Transferrin-modified c[RGDfK]-paclitaxel loaded hybrid micelle for sequential blood-brain barrier penetration and glioma targeting therapy

Article abstract of DOI:10.1021/mp200600t

The effective chemotherapy for glioblastoma multiform (GBM) requires a nanomedicine that can both penetrate the blood-brain barrier (BBB) and target the glioma cells subsequently. In this study, Transferrin (Tf) modified cyclo-[Arg-Gly-Asp-d-Phe-Lys] (c[RGDfK])-paclitaxel conjugate (RP) loaded micelle (TRPM) was prepared and evaluated for its targeting efficiency, antiglioma activity, and toxicity in vitro and in vivo. Tf modification significantly enhanced the cellular uptake of TRPM by primary brain microvascular endothelial cells (BMEC) to 2.4-fold of RP loaded micelle (RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM) (39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising nanomedicine for glioma chemotherapy.

Full text of DOI:10.1021/mp200600t

Article
pubs.acs.org/molecularpharmaceutics
Transferrin-Modified c[RGDfK]-Paclitaxel Loaded Hybrid Micelle for
Sequential Blood-Brain Barrier Penetration and Glioma Targeting
Therapy
Pengcheng Zhang,†,§ Luojuan Hu,‡,§ Qi Yin,† Linyin Feng,*,‡ and Yaping Li*,†
‡
†Center of Pharmaceutics and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
ABSTRACT: The effective chemotherapy for glioblastoma
multiform (GBM) requires a nanomedicine that can both
penetrate the blood-brain barrier (BBB) and target the glioma
cells subsequently. In this study, Transferrin (Tf) modified
cyclo-[Arg-Gly-Asp-D-Phe-Lys] (c[RGDfK])-paclitaxel conju-
gate (RP) loaded micelle (TRPM) was prepared and evaluated
for its targeting efficiency, antiglioma activity, and toxicity in
vitro and in vivo. Tf modification significantly enhanced the
cellular uptake of TRPM by primary brain microvascular
endothelial cells (BMEC) to 2.4-fold of RP loaded micelle
(RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous
injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin
overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most
importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG
glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM)
(39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead
to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising
nanomedicine for glioma chemotherapy.
KEYWORDS: glioma, blood-brain barrier, micelle, sequential targeting
drugs in the whole brain after BBB penetration may cause
neurotoxicity, which affects all cognitive function.12
INTRODUCTION
■
Glioblastoma multiform (GBM) is one of the most aggressive
and lethal cancers, with a median survival of about 14 months
and a 5 year survival rate of less than 5%.1 Chemotherapeutic
drugs such as carmustine, lomustine, and Temozolomide have
shown some promise, but the patients’ survival improvement is
usually limited mainly due to overexpression of the DNA repair
enzyme O6-methylguanine DNA methyltransferase (MGMT),
which repairs the DNA injury caused by the above-mentioned
alkylator.2 Several widely used chemotherapeutic agents with
distinct antitumor mechanisms such as paclitaxel (PTX),
doxorubicin and topotecan are unable to penetrate the blood-
brain barrier (BBB), which is still functional at the outer rim of
the tumor and the malignant cells infiltrated peritumoral
tissue.3,4 In the past decade, several brain targeting ligands were
developed with the improvement of brain drug accumulation,5
but targeting GBM tumor or infiltrated glioma cells after
penetrating BBB remains challenging. Recently, dual-targeting
nanocarriers were developed by modifying nanocarriers with
ligands such as wheat germ agglutinin (WGA),6 angiopep,7 and
lactoferrin (Lf),8 the receptors of which are expressed on both
BBB and glioma cells. However, these receptors are also highly
expressed throughout the brain,9,10 and the degradation of
ligands during transportation across BBB would further reduce
the selectivity of nanocarriers.11 The nonspecific distribution of
To solve this issue, we designed and constructed a novel
sequential-targeting nanomedicine by encapsulating a drug
conjugate that can target glioma cells into a BBB-penetrating
micelle. Transferrin (Tf), an endogenous protein that can be
effectively transcytosed across the BBB and can direct carrier
systems with encapsulated drugs across the BBB by receptor
mediated transcytosis (RMT),13,14 was grafted onto the micelle.
cyclo-[Arg-Gly-Asp-D-Phe-Lys] (c[RGDfK]), a peptide that
specifically binds to integrin overexpressed glioma cells,15,16
was conjugated to PTX to obtain c[RGDfK]-paclitaxel con-
jugate (RP) (Scheme 1).The targeting efficiency of Tf modified
RP loaded hybrid micelle (TRPM) was evaluated in the aspects
of internalization by primary brain microvascular endothelial
cells (BMECs) and U-87 MG cells in vitro and the brain
distribution in vivo. The general toxicity and antiglioma activity
of TRPM was evaluated on nude mice bearing intracranial U-87
MG gliomablastoma.
