A paclitaxel prodrug with bifunctional folate and albumin binding moieties for both passive and active targeted cancer therapy

Article abstract of DOI:10.7150/thno.24382

Folate receptor (FR) has proven to be a valuable target for chemotherapy using folic acid (FA) conjugates. However, FA-conjugated chemotherapeutics still have low therapeutic efficacy accompanied with side effects, resulting from complications such as sho

Full text of DOI:10.7150/thno.24382

Theranostics 2018, Vol. 8, Issue 7
2018
Ivyspring
International Publisher
Theranostics
2
018; 8(7): 2018-2030. doi: 10.7150/thno.24382
Research Paper
A paclitaxel prodrug with bifunctional folate and albumin
binding moieties for both passive and active targeted
cancer therapy
1
,2
1
2
2
2
2
2
Lingling Shan , Xin Zhuo , Fuwu Zhang , Yunlu Dai , Guizhi Zhu , Bryant C. Yung , Wenpei Fan , Kefeng
1
2
2
2
1
2
Zhai , Orit Jacobson , Dale O. Kiesewetter , Ying Ma , Guizhen Gao , Xiaoyuan Chen
1
2
.
.
Institute of Pharmaceutical Biotechnology, School of Biology and Food Engineering, Suzhou University, Suzhou 234000, China
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health,
Bethesda 20892, USA

Corresponding authors: Xiaoyuan Chen, Email: shawn.chen@nih.gov and Guizhen Gao, Email: szxyggz@163.com
©
Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.12.25; Accepted: 2018.02.02; Published: 2018.02.16
Abstract
Folate receptor (FR) has proven to be a valuable target for chemotherapy using folic acid (FA)
conjugates. However, FA-conjugated chemotherapeutics still have low therapeutic efficacy
accompanied with side effects, resulting from complications such as short circulation half-life, limited
tumor delivery, as well as high kidney accumulation. Herein, we present a novel FA-conjugated
paclitaxel (PTX) prodrug which was additionally conjugated with an Evans blue (EB) derivative for
albumin binding. The resulting bifunctional prodrug prolonged blood circulation, enhanced tumor
accumulation, and consequently improved tumor therapeutic efficacy.
Methods: Fmoc-Cys(Trt)-OH was coupled onto PTX at the 7’-OH position for further synthesis of
ester prodrug FA-PTX-EB. The targeting ability was investigated using confocal microscopy and flow
cytometry. The pharmacokinetics of this bifunctional compound was also studied. Meanwhile, cell
viability was evaluated in normal cells and three cancer cell lines by MTT assay. In vivo therapeutic
effect was tested on FR-α overexpressing MDA-MB-231 tumor model.
Results: Compared with free PTX, the FA-PTX, PTX-EB and FA-PTX-EB prodrugs increased
circulation half-life in mice from 2.19 to 3.82, 4.41, and 7.51 h, respectively. Pharmacokinetics studies
showed that the FA-PTX-EB delivered more PTX to tumors than FA-PTX and free PTX. In vitro and
in vivo studies demonstrated that FA-EB-conjugated PTX induced potent antitumor activity.
Conclusion: FA-PTX-EB showed prolonged blood circulation, enhanced drug accumulation in
tumors, higher therapeutic index, and lower side effects than either free PTX or monofunctional
FA-PTX and EB-PTX. The results support the potential of using EB for the development of
long-acting therapeutics.
Key words: Evans blue; circulation half-life; paclitaxel prodrug; tumor therapy.
Introduction
Targeted anticancer drug delivery is a promising
method to improve the efficacy and safety of
therapeutic agents. Among various cell surface
targets, folic acid receptor (FR) has been demonstrated
as one of the most promising candidates [1-4]. FR is
over expressed on the cell membrane of a series of
solid tumor cells, including breast, ovarian, and
non-small cell lung cancers [5-8]. Conjugation of folic
acid, i.e., folate, is a valuable approach to target
FR-expressing tissues for chemotherapy.
However, folate-conjugated drugs have poor
therapeutic performance and dose limiting side
effects, attributing to the poor water solubility, limited
blood circulation time, and extremely high kidney
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accumulation [9-11]. Indeed, poor water solubility has
been considered as a causative factor in the slow
release of drug molecules from a conjugate and had
been previously shown to lead to low therapeutic
efficacy [12, 13]. In addition, suboptimal pharmaco-
kinetics or limited blood circulation time likely cause
poor therapeutic outcomes [14]. All these
considerations highlight the need to understand the
pharmacokinetic nature of folate-conjugated small
molecule drugs as well as the folic acid
receptor-mediated endocytic pathways to enhance
folate-conjugated compound drug release[15, 16].
One promising approach to improve drug
pharmacokinetics is by hitchhiking albumin to further
prolong blood circulation time and enhance drug
accumulation in tumors. As one of the most abundant
circulating proteins in the blood, albumin is a perfect
drug delivery carrier for many endogenous and
exogenous compounds [17, 18]. Conjugating drugs to
albumin-binding moieties is one of the most
successful approaches due to its high efficiency in
antitumor drug delivery and low side effects. Among
small molecule albumin-binding moieties, Evans blue
the delivery of PTX remain to be solved.
In this study, PTX derivatives were developed at
the 7’-hydroxyl position using Fmoc-Cys(Trt)-OH as a
linker. The amino group of the linker was covalently
attached to folic acid through an amide bond to
synthesize FA-PTX. An EB derivative was further
coupled to synthesize FA-PTX-EB prodrug using a
maleimide bond. The pharmacokinetic properties of
this bifunctional PTX prodrug were significantly
enhanced. Consequently, the therapeutic efficacy of
the prodrug was augmented in a breast cancer model.
