Disulfide bond reduction-triggered molecular hydrogels of folic acid-Taxol conjugates

Article abstract of DOI:10.1039/c3ob40969d

Molecular hydrogels of therapeutic agents are a novel kind of self-delivery system that can sustain release of drugs or pro-drugs. We have previously developed a molecular hydrogelator of folic acid (FA)-Taxol conjugate triggered by phosphatase. In this paper, we report a novel molecular hydrogelator system of FA-Taxol conjugates with improved synthetic strategy. The hydrogels are formed by the reduction of disulfide bond by glutathione (GSH). These hydrogels could sustain release of Taxol through ester bond hydrolysis. Compared with intravenous (i.v.) injection of clinically used Taxol with four times the dosage, our hydrogel could inhibit tumor growth more efficiently by a single dose of intra-tumor (i.t.) administration. These observations suggested the big potential of this novel gelation system of Taxol for cancer therapy.

Full text of DOI:10.1039/c3ob40969d

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Disulfide bond reduction-triggered molecular hydrogels of Folic acid-
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DOI: 10.1039/C3OB40969D
Taxol conjugates
Chengbiao Yang,a Dongxia Li,a Qianqi FengZhao,b Lianyong Wang,*, a Ling Wang,b and Zhimou Yang*, a
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
5
Molecular hydrogel of therapeutic agents is a novel kind of self-delivery system that can
sustainedly release drugs or pro-drugs. We have previously developped a molecular hydrogelator
of Folic acid (FA)-Taxol conjugate triggered by phosphatase. In this paper, we reported on a novel
10 molecular hydrogelator system of FA-Taxol conjugates with improved synthetic strategy. The
hydrogels are formed by the reduction of disulfide bond by glutathione (GSH). These hydrogels
could sustainedly release Taxol through ester bond hydrolysis. Compared with intraveneous (i.v.)
injection of clinically used Taxol® with four times of dosage, our hydrogel could inhibit tumor
growth more efficiently by a single dose of intra-tumor (i.t.) administration. These observations
15 suggested the big potential of this novel gelation system of Taxol for cancer therapy.
morphology.60 In the system, FA was connected with Taxol
through a phosphorylated tripeptide of GpYK (G: glycine, pY:
phosphorylated tyrosine, and K: lysine), which needed five
55 steps of synthesis to obtain it in a relative low yield (total
yield in five steps lower than 10%). We recently found that
Folic acid (FA) could be a general motif to construct
1. Introduction
Molecular hydrogels are formed by small molecules
(hydrogelators) with molecular weight usually lower than
2,000.1-7 Many small molecules of sugar derivatives, amino
20 acid derivatives, and peptides have been demonstrated to be
efficient hydrogelators.8-18 Molecular hydrogels have shown
big potential for sensing of bioactive molecules,19-22 synthesis
of organic and inorganic materials,23-29 cell culture,30-33 drug
delivery,34-38 etc. Recent research interests in molecular
25 hydrogel focus on the development of novel gelation systems
(especially responsive ones39-41), novel strategies to improve
the mechanical property of molecular hydrogels,42-46 and
novel applications of them. For example, Adams and McNeil
groups independently applied polymeric additives to improve
30 the property of short peptide-based molecular hydrogels.42, 44,
molecular
hydrogelators
through
conjugation
with
hydrophobic therapeutic agents of triamcinolone acetonide
60 (TA) and rapamycin.62 The hydrogels of FA-TA and FA-
rapamycin conjugates were formed by disulfide bond
reduction triggered by glutathione (GSH). Based on these
information, we planed to develop a GSH-triggered gelation
system based on FA-Taxol conjugates.