Received: November 24, 2011
Revised: April 6, 2012
Accepted: April 12, 2012
Published: April 12, 2012
© 2012 American Chemical Society
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a
4-dimethylamino-pyridine (93 mg, 0.762 mmol), succinic
anhydride (76 mg, 0.762 mmol), and triethylamine (77 mg,
0.762 mmol) were dissolved in 15 mL anhydrous dioxane and
stirred overnight at room temperature under an N2 atmosphere.
The reaction solution was evaporated and redissolved in
chloroform. The solution was sequentially washed with 10 mM
HCl and saturated brine solution, dried over Na2SO4, and
evaporated to yield PTXS as an off-white solid.
Scheme 1. Synthesis Scheme of RP
PTXS was activated by dicyclohexylcarbodiimide/N-hydroxy-
succinimide (1:1.2:1.2, mol/mol) in anhydrous tetrahydrofuran
(THF) for 12 h at room temperature under an N2 atmosphere.
The reaction solution was filtered, and the filtrate was
evaporated to yield PTXS succinimide ester (PTXSSE).
PTXSSE, c[RGDfK] and diisopropylethylamine (1:1.2:1.2,
mol/mol) were dissolved in anhydrous N,N-dimethylforma-
mide and stirred overnight at room temperature under an
N2 atmosphere. The reaction solution was evaporated under
vacuum to yield crude RP. Purification of crude RP was carried
out on a HPLC system (Waters 1525 chromatography system
with a 2489 UV−vis detector) with the following conditions:
Waters XBridge BEH130 Prep C18 (5 μm, 10 × 150 mm); the
flow rate was 3 mL/min with the mobile phase starting from
55% solvent A (0.05% trifluoroacetic acid in water) and 45%
solvent B (acetronitrile) (0−4 min) to 90% solvent A and 10%
solvent B at 10 min; the monitored wavelength was 237 nm.
The fractions containing RP were collected and lyophilized
(RD85, Millrock). Analytical HPLC was performed on an
Agilent 1100 with the following conditions: Agilent HC-C18
column (5 μm, 4.6 × 250 mm); the flow rate was 1 mL/min
with the mobile phase starting from 70% solvent A (0.05%
trifluoroacetic acid in water) and 30% solvent B (acetronitrile)
(0−5 min) to 30% solvent A and 70% solvent B at 40 min; the
monitored wavelength was 237 nm. The retention time of RP
on analytical HPLC was 23.1 min. The retention time of PTX
under the same condition was 29.3 min. RP was also deter-
mined using a 300 MHz 1H NMR spectrometer (Varian, USA)
and an electrospray ionization time-of-flight (ESI-TOF) mass
spectrometer (Finnigan MAT-9, Thermo), respectively.
Preparation of RP and PTX Loaded Micelles. RP loaded
micelle (RPM) was prepared using a blend of PCL-PEEP and
Mal-PEG-PCL by a dialysis method. Briefly, 4 mg RP, 45 mg
PCL-PEEP, and 5 mg Mal-PEG-PCL were dissolved in 250 μL
THF, and then 1.25 mL deionized water was added dropwise
over 10 min. The solution was stirred gently for 30 min
followed with dialysis against 2 L of deionized water overnight
to remove THF. The RPM was then purified and concentrated
using Amicon Ultra-15 (MWCO: 100 kDa, Millipore). For Tf
conjugation, Tf was first thiolated and characterized as
previously described.20 The thiolated Tf was then incubated
with RPM at a thiolated Tf to maleimide group molar ratio of
1:10 for 8 h to obtain Tf conjugated RPM (TRPM). The
TRPM was then purified and concentrated using Amicon Ultra-
15 (MWCO: 100 kDa, Millipore). PTX loaded micelle (PM)
and Tf conjugated PM (TPM) were prepared using the same
procedure as described above and were used as control. Finally,
all of the micelles were sterilized by 0.22 μm filtration.
a
DMAP, 4-(dimethylamino)pyridine; DCC, dicyclohexylcarbodiimide;
NHS, N-hydroxysuccinimide; DMF, dimethylformamide.