Material and methods
General methods
PTX, folic acid, Fmoc-Cys(Trt)-OH, dicyclohex-
ylcarbodiimide (DCC) and, N-hydroxysuccinimide
(NHS) were purchased from Sigma-Aldrich (St. Louis,
MO). All other chemicals were purchased from Fisher
Scientific (USA). The synthesized compound was
purified by using a semi-preparative C18 HPLC
column (XTerra Prep RP18, 10 μm, 7.8 × 300 mm,
Waters). The purified compound was characterized
1
by a Waters LC-MS system (Milford, MA). The H
(
EB) exhibits strong binding affinity for serum
NMR spectra was tested on
spectrophotometer at 500 MHz.
a Varian NMR
albumin. EB has been extensively applied in
physiologic and clinical investigations, and has been
used in routine clinical practice as a way of
determining patient plasma volume. By exploiting the
high binding affinity of EB to blood albumin in vivo,
EB can be used as an effective moiety to improve
EB-conjugated drug pharmacokinetic properties
Synthesis of FA-PTX-EB
FA-PTX-EB was prepared in four steps (Scheme
S1). 2’-TBSCl-PTX was synthesized as previously
reported with slight modifications [30]. Tert-butyldi-
methylsilyl chloride (TBSCl) (30 mg, 0.234 mmol) and
imidazole (82 mg, 1.2 mmol) were dissolved into a dry
Dimethylformamide (DMF) (2.0 mL) solution
containing PTX (100 mg, 0.117 mmol). The mixture
reacted overnight at room temperature under
nitrogen. After reaction completion, 15 mL ethyl
acetate was added into the mixture and the crude
[
19-21]. Our previous work demonstrated EB
conjugates for the realization of long-acting
therapeutics and diagnostic molecular probes [22, 23].
In this work, we conjugated truncated EB to FA-PTX
to prolong the PTX blood circulation and improve the
antitumor therapeutic efficacy.
Pharmacokinetics play
a
vital role in
product was washed with 20 mL H O and 20 mL
2
pharmaceutical research and development [24, 25].
The development of many pharmacologically active
saturated sodium chloride (NaCl) solution. The
resulting compound was collected and dried by
compounds
has
been
hindered
by
poor
Na
2
SO and filtered. After evaporation under reduced
4
pharmacokinetics, low solubility, and formulation
difficulties. For instance, PTX, one of the most widely
used and effective antitumor agents derived from
natural sources for a wide spectrum of tumors is
highly lipophilic with very poor pharmacokinetics
pressure, the compound was purified using a silica
gel column. The purified compound was collected
under reduced pressure to yield a white solid (102.5
mg, yield: 80 %). 100.27 mg 2’-TBSCl-PTX (0.1 mmol, 1
eq.) and 82.23 mg Fmoc-Cys(Trt)-OH (0.14 mmol, 1.2
[26, 27]. Abraxane®,
a
PTX albumin-bound
eq.) were mixed in 10 mL CH Cl2, and added to 14.27
2
nanoparticle delivery system with particle diameters
of ~130 nm, was approved by the US FDA for the
treatment of metastatic breast cancer. This
nanoparticle formulation has yielded advantages
based on decreased toxicity compared to Taxol, a free
PTX formulation. However, whether Abraxane® can
increase survival and address drug resistance is still
unclear [28, 29]. Therefore, substantial challenges for
mg 4-dimethylaminopyridine DMAP (0.117 mmol, 1
eq.). Then 57.51 mg cold EDC was dissolved in 10 mL
CH
mixture over 25 min under room temperature. After
4 h, the reaction mixture was washed by saturated
2
Cl
2
(0.3 mmol) and added dropwise to the
2
aqueous NaHCO
3
, and further purified by HPLC
+
(
110.5 mg, yield: 82 %). LC-MS: [M+H] = 1347.11 calc:
1
348.11. 440 mg of folic acid (0.1 mmol) in 5 mL DMF
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was reacted with DCC and NHS (molar ratio of FA:
DDC: NHS is 1: 2 : 2) under room temperature
overnight. The residue was washed with water and
anhydrous acetone. The extracted FA-NHS was then
filtered under reduced pressure (220 mg, yield: 50%).
(293T) cell lines were purchased from the American
Type Culture Collection (ATCC, Manassas, VA). All
cell lines were incubated with different medium at 37
°C under 5% CO
2
atmosphere L-15 (MDA-MB-231),
.
DMEM (OVCAR3, 293T) and MEM (U87MG)
medium included 10% fetal bovine serum, 100 U/mL
penicillin and 100 μg/mL streptomycin, respectively.
2
’-TBSCl-7’PTX-Cys(Trt)-NH (134.81 mg, 0.1 mmol)
2
was mixed in FA-NHS solution (80.7 mg, 0.2 mmol) in
dry DMF and reacted under room temperature
overnight with monitoring by HPLC (122.5 mg, yield:
Cellular uptake of FA-PTX-EB prodrugs
MDA-MB-231, OVCAR3, U87MG, and 293T
were evaluated for FA-α receptor protein expression
by Western blot analysis. All cell lines were handled
using a reported method [32].
5
6%). FA-2’TBSCl-7’PTX-Cys(Trt) was dissolved in
TFA (5 mL) to remove the protection group for 12 h.
When HPLC showed complete conversion to the
desired compound, it was filtered under reduced
pressure. The FA-7’PTX-Cys-SH compound was
further purified by HPLC (111.2 mg, yield: 90%).
EB-conjugated PTX prodrugs were observed
using EB fluorescence for confocal microscopy and
flow cytometry experiments. Cell lines were cultured
in confocal plates with medium for 4 h, respectively.
After 4 h, the cells were fixed on confocal plates
directly and observed by confocal laser scanning
microscope. PTX-EB and FA-PTX-EB (200 μL, 1
mg/mL) were incubated in 6-well plates with 2 × 105
cells for 4 h, respectively, and then washed with PBS
twice. 500 μL suspension in PBS was analyzed by flow
cytometry.
+
LC-MS (ESI, m/z): 1379.48 ([M + H] ), calc: 1380.48.