65
2. Results and discussions
47, 48
2.1 Molecular design and synthesis
Banerjee and co-workers had incorporated carbon
materials to endow amino acid-based hydrogels with novel
properties.49, 50
In our previous study, we had shown that the FA-GpYK(Taxol)
could be converted to FA-GYK(Taxol) by the enzyme of
70 phosphatase, resulting in a molecular hydrogel with a three
dimensional nanosphere network.60 The relative low yield to
obtain FA-GpYK(Taxol) would hinder the futher development
and application of this system. We therefore planed to design
a novel hydrogel system of FA-Taxol conjugates by disulfide
75 bond reduction. We had demonstrated that disulfide bond was
a useful cleavable linker to construct precursors of gelators.63
The reduction of disulfide bond by Dithiothreitol (DTT) or
GSH could release gelators and result in the formation of
hydrogels of peptides and conjugates of FA and hydrophobic
80 therapeutic agents.60, 63
We firstly designed FA-GFK(Taxol)EssEE (Scheme 1) as a
precursor of gelator. The peptide part of GFKE was used to
connect FA and Taxol and disulfide bond reduction could
generate FA-GFK(Taxol)E-s with a similar chemical structure
85 of FA-GYK(Taxol)-COOH produced by phosphatase in our
previous study.60 The FA-GFKEssEE was obtained by
standard Fmoc- solid phase peptide synthesis (SPPS) and then
Most recently, the application of molecular hydrogels in
35 drug delivery attracted extensive research efforts.51-57 Besides
using hydrogels as physically carriers to deliver therapeutic
agents, molecular hydrogel of therapeutic agents was a self-
delivery system that hold advantages. This recently
developped drug delivery system usually had high drug
40 loadings. Importantly, the drug loading could be designable
and fixed through conjugation with different compounds such
as different peptides.51 This system could constantly release
therapeutic agents without burst release phenomenon.58
Several examples of molecular hydrogels of therapeutic
45 agents including Taxol,56, 58-60 Camptothecin,51, 61 Naprofen,52
and Olsalazine53 had been rationally designed and reported up
to now.
Our group focuses on the development of molecular
hydrogels of hydrophobic therapeutic agents,57 typically Taxol.
50 We had reported a phosphatase-triggered hydrogel system of a
Folic acid (FA)-Taxol conjugate with nanosphere
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Organic & Biomolecular Chemistry
Page 2 of 6
used to couple with N-hydroxylsuccinimide (NHS)-activated
succinated Taxol. The resulting compound was purified by
in combine with our previous results of hydrogels of
conjugates of FA and TA/rapamycin, sugDgeOsI:te1d0.1th0a3t9/FCA3OwB4a0s9a69D
View Article Online
reverse phase high performance liquid chromatography 35 useful motif to construct hydrogelators through conjugating
(HPLC). The FA-Taxol conjugates could be obtained in
moderate yields of 40% approximately over two steps. We
then planed to make other FA-Taxol conjugates with shorter
peptide sequences. The use of shorter peptide linkers would
make the synthesis of FA-peptides easier and improve the
yield of synthesis. Different peptide sequences might also lead
with other drug molecules.
5
10 to different release profile of resulting gels. What’s more,
application of shorter peptide linkers would improve the
loading of Taxol in the resulting self-assembled
nanostructures. In this study, peptides of GFKE, GKE, KE,
and K were designed and used (Scheme 1).
15
HO
O
H
O
O
O
CH3
O
COOH
O
H
O
O
NH
OH
N
O
N
O
OH
O
H
O
O
N
N
O
N
N
H
O
H2N
FA
succinated Taxol
O
Taxol
Taxol
HN
HN
Fig. 1. A) Optical images of the hydrogels from PBS solution containing
40 0.5 wt% of the precursors with 4 equiv. of GSH (from left to right:
GFKEgel, GKEgel KEgel, Kgel, respectively) and B) Dynamic frequency
