Synthesis, Characterization, and Evaluation of Triptolide Cell-Penetrating Peptide Derivative for Transdermal Delivery of Triptolide

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00914

Triptolide (TP) has been used as one of the most common systemic treatments for various diseases since the 1960s. However, TP displays diverse side effects on various organs, which limits its clinical application. To overcome this issue, numerous C-14-hydroxyl group derivatives of TP have been synthesized. In this research, the C-14-hydroxyl group of TP is modified by a cell-penetrating peptide polyarginine (R₇). The derivative TP-disulfide-CR₇ (TP-S-S-CR₇) containing a disulfide linkage between TP and R₇ possesses less toxicity at various concentrations on the immortal human keratinocyte (HaCaT) cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay compared with free TP. Treating HaCaT cells with TP (100 nM) could increase intracellular ROS (reactive oxygen species) and decrease the activity of SOD (superoxide dismutase). Meanwhile, treating HaCaT cells with equimolar concentration of TP-S-S-CR₇ did not cause both of the above TP-induced alterations. In addition, TP-S-S-CR₇ did not show significant dermal toxicity on Guinea pigs and could efficiently overcome the barrier of corneum and then reach epidermis and dermis within 2 h of transdermal administration. In addition, there was a relatively lower concentration of TP in blood indicating less toxicity on organs. Such results suggest that topical therapy using polyarginine is possible by the transdermal delivery of TP.

Full text of DOI:10.1021/acs.molpharmaceut.7b00914

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Article
Synthesis, Characterization and Evaluation of Triptolide-Cell
Penetrating Peptide Derivative for Transdermal Delivery of Triptolide
Tian Tian, Yuming Song, Ke Li, Yuming Sun, and Qing Wang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00914 • Publication Date (Web): 08 Jan 2018
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Synthesis, Characterization and Evaluation
of Triptolide-Cell Penetrating Peptide
Derivative for Transdermal Delivery of
Triptolide
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Tian Tian†, Yuming Song†, Ke Li†, Yuming Sun‡, Qing Wang†, §,
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† School of Pharmaceutical Science and Technology, Dalian University of
Technology, No. 2 Linggong Road, Dalian 116024, China
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§ State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2
Linggong Road, Dalian 116024, China
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‡ Chemical Analysis and Research Center, Dalian University of Technology, No. 2
Linggong Road, Dalian 116024, China
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Triptolide (TP) has been used as one of the most common systemic treatments for
various diseases since 1960s. However, TP displays diverse side effects on various
organs which limits its clinical application. To overcome this issue, numerous Cꢀ14
hydroxyl group derivatives of TP have been synthesized. In this research, the Cꢀ14
hydroxyl group of TP is modified by a cell penetrating peptide poly arginine (R7). The
derivative TPꢀdisulfideꢀCR7 (TPꢀSꢀSꢀCR7) containing a disulfide linkage between TP
and R7 possesses less toxicity at various concentrations on immortal human
keratinocyte line (HaCaT) cell line by 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,
5ꢀdiphenyltetrazolium bromide (MTT) assay compared with free TP. Treating HaCaT
cells with TP (100 nM) could increase intracellular ROS (reactive oxygen species)
and decrease the activity of SOD (Superoxide Dismutase). Meanwhile, treating
HaCaT cells with equimolar concentration of TPꢀSꢀSꢀCR7 did not cause both of the
above TPꢀinduced alterations. In addition, TPꢀSꢀSꢀCR7 didn’t show significant dermal
toxicity on guinea pigs and could efficiently overcome the barrier of corneum then
reached epidermis and dermis within 2 h of transdermal administration. In addition,
there was a relatively lower concentration of TP in blood indicates a less toxicity on
organs. Such results suggest that topical therapy using poly arginine is possible by the
transdermal delivery of TP.