EB-maleimide (69.37 mg, 0.1 mml) was added into 5
mL DMF FA-PTX-Cys-HS (207.07 mg, 0.15 mmol)
solution and reacted for 24 h in the dark at room
temperature [22]. The compound was purified using
HPLC. The purified FA-PTX-EB was collected as a
yellow solid with 75% yield (156.3 mg). LC-MS: [M +
+
H] = 2073.13, calc: 2074.13. The chemical structures of
’-TBSCl-PTX 2’-TBSCl-7’PTX-Cys(Trt)-NH , FA-2’TB
2
2
SCl-7’PTX-Cys-SH, and FA-PTX-EB prodrugs were
supported by H NMR spectroscopy and electrospray
Cell viability assay
1
MDA-MB-231, OVCAR3, U87MG, and 293T cell
lines were used to measure the cytotoxicity of
FA-PTX-EB, and a standard protocol was followed
ionization mass spectroscopy analysis (Figures S2-S8).
Characterization of FA-PTX-EB
[
33]. The same concentration of PTX, FA-PTX, PTX-EB
The physicochemical properties of PTX prodrugs
were determined, including aqueous solubility and
stability. The solubility of FA-PTX, PTX-EB, and
FA-PTX-EB were analyzed using a reported method
and FA-PTX-EB from 3 pM to 1.6 nM was added to 5 ×
3
1
0 cells/well in 96-well plates for 24 h, with six
replicates. Then 10 μL MTT solution (5 mg/mL) was
added into each well and incubated for 4 h. After
changing to 150 μL DMSO, the absorbance of each
well was tested by a microplate reader at 490 nm.
[
31]. At room temperature, 100 mg of the three
compounds were added into volumetric flasks (10
mL), respectively, and 50 µL of water was injected
slowly. When the solution was saturated, the aqueous
solubility of FA-PTX, EB-PTX and FA-PTX-EB were
measured, respectively. To investigate the release of
PTX from different prodrugs in vitro, the three
PTX-conjugated prodrugs were incubated in mouse
plasma at 37 °C. The sampling times were at 0.5, 1, 2,
Apoptosis study
In vitro effect of FA-PTX-EB prodrugs were
measured on three tumor cell lines qualitatively and
quantitatively. The change in cell morphology during
apoptosis was observed by confocal microscopy. Flow
cytometry was used to quantify cell apoptosis [34].
Cells were incubated with 200 μL PTX, FA-PTX,
PTX-EB, and FA-PTX-EB (1 mg/mL) for 24 h in
confocal plates and washed using PBS (7.4 pH) twice.
After adding 500 L binding buffer, 5 μL propidum
iodide and 1 μL Annexin V-FITC were added to the
mixture and visualized by confocal microscopy. In the
same way, cells were incubated with PTX prodrugs
for 24 h in 6-well plates to quantify apoptosis using
flow cytometry.
4
, 8, 12, 24, 48 and 72 h. 50 μL PTX conjugated
prodrugs in 10 nM Tris buffer (5 mg/mL) were
incubated in 200 μL mouse plasma at 37 °C,
respectively. Then, 200 µL of each sample was
collected at each designated time point and free PTX
was extracted using 2 mL ethyl acetate and analyzed
by reversed-phase HPLC with acetonitrile/water
gradient.
Cell culture
Human breast cancer (MDA-MB-231), human
ovarian cancer (OVCAR3), human primary
glioblastoma (U87MG), and human renal epithelial
Animal subjects and tumor model
All animals including normal (Kunming) mice
and BALB/c athymic nude mice (female) were
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Theranostics 2018, Vol. 8, Issue 7
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purchased from Charles River Laboratories
equal to PTX 6 mg/kg) were administered to mice by
tail vein injection, respectively. At 4 h after injection,
the tissues (liver, kidneys, tumor) of sacrificed mice
were directly visualized by fluorescence microscopy.
The tissues were further analyzed using flow
cytometry.
(
Shanghai, China). All animals were maintained
based on the Animal Management Rules of the
Ministry of Health of the People's Republic of China
(
Document NO. 55, 2001) and the guidelines for the
Care and Use of Laboratory Animals of Suzhou
University.
Antitumor activity
Following the animal protocol, a folate-deficient
rodent diet was fed to the nude mice for 6 days, and
MDA-MB-231 tumor cells were inoculated at the right
axillary fossa of nude mice [9]. When the tumor
volumes reached 80 ~ 150 mm , the nude mice were
used to investigate the targeting ability,
MDA-MB-231 tumor bearing mice were
randomly assigned to five groups (n = 5 per group),
respectively. Each group was injected PTX prodrugs
by tail vein: (A) PBS buffer (pH 7.4, control), (B) pure
PTX solution (6 mg/kg), (C) FA-PTX (6 mg/kg PTX
equivalent), (D) PTX-EB (6 mg/kg PTX equivalent),
and (E) FA-PTX-EB (6 mg/kg PTX equivalent). Mice
experienced three i.v. injections, administered every
other day. The PTX prodrug therapeutic efficacies and
toxicities on MDA-MB-231 tumor-bearing mice were
evaluated by measuring tumor volume and body
weight of each mouse every other day. To further
evaluate the anti-tumor effect of PTX prodrugs, the
liver, kidneys and tumor tissues were excised for
pathology and measurement of CD46 protein levels
[35].
3
pharmacokinetics, and therapy of FA-PTX-EB.
Pharmacokinetics studies
Ten days after MDA-MB-231 tumor cell
implantation, mice were randomized into PTX (pure
PTX solution), FA-PTX, PTX-EB and FA-PTX-EB
3
groups with an average tumor volume of 98 ± 34 mm .
All mice received 6 mg/kg PTX or 6 mg/kg PTX
equivalent of prodrug via a single tail vein injection.
After drug injection, three mice per dosing group
were sacrificed at 0.12, 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48
h. Under anesthesia, the blood (500 μL) was collected
using terminal cardiac puncture and centrifuged (1500
g, 5 min). Collected tissue samples (liver, kidneys, and
tumor) were rinsed with PBS and immediately frozen
in liquid nitrogen. All samples were stored at -80 °C
before drug analysis.
Statistical analysis
All data are reported as the mean ± SD of n
independent measurements. Statistical analysis was
performed by using Student’s t-test with statistical
significance assigned at a p value of < 0.05.