EXPERIMENTAL SECTION
■
Materials. Paclitaxel (PTX) was purchased from Shanghai
Sunve Pharmaceutical Co., Ltd. (China). cyclo-[Arg-Gly-Asp-D-
Phe-Lys] (c[RGDfK]) (purity >98%) was synthesized by GL
Biochem Ltd. (China). Transferrin (Tf), N-(2-hydroxyethyl)-
piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-iminothio-
lane hydrochloride (ITH), thiazolylblue tetrazoliumbromide
(MTT), and Ellman’s reagent were obtained from Sigma-
Aldrich (USA). BODIPY TR-X SE was purchased from
Invitrogen (USA). Taxol was purchased from Bristol-Myers-
Squibb Company (USA). Poly(ε-caprolactone)-block-poly-
(ethyl ethylene phosphate) (PCL-PEEP) was synthesized and
characterized as previously described,17 and the degrees of
polymerization (DP) of PCL and PEEP were 31 and 35,
respectively. Maleimide-poly(ethylene glycol)-block-poly(ε-cap-
rolactone) (Mal-PEG-PCL) was synthesized by ring-opening
polymerization using maleimide-poly(ethylene glycol)-OH as
initiator in the presence of stannous octoate,18 and the DP of
PEG and PCL were 77 and 28 determined by 1H NMR, respec-
tively. All other reagents were of analytical grade and used as
received without further purification.
Cells and Animals. U-87 MG cell line was obtained from
the American Type Culture Collection (ATCC, USA), and
HEK-293 cells stably expressing enhanced green fluorescence
protein (HEK-293-EGFP) were kindly provided by National
Laboratory of Oncogenes and Related Genes, Shanghai Cancer
Institute. Both cell lines were grown in DMEM containing 10%
fetal bovine serum (FBS). Primary brain microvascular
endothelial cell (BMEC) was obtained from Sprague−Dawley
rats brain and was grown in EMB-2 (Lonza, Switzerland)
containing 10% FBS. All the cells were incubated at 37 °C in a
humidified and 5% CO2 incubator.
ICR mice (18−22 g, ♂), Sprague−Dawley (SD) rats (100−
120 g, ♂), and BALB/c-nu mice (18−22 g, ♂) were obtained
from Shanghai Experiment Animal Center, Chinese Academy
of Sciences, and maintained at 25 2 °C on a 12 h light-dark
cycle with free access to food and water. All animal procedures
were performed according to the protocols approved by the
Institutional Animal Care and Use Committee at Shanghai
Institute of Materia Medica, Chinese Academy of Sciences.
c[RGDfK]-Paclitaxel Conjugate (RP) Synthesis. To
synthesize RP, PTX was first reacted with succinic anhydride
to obtain PTX-2′-succinate (PTXS) as previously described
with minor modification.19 Briefly, PTX (500 mg, 0.586 mmol),
Hydrolysis. The RP solution was added to a phosphate
buffer solution (PBS) at pH 5.0, 7.4, and 8.4 to evaluate the
effect of pH on RP hydrolysis (final concentration was 10 μM).
The solutions were incubated (37 °C, 150 rpm) and sampled at
scheduled times for HPLC analysis to determine RP content.
Each experiment was repeated in triplicate. To evaluate the
stability of RP in plasma, RP solution or RPM were added to
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1
Figure 1. HPLC chromatogram (A), H NMR spectrum (B), and low- and high-resolution ESI-TOF mass spectrum (C) of RP.
rat plasma (final concentration was 10 μM). The solutions were
incubated (37 °C, 150 rpm) and sampled at predetermined
intervals for RP content determination using HPLC.
In Vitro Release. The release profiles of RP from RPM and
TRPM were investigated in 1 M sodium salicylate, which has
been reported to aid in solubilizing PTX without destabilizing
the PEG-b-poly(phenylalanine) micelle.21 Briefly, dialysis bags
(MWCO: 6−8 kDa, Spectrum Lab) containing RPM or TRPM
(containing 0.3 μmol RP) were incubated with 5 mL of 1 M
sodium salicylate (37 °C, 150 rpm). At scheduled time in-
tervals, the release medium was withdrawn and replaced with a
predetermined volume of fresh medium. The concentrations of
RP and PTX resulting from RP hydrolysis were measured by
HPLC as described above.
