Self-assembled nanospheres as a novel delivery system for taxol: A molecular hydrogel with nanosphere morphology

Article abstract of DOI:10.1039/c1cc10506j

Here we reported on the first example of a Folic acid-based molecular hydrogel with nanosphere morphology as a delivery system for Taxol.

Full text of DOI:10.1039/c1cc10506j

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Self-assembled nanospheres as a novel delivery system for taxol:
a molecular hydrogel with nanosphere morphologyw
Huaimin Wang,a Cuihong Yang,a Ling Wang,b Deling Kong,a Yongjun Zhangc and
Zhimou Yang*a
Received 25th January 2011, Accepted 22nd February 2011
DOI: 10.1039/c1cc10506j
Here we reported on the first example of a Folic acid-based
molecular hydrogel with nanosphere morphology as a delivery
system for Taxol.
We believed that the strategy capable of making Taxol
derivatives self-assemble into stable nanospheres would be
an alternative approach for the delivery of Taxol because of
the well-controlled synthetic pathways for small molecular
Taxol derivatives and the much higher loading amount of
the Taxol within the self-assembled nanospheres than that in
other delivery systems. Stimulated by the first example of
molecular hydrogel bearing Taxol,13 we designed and synthesized
a FA–Taxol conjugate of FA-GpYK-Taxol (Scheme 1). It
has several features: (1) it is a Taxol derivative which can
be considered as a prodrug releasing the Taxol upon ester
cleavage; (2) it has the FA moiety, which has a strong tendency
to form a stable tetramer14 and offers the FA–Taxol conjugate
the targeting effect to cancer cells;15 (3) phosphorylated and
succinated tripeptide of Glycine-Tyrosine-Lysine (GYK) as a
linker to connect the Taxol and FA not only provides the
sufficient water solubility to the FA-GpYK-Taxol but also
acts as the substrate of the phosphatase.16 Upon the treatment
of the phosphatase, the FA-GpYK-Taxol was converted to the
FA-GYK-Taxol with less solubility in aqueous solutions. Due
to the self-assembled ability of the FA, the FA-GYK-Taxol
might be able to self-assemble into nanospheres.
Nanospheres,1 especially those formed by the self-assembly of
amphiphilic polymers, dendrimers, and biomacromolecules
such as DNA molecules and peptides, have been extensively
investigated in the fields of drug delivery,2 patterning,3
lithography,4 molecular sensing,5 etc. What’s more, the morpho-
logy and the size of their self-assembled structures can be
well controlled. However, the nanospheres formed by small
molecules (molecular weight less than 2000) in aqueous
solutions are relatively less explored and their morphology is
hard to be controlled due to the more dynamic property of
small molecules. Up to now, only several examples of nano-
spheres formed by small molecules have been reported.6
Among them, the di-phenylalanine and its derivatives are
probably the most investigated system.7 The nanospheres
formed by di-phenylalanine derivatives can be used for the
delivery of ss-DNA and quantum dots.8,9 Most recently, the
nanospheres formed by the derivative of di-phenylalanine had
been demonstrated to be as rigid as metal nanoparticles.10
Besides the di-phenylalanine system, Hamachi’s group demon-
strated that the nanospheres formed by amphiphilic small
molecules could be used for the detection of biomacromolecules
by turning on signals of F-NMR or fluorescence.11
After the successful synthesis of the FA-GpYK-Taxol,
its self-assembly was evaluated by the treatment with the
phosphatase. The FA-GpYK-Taxol was highly soluble in
aqueous solutions with a solubility of higher than 4 wt% in
the phosphate buffer saline (PBS) solutions (pH = 7.4). The
phosphatase was then added to the clear and slightly
yellow solution containing 0.2 wt% of the FA-GpYK-Taxol
(final concentration of the phosphatase = 90 U mLꢀ1). We
observed the formation of a transparent hydrogel (Fig. 1A, insert)
after incubation of the above solution at room temperature
Taxol, as an effective anti-cancer chemotherapy drug, is
widely used in the clinic.12 Due to its limited water solubility, it
is usually administrated in the forms of liposome, conjugates
with proteins and hydrophilic polymers, etc. These adminis-
tration forms, however, frequently suffer from the low
loading amounts of Taxol, difficult synthetic pathways, etc.