The 100 μL plasma was extracted with 2 mL of
ethyl ether and added to 20 μL of internal standard
diazepam (500 ng/mL) with centrifugation at 12,000
rpm for 5 min. The mixture was resolved in 150 μL
methanol and analyzed by HPLC. In the same way,
the 0.2 g tissues including liver, kidneys, and tumor
were homogenized in 1 mL saline solution. Then, the
Results
Synthesis and characterization of
EB-conjugated PTX targeted prodrugs
The purified FA-PTX-EB and intermediate
products (structures shown in Figure 1, synthetic
scheme in Scheme S1) were characterized by LC-MS,
2
00 μL homogenate was added to the same volume of
1
H NMR, and HPLC analysis (Figure S1-S7). As
acetonitrile and vortexed for 6 min. After the mixture
was centrifuged at 12,000 rpm for 10 min, a 20 μL
sample was subjected to HPLC analysis (Figure S8).
The mobile phase of HPLC contained methanol and
water (80 : 20, v : v). PTX was measured at 227 nm.
Pharmacokinetic parameters were calculated by DAS
controls, we also synthesized control prodrugs, FA-
PTX and PTX-EB, both of which were monofunctional
in contrast to bifunctional FA-PTX-EB. To investigate
the aqueous solubility of EB-conjugated FA-PTX
prodrugs, the direct observations were strictly based
on the solubility calculations of PTX prodrugs. The
solubility of FA-PTX, PTX-EB, and FA-PTX-EB were
2
.0 Pharmacokinetics Software (Chinese Society of
Mathematical Pharmacology) with compartmental or
non-compartmental modeling approaches. Results
were presented as mean ± S.D.
1
.121 ± 0.12, 5.03 ± 0.09, and 7.02 ± 0.18 mg/mL,
respectively (n = 4). (Figure S8)
In vitro release studies
Targeting ability of EB-conjugated FA-PTX in
tumor-bearing mice
The targeting ability of PTX-EB and FA-PTX-EB
prodrugs was investigated by MDA-MB-231
xenograft tumors (n = 3/group). PTX-EB (0.2 mL, 6
mg/kg PTX equivalent) and FA-PTX-EB (0.2 mL,
The release of PTX from these prodrugs
FA-PTX, PTX-EB and FA-PTX-EB) was measured by
(
quantifying the amount of free PTX via HPLC, after
incubating the prodrugs in mouse plasma at 37 °C.
Figure 1B and Figure S7 showed PTX release from
prodrugs as measured by HPLC. The FA-PTX
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Figure 1. (A-C) Chemical structures of PTX-EB (A), FA-PTX-EB (B), and FA-PTX (C). (D) In vitro release of PTX in mouse plasma. PTX concentration was measured
by HPLC. The release was expressed as mean ± standard deviation based on triplicate experiments.
displayed fast release of PTX in vitro, resulting in
precipitation of PTX (t1/2 ˂ 4 h). By comparison, the in
vitro PTX release from PTX-EB exhibited relatively
lower release rates, with t1/2 of 6.82 h. The release of
PTX from FA-PTX-EB prodrug had a t1/2 of 9.15 h.
Interestingly, EB conjugation enabled more
sustainable drug release from prodrugs.
about 23.2% and 32.1% in U87MG cells. In contrast,
these two prodrugs were taken up by 293T cells at
merely 1.5% and 1.8% (Figure 2C). These findings
suggest that FA conjugation increased the targeting
selectivity of PTX prodrugs to tumor cells with FR-α
expression. The uptake of EB-conjugated prodrugs in
the different tumor cells were in agreement with their
FR-α expression levels.
In Vitro Cellular Uptake
In vitro antitumor activity of EB-conjugated
PTX prodrugs
To study receptor-mediated cellular uptake of
PTX-EB and FA-PTX-EB, the FR-α expression levels
on a panel of cells were first evaluated by Western
blot. The results showed that the FR-α expression
levels were higher in MDA-MB-231 cells compared to
OVCAR3 and U87MG tumor cell lines, as shown in
Figure 2A, and the FR-α protein expression level was
hardly detectable in 293T cells.
The cellular uptake of EB-conjugated prodrugs
was studied by fluorescence microscopy in cultured
cancer cells at 4 h (Figure 2B). The EB-conjugated PTX
prodrug, FA-PTX-EB, displayed increased uptake in
FR-α overexpressing MDA-MB-231, OVCAR3, and
U87MG tumor cells. Flow cytometry analyses
indicated that PTX-EB and FA-PTX-EB were taken up
in MDA-MB-231 tumor cells at 35.6% and 65.9%,
respectively. 29.1% and 38.1% PTX-EB and FA-PTX-
EB were retained in OVCAR3 cells, respectively, and
The antitumor effect of four cell lines including
tumor cell lines MDA-MB-231, OVCAR3, U87MG,
and normal cell line 293T were studied in vitro. As
shown in Figure 3, for all cell lines, prodrugs
displayed dose-dependent antitumor activity.
Especially, in all the three different tumor cell lines,
FA-PTX-EB prodrug showed inhibition ratios greater
than 85%, however, PTX inhibited only a maximum of
63% of tumor cells. In addition, the inhibition ratios of
PTX-EB were about 75% in MDA-MB-231, OVCAR3,
and U87MG cell lines. The cell inhibition ratio of
FA-PTX without EB was only about 68% at the highest
dose. The half maximal inhibitory concentration (IC50
)
of free PTX exhibited higher concentration compared
to the EB conjugated FA-PTX. The MDA-MB-231
tumor cells were inhibited to a greater extent over
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Figure 2. Cellular uptake of FA-PTX-EB in a series of tumor cells with different expression levels of FR-α. (A) Western blot results verify the different expression
levels of FR-α on tumor cells and normal cell lines. (B) Confocal microscopy images showing the intracellular uptake ability of FA-PTX-EB and PTX-EB prodrugs in
2
93T, U87MG, OVCAR3, and MDA-MB-231 cells. The nucleus is stained by DAPI, and the red corresponds with EB fluorescence from EB-conjugated compounds.