Cellular Uptake. Primary BMECs were seeded on a 12-well
plate and allowed to reach confluence.17 The cells were then
treated with 10 μM RP containing RPM or TRPM for 1 h at
37 °C. To prove the transferrin receptor (TfR)-mediated
clathrin-dependent endocytosis, 5 mg mL−1 free Tf or 10 μM
chlorpromazine (Chl) were added 30 min prior to TRPM
addition in 3 wells, respectively. The cells were then washed 3
times with ice-cold PBS and lysed with Triton X-100 (1%, v/v)
for 30 min. The RP and PTX in cell lysates were extracted
with methanol and determined by HPLC. The amount of
endocytosed drugs were then normalized with the amount of
cellular protein quantified by the Coomassie brilliant blue
method.
Figure 2. Sizes (A) and zeta potentials (B) of PM (black line), TPM
(red line), RPM (green line), and TRPM (blue line).
To investigate whether c[RGDfK] conjugate could target the
integrin overexpressing U-87 MG cells, a c[RGDfK]-BODIPY-
TR conjugate (RB, m/z = 1123.4904 corresponding to the
[M + H]+) was synthesized and purified following the same
procedure as described for RP synthesis. U-87 MG cells (3 ×
104 cells/well) and HEK-293-EGFP cells (2 × 104 cells/well)
were cocultured on 10 mm2 glass coverslips coated with poly-L-
lysine (PLL) in 24-well plates for 24 h. The coculture model
was incubated for 1 h with BODIPY-TR or RB (5 μM), washed
3 times with ice-cold PBS, fixed with 4% polyformaldehyde,
stained with DAPI (10 μg mL−1) for 10 min, and washed with
PBS twice again. The cells were then mounted on glass slides
with 3 μL of MobiGlow (MoBiTec, Germany) and visualized
by confocal microscopy (FluoView FV1000, Olympus).
Cytotoxicity Assay. U-87 MG cells were seeded in a 96-
well plate at 5 × 103 cells/well and incubated for 24 h. The
medium was replaced with fresh medium containing various
concentrations of PTX, RP, PM, and RPM. At 72 h, the cell
viability was measured by SRB assay according to the
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manufacturer’s instruction (Sigma, USA), and the dose-effect
curves were plotted.
Cell Cycle Analysis. U-87 MG cells were seeded in a 6-well
plate at 2 × 105 cells/well and incubated for 12 h. The
exponentially growing U-87 MG cells were treated with 20 nM
PTX, PM, RP, RPM, c[RGDfK], a mixture of PTX and
c[RGDfK], or mixture of PM and c[RGDfK] for 24 h,
respectively. Then, cells were harvested and fixed in 70%
ethanol for 12 h at 4 °C. The cells were collected by
centrifugation (1 × 103 rpm, 4 min, 4 °C), washed twice with
cold PBS to remove residual ethanol, resuspended in 0.5 mL
PBS containing 0.5 mg/mL RNase A, 0.02 mg/mL PI, and
0.1% Triton X-100, and incubated at 37 °C for 30 min. Cell
cycle profiles of treated U-87 MG cells were studied using a
FACS calibur flow cytometer (Becton Dickinson, USA). The
data were analyzed through Mod-Fit 2 software (Becton
Dickinson, USA).
Western Blot and Antibodies. The mice bearing
intracranial U-87 MG glioma were established as follows:
U-87 MG cells (1 × 105 cells in 1 μL PBS) were injected into the
right striatum (1.8 mm lateral to the bregma, 0.6 mm anterior
to the bregma, and 3 mm of depth) of male BALB/c-nu mice
(18−22 g) at 1 μL/min using a stereotactic fixation device with
mouse adaptor. Four weeks after implantation, three mice were
anaesthetized and sequentially perfused with 0.9% saline to
remove the blood. The glioma, peritumoral, and normal brain
tissue were collected. The tissues were weighed and
homogenized in ice-cold RIPA lysis buffer (20 μL/mg tissue).
The resulting homogenates were centrifuged under 1.5 × 104 g
at 4 °C for 15 min, and the protein concentrations in
supernatant were determined using the Coomassie brilliant
blue method. Primary antibodies used for Western blot analysis
were rabbit anti-integrin αv antibody (1:1000, Cell signaling,
USA) and mouse anti-β-actin antibody (1:15000, Sigma-
aldrich, USA).
Application of Micelles in Animals. Intracranial glioma
bearing mice were established as described above. Four weeks
after implantation, the mice were randomly assigned into one of
the following groups (n = 12 per group): Taxol, PM, TPM, and
TRPM, and mice were administrated via tail vein at the
equivalent 10 mg kg−1 dose of PTX. At 1, 4, 12, and 24 h after
the administration, the blood samples were collected and
centrifuged under 3 × 103 g at 4 °C for 5 min immediately to
harvest plasma. Then, the mice were killed and tissues including
heart, liver, spleen, lungs, kidneys, and tumor, peritumoral, and
normal brain tissue were collected, washed with saline, blotted,
and weighed. All the samples were stored at −80 °C until
HPLC analysis. The tissues were homogenized (Precellys 24,
Bertin technology), and drugs in the homogenate and plasma
were extracted and analyzed by HPLC.
