Kinetic control over supramolecular hydrogelation and anticancer properties of taxol

Article abstract of DOI:10.1039/c7cc08041g

We reported a kinetic control over supramolecular hydrogelation and more importantly over the anticancer properties of taxol.

Full text of DOI:10.1039/c7cc08041g

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ARTICLE TYPE
Kinetic Control over Supramolecular Hydrogelation and Anticancer
Property of Taxol
Xiaoli Zhang,a Youzhi Wang,b Yongquan Hua,c Jinyou Duan,a Minsheng Chen,c Ling Wang,b and
Zhimou Yangb, *
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
We reported
a
kinetic control over supramolecular
hydrogelation and more importantly the anticancer property
of taxol.
Supramolecular hydrogels have shown big potential in tissue
2
engineering,1,
sensing,3ꢀ6 drug delivery,7ꢀ11 cancer cells
inhibition,12ꢀ16 and immune modulation.17ꢀ20 In order to trigger the
selfꢀassembly of the small molecules (gelators), external stimuli
should be applied including heatingꢀcooling process, pH
adjustment, ionic strength increase, chemical reaction, enzymatic
reaction, etc.21ꢀ27 Among these methods, those using (auto)
catalytic28ꢀ30 or enzymatic reactions31ꢀ34 attracted increasing
research interests, because they provide more opportunities to
manipulate the property of hydrogels and have led to dynamic,35
dissipative36 or nonꢀequilibrium37ꢀ39 nanomaterials. Besides,
using enzymatic reactions to trigger molecular selfꢀassembly can
lead to more functional nanomaterials than conventional
methods.40, 41 Recent studies clearly indicated that the kinetics of
reactions had pronounced effects on the properties of resulting
hydrogels. For example, a pioneering work reported by Eelkema
and van Esch demonstrated the big influence of reaction kinetics
on the properties of resulting gels including the mechanical
property, appearance, and morphology of nanostructures.42 In this
study, we showed that the kinetic of hydrogelation affected the
mechanical property and more importantly the antiꢀcancer
property of a molecular hydrogel formed by taxol itself.
Figure 1. A) Chemical structure of TaxolꢀGnE4ꢀn and their autohydrolysis
route to generate taxol and B) Optical images of gels formed by PBS
solutions containing 4 mM (about 5 mg/mL) of taxolꢀGGGE (compound
1), taxolꢀGGEE (compound 2), and taxolꢀGEEE (compound 3),
respectively.
Three compounds were synthesized by a combination of
solid phase peptide synthesis (SPPS) and solution phase organic
synthesis (Scheme Sꢀ1). We firstly obtained the peptides by
standard FmocꢀSPPS and then reacted with Nꢀhydroxyl
succinimide (NHS) activated succinated taxol to afford designed
compounds 1, 2, and 3. The pure compounds were obtained by
high performance liquid chromatography (HPLC). They
exhibited different critical micelle concentration (CMC) values of
0.41, 0.71, and 1.07 mg/mL, respectively (Figure Sꢀ7), suggesting
their different amphiphilicity and selfꢀassembling property. We
then dissolved them in phosphate buffer saline solution (PBS, pH
= 7.4, adjusted by adding Na2CO3) at a final concentration of 0.5
wt% (5 mg/mL). They formed clear solutions in PBS and then
rapidly converted to hydrogels (gel1, gel2, and gel 3) upon
We found that hydrophobic small molecules could also form
molecular hydrogels through hydrolysis processes from their
hydrophilic precursors,43 and we reported a molecular hydrogel
of the very hydrophobic antiꢀcancer drug of taxol.44 The hydrogel
of taxol was formed through an ester bond autohydrolysis process
from its precursor of taxolꢀsuccꢀGSSG. Since the kinetics of
hydrogelation dramatically affect the property of resulting gels,
we opted to investigate the effect of kinetics of autohydrolysis on
the property of hydrogels of taxol. We therefore designed and
synthesized three precursors of taxol. As shown in Figure 1A, the
three precursors contained 1, 2, and 3 glutamic acids (E),
respectively (taxolꢀGGGE, taxolꢀGGEE, and taxolꢀGEEE as
compounds 1, 2, and 3, respectively). We speculated that the
amphiphilicity of three precursors might affect the autohydrolysis
speed of ester bond between taxol and succinated peptides,
leading to the kinetic control of molecular hydrogelations of taxol.
0
incubation at room temperature (20ꢀ25 C) within about 4, 8, and
30 minutes, respectively (Figure 1B). The resulting three gels
shown different appearances. As shown in Figure 1B, gel1, gel2,
and gel3 was clear, opalscent, and turbid, respectively, suggesting
that the kinetics of hydrogelation had significant effects on the
properties of the resulting gels.
The hydrogel was formed through the ester bond
autohydrolysis. The transformation process from the
precursors to Taxol could be monitored by LCꢀMS. As shown
in Figure 2A, three compounds exhibited different hydrolysis
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kinetics. Compound 1 showed the highest rate of hydrolysis
and compound 3 possessed the lowest one. The percentage of
compound hydrolysed was about 74, 66, and 57 % for
compounds 1, 2, and 3, respectively at 1h time point. After 5
hours (300 minutes), more than 98% of the precursors had
been converted to taxol. We then characterized the mechanical
Therefore, gel1 showed the highest crossꢀlinking density, thus
leading to its relatively better mechanical property. The
observations in TEM images correlated well with the appearance
and mechanical properties of the gels. Furthermore, nanofibers in
three hydrogels exhibited negative zeta potentials in PBS with the
value of ꢀ44, ꢀ48 and ꢀ58 eV for gel1, gel 2, and gel3,
respectively. Nanofibers with bigger zeta potential values might