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Key words: Triptolide, Cell penetrating peptide, Toxicity reduction, Drug
modification
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51 INTRODUCTION
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Triptolide (TP), an active compound isolated from Tripterygium wilfordii Hook F
(TWHF), commonly called lei gong teng or thunder god vine, which displays multiple
bioactivities such as immunosuppressive, antiꢀfertility, antiꢀcystogenesis,
antiꢀinflammatory, antiꢀcancer activities1,2. Currently, TP has been used for the
treatment of rheumatoid arthritis, psoriasis and leukemia by oral or intravenous route.
However, due to its poor water solubility and severe side effects, there are some
roadblocks to be applied systemically in clinic. The tissues and organs being inflicted
mainly include reproductive system3, liver4, kidney5 as well as heart6. Over a long
time, the derivatives aimed at the Cꢀ14 hydroxyl group of TP were designed and
synthesized to overcome this issue. For example, TRC4, a TP derivative modified
with amine ester is water soluble, which remains potent anticancer activity without
affecting the growth of normal cells7. Yutaka Aoyagi et al also synthesized a series of
Cꢀ14ꢀhydroxyl group derivatives, which are more effective than free TP against A549
and HT29 cell lines8. Furthermore, TPꢀLZM, a C14ꢀhydroxyl group derivative, can
solute in water, more importantly, this derivative causes less toxicity in Normal Rat
Kidney cell line (NRKꢀ52E)9. Moreover, transdermal delivery system can bypass
hepatic first pass metabolism and reduce the incidence or severity of gastrointestinal
reactions10. Therefore, transdermal delivery system could also reduce the toxicity of
TP. Owing to these reasons, there is no doubt that developing the transdermal delivery
of TP is imperative. Gui Chen et al. used microneedles to break the barrier of skin and
successfully delivered TP through the skin of rat11. However, microneedles could
cause micrometerꢀscale openings on corneum thus causing skin irritation. Based on
these results, developing a biological enhancement technology for TP is necessary.
Cellꢀpenetrating peptides (CPPs) such as Antp, TAT, PEPꢀ1 and polyarginine12, is a
new kind of drug carrier, could enhance the transdermal drug delivery without skin
irritation. CPPs are first discovered in 198813, 14 and have successfully delivered
therapeutic molecules (nucleic acids, drugs, imaging agents) into cells and tissues in a
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nontoxic manner. Recently, a number of interdisciplinary studies proved that CPPs
have successfully carried various cargos such as nucleic acids15, polymers16,
oligonucleotides17, liposomes18, nanoparticles19, and low molecular drugs20 across
cytoꢀmembrane or other bioꢀmembrane21. Moreover, it is reported that a chain of
arginines compose one of the most widely used CPPs and more effective than others22.
Poly arginine display many structural and functional advantages over other CPPs may
because the peptides are positively charged and they interact with critical membrane
components needed for penetration. Poly arginine (11R) covalently attaching with
hydroquinone successfully cured UVꢀinduced pigmentation by transdermal delivery.
Such results demonstrates that topical therapy using the conjugation with poly
arginine is thought to be possible for the delivery of small molecular drugs by
transdermal route23.
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The aim of this work is to design and synthesize a TPꢀpoly arginine derivative
(TPꢀSꢀSꢀCR7) using succinic anhydride and pyridyl disulfide cysteamine
hydrochloride as linkers. TPꢀSꢀSꢀCR7 showed lower cell toxicity on immortal human
keratinocyte (HaCaT) cell line at different concentrations than TP, meanwhile,
TPꢀSꢀSꢀCR7 could also cause less dermal toxicity on guinea pigs compare to free TP.
97 EXPERIMENTAL SECTION
98 2.1 Animals
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Male SpragueꢀDawley (SD) rats and guinea pigs and Kunming (KM) mice used in all
experiments were supplied by Dalian Medical University (Dalian, China). The
animals were kept in an animal room with regulated temperature of 20 ± 2 °C and
relative humidity of 60 ± 10%. All of them were given free access to food and water.
103 2.2 Synthesis of pyridyl disulfide cysteamine hydrochloride
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The synthesis procedure of pyridyl disulfide cysteamine hydrochloride is described as
follow. An amount of 10 ml methanol containing 1.04 g cysteamine hydrochloride
was added to a solution of 2, 2’ꢀdithiodipyridine (4 g) in 16 ml of methanol and 0.6 ml
acetic acid dropwisely. The yellow solution was stirred overnight at room temperature.