Figure 3. Stability of free and encapsulated RP in PBS and plasma.
The effect of pH (A) and plasma (B) on the stability of RP (n = 3 for
each group at each time point). All error bars reflect SD.
Mice bearing intracranial U-87 MG glioma were established
as described above. The mice were randomly assigned into one
of the following groups (n = 8): saline, Taxol, PM, TPM, and
TRPM, and mice were administrated via tail vein at the
equivalent 10 mg kg−1 dose of PTX at 7, 12, 17, 22, and 27 days
after tumor implantation. The body weights of mice were
monitored twice a week for 4 weeks after tumor implantation.
The survival time of mice were recorded.
Figure 4. Cumulative release profile of RP from RP loaded micelle
(RPM) and transferrin modified RPM (TRPM) in 1 M sodium
salicylic acid at 37 °C (n = 3 for each group at each time point). All
error bars reflect SD.
Statistics. Statistical analysis was performed using the
Student’s t-test. Survival data were presented using Kaplan−
Meier plots and were analyzed using a log-rank test using SPSS
program 19.0. The differences were considered significant for
P < 0.05 and very significant for P < 0.01.
RESULTS
■
Synthesis and Characterization of RP. To confer the
specificity of PTX targeting glioma cells, we conjuga-
ted targeting peptide c[RGDfK] to PTX by derivatizing the
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Figure 5. In vitro targeting efficiency. Cellular uptake of transferrin modified c[RGDfK]-paclitaxel loaded micelle (TRPM) by primary brain
microvascular endothelial cells (BMEC) in the presence or absence of free transferrin (Tf) and chlorpromazine (Chl) in the comparison with
c[RGDfK]-paclitaxel loaded micelle (RPM) (n = 3) (A). *P < 0.05 compared with RPM; **P < 0.01 compared with TRPM. High resolution ESI-
TOF spectrum of RB (B). Cellular uptake of BODIPY-TR and c[RGDfK]-BODIPY-TR conjugate (RB) by the coculture of U-87 MG and HEK-
293-EGFP cells after 1 h incubation (C). Red, BODIPY-TR; green, HEK-293-EGFP cells expressing enhanced green fluorescent protein (EGFP);
blue, nuclei stained by DAPI. Bar: 50 μm. Outlines of HEK-293-EGFP cells were drawn in white line.
2′-hydroxyl function of PTX with succinic anhydride, activating
with dicyclohexylcarbodiimide/N-hydroxysuccinimide, and
coupling with c[RGDfK] peptide through the lysine ε-amino
group (Scheme 1). The purity of RP was >98% as verified by
HPLC (Figure 1A), and the structure of RP was confirmed by
1H NMR spectroscopy where the characteristic peaks of PTX
and c[RGDfK] could be found (Figure 1B). In addition, the
low and high resolution ESI-MS spectrum of RP exhibited a
peak at m/z = 1539.6589 corresponding to the [M + H]+,
which indicated that the molecular formula of RP was
C78H94N10O23 (Figure 1C).
Micelle Preparation and Characterization. We encapsu-
lated RP into polyphosphoester based hybrid micelle to
construct RP loaded micelle (RPM), and then conjugated Tf
onto RPM to obtain BBB permeable nanomedicine (transferrin
modified RPM, TRPM) using Traut’s reagent.20 The particle
sizes of RPM and TRPM determined by dynamic light
scattering (DLS) were 38.49 1.97 nm (n = 3) and 98.44
2.67 nm (n = 3) with an acceptable polydispersity index (PDI <
Figure 6. Cell viability of U-87 MG cells after incubation with various
concentration (from 0.1 to 2500 nM) of paclitaxel (PTX), paclitaxel
loaded micelle (PM), RP, and RPM for 72 h.
0.22 for both micelles) (Figure 2A). The ζ potential of RPM
and TRPM were 1.38
0.27 mV and −8.71
0.78 mV,
efficiencies of RPM and TRPM were 82.6 3.0% (n = 3) and
82.9 2.9% (n = 3), which were a bit lower than that of PM
(91.2 3.5%) and TPM (89.9 3.4%), reflecting the relatively
high water solubility of RP compared with PTX.