sweep of four gels formed at 2 hour time point at the strain of 1% and at
25 ℃ (the solid symbols represent elasticity (G’) and the hollow ones
represent viscocity (G’’))
O
O
O
O
H
H
N
H
H
N
N
FA
N
FA
N
ssEE
N
H
N
ssEE
H
H
O
O
O
COOH
COOH
FA-GK(Taxol)EssEE
FA-GFK(Taxol)EssEE
45
Taxol
Taxol
HN
HN
We then characterized the mechanical property of the
hydrogels by a rheometer. As shown in Fig. 1B, a PBS
solution containing 0.5 wt% of the compound with 4 equiv. of
GSH was transferred to the rheometer. After two hours’
50 incubation, a dynamic frequency sweep was then performed at
the strain of 1% and at 25 ℃. The results showed that G’
value of the gel was about 130, 230, 360, and 800 Pa for
GFKEgel, GKEgel, KEgel, and Kgel, respectively. The G’
value was at least one magnitude bigger than G’’ value for
55
O
H
FA
N
FA
ssEE
N
ssEE
N
H
H
O
O
COOH
FA-K(Taxol)EssEE
FA-K(Taxol)ssEE
COOH
O
O
H
H
glutathione
S
N
N
SH
N
S
N
OH
N
H
H
H
O
O
s
COOH
ssEE
Scheme 1. Chemical structures of all Folic acid (FA)-Taxol conjugates
used for GSH-triggered hydrogelations
20 2.2 Gelation test and characterization of hydrogels
The gelation ability of obtained compounds were tested. As
presented in Fig. 1A, we found that all of them could form
hydrogels in the phosphate buffer saline (PBS, pH = 7.4) upon
the treatment of 4 equiv. of GSH. The gels formed by the
25 compounds were named as the GFKEgel, GKEgel, KEgel, and
Kgel for compound of FA-GFK(Taxol)EssEE, FA-
GK(Taxol)EssEE, FA-K(Taxol)EssEE and FA-K(Taxol)ssEE,
respectively. The minimum gelation concentration (MGC)
was about 0.25 wt%, 0.175 wt%, 0.3 wt%, and 0.4 wt% for
30 GFKEgel, GKEgel, KEgel, and Kgel, respectively. The
different colors of these gels might be due to the presence of
different amounts of free thiol compounds. The observations,
Fig. 2. Cryo-TEM images of A) GFKEgel, B) GKEgel, C) KEgel, and D)
Kgel (the scale bars represent 50 nm)
2
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Organic & Biomolecular Chemistry
each gel, indicating the formation of true gels.64 The
2.4 Tumor inhibition
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morphology of self-assembled structures in the hydrogels was
characterized by Cryo-transmission electron microscopy
(Cryo-TEM). As shown in Fig. 2, all hydrogels exhibited
filamentous structures. The diameter of the fibril was about 4-
6 nm for all hydrogels (about 5.1, 4.9, 4.8, and 4.6 nm for
GFKEgel, GKEgel, KEgel, and Kgel, respectively). This
observation suggested that shorter peptide linkers would lead
to smaller sized fibrils in the gels. The small fibrils entangled
DOI: 10.1039/C3OB40969D
The anti-tumor efficacy of our hydrogels in mice tumor model
45 (4T1-luciferase breast tumors in mammary fat pad of female
mice) was also evalutaed in vivo. We choose Kgel for the
evaluation because of the highest drug loading and the lowest
release speed in the four gels. When the volume of breast
tumors reached about 500 mm3, different dosages (10, 20, and
50 40 mg/kg of Taxol) of the hydrogel was injected directly into
tumors with the same volume (100 μL) by variation of the
concentration of the compound in gels. As shown in Fig. 4,
our hydrogels could efficiently hinder tumors growth in the 14
days. The final volume of tumors was about 376%, 353%,
55 and 398% bigger than the original volume of tumors (~500
mm3) and there were not obvious difference among different
dosages (10, 20, and 40 mg/kg of Taxol) of our hydrogels (Fig.
S-10). However, the final relative tumor volumes were 756%
and 832% in groups administrated with intravenous (i.v.)
60 injection of 40 mg/kg of Taxol® (10 mg/kg * 4 every other
5
10 with each other and formed three dimensional network
structure that served as the matrix of the hydrogels.