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Then the solvent was evaporated off. The product was obtained by precipitation in
500 ml diethyl ether. After filtration, the residue was dissolved in 15 ml of methanol
and again precipitated in 500 ml of diethyl ether. The yellow solid was obtained after
drying under reduced pressure. The structure of pyridyl disulfide cysteamine
hydrochloride was characterized by 1HꢀNMR.
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113 2.3 Synthesis of TPS
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An amount of TP 100 mg and succinic anhydride 320 mg were dissolved in 2 ml
anhydrous methylene chloride (CH2Cl2) followed by the addition of 1.5 ml CH2Cl2
containing 4ꢀdimethylaminopyridine (DMAP) 360 mg and trimethylamine 0.5 ml
dropwisely. The reaction stirred at room temperature for 24 hours. The reaction was
monitored by TLC with the solvent system of ethyl acetateꢀpetroleum ether (1:1, v/v),
and the light purple spot was detected by soaking in 2% 3,5ꢀdinitrobenzene in ethanol
and 10% potassium hydroxide in methanol9. After evaporating the solvent away, the
crude product was washed by cold carbon tetrachloride (CCl4) three times. The CCl4
was removed and then dried under reduced pressure, yellow powder was obtained.
The structure of TPS was characterized by 1HꢀNMR, 13CꢀNMR and HRMS.
124 2.4 Synthesis of TPSP
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Amount of TPS 120 mg, pyridyl disulfide cysteamine hydrochloride 61 mg were
dissolved in 0.8 ml tetrahydrofuran containing 0.4 ml triethylamine. Then 54 mg
4ꢀ(4,6ꢀDimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀmethylmorpholinium chloride (DMTMM)
was added to the solution. At last, 1.8 ml tetrahydrofuran was added. The reaction
mixture was stirred at room temperature for 24 hours. After removing the solvent
away, the crude product was purified by preparative chromatography (LCꢀ20AP,
Shimadzu, Japan) using C18 chromatographic column (ZORBAX SBꢀC18, 21.2×
150 mm，Agilent, America, 10ꢀ100% B for 30min, A: 0.1% trifluoroacetic acid in
water, B: 0.1% trifluoroacetic acid in acetonitrile, flow rate = 7ml/min). After
removing the solvent away light yellow spongy solid was obtained. The structure of
TPSP was characterized by 1HꢀNMR, 13CꢀNMR and HRMS. The purity of TPSP was
determined by HPLCꢀUV.
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Amounts of TPSP 60 mg, R7C (adding a cysteine on the Nꢀterminal of R7)·8TFA 203
mg were dissolved in 3 ml DMF. Then the reaction solution was stirred for 48 hours at
room temperature. After completion of the reaction, the solvent was evaporated off,
the crude product was purified by preparative chromatography using SBꢀC18
chromatographic column (10ꢀ100% solvent B; 30min, flow rate = 7ml/min; solvent A:
0.01% trifluoroacetic acid in water, solvent B: 0.01% trifluoroacetic acid in
acetonitrile). After removing the solvent away, white powder was obtained. The
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structure of TPꢀSꢀSꢀCR7 was characterized by HꢀNMR, HRMS and HRMS2. The
proposed fragmentation pathway of TPꢀSꢀSꢀCR7 was simulated using Mass Frontier
7.0. The purity of TPꢀSꢀSꢀCR7 was determined by HPLCꢀUV.
148 2.6 Cell culture
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HaCaT cell line was kindly offered by Professor Wenli Li (School of Life Science and
Biotechnology, Dalian University of Technology) was cultured in Dulbecco modified
Eagle medium (DMEM) high glucose containing 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin solution (100 IU/ml penicillin; 100 ꢁg/ml streptomycin) in an
incubator at 37 °C with 5% CO2, 95% air, and 90% relative humidity and offered
fresh medium every other day.