Hydrolysis. To assess whether micelle could protect RP
from degradation, we investigated the hydrolysis of RP in PBS
and plasma in vitro. According to HPLC analysis, the hydrolysis
product of RP was PTX through the hydrolysis of ester bond
between PTX and succinate. The hydrolysis of RP was found to
accelerate with increasing pH of PBS (Figure 3A), such that
66% of RP was hydrolyzed into PTX at pH 8.4 in 2 h, while it
respectively (Figure 2B). Compared with RPM, TRPM showed
increased particle size and decreased ζ potential, owing to the
coupling of hydrophilic, nanosized, and negatively charged Tf
onto the micelle.22,23 Control formulations, PTX loaded micelle
(PM, 33.31
0.61 nm, −2.02
0.46 mV), and transferrin
modified PM (TPM, 87.85 2.32 nm, −12.33 1.46 mV),
showed comparable size and ζ potential with RPM and TRPM,
respectively (Figure 2). The drug loading capacities of RPM
and TRPM as determined by HPLC were 3.67 0.12% (n = 3)
and 3.18
0.10% (n = 3), respectively. The encapsulation
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Figure 7. Effects of treatment with paclitaxel (PTX), PTX loaded micelle (PM), c[RGDfK]-paclitaxel conjugate (RP), RP loaded micelle (RPM),
and a physical mixture of c[RGDfK] and PTX or PM on the cell cycle of U-87 MG cells after 24 h incubation.
took 8 h to degrade 51% of RP at pH 7.4. The hydrolysis of RP
was greatly enhanced in plasma due to the presence of plasma
esterase but could be significantly inhibited by encapsulating
RP into the micelle (Figure 3B). After 8 h of incubation, more
than 90% of free RP was degraded, but 92% of encapsulated RP
remained intact. These results demonstrate that the micelle can
protect the drug from degradation possibly by limiting the
interaction of RP with esterase in plasma.
Figure 8. Expression of integrin αv in the normal brain tissue (1),
peritumoral brain tissue (2), and glioma (3) of intracranial glioma
bearing mice.
In Vitro Release. We investigated the in vitro cumulative
release profiles of RP from RPM and TRPM. RPM and TRPM
showed similar biphase release behaviors (Figure 4), which
indicated that the Tf modification did not affect the release
behavior of RP. For the first 12 h, the release of RP was fast, but
no burst release was observed. Then, it slowed down, and most
of the encapsulated RP was released within 72 h.
showed no difference between U-87 MG and HEK-293-EGFP
cells, but RB was found to preferentially accumulate in U-87
MG cells (Figure 5C). This result implies that c[RGDfK]
conjugation could increase the drug accumulation in glioma
after transport across the BBB thereby decreasing the
neurotoxicity of drug.
Cellular Uptake. To assess whether TRPM would be able
to specifically deliver RP to glioma cells in vitro, we used
primary BMEC to establish the in vitro BBB model. TRPM
exhibited enhanced cellular uptake of 2.4-fold (P < 0.05)
compared with RPM after 1 h incubation (Figure 5A) and
could be significantly inhibited by excess free Tf (P < 0.01). It
was reported that the endocytosis of Tf was clathrin-
dependent,24 a process that can be inhibited by chlorproma-
zine.25 Significant TRPM endocytosis inhibition was observed
in the presence of 10 μM chlorpromazine (P < 0.01), indicating
that clathrinis was indeed involved in the endocytosis of
TRPM.
In order to prove our assumption that c[RGDfK] conjugated
molecules can target the integrin overexpressed cells, we
synthesized a conjugate of c[RGDfK] and BODIPY-TR (RB,
m/z = 1123.4904) (Figure 5B), and the cellular uptakes of RB
and BODIPY-TR were evaluated on the coculture of U-87 MG
and HEK-293-EGFP cells, which express high and low level
integrin, respectively.26,27 The cellular uptake of BODIPY-TR
Cytotoxicity. Since the chemical modification of drug
molecules can sometimes lead to loss of activity, the
cytotoxicity of PTX, RP, PM, and RPM were evaluated on
U-87 MG cells. The study showed that the antiproliferative
effects of RP and RPM were not significantly different from that
of PTX and PM (Figure6), indicating that the conjugation of
PTX to c[RGDfK] and encapsulation of the conjugate into
polymeric micelle did not affect its cytotoxicity.