2.3 Release profile
The releasing profile of Taxol or Taxol conjugates from gels
15 at physiological temperature condition (37 ℃) in vitro was
then studied. We added a 0.25 mL of PBS buffer solution on
top of 0.2 mL of gels (original concentration of all compounds
was 0.5 wt%). The 0.2 mL of upper solutions were taken out
at desired time points for measurement of accumulating
20 release amount of Taxol from gels, and a fresh PBS buffer
solution (0.2 mL) was then added. As shown in Fig. 3, the
four gels exhibited different release profiles. They released
Taxol at a mean rate of 0.16, 0.25, 0.34, and 0.11 μg/mL per
hour for GFKEgel, GKEgel, KEgel, Kgel respectively for 24h,
25 and at a mean rate of 40.00, 54.41, 52.79, and 35.24 μg/mL
per hour for GFKEgel, GKEgel, KEgel, Kgel respectively for
14 days (Fig. S-12). The results indicated that gels formed by
gelators with hydrophilic amino acid of E could obviously
release Taxol at higher rates, and the presence of hydrophobic
30 F could result in the release of Taxol at lower rates. We only
observed Taxol itself got released from gels, indicating that
the peptide derivatives were trapped in the matrix of gels and
Taxol was released through ester bond hydrolysis. The
accumulating released percentage of Taxol were about 0.13%,
35 0.22%, 0.30%, 0.40% correspond to Kgel, GFKEgel, GKEgel,
KEgel, respectively during the 24h experimental period.
These observations implied that our Taxol hydrogels could be
used for sustainedly release of Taxol for cancer therapy.
day) and i.v. injection of
a PBS solution (100 μL),
respectively. These observations clearly indicated that our
hydrogel of Taxol could efficiently hinder the growth of
breast tumors. We also monitored the body weight of the mice.
65 There were no obviously body weight losses in groups of mice
administrated with our hydrogels (Fig. S-11), compared to the
control group of mice without any treatments. These results
suggested low toxicity and high efficiency to inhibit tumor
growth of our hydrogels.
70
Fig. 4. Kgel inhibits xenografted mouse breast tumor (4T1-luciferase)
growth in vivo (gels were administrated into i.t. spaces after tumor sizes
reaching ~500 mm3, n=5, data were represented as Mean±SEM)
75
3. Experimental
3.1 Peptide derivate synthesis: Peptide derivates of FA-
GFKEssEE, FA-GKEssEE, FA-KEssEE, FA-KssEE were
prepared by solid phase peptide synthesis (SPPS) using 2-
80 chlorotrityl chloride resin and the corresponding N-Fmoc
protected amino acids with side chains properly protected by a
tert-butyl group. The first amino acid (Fmoc-Glu(OtBu)-OH)
was loaded on the resin at the C-terminal with the loading
40
Fig. 3. Release profile of Taxol from hydrogels to PBS buffer solution at
37 0C
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efficiency about 1.0 mmol/g. 20% piperidine in anhydrous
N,N’-dimethylformamide (DMF) was used during the 60 The vitrified samples were then stored inDlOiqI:u1i0d.1n0i3t9ro/Cg3eOn.B4T0h9e69D
for about 10 s to relax any stresses induced during the blotting.
View Article Online
deprotection of Fmoc group. Then the next Fmoc-protected
amino acid was coupled to the free amino group using O-
(Benzotriazol-1-yl)-N,N,N’,N’-
samples were then transferred to a cryogenic sample holder
(Gatan 626) were examined with a JEM 2200FS TEM (200 keV)
at about -174 ℃. The phase contrast was enhanced by under
focus. The images were recorded on a Gatan multiscan CCD and
5
tetramethyluroniumhexafluorophosphate (HBTU) as the
coupling reagent. The growth of the peptide chain was 65 preceded with Digital Micrographs. Transmission electron
according to the established Fmoc SPPS protocol. At the final
step, Folic acid was used to couple with the peptide. After the
microscopy (TEM) was performed on a MODEL H-800 Electron
microscope operating at an acceleration voltage of 100 kV.
10 last coupling step, excessive reagents were removed by a
single DMF wash for 5 min (5 ml per gram of resin), followed
3.5 Release profile: A hydrogel in PBS (PH=7.4) solution
by five steps of washing using dichloromethane (DCM) for 2 70 containing 0.5 wt% of compound was formed in Eppendorf
min (5 ml per gram of resin). The peptide derivatives were
cleaved from the resin by ice-cold reagent (95%
15 Trifluoroacetic acid, 2.5% Triisopropylsilane, 2.5% double-
distilled water) and the mixture was stirred at room
tube at 25℃. After 24h, we added 0.25 mL of PBS on the
surface of the hydrogels, 0.2 mL solution was taken out at the
desired time point and 0.2 mL PBS was added back. For the
following time points, 0.2 mL of PBS was taken out and 0.2
B
temperature for 30 min, filtered, and poured into ice-cold 75 mL of PBS was added back at each point. We then monitored
diethylether. The resulting precipitate was centrifuged for 10
min at 4℃ at 10,000 rpm. Afterward the supernatant was
20 decanted and dissolved in double-distilled (dd) water and
lyophilized.
and calculated the release profile from the gel formed by a
LCMS-20AD (Shimadzu) system. The experiment was
performed at 37 0C.