155 2.7 Cytotoxicity assay of TP and TP-S-S-CR7 on HaCaT cell line
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The effect of TPꢀSꢀSꢀCR7 and TP on HaCaT cell viability was measured at different
concentrations using MTT assay. HaCaT cells (7×103 cells/well) were seeded in
96ꢀwell tissue culture plates. After 24 h, the medium was replaced with fresh medium
containing various concentrations of TP, TPꢀSꢀSꢀCR7 (10, 1, 0.1, 0.01, 0.001 ꢁM) or
medium containing 0.5% dimethyl sulfoxide (DMSO). After incubation for 24 h, 110
ꢁL of 500 ꢁg/ml MTT was added, and the plates were incubated for another 4 hours.
Thereafter, the medium was removed and replaced with 150 ꢁL of DMSO, the optical
density (OD) was measured at 490 nm using a microplate reader. The cytotoxic
effects of tested agents were expressed as the 50% inhibiting concentration (IC50), and
the IC50 values were calculated using SPSS.
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166 2.8 The stability of TP-S-S-CR7 in HaCaT cell homogenate, rat skin
167 homogenate and rat plasma
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The HaCaT cells were seeded in the culture plate (diameter was 10 cm), after 80% ꢀ
90% confluent, cells and cell culture medium were then separated. Cells were then
washed with cold PBS 3 times and collected using cell scraper with 1 ml cold PBS in
a 10 ml Eppendorf tube. Then 500 ꢁl (1 mg/ml) TPꢀSꢀSꢀCR7 was added to 500 ꢁl of
the cell homogenate and mixed then incubated at 37 . After 24 h, an aliquote of the
sample (100 ꢁl) was withdrawn from the solution and equal volume acetonitrile was
added to stop the reaction. The samples were analyzed by HPLCꢀHRMS. (n=3)
The abdominal skin was obtained soon after the rat was executed. Then the skin
samples (200 mg) were cut into small pieces in a 10 ml centrifuge tube, 2.0 ml of PBS
was added. The samples were homogenized at 18000 rpm for about 1 min, and the
supernatant was obtained by centrifugation at 6×103 g for 5 min. Then 1 ml
TPꢀSꢀSꢀCR7 (1 ml) was added to 1ml of the skin homogenate and mixed then
incubated at 37℃. Then after 1h, an aliquote of the sample (100 ꢁl) was withdrawn
from the solution and equal volume acetonitrile was added to stop the reaction. The
amount of TPꢀSꢀSꢀCR7 was detected by HPLCꢀHRMS. (n=3)
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Blood was collected from adult male SD rats from orbital plexus to heparinized tubes,
then the plasma was separated immediately by centrifugation (6×103 g, 10 minutes).
Immediately after the collection of plasma, 1 ml TPꢀSꢀSꢀCR7 (1 mg/ml) was added to
1ml plasma. Then an aliquote of the sample (100 ꢁl) was withdrew from the solution
and equal volume acetonitrile was added to stop the reaction. The amount of
TPꢀSꢀSꢀCR7 was detected by HPLCꢀHRMS. (n=3)
189 2.9 The stability of TP-S-S-CR7 in glutathione (GSH) solution
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1 ml TPꢀSꢀSꢀCR7 (1 mg/ml) was mixed with 1 ml (200 ꢁg/ml or 2 ꢁg/ml) GSH in
PBS and incubated at 37°C for 24 h. Aliquots were withdrawn from the solution at
specified time points (0, 1, 3, 6, 12 and 24 h) and analyzed by HPLCꢀUV method, and
the corresponding degradation products were identified by HPLCꢀHRMS method.
(n=3)
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2.10 Intracellular ROS determination
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DCFHꢀDA, a lipophilic dye was used to determine the intracellular accumulation of
ROS. HaCaT cells were seeded on 6ꢀwell plate until about 80%ꢀ90% confluent.