Cell Cycle. To further investigate whether c[RGDfK]
conjugation affects the mechanisms of action of PTX, we
analyzed the cell cycle distribution of U-87 MG cells using flow
cytometry. Free PTX and PM were shown to induced potent
G2/M arrest on U-87 MG cells, consistent with the report that
disturbance of microtubule dynamics was the antitumor
mechanism of PTX.28 As RP converted to PTX efficiently in
the presence of esterase, the antitumor mechanism of RP
should be the same as PTX. RPM induced the most potent
G2/M arrest in the cell cycle, while cells treated with a mixture
of c[RGDfK] and PTX (or PM) did not show any discernible
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Figure 9. Biodistribution of Taxol, paclitaxel loaded micelle (PM), transferrin modified paclitaxel loaded micelle (TPM), and TRPM in intracranial
U-87 MG glioma bearing mice at 1, 4, 12, and 24 h after intravenous injection (n = 3 for each group at each time point). *P < 0.05 and **P < 0.01
†
compared with TPM. P < 0.05 and ††P < 0.01 compared with PM. All error bars reflect SD.
improvement compared to cells treated with PTX or PM alone
(Figure 7).
the main limitations for clinical application of PTX is its
toxicity. The BWC of TRPM treated mice was similar to that of
saline treated ones. On the contrary, the body weight loss was
observed in Taxol, PM, and TPM treated mice since the first
dose. Despite the obvious increase of drug accumulation in the
liver, lungs, spleen, and kidneys, TRPM displayed substantially
less toxicity than Taxol, PM, and TPM.
Integrin αv Expression. The expression of integrin αv in
the brain of intracranial glioma bearing mice was investigated
(Figure 8). U-87 MG glioma tumor were found to express a
very high level of integrin αv, which was in accordance with a
report by Skuli et al.26 Integrin αv was barely expressed in
normal brain tissue, but the amount of integrin αv in
peritumoral tissue increased due to the infiltration of U-87
MG cells.29,30
Biodistribution. To evaluate the BBB penetration and
glioma targeting efficiency of TRPM in vivo, the distribution of
TRPM in tumor and other tissues was investigated. The accu-
mulation of TRPM in glioma, peritumoral, and normal brain
tissue was significantly higher compared with TPM at 1 h
postadministration (P < 0.01), and a longer retention of TRPM
was achieved in tumor and peritumoral tissues where the high
level of integrin was expressed when compared with TPM
(Figure 9). The clearance of TRPM from other tissues was also
the slowest among all of the formulations, indicating that
c[RGDfK] conjugation affects the pharmacokinetic properties
of PTX.
Antiglioma Activity and Toxicity. We next examined
whether increased glioma accumulation and specificity could
lead to an enhanced antiglioma effect by recording the sur-
vival time of intracranial U-87 MG glioma bearing mice
(Figure 10A). The mean survival times of TRPM, TPM, PM,
Taxol, and saline treated mice were 42.8, 39.5, 34.8, 33.6, and
34.5 days, respectively. Compared with saline, TPM (P < 0.01)
and TRPM (P < 0.01) significantly prolonged the survival time,
but PM showed no therapeutic effect. TRPM was significantly
superior to TPM (P < 0.01), which is rational considering
its higher accumulation and longer retention in glioma and
peritumoral tissue.
DISSCUSSION
■
The efficacy of glioblastoma chemotherapy is hindered by low
brain drug accumulation and low selectivity. Therefore, we
developed a novel micelle that could penetrate the BBB and
target the glioblastoma tumor subsequently by encapsulating
RP into a Tf modified micelle.
The first step of our research was the synthesis of RP. An
important goal in drug conjugate design is to retain a suitable
balance between biological stability of the drug conjugate and
efficient conversion to the active parent drug in vivo.31,32 RP
degraded quickly into parent drug PTX in plasma due to the
hydrolysis of the ester bond between PTX and succinate
(Figure 3B). However, as the linkage with c[RGDfK] is crucial
for leading PTX to glioma cells, the hydrolysis would impair the
targeting ability. So, we encapsulated RP into polymeric
micelles. Our result confirmed the general consideration that
nanocarriers could protect encapsulated drug from premature
degradation (Figure 3B),33 implying that the targeting
capability of RP could be preserved in physiological solution.
The release kinetic of RP from micelles was similar to Cho’s
report (Figure 4).34 As the hydrolysis of free RP was fast in the
presence of esterase, the sustained release of RP from RPM or
TRPM could help to retain a relatively high level of RP in
biological fluid such as plasma and cerebrospinal fluid, which
may favor the targeting of RP to the glioma.
According to our result (Figure 5A), the endocytosis of
TRPM was TfR-dependent and involved clathrin as the
report,13,14 which was more efficient than that of RPM.
Finally, we investigated the toxicity of TRPM in terms of
body weight change (BWC) of mice (Figure 10B), as one of
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Article
peritumoral tissue could facilitate the accumulation of
c[RGDfK] modified drug and inhibit the invasion of glioma
subsequently. Compared with other receptors such as LfR and
LRP, which are also highly expressed in normal brain tissue,9,10
integrin provids much higher specificity.
The pharmacokinetic properties of TRPM were different
from TPM (Figure 9), as c[RGDfK] modification not only
increases the affinity of drug to integrin overexpressed tumor
but may also alter the drug interaction with transporters such as
P-glycoprotein (P-gp).35 After being transcytosed across the
BBB through a TfR dependent pathway, the released RP could
not be exfluxed as efficiently as PTX, leading to its high
accumulation in the brain. Chemical modification to PTX also
slowed the excretion of the RP from mice (Figure 9), and a
similar phenomenon to that observed when the biodistributions
of poly-(L-γ-glutamylglutamine)-paclitaxel conjugate and lino-
leic acid-paclitaxel conjugate were evaluated.36,37
The in vivo antitumor activity of TRPM was the most
potent among all the formulations without significant toxicity
(Figure 10). Therefore, its efficacy could be improved further
by escalating the dosage because it has been proved that a
higher dose will result in better clinical effect when Abraxane
was used for tumor chemotherapy.38 As no body weight loss
was observed after the administration of TRPM, the frequency
of administration could also be increased to maintain high drug
concentration in the tumor, which should lead to better
antitumor activity.
In conclusion, we have provided a novel sequential targeting
nanoscale formulation of PTX, TRPM, for targeted glioma
chemotherapy. Tf conjugation significantly enhanced the ability
of a hybrid micelle to transport RP into primary BMECs in
vitro and brain in vivo, while c[RGDfK] modification improved
the drug selectivity to integrin overexpressed glioma cells in
vitro and prolonged the retention of drug in glioma and
peritumoral tissue in vivo. Most importantly, TRPM signifi-
cantly prolonged the survival time of intracranial glioma bearing
mice without showing obvious toxicity. These results indicated
that this sequential targeting nanomedicine could be promising
therapeutics for future glioma treatments.
Figure 10. Antiglioma activity and toxicity of micelles. Kaplan−Meier
survival curves of mice treated with saline, Taxol, paclitaxel loaded
micelle (PM), transferrin modified PM (TPM), and transferrin
modified c[RGDfK]-paclitaxel loaded micelle (TRPM) (n = 8 for
each group) (A), and the body weight change of the treated mice (n = 8)
(B). All error bars reflect SEM.*P < 0.05 and **P < 0.01 compared
with TPM; ††P < 0.01 compared with saline.
However, more drug exposure in the brain will cause higher
neurotoxicity if glioblastoma specificity is not achieved.
BODIPY-TR was chosen as a fluorescent indicator because of
its hydrophobicity and cell membrane permeability, which was
much like PTX. The enhanced selectivity of TB could be
attributed to the decreased cellular uptake through diffusion
and increased endocytosis through integrin-mediated pathway
after c[RGDfK] conjugation.
AUTHOR INFORMATION
■
Corresponding Author
*Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 501 Haike Road, Shanghai 201203, China. Tel/Fax:
+86-21-2023-1979. E-mail: ypli@mail.shcnc.ac.cn (Y.L.);
lyfeng@mail.shcnc.ac.cn (L.F.).
RPM showed similar cytotoxicity but more potent cell cycle
arrest activity compared with PTX, RP, and PM (Figures 6
and 7). The difference in incubation time may be the most
possible explanation. The enhanced cell cycle arrest activity of
RP may result from its increased cellular uptake through
integrin-mediated endocytosis because the cell cycle arrest
activity (RPM > RP > c[RGDfK] + PTX ≈ c[RGDfK] + PM)
correlated closely with the content of RP in the medium (RPM
> RP > c[RGDfK] + PTX = c[RGDfK] + PM). However, RP
was converted into PTX efficiently, and during the 72 h
incubation period, the cellular drug accumulation of the four
formulations was not significantly different.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The National Basic Research Program of China (2010CB934000
and 2007CB935804), the National Natural Science Foundation of
China (30925041 and 30901866), and Shanghai Elitist Program
(11XD1406200) are gratefully acknowledged for financial support.
The expression of integrin in the brain of mice bearing
intracranial glioblastoma was shown in Figure 8, indicating a
very significant difference in integrin αv expression between
normal brain tissue and glioma tumor, so active targeting of RP
to glioma was possible. The increased integrin αv expression in
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