lead to stronger repulsion force between them, thus resulting in
mechanically weaker gels and more rigid morphology of
nanofibers.
property of the hydrogels by
a rheometer. We firstly
performed the dynamic time sweep to study the hydrogelation
process. For solution of 1 (Figure 2B), the value of G’ was
bigger than that of G” after about 3 minutes, suggesting a very
rapid solꢀgel phase transition. This observation was consistent
with the observation that gel1 would form within 4 minutes
upon standing without disturbance. For solutions of 2 and 3,
the time point at G’ value dominating G” value was about 7
and 25 minutes, respectively (Figures 2C and 2D). For three
samples, both G’ and G” values increased rapidly after
hydrogel formation and reached plateaus within 3 hours. After
that, the dynamic frequency sweep was performed at the strain
of 0.5%. The gels exhibited weak frequency dependences
within the frequency range from 0.1 to 100 rad/s, suggesting
high elasticity of the hydrogels (Figure 3A). Meanwhile, the
G’ value of the resulting gels was followed the trend of
gel1>gel2>gel3. For example, the G’ value was about 3584,
2599, and 1968 Pa at the frequency of 1 rad sꢀ1 for gel1, gel2
and gel3, respectively. These observations clearly indicated
that, besides the appearance, the mechanical properties of
hydrogels would also be affected by the kinetics of hydrogel
formation.
Figure 3. A) Rheological measurements with the mode of dynamic
frequency sweep at the strain of 0.5% for three gels (square in black: gel1,
circle in gray: gel2, diamond in blue: gel3, closed symbols: G’ and open
symbols: G”) and TEM images of B) gel1, C) gel2, and D) gel3
(concentration of precursors of gelators in PBS = 0.5 wt%, gelation time
= 5 hours).
We therefore evaluated the IC50 value of the gel1, gel2, gel3
and taxol against a cancer cell line of HepG2 cells. After
incubating cells with different compounds at different
concentrations for 48h, the MTT assay was performed. As shown
in Figure 4A, taxol, gel1, gel2, and gel3 exhibited an IC50 value
of 13.14, 6.11, 7.39, 10.66 ꢁM against HepG2, respectively.
These observations clearly indicated better inhibition capacity of
gels than taxol to HepG2 cells in vitro. The antiꢀtumor efficacy of
our hydrogels in the mice tumor model (4T1ꢀluciferase breast
tumors in mammary fat pad of female mice) was then studied in
vivo. When the volume of breast tumors reached about 50 mm3,
we injected the same dosages (10mg Kgꢀ1 of taxol*4 every other
day) of different formulations of taxol into the mice through
caudal vein. As shown in Figure 4, gel3 exhibited a similar antiꢀ
tumor growth efficacy to Taxol®. While both gel1 and gel2
showed enhanced antiꢀtumor growth capacities over gel3 and
Taxol®. The final volume of tumors was about 1114, 950, 793,
648, and 565% bigger than the original volume of tumors for the
PBS control, Taxol®, gel3, gel2, and gel1, respectively. Besides,
mice administrated with Taxol® showed a slight body weight
loss during the experimental time (Figure 4D), probably due to
the presence of organic solvents in clinically used Taxol®. While
the administration of our nanofibers wound not significantly
decrease the body weight of mice. The in vivo images of tumors
(Figures 4E and Sꢀ11) clearly indicated that mice receiving gel1
Figure 2. A) Hydrolysis percentage of different compounds determined
by LCꢀMS and rheological measurements with the mode of dynamic time
sweep at a frequency of 0.5 rad sꢀ1 and strain of 0.5% for PBS solutions
containing 0.5 wt% of: B) compound 1, C) compound 2, and D)
compound 3 (closed symbols: G’ and open symbols: G’’).
We then used transmission electron microscopy (TEM) to
investigate the morphology of nanostructures in the resulting gels.
We observed the dense and three dimensional (3D) networks of
nanofibers in all gels. However, the diameter and morphology of
the nanofibers in these gels were different. As shown in Figures
3Bꢀ3D, the diameter of the nanofibers in gel1, gel2 and gel3 was
about 20, 28 and 34 nm, respectively (at least 50 nanofibers were
measured to calculate their diameter). Since the three gels had the
same concentration of taxol, gel1 with the smallest nanofibers
should possess largest number of nanofibers among three gels.
2
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bState Key Laboratory of Medicinal Chemical Biology, College of Life
Sciences, Key Laboratory of Bioactive Materials, Ministry of Education,
and Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China;
E-mail: yangzm@nankai.edu.cn
or gel2 showed smaller size of light spots, which correlated well
with the results of tumor volumes obtained in Figure 4B. These
results indicated that the kinetic process of hydrogel formation
influence the in vitro and in vivo anticancer property of
nanofibers in resulting hydrogels.
cDepartment of Cardiology, Zhujiang Hospital of Southern Medical
University, and Guangdong Provincial Center of Biomedical Engineering
for Cardiovascular Diseases, Guangzhou 510280, P. R. China
†Electronic Supplementary Information (ESI) available: [Synthesis and
characterization, Protein expression and purification, rheology, Emission
spectrum,
confocal
images,
and
TEM
images].
See
DOI: 10.1039/b000000x/
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This work is supported by the Science and Technology
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Chinese Universities Scientific Fund (2452016192), Science
&
Technology
Projects
of
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DOI: 10.1039/C7CC08041G
The anticancer property of supramolecular nanofibers of taxol in hydrogels could be manipulated
by the kinetics of hydrogel formation.