(C) Flow cytometric analysis of FA-PTX-EB and PTX-EB prodrug in 293T, U87MG, OVCAR3, and MDA-MB-231 cells.
other cancer cell lines owing to the higher expression
levels of FR-α receptor. Interestingly, the two
EB-conjugated prodrugs exhibited comparatively low
cell inhibition ratios relative to normal cells (293T),
even at the highest concentration (55%). This result
supports the hypothesis that EB-conjugated PTX
compound shows lower cytotoxicity to normal cells,
and enhances the antitumor ability of free PTX to
tumors that overexpress FR.
The morphological changes of MDA-MB-231,
OVCAR3, and U87MG tumor cells after the
incubation of FA-PTX, PTX-EB, and FA-PTX-EB, were
observed under confocal microscopy, as shown in
Figure 4A. Using annexin V and propidium iodide
4B, the late apoptotic cells (Q4) were evident in
different tumor cells treated with PTX, FA-PTX,
PTX-EB, and FA-PTX-EB prodrugs. Quantification of
flow cytometry results indicated the portions of cells
undergoing early apoptosis (Q2) of U87MG,
OVCAR3, and MDA-MB-231 were 29.8%, 21.8%, and
33.8% (PTX), 37.1%, 28.8%, and 36% (FA-PTX), 36.8%,
31.5%, and 44.7% (PTX-EB), 47.9%, 50.9%, and 62.3%
(FA-PTX-EB), respectively. The percent of Q4 was
3.1%, 2.5%, and 1.3% (PTX), 3.7%, 23.5%, and 34.5%
(FA-PTX), 13.2%, 36.5%, and 27.5% (PTX-EB), 12.3%,
22.2%, and 22.7% (FA-PTX-EB), respectively (Figure
4B). The above analyses prove that the prodrugs with
EB greatly increased the antitumor effect of low-dose
PTX.
(PI) staining, we further studied cell apoptosis and
necrosis upon treatment with prodrugs [10]. In Figure
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Figure 3. In vitro cytotoxicity of FA-PTX-EB, FA-PTX, PTX-EB, and free PTX against normal cells 293T (A), U87MG (B), OVCAR3 (C), and MDA-MB-231 tumor
cells. The numbers in parenthesis are the corresponding IC50 values calculated by GraphPad Prism.
Figure 4. In vitro apoptosis study. (A) The cells were incubated with free PTX, FA-7PTX, 7PTX-EB, or FA-7PTX-EB. One day later, morphology of cell apoptosis was
observed by confocal microscopy. Blue: DAPI fluorescence (nucleus stain); Green: Annexin V-FITC (apoptotic cell membrane); and red: Propidium Iodide, PI
(apoptotic cell nucleus). (B) The apoptosis cycle percent (%): Q2 + Q4.
levels in plasma and tissues represented the total
concentration of free PTX and PTX released from
prodrugs.
Concentration vs. time curves of PTX were
generated for plasma, liver, kidneys and tumor from
mice receiving treatment of FA-PTX, PTX-EB and
FA-PTX-EB, and the pharmacokinetic parameters
were then determined (Table 1-4). Cmax levels attained
after i.v. injection of prodrugs were 7854 ng/mL
Pharmacokinetics and tissue distribution in
plasma, liver, kidneys, and tumor
The pharmacokinetics and tissue distribution of
PTX and PTX prodrugs were studied by quantifying
total amount of PTX in the plasma and different tissue
samples from tumor-bearing mice. Figure 5 shows the
representative HPLC chromatograms of PTX in
mouse plasma and different tissues after i.v. injection
of PTX and PTX prodrugs at an equivalent PTX dose
of 6 mg/kg. As shown in Figure 5A, following i.v.
administration of PTX and PTX prodrugs, the PTX
(
9
PTX), 8034 ng/mL (FA-PTX), 8396 (PTX-EB), and
132 ng/mL (FA-PTX-EB), respectively (Table 1). The
highest AUC PTX values were observed with
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Theranostics 2018, Vol. 8, Issue 7
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FA-PTX-EB and were at least 8.6 times greater than
those of FA-PTX. Similarly, in PTX-EB, higher AUC
values were observed, at least 4.2 times greater than
those of free PTX.
Figure 5. In vivo pharmacokinetic studies. (A) Mean concentration-time profile of PTX in plasma following i.v. administration of a single dose of 6 mg/kg of PTX,
FA-PTX, PTX-EB, or FA-PTX-EB in mice. Time courses of PTX levels in tissues of MDA-MB-231 inoculated mice following i.v. administration of a 6 mg/kg dose of PTX
or equivalent formulated PTX. (B) Liver. (C) Kidneys. (D) Tumor. Data are shown as the mean concentration ± S.D. (n = 5/group).
