosis was involved in the results of flow cytometry.
cellular uptake of OCT-modified polymeric drug. PTX is a mi-
It should be noted that that as PTX molecules possess
crotubule stabilizer, and enhanced localization of PTX in the no fluorescent characteristics, the hydrophobic fluorescence
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2025
Figure 4. Confocal microscopy images of NCI-H446 cells after 4 h of in vitro exposure to mPEG–TRITC (a) and OCT(Phe)–PEG–TRITC
b) in the absence of free OCT in the medium and OCT(Phe)–PEG–TRITC in the presence of free OCT in the medium (c) at 37°C. For each panel,
(
images from left to right show cell nuclei stained with Hoechst 33342 (blue), TRITC fluorescence in cells (red), and overlay of the two images.
probe TRITC was chosen as an alternative of PTX to for 1.22-fold higher than that of mPEG–PTX and Taxol. Inhibi-
visualization under CLSM. However, TRITC does not pos- tion ratios calculated using tumor weight were consistent with
sess similar pharmacological activities as PTX, such as micro- the results of tumor volume measurements. The remarkable
tubule targeting. Moreover, the physicochemical prosperities effect of OCT(Phe)–PEG–PTX on the suppression of tumor
of TRITC and PTX are not identical. Therefore, the observed growth was also well supported by the histological analysis
cellular uptake and distribution of OCT(Phe)–PEG–TRITC or of the tumor cross-sections. Microscopic analysis of the H&E-
mPEG–TRITC cannot entirely reflect the profiles of OCT(Phe)– stained section of the tumor tissue from the saline control
PEG–PTX or mPEG–PTX. In future studies, a more reasonable group showed typical pathological characteristics of the tumor,
method for detecting intracellular behavior of prodrugs will be such as closely arranged tumor cells (Fig. 5c). In contrast, the
of benefit.
groups injected with Taxol, OCT(Phe)–PEG–PTX, and mPEG–
PTX showed spotty necrosis and intercellular blank. Among the
three therapeutic groups, tumor tissues from animals treated
with OCT(Phe)–PEG–PTX showed the least number of tumor
cells and the highest antitumor efficacy. The best therapeu-
tic effect of OCT-modified PTX polymeric prodrug could be ex-
In Vivo Antitumor Activity and Toxicity
In Vivo Antitumor Activity
In vivo antitumor efficacies of Taxol, mPEG–PTX, and plained by the following aspects. First, PEGylation prevents
OCT(Phe)–PEG–PTX were evaluated in NCI-H446 xenograft the rapid renal clearance of drugs and their receptor-mediated
models. All PTX formulations were effective in preventing tu- uptake by the reticuloendothelial system, thus prolonging their
mor growth compared with saline (Fig. 5). On the basis of in vivo half-life. Second, long-circulating polymers passively ac-
the tumor volume (Fig. 5a), mPEG–PTX showed a comparable cumulate in the tumor tissue by the well-known EPR effect.40
tumor growth suppression than Taxol. Moreover, OCT(Phe)– Third, modification of OCT results in fast cellular internaliza-
PEG–PTX exhibited the highest tumor growth inhibition ef- tion through receptor-mediated endocytosis, as confirmed in
ficacy among all PTX formulations. TIR values of OCT(Phe)– cellular uptake studies, which further facilitates tumor tar-
PEG–PTX, mPEG–PTX, and Taxol were calculated based on geting of polymeric prodrugs. Thus, OCT(Phe)–PEG–PTX has
the tumor weight at the end of experiment (Fig. 5b). The TIR a promising potential to be used as a targeting carrier for
value of OCT(Phe)–PEG–PTX was 66.3%, which was 1.54- and efficient cancer therapy.
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Figure 5. (a) Tumor growth inhibition in nude mice-bearing NCI-H446 cells after multiple injections of different PTX formulations. Data are
expressed as mean ± SD (n = 5). (b) Tumor weight in NCI-H446-bearing nude mice treated with saline, Taxol, mPEG–PTX, and OCT(Phe)–
PEG–PTX (n = 5). (c) H&E staining shows growth inhibition of NCI-H446 cells in tumor tissue after treatment.
Evaluation of In Vivo Toxicity
cence probe was selectively taken into tumor cells by SSTR-
mediated endocytosis and exhibited significantly stronger cy-
totoxicity against NCI-H446 cells (SSTR overexpression) but
comparable cytotoxicity against WI-38 cells (no SSTR expres-
sion) compared with the nontargeting control. In addition, in
vivo investigation of micelles in nude mice-bearing NCI-H446
cancer xenografts confirmed that OCT(Phe)–PEG–PTX exhib-
ited stronger antitumor efficacy and lower systemic toxicity
than mPEG–PTX and commercial Taxol. Thus, the results of
the present study indicated that OCT(Phe)–PEG–PTX had a
great potential for use in targeted cancer therapy. Yet, signifi-
cant refinement in terms of safety and activity in a more clini-
cally accurate tumor model is required before the product can
be applied to patients. This is the focus of our future work.
Body weight is a crucial criterion to evaluate the systemic toxi-
city of PTX formulations. Figure 6a shows the variations in the
relative body weights of mice with time. Compared with the ini-
tial body weights of tumor-bearing mice, no significant weight
loss was observed after the administration of PTX-polymeric
prodrugs, indicating that PTX-polymeric prodrugs were well
tolerated at the tested dose. However, mice treated with the
same dose of commercial Taxol showed significant weight loss
during the first 10 days, after which the weight loss recovered
slowly.
To evaluate the changes in hepatic and renal functions af-
ter PTX treatment, tumor-bearing mice were euthanized on
day 20. Biochemical parameters such as ALT, AST, ALP, BUN,
and SCr levels were determined (Figs. 6b and 6c). These levels
were significantly elevated in the group treated with Taxol com-
pared with those in the group receiving saline (p < 0.001). How-
ever, treatment with PTX-polymeric prodrug induced negligible
changes in all the parameters, suggesting that PTX-polymeric
prodrugs had lower hepatic and renal toxicity than Taxol.
ACKNOWLEDGMENTS
This work was supported by the project of the National Nat-
ural Science Foundation of China (no. 81102397), the Natu-
ral Science Foundation of Jiangsu Province (no. BK2012761),
Qing Lan Project of Jiangsu Province (no. 02432009), Fos-
tering Plan of University Scientific and Technological Inno-
vation Team of Jiangsu Qing Lan Project (2014). Innova-
tive Project for Graduate Cultivation of Jiangsu Province (no.
CONCLUSIONS
In this study, OCT(Phe)–PEG–PTX was developed for targeted CXZZ11-0807), the Project Program of State Key Laboratory
cancer therapy. The nontargeting prodrug mPEG–PTX was also of Natural Medicines, China Pharmaceutical University (no.
synthesized and used as control. Drug content in OCT(Phe)– JKGQ201107 and JKPZ2013004), Key New Drug Innovation
PEG–PTX and mPEG–PTX was 12.0% and 14.0%, respectively, Project from the Ministry of Science and Technology of China
and the aqueous solubility was 20,000- and 30,000-fold higher (no. 2009ZX09310004) and National Basic Research Program
than that of the parent drug, respectively. In vitro release pro- of China (973 Program, no. 2009CB903300). The authors also
files showed that the release of PTX from mPEG–PTX and acknowledge Michael Lin in the University of North Carolina
OCT(Phe)–PEG–PTX was much faster at pH 7.4 than at pH at Chapel Hill for his kind editorial help in revising the English
5
.8. Moreover, the targeting prodrug labeled with the fluores- of this article.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2027
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