80 3.4 Tumor inhibition assay: Female Balb/c mice were
inoculated with 2×105 4T1-luciferase cells in the mammary
fat pad for building breast tumor model. The tumor growth
was monitored every other day. Tumor volume was recieved
through calculating by the formula: length × width × (Length
3.2 Synthesis of FA-Taxol conjugates: 0.15 mmol of
compounds
25 Hydroxysuccinimide (NHS) active ester (0.1 mmol) were
dissolved in mL of DMF, 41.25 μL of
and 95.4 mg of Taxol-succinic acid N-
5
diisopropylethylamine (DIPEA, 0.25 mmol) was then added. 85 + Width)/2. When tumors size reached ׽500 mm3, mice were
The resulting reaction mixture was stirred at room
temperature overnight. FA-Taxol conjugates were obtained by
30 HPLC (yields of 40-50%).
randomly divided into different treatment groups. The day of
giving drugs was designated as day 0. Mice weight was also
monitored after receiving treatment.
3.3 Hydrogel formation: 1 mg of compond was dissolved in
0.15 mL of PBS buffer solution containing 6 equiv. of
Na2CO3 (2 equiv. of Na2CO3 were used to neutralize the
35 compounds and the additional 4 equiv. of Na2CO3 were used
to neutralize GSH to make the final pH value to 7.4). 0.05 mL
of PBS buffer solution containing 4 equiv. of GSH was then
added. Gels would form after being kept at room temperature
25℃for less than 10 minutes.
90 4. Conclusions
In summary, we have developed a series of molecular
hydrogels of FA-Taxol conjugates formed by disulfide bond
reduction by GSH. All designed FA-Taxol conjugates could
form hydrogels by GSH, demonstrating in a further step that
95 FA could be a versatile molecular to construct molecular
gelators through conjugation with other drug molecules. The
hydrogels of FA-Taxol conjugates in this study could
sustainedly release Taxol through ester bond hydrolysis.
Compared with i.v. injection of Taxol with four times of
100 dosage, our hydrogel could inhibit tumor growth more
efficiently by a single dose of i.t. administration. The prolong
and sustainedly release profile, good biocompatibility, and
enhanced anti-cancer efficacy of our hydrogels suggested their
big potential for cancer therapy.
40
3.4 Characterization of hydrogels:
Rheology: Rheology test was done on an AR 2000ex (TA
instrument) system, 40 mm parallel plates was used during the
experiment at the gap of 500μm. For the dynamic time sweep,
45 the solution of compounds upon adding 4 equiv. of GSH was
directly transferred to the rheometer and it was conducted at
the frequency of 1 rad/s and the strain of 1% after 1 hour.
Before dynamic sweep, dynamic strain sweep was performed
to find out the linear region. The gels were characterized by
50 the mode of dynamic frequency sweep in the region of 0.1-
100 rad/s at the strain of 1%.
105
The authors acknowledge NSFC (51203078) for the financial support.
Notes and references
Cryo-Transmission Electron Microscopy (Cryo-TEM): Cryo-
TEM samples (hydrogels containing 0.5%wt compounds) were
55 prepared at 25℃ and stored at 4℃. Sample was loaded onto a
lacey support TEM grid by using a micropipette which was held
by tweezers. The samples were quickly plunged into a reservoir
of liquid ethane at its melting temperature (-183 ℃) upon waiting
a State Key Laboratory of Medicinal Chemical Biology and College of
110 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.
4
| Journal Name, [year], [vol], 00–00
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Organic & Biomolecular Chemistry
† Electronic Supplementary Information (ESI) available: Synthesis and
characterization of compounds, dynamic time sweep, and body weight
change of mice. See DOI: 10.1039/b000000x/
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