a Key Laboratory of Bioactive Materials, Ministry of Education,
College of Life Sciences, Nankai University, Tianjin 300071,
P. R. China. E-mail: yangzm@nankai.edu.cn
b College of Pharmacy and Tianjin Key Laboratory of Molecular
Drug Research, Nankai University, Tianjin 300071, P. R. China
c Key Laboratory of Functional Polymer Materials,
Institute of Polymer Chemistry, College of Chemistry,
Nankai University, Tianjin 300071, P. R. China
w Electronic supplementary information (ESI) available: Synthesis
and characterization of FA-GpYK-Taxol, formation of the hydrogel,
conversion and stability of different compounds in gels, determination
of IC50 values. See DOI: 10.1039/c1cc10506j
Scheme 1 Chemical structure of the FA-GpYK-Taxol (purple: Folic
acid (FA), black: phosphorylated and succinated tripeptide of
Glycine-Tyrosine-Lysine (GYK), and red: Taxol).
c
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10% of FBS (final concentration of FA-GYK-Taxol was
about 0.003%, 30 mL of gels were diluted to 1.8 mL by these
two solutions). As shown in Fig. 3A and B, the as formed
nanospheres exhibited small aggregates in both solutions—
their average sizes were 183 nm and 252 nm in the PBS
buffer solution and the DMEM solution with 10% of FBS,
respectively. The relative larger size of the small aggregates
in DMEM solution than that in PBS buffer solution was
probably due to the existence of Taxol binding proteins and
FA binding proteins in FBS. The size of the small aggregates
in both solutions decreased slightly after being incubated at
room temperature for 24 h (about 136 nm and 120 nm in both
solutions, respectively). These results clearly indicated that the
self-assembled nanospheres possessed good stability in both
solutions with an anti-dilution property. We then studied the
chemical stability of the FA-GYK-Taxol in gels. The gels
were firstly incubated at room temperature overnight for the
enzymatic conversion to reach the balance, gels were then
incubated in a water bath at 37 1C. The molar percentage of
the FA-GYK-Taxol remained in the gels was determined
by the LC-MS—there was 92, 78, and 68% molar of
FA-GYK-Taxol remained in the gels at 12, 24, and 48 h time
points, respectively. The degradation of FA-GYK-Taxol
conjugate underwent complex pathways—we observed a small
peak from Taxol formed by the ester bond hydrolysis18 and
several other peaks from unknown compounds probably due
to the decomposition of FA.17 When calculating the weight
percentage of the Taxol in the nanospheres, the value was
49.4%, which was much higher than that in liposome systems
or in Taxol–polymer/dendrimer conjugates. The high stability
of the self-assembled nanospheres and the high weight
percentage of Taxol in the nanospheres suggested that they
could be developed into a novel delivery system for Taxol and
for cancer therapy.
Fig. 1 The rheological measurements with the mode of (A) dynamic
time sweep at the frequency of 2 rad sꢀ1 and the strain of 2% for the
PBS solution containing 0.2 wt% of FA-GpYK-Taxol and 90 U mLꢀ1
of phosphatase (insert: optical image of the gel) and (B) dynamic
frequency sweep at the strain of 2% for the gel formed 2 h after the
addition of the phosphatase.
(22–25 1C) within 3 min (66% mol of FA-GpYK-Taxol
was converted to the FA-GYK-Taxol at the gelling point,
determined by the HPLC). The hydrogel was stable at room
temperature for at least 6 months without disturbing. The
minimum concentration of FA-GpYK-Taxol needed for
gelation was about 0.08 wt% in the PBS buffer solutions.
These results indicated that FA-GYK-Taxol was an effective
gelator and it had an excellent self-assembly ability.