Table 1. Pharmacokinetic parameters of PTX prodrugs compared with free PTX. Results represent mean ± S.E. (n = 5)
Parameter
Units
ng/mL
ng.h/mL
h
PTX
FA-PTX
PTX-EB
FA-PTX-EB
9132 ± 412
134922 ± 624
0.033 ± 0.001
7.51 ± 0.68
0.29 ± 0.08
C
max
7854 ± 403
7630 ± 114
0.201 ± 0.01
2.19 ± 0.32
1.31 ± 0.53
8034 ± 512
15657 ± 368
0.129 ± 0. 01
3.82 ± 0.53
1.08 ± 0.46
8396 ± 422
59570 ± 428
0.068 ± 0.001
4.41 ± 0.48
0.58 ± 0.32
AUC
t
t
1/2ɑ
1/2β
h
CL
mL/h/kg
Pharmacokinetic parameters were calculated using a two-compartment model. Values are the calculated pharmacokinetic parameter ± the standard error of the estimated
single model-dependent calculations
Table 2. Pharmacokinetic parameters of PTX prodrugs compared with free PTX. Results represent mean ± S.E. (n=5)
Parameter
Units
PTX
FA-PTX
PTX-EB
FA-PTX-EB
4210 ± 602
15846 ± 1610
0.395 ± 0.68
14.86 ± 0.98
C
max
ng/mL
ng.h/mL
h
8890 ± 432
11388 ± 1332
8.455 ± 1.08
37.31 ± 4.03
7412 ± 422
61354 ± 1006
5.22 ± 0.92
33.84 ± 5.46
6037 ± 511
26453 ± 1315
1.67 ± 0.48
22.23 ± 1.32
AUC
t
1/2
CL
mL/h/kg
Liver tissue pharmacokinetics were calculated using non-compartmental analysis
Table 3. Pharmacokinetic parameters of PTX prodrugs compared with free PTX. Results represent mean ± S.E. (n = 5)
Parameter
Units
PTX
FA-PTX
PTX-EB
FA-PTX-EB
4285 ± 405
118971 ± 1256
3.29 ± 0.56
7.17 ± 2.09
C
max
ng/mL
ng.h/mL
h
5769 ± 712
46821 ± 1025
2.15 ± 0.62
28.57 ± 3.03
9181 ± 639
234286 ± 2204
6.82 ± 0.53
10.21 ± 4.25
3917 ± 233
8659 ± 1062
1.41 ± 0.48
15.07 ± 2.02
AUC
t
1/2
CL
mL/min
Kidney tissue pharmacokinetics were calculated using non-compartmental analysis
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Theranostics 2018, Vol. 8, Issue 7
2026
Table 4. Pharmacokinetic parameters of PTX prodrugs compared with free PTX. Results represent mean ± S.E. (n=5)
Parameter
Units
PTX
FA-PTX
PTX-EB
FA-PTX-EB
12948 ± 1766
391016 ± 1232
124.51 ± 4.68
29.86 ± 2.98
C
max
ng/mL
ng.h/mL
h
3938 ± 304
21511 ± 1244
6.19 ± 0.32
13.11 ± 2.14
7108 ± 611
131053 ± 5462
44.21 ± 2.53
16.88 ± 5.09
6503 ± 455
31825 ± 1102
34.41 ± 3.48
22.23 ± 4.32
AUC
t
1/2
CL
mL/h/kg
Tumor tissue pharmacokinetics were calculated using non-compartmental analysis
The mean PTX tissue concentration-time profiles
after i.v. injection of all PTX prodrugs are shown in
Table 2-4. For free PTX, the highest concentration of
drugs was found in the liver. For FA-PTX prodrugs,
the highest concentrations were found in the kidneys,
and the maximal PTX concentrations of FA-PTX-EB in
tumor were 12948 ng/g. In the kidney, the highest
concentrations of PTX were 5769, 9181, 3917 and 4285
ng/g for PTX, FA-PTX, PTX-EB, and FA-PTX-EB
prodrugs, respectively (Table 3). The highest
concentration of PTX displayed in tumor for various
prodrugs were as follows: 12948 ng/g (FA-PTX-EB),
prodrug. In contrast, the highest PTX concentration of
free PTX in tumor was only 3938 ng/g (Table 4).
Targeting ability of FA-PTX-EB in tumor
bearing mice
Based on the above pharmacokinetics studies,
after injection of PTX-EB and FA-PTX-EB at 4 h, we
sacrificed mice bearing MDA-MB-231 tumor
xenografts, and excised tissues including liver,
kidneys, and tumor for tissue sectioning. Tissue
sections were visualized under fluorescence
microscopy. In Figure 6A, in comparison with other
excretion organs such as the liver and kidneys, the
highest fluorescence signal was observed in tumor
tissues for FA-PTX-EB. At
7
108 ng/g (FA-PTX), and 6503 ng/g (PTX-EB)
the same time, tumor
tissues from FA-PTX-EB
treatment
exhibited
stronger EB fluorescence
signals than PTX-EB,
suggesting higher uptake
of
FA-PTX-EB
than
PTX-EB.
To further study the
tumor targeting ability of
the
prodrugs,
the
fluorescence intensity of
organ
analyzed
tissues
by
was
flow
cytometry (Figure 6B).
The pharmacodynamics
of
PTX-EB
and
FA-PTX-EB signals at 4 h
in tissues and tumors are
described in Figure 6B. At
4
h after injection of
FA-PTX-EB,
the
fluorescence
intensity
ratio of MDA-MB-231
tumor was 65.98 ± 0.125
%
.
In contrast,
the
MDA-MB-231
tumor-bearing mice with
administration of PTX-EB
prodrugs showed a ratio
of 25.12 ± 0.64 % at 4 h
post injection.
Figure 6. Tissue uptake of FA-PTX-EB and PTX-EB. MDA-MB-231 tumor xenografts at 4 h post-injection of
EB-conjugated PTX prodrugs. Tissues (liver, kidneys, and tumor) were immediately excised for sectioning and were
visualized by fluorescence microscopy. The red fluorescence from EB-conjugated compounds (A) and corresponding flow
cytometry values (B) were observed in liver, kidneys and tumor, respectively. Data represent mean ± standard deviation
(SD), n = 5/group.
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Theranostics 2018, Vol. 8, Issue 7
In vivo therapeutic experiment
2027
FA-PTX (45.13%), and PTX (41.04%). Based on tumor
weight, the relative inhibition rates were consistent
with the results from tumor volume measurements as
shown in Figure 7C.
The change in body weight was monitored
throughout the study period and is presented in
Figure 7B. The body weight of mice treated with free
PTX showed significant decrease attributed to severe
side effects and toxicity of PTX. The body weight loss
of the PTX group was 0.65 ± 0.02 g on day 21 post
initial treatment, about
Mice bearing MDA-MB-231 tumors were used to
evaluate the in vivo anti-tumor efficacy of PTX,
FA-PTX, PTX-EB, and FA-PTX-EB. As shown in
Figure 7, the change of mice body weight and tumor
volume was depicted in different treatment groups.
FA-PTX-EB most effectively inhibited tumor growth
among all treatment regimens (Figure 7A). The results
show that FA-PTX-EB inhibited tumor volume most
prominently (74.82%), followed by PTX-EB (50.08%),
3
.5% of the initial weight.
However, the data
showed that the body
weight of mice was
significantly
increased
following treatment with
FA-PTX, PTX-EB, and
FA-PTX-EB
groups.