Subsequently, the cells were treated with 100 nM TP, TPꢀSꢀSꢀCR7 or fresh medium
for 12 h respectively. After that, the medium was removed; the cells were washed
twice with PBS. Then, cells were incubated with DCFHꢀDA (20 ꢁM) for about half an
hour at 37 ℃ in dark. Then washed with serum free fresh DMEM ３times.
Fluorescence was detected with flow cytometry. Excitation: λ 488 nm and Emission:
λ 520 nm. (n=3)
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204 2.11 SOD activity determination
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HaCaT cells were seeded in a 6ꢀwell plate until about 80% ꢀ 90% confluent. Then
cells were exposed to TP, TPꢀSꢀSꢀCR7 or fresh medium (as negative control) for 12h.
After that, the medium was removed, and then the cells were washed twice with cold
PBS (pH = 7.4, NaCl: 8 g/L, Na2HPO4: 2.86 g/L, KH2PO4: 0.2 g/L, KCl: 0.19 g/L).
Through tissue homogenized on ice, centrifuged at 1×104 g for 15min at 4℃, the
total intracellular protein could be obtained. Total protein of each sample was
determined by the BCA Protein Assay Kit. SOD activity was determined using SOD
Detection Kit. (n=3)
213 2.12 Skin irritation test in guinea pigs
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6 guinea pigs (200ꢀ300 g) were used for this skin irritation test. Three days prior to
application, the hair on the dorsal area was removed using clipper and shaver, then the
belly skin of each guinea pig was divided into three areas (3 × 3 cm2). Then 100
ꢁg/kg/day TP or 722 ꢁg/kg/day TPꢀSꢀSꢀCR7 (equimolar concentration) and equal
volume of PBS were smeared on each depilated skin areas for seven days
successively24. 24 h after the last administration, collect each area then fixed in 4%
paraformaldehyde for at least 24 h. Tissue sections (5 ꢁm) were prepared by mounted
on common slides, and then stained with hematoxylin and eosin.
222 2.13 In vivo penetration experiment
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Six male SD rats (weighing 150ꢀ200 g) were randomly divided into two groups. 24
hours prior to the experiment, the hair on the dorsal area was removed using clipper
and shaver. Then TP at a dose of 3 mg/kg or TPꢀSꢀSꢀCR7 at a dose of 21 mg/kg
(equimolar concentration) were smeared onto the dorsed areas of rats (3×3 cm2).
Blood samples were collected from the retroꢀorbital plexus into heparinized tubes at
designated time intervals (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 hours) post
administration then plasma was separated immediately by centrifugation (6×103 g, 10
minutes). The plasma samples were stored at ꢀ40℃until analysis. Plasma sample 50
ꢁL was mixed with 50 ꢁL of internal standard solution (200 ꢁg/ml triamcinolone
acetonide in methanol), then 50 ꢁL of methanol was added and vortexed for 1 minute.
After centrifugation at 6×103 g for 5 min, the supernatant was transferred to a new
tube. 20 ꢁL of each sample was injected into the LCꢀMS system for quantitative
analysis.
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Separation was performed on an Agilent Zorbax SBꢀC18 column (4.6×150 mm, 5
ꢁm, Agilent Technologies, USA). The mobile phase was composed of 40ꢀ80% B for 6
min, then 80ꢀ100% B for 1min, then 100% B for 1min (A: water, B: acetonitrile),
delivered at a flow rate of 0.5 ml/min.
Thermo LTQ orbitrapXL mass spectrometer with high resolution was operated in ESI
positive mode. The parameters were as follows: vaperizer temperature 350 ℃; sheath
gas flow rate 30 Arb; auxiliary gas flow rate 10 Arb; ion spray voltage 3.5 kV;
capillary temperature 350 ℃; capillary voltage 39 V. The resolution was set at 30000
with mass error tolerance of ±5 ppm. The retention times of TP and internal standard
(triamcinolone acetonide) were 7.01 and 9.69 minutes. Over the range of 1.25ꢀ200
ng/ml, TP concentrations were linearly proportional to the area ratio of TP/internal
standard. Calibration curves were constructed for TP is y=ꢀ0.000136353 +
0.00144784x (R=0.999).