A rheological measurement with the mode of dynamic time
sweep was used to characterize the kinetics of enzymatic
hydrogelation. As shown in Fig. 1A, the gel formed immediately
after the addition of the enzyme, as indicating by the value of
elasticity (G0) dominating than that of viscosity (G0 0). The
enzyme of phosphatase kept converting the FA-GpYK-Taxol
to the FA-GYK-Taxol post the gelling point, thus leading to
the increase of the values of both G0 and G0 0 and the forma-
tion of a more rigid hydrogel. The conversion was recorded at
a low rate 1 h post the incubation, as indicating by the
observation in the rheological measurement that there was
only a slightly increase of the values of both G0 and G00 from
one to one and a half hour time points (the molar percentage
of FA-GpYK-Taxol converted was similar at these two time
points, both were about 86%). And then the rheological
measurement with a mode of dynamic frequency sweep was
used to characterize the resulting hydrogel. As shown in
Fig. 1B, the gel showed a slight dependence to the frequency—
the value of G0 changed from about 30 Pa to about 80 Pa at
the frequency of 0.1 and 100 rad sꢀ1, respectively, which
implied the existence of a weak matrix in the gel.
We then evaluated the activity of the nanospheres by
treating HepG2 cells at serials of concentrations. After 72 h
of incubation with the cells, the FA-GYK-Taxol in the nano-
sphere exhibited an IC50 value of 27.3 nM (the gel dispersed in
the DMEM medium with 10% of the FBS was used directly
for the test), which is comparable to that of Taxol (24.8 nM).
We proposed that the cellular uptake of the nanospheres
occurred by the endocytosis, which was a well-known pheno-
menon for many delivery systems.8,19 The phosphorylated
peptide without the Taxol showed no inhibitions on HepG2
cells at 250 mM. FA-GpYK-Taxol exhibited an IC50 of
19.6 nM, which was also comparable to those of Taxol and
FA-GYK-Taxol. In addition, the shapes of the regression
curves of the three compounds were similar. These results
indicated that the activity of Taxol was conserved successfully
in the precursor and the hydrogelator, which conferred the
great potentials for the system here to be used as a novel
delivery system for Taxol.
The atomic force microscopy (AFM) was then used to
characterize the nanostructures in the gel. Unlike most of small
molecular hydrogels with nanofibers and tubular structures,
highly uniform nanospheres with the size of about 50 nm were
observed in the gel (Fig. 2A). This result clearly proved the
success of our design. Though the reason for why FA-GYK-Taxol
could self-assemble into nanospheres remained unknown, we
proposed that both the strong tendency of the FA to form the
tetramer structure (Fig. 2C and D) and the hydrophobic
interaction between Taxol molecules might help to extend
the supramolecular chain and form a dendrimer-like structure
(Fig. 2B), thus resulting in the formation of the nanospheres.
To evaluate the stability of the nanospheres in different
kinds of aqueous solutions, the gels were dispersed by vortex
in both the PBS buffer solution and the DMEM solution with
In summary, we have successfully obtained nanospheres of
a Taxol derivative without comprising its activity and reported
a novel gelation system based on the FA. We are unable to
predict performance of the self-assembled system in this study
because of the existence of Taxol binding proteins and FA
binding proteins in blood stream. And it needs to be studied by
in vivo experiments future. Aside from Taxol which was chosen
c
4440 Chem. Commun., 2011, 47, 4439–4441
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Excellent Talents in University (NCET-08-0698), and the
Fundamental Research Funds for the Central Universities
(65011041).
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Fig. 2 (A) The atomic force microscopy (AFM) image of the nano-
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FA-GpYK-Taxol with 90 U mLꢀ1 of phosphatase (the scale bar
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nanospheres in Fig. 2A), (C) a cartoon diagram of FA-GYK-Taxol
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molecules could also be used for the construction of functional
hydrogels for biomedical applications. We believed that the
hydrogels with nanospheres would have big potentials to be
developed into a novel kind of delivery system for hydrophobic
drugs to treat different diseases.
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This work is supported by National Outstanding Youth Fund
(30725030), NSFC (20974054), Program for New Century
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This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4439–4441 4441