Results suggest that these
PTX conjugations could
significantly reduce the
systemic toxicity of PTX.
After treatment with
PTX, FA-PTX, PTX-EB,
and FA-PTX-EB, liver,
kidneys and tumor tissues
were excised and further
evaluated
antitumor
toxicity
for
effect
the
and
by
histopathological
examination.
representative
Figure 7. FA-PTX-EB significantly inhibited tumor growth in a breast cancer mouse model. (A) Tumor volumes of
MDA-MB-231-bearing mice as a function of time (d) (P values, *P ˂ 0.05, ***P ˂ 0.001, are calculated by t-test). (B) Body
weights of MDA-MB-231-bearing mice as a function of time (d). Data were given as mean ± SD (n = 6/group). (C) The
relative inhibition rates (%). (D) Kaplan-Meier survival plot.
The
tumor
from
of
tissue
different
sections
groups
treatments were shown in
Figure 8. Tumor tissues in
the PTX, FA-PTX, and
PTX-EB groups exhibited
different populations of
spherical
intercellular blank spots,
and necrosis. The
cells,
FA-PTX-EB group showed
larger intercellular empty
spaces in tumor tissue that
indicate efficacious tumor
therapy. Compared with
the control group, no
distinct
pathologic
changes were found in the
liver and kidneys of the
Figure 8. (A) H&E staining of tumor tissues excised from nude mice 21 days after administration of PTX, FA-PTX, mice injected with PTX,
PTX-EB and FA-PTX-EB. (B) The level of CD46 protein in tumor tissues was measured by ELISA assay kit (n = 5/group).
FA-PTX, PTX-EB, and
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Theranostics 2018, Vol. 8, Issue 7
2028
FA-PTX-EB (Figure S9). In addition, we measured the
expression levels of membrane cofactor protein CD46
on the tumor tissue. The CD46 values of the
FA-PTX-EB treated group were significantly
decreased compared with other groups. Overall, these
results demonstrated the potent and specific
therapeutic effect of FA-PTX-EB.
without EB (Table 1). The plasma AUC value of PTX
from the PTX-EB group was seven times higher
compared to the group administered with free PTX,
and the AUC value of FA-PTX-EB in plasma was eight
times higher than that of FA-PTX. Similarly, the PTX
plasma half-life t1/2β value after PTX-EB administrat-
ion was two times higher than that of free PTX, and
the plasma half-life t1/2β value of FA-PTX-EB was two
times higher than that of FA-PTX. These results
suggested that PTX-conjugated EB prodrugs may
circulate for a longer time in blood than PTX prodrug
without EB. In addition, EB modification resulted in
slower release of PTX from the FA-PTX-EB prodrug
over a prolonged period.
Discussion
Optimal pharmacokinetics is key to drug
development. The limited efficacy of many drugs is
often attributed to poor pharmacokinetics that leads
to low bioavailability, for example, many folate-
conjugated chemotherapeutics failed in clinical trials
[
36]. Recently, we developed a new class of small
Notably, the PTX concentration of EB-PTX
prodrugs in the tumor was significantly higher than
those of the PTX prodrugs without EB, especially
FA-PTX-EB. The primary FR populated tissue
(kidneys) comprises the major sites of accumulation
for circulating FA conjugates. Therefore, one would
expect drug clearance in the FR rich tissues to be
significantly greater for FA-PTX conjugated EB than
for FA-PTX. The PTX AUC of FA-PTX-EB was lower
in the liver and kidneys; and higher in plasma and
tumor compared to FA-PTX group. Similar results
were observed between the PTX AUC of PTX-EB and
PTX groups. Importantly, there were statistically
molecule albumin binding entities based on the
long-circulating EB. EB displays a strong noncovalent
binding with both murine and human serum albumin
[
37-40]. In our previous work, we have extensively
studied the albumin binding abilities of EB conjugates
with various chemical agents, and have confirmed
their strong binding abilities [41, 42]. In this study, we
functionalized folate-PTX prodrug with truncated EB
as an albumin binding moiety for targeted tumor
therapy. The resulting prodrug conjugate, namely
FA-PTX-EB, which greatly enhanced aqueous
solubility of PTX, dramatically increased blood
circulation half-life and tumor uptake of the drug, and
decreased the drug accumulation in the kidneys. The
fluorescence signal of EB was used to study the
dynamic distribution of the prodrugs at the cellular
level and the tissue level.
significant
formulation-dependent
differences
observed in the liver and kidneys exposure profiles.
This finding may be rationalized by the fact that small
molecular albumin binding moieties can extend the
blood-circulation time of PTX prodrugs and decrease
its accumulation in the liver and kidneys.
The vitamin B9 (folic acid) is one of the
well-proven ligands for targeted delivery of imaging
or therapeutic agents to tumors. Therefore, in this
paper, the cellular uptake ability of FA-PTX
conjugated EB was evaluated in vitro and in vivo
(Figure 2 and Figure 6). The percentage of cell uptake
of FA-PTX-EB (23.09%~65.91%) treated tumor cell
groups was far higher than normal cells with low
FR-α expression (1.5%, 1.8%, respectively). Compared
with prodrug without FA, FA-PTX-EB significantly
enhanced targeting ability for tumors via
Physicochemical properties of these PTX
prodrugs, including FA-PTX, PTX-EB, and FA-PTX-
EB, were assayed by HPLC. The water-soluble EB and
amino acid cysteine increased the water solubility of
PTX prodrug from ∼0.4 μg/mL for PTX to 7.02
mg/mL for the prodrug, and the increase in the water
solubility of prodrug with EB was significantly
different from prodrug without EB (Figure S8).
Interestingly, PTX was sustainably released from
PTX-EB and FA-PTX-EB prodrugs, with t1/2 = 6.82 h
and 9.15 h, respectively. These results suggest that EB
acted as a compatibilizer in PTX prodrugs, resulting
in improvements in water solubility, physical
stability, and drug release.