249 2.14 In vivo skin retention experiment
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Twentyꢀfour KM male mice weighing 20ꢀ25 g were randomly divided into two groups.
Sodium sulfide 4 g, Amidulin 3.5 g, sucrose 2 g, Sodium tetraborate 0.5 g, Glycerin
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2.5 g were carefully weighed and dissolved in 50 ml water and stirred to obtain a
homogeneous solution. The hairs on the back of the mice were lightly rubbed with a
pledget preꢀwetted in the above solution. After all the hairs were removed, the
remaining solution on the back of the animal was washed away with warm water. The
treated animal was kept for at least 48 h to ensure that no visible defect was present25.
Then TP at a dose of 4.3 mg/kg or TPꢀSꢀSꢀCR7 at a dose of 30 mg/kg (equimolar
concentration) were smeared onto the dorsed areas of mice (2 cm2). After 2, 6, 12
hours of administration, the animals were executed and the administration areas of
skin were collected. (n=3)
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The corneum of the skin samples were removed and then the skin samples (100 mg)
were cut into small pieces in a 10 ml centrifuge tube, 1.0 ml of methanol was added.
The samples were homogenized at 18000 rpm for about 1 min, and the supernatant
was obtained by centrifugation at 6×103 g for 5 min26. The supernatant was treated
using the protocol used for the plasma samples and analyzed by HPLCꢀMS method
which was the same as that for plasma samples.
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RESULTS
3.1 Synthesis and characterization of pyridyl disulfide cysteamine
hydrochloride
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To make the amino acid cysteine at the Nꢀterminal of poly arginine accessible for TP,
the corresponding sulfydrylꢀreactive functionalities had to be introduced. In this
research, we choose pyridyl disulfide cysteamine hydrochloride because it is widely
used and easy to be synthesized27. The synthesis procedure is described on Scheme 1.
1
The productivity is 74%. H NMR (400 MHz D2O): δ = 3.15ꢀ3.08 (2H, t), 3.40ꢀ3.33
(2H, t), 7.37ꢀ7.31 (1H, m), 7.77ꢀ7.72 (1H, d), 7.87ꢀ7.80 (1H, tꢀd), 8.49ꢀ8.44 (1H, m).
The spectrum of pyridyl disulfide cysteamine hydrochloride is shown in supporting
information.
279 3.2 Synthesis and characterization of TPS
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TP has an active hydroxyl group at Cꢀ14 position, which can be conjugated to poly
arginine via spacers. In this research, we choose succinic anhydride as the first spacer.
The schematic synthetic route is shown in Scheme 2. The productivity is 94%.
1HꢀNMR (400 MHz, CDCl3): 5.06ꢀ5.05 (1H, s), 4.72ꢀ4.61 (2H, s), 3.83ꢀ3.80 (1H, d),
3.54ꢀ3.51 (1H, d), 3.47ꢀ3.42 (1H, d), 2.84ꢀ2.62 (5H, m), 2.34ꢀ2.25 (1H, d), 2.21ꢀ2.04
(2H, m), 1.95ꢀ1.82 (2H, m), 1.61ꢀ1.47 (1H, dd), 1.30ꢀ1.13 (1H, m), 1.05ꢀ1.01 (3H, s),
0.95ꢀ0.90 (3H, d), 0.85ꢀ0.79 (3H, d). HRMS m/z 461.1807 [M+H]+. The spectrums of
TPS are shown in supporting information.
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288 3.3 Synthesis and characterization of TPSP
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In order to modify TPS with 2ꢀ(methyldisulfanyl) pyridine residue, pyridyl disulfide
cysteamine hydrochloride is chosen as the second spacer. DMTMM is chosen as
catalyst, because it is insoluble in THF, thus easy to remove after the reaction. The
1
reaction procedure was described in scheme 3. The productivity is 50%. HꢀNMR
(400 MHz, CDCl3): 8.56ꢀ8.48 (1H, s), 7.67ꢀ7.58 (1H, s)7.58ꢀ7.50 (1H, d), 7.21ꢀ7.07
(2H, s), 5.11ꢀ5.04 (1H, s), 4.71ꢀ4.62 (2H, s), 3.88ꢀ3.78(1H, s), 3.62ꢀ3.43 (4H, t),
2.96ꢀ2.52 (8H, m), 2.39ꢀ2.23 (1H, m), 1.97ꢀ1.79 (2H, d), 1.56ꢀ1.50 (1H, m), 1.34ꢀ1.19
(1H, m), 1.06ꢀ1.00 (3H, s), 0.96ꢀ0.91 (3H, d), 0.84ꢀ0.79 (3H, d). HRMS m/z 629.1984
[M+H]+. The spectrums of TPSP are shown in supporting information.
298 3.4 Synthesis and characterization of TP-S-S-CR7
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As previously studied, adding a cysteine to the Cꢀterminal of CPPs could not affect
their activities28. So, in this study, we add a cysteine to the Nꢀterminal of poly arginine
to offer a sulfydryl in order to react with TPSP. The reaction procedure is described on
scheme 4. The productivity is 60% and the purity is 98%. HRMS m/z 289.4912 (z=6),
347.1882 (z=5), 433.7333 (z=4), 577.9754 (z=3), 866.4596 (z=2). The structure of
TPꢀSꢀSꢀCR7 is shown in Scheme 5. The spectrums of HRMS2, the proposed
1
fragmentation pathways and HꢀNMR of TPꢀSꢀSꢀCR7 are shown in supporting
information.
307 3.5 Cytotoxicity of TP and TP-S-S-CR7 on HaCaT cell line
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choose HaCaT cell line as a cellular model to investigate the dermal toxicity of TP
and TPꢀSꢀSꢀCR7 in vitro using MTT assay. The cells were treated with
various concentrations of TP and TPꢀSꢀSꢀCꢀR7 for 24 h. A doseꢀdependent effect of
TP and TPꢀSꢀSꢀCR7 could be observed on HaCaT cell line (Figure 1). The IC50 value
of TP on HaCaT cell line for 24 h was 1.00 ꢁM, while the IC50 value of TPꢀSꢀSꢀCR7
was 9.06 ꢁM indicating that TPꢀSꢀSꢀCR7 can reduce dermal toxicity in vitro,
especially at 10, 1 and 0.1 ꢁM cell viability can be significantly improved. Moreover,
polyꢀarginine has no cell toxicity (data not shown). Moreover, from the result of the
stability of TPꢀSꢀSꢀCR7 in HaCaT cell homogenate, TPꢀSꢀSꢀCR7 was found to
degrade into TP. But only part of TPꢀSꢀSꢀCR7 was degraded. This perhaps was
because the lower esterase concentration in HaCaT cell line and further led to the
result of lower cytotoxicity.
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321 3.6 Induction of oxidative stress in HaCaT cells by TP and
322 TP-S-S-CR7
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As previously reported, TP could induce intracellular ROS accumulation and then
lead to tissue damage. So in this research, we attempt to investigate whether
TPꢀSꢀSꢀCR7 could cause oxidative damage on HaCaT cells as TP does. The cells were
treated with 100 nM TP, TPꢀSꢀSꢀCR7 and fresh medium for 12 h. Then, intracellular
ROS level as well as SOD activity were measured. As shown in Figure 2, compared
with the control group, TP exposure increased the accumulation of intracellular ROS.
Meanwhile, the intracellular SOD activity decreased significantly. However,
TPꢀSꢀSꢀCR7 treated cells have approximated the same intracellular ROS level and
SOD activity as the control group (Figure 2 and 3). Such results indicated that
TPꢀSꢀSꢀCR7 didn’t show significant oxidative damage on HaCaT cell line.
333 3.7 Skin irritation test in guinea pigs
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To further investigate whether TPꢀSꢀSꢀCR7 has less skin irritation, 0.1 mg/kg/day TP,
0.722 mg/kg/day TPꢀSꢀSꢀCR7 and equal volume of PBS（pH=7