The purpose of EB-conjugation is to enhance the
blood circulation and tumor tissue retention time of
PTX, and to reduce the toxicity of free PTX to normal
tissue. Plasma pharmacokinetics and tissue
distribution behavior were analyzed, which provided
strong evidence of enhanced delivery [43, 44]. In the
pharmacokinetics study, pharmacokinetic parameters
of PTX from the PTX with EB formulation were
significantly better than those from the PTX group
receptor-mediated
uptake
as
evidenced
by
fluorescence microscopy. In vitro data clearly proved
that FA-PTX-EB prodrugs enhanced the cellular
uptake and antitumor activity through FR-α targeting.
The same results were also displayed in the tissue
sections at 4 h post injection of FA-PTX-EB (Figure 6).
These results support the notion that FA-PTX-EB
improved the targeting ability of PTX to FR-positive
tumors. Taken together, this bifunctional drug
delivery system enhanced the drug retention time and
drug concentration in tumor tissues.
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Theranostics 2018, Vol. 8, Issue 7
2029
PTX is a potent anticancer agent as a microtubule
stabilizer, and it plays a central role in the treatment of
breast cancer. Accordingly, PTX has a low IC50 value
of 4.5 nM for the human MDA-MB-231 breast cancer
cell line (Figure 3B). In comparison to free PTX or
prodrug without EB, the IC50 value of PTX-EB or
FA-PTX-EB had a lower value of 0.98-2.29 nM,
reflecting the enhancement of cytotoxicity via EB
conjugation. The IC50 values of FA-PTX-EB as a
prodrug were about 3-fold higher, 12 nM in healthy
cells, suggesting the specificity of drug cytotoxicity.
Notably, FA-PTX-EB or PTX-EB were more effective
than FA-PTX or free PTX in vitro. Thus, truncated EB
enhanced the intracellular delivery of PTX prodrugs
that further led to stronger cytotoxicity in tumor cells.
water soluble cysteine, FA, EPR effect, the “stealth”
character of amino acid ester, FA/FR-α mediated
endocytosis, and the EB enhanced blood circulation/
tumor retention. PTX ester prodrugs are often
water-soluble and less toxic, but less active as
anticancer agents. However, FA-PTX prodrug with
EB prodrug is a unique anticancer prodrug in terms of
physical stability, lower systemic toxicity, and higher
antitumor efficacy in an MDA-MB-231 xenograft
model. Given slower in vitro release and improved
physical stability of FA-PTX prodrug with EB vs.
FA-PTX without EB prodrug, the EB containing
formulation may circulate longer in the bloodstream,
increase AUC, reduce the distribution of PTX in
non-target tissue, and increase tumor retention time.
+
The similar percentage of late apoptotic cells (PI ) in
Conclusions
tumor groups was higher for the EB formulations. The
percentage of necrotic/dead cells (Q2 + Q4) was 85%
for FA-PTX-EB compared with only 34.6% in free PTX
treated cells (MDA-MB-231).
Taken together, we have harnessed the
advantages of an albumin binding moiety and folate
to establish FA-PTX-EB as a bifunctional prodrug for
both passive and active targeted delivery of PTX,
resulting in long blood circulation, improved
pharmacokinetics, high antitumor activity, and low
toxicity. Moreover, in comparison with previous PTX
formulations, EB-conjugated FA-PTX prodrugs also
exhibited higher water solubility, prolonged tumor
retention, and potentiated antitumor activity, as well
as reduced toxicity. We believe that the strategy of
bifunctional targeting will expand the clinical
implications of PTX-based cancer therapy and spur
the development of similar bifunctional prodrug
platforms.
The in vivo anticancer efficacy of PTX prodrugs
was evaluated in a MDA-MB-231 cancer model after
tail vein injection at an equivalent dose of 6 mg/kg
PTX (Figure 7). The tumor growth in the FA-PTX-EB
group was remarkably inhibited compared to free
PTX. Importantly, the tumor growth inhibition
correlated highly with significant apoptosis observed
in tumor tissues (Figure 8A). No obvious changes of
mouse body weights were observed after treatment
with prodrugs. In the MDA-MB-231 cancer model, the
antitumor effect of FA-PTX-EB was significantly
stronger than other PTX prodrug or PTX. The
tumor-inhibition of FA-PTX-EB reached 71.6%, which
was about 1.2 times higher than FA-PTX and 2 times
higher than PTX. These data confirmed that EB and
FA cooperatively enhanced the antitumor effect of
FA-PTX-EB.
Acknowledgements
The authors are grateful to the Anhui Province
Educational Committee Major Project (KJ2016SD62),
Key Project of Anhui Educational Committee
The decrease of mouse weight in the free PTX
treatment group demonstrated that free PTX had
severe side effects. The mean body weight loss of the
PTX group was 0.65 ± 0.02 g, representing a 3.5% loss
of the original weight. On the contrary, the increase of
body weight of other groups treated with PTX-EB and
FA-PTX-EB indicated that the small molecular
binding moiety EB improved PTX’s water solubility,
pharmacokinetic properties, and biodistribution,
which collectively contributed to low toxicity. The
hematoxylin and eosin (H&E) staining showed no
histological signs of toxicity in major organs with all
PTX prodrugs (Figure S9). Nevertheless, necrotic
areas were clearly shown in the FR-α positive tumors
treated with FA-PTX-EB. Similar antitumor effects
were observed by CD46 analysis (Figure 8B). The
improved therapeutic effect of the FA-PTX-EB
prodrug is likely a result of the synergistic function of
(
KJ2013A262, KJ2015A220, and gxfxZD2016266),
Natural Science Foundation Committee of China
NSFC 81000666), Quality Project of AnHui Education
Department -The plan of “ChuangKe” Laboratory
2015ckjh109), Research Platform Project of Suzhou
(
(
University (2016kyf08), and the Intramural Research
program, National Institute of Biomedical Imaging
and Bioengineering, National Institutes of Health.
Lingling Shan was partially funded by the China
Scholarship Council (CSC).
Supplementary Material
Supplementary figures.
http://www.thno.org/v08p2018s1.pdf
Competing Interests
The authors have declared that no competing
interest exists.
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Theranostics 2018, Vol. 8, Issue 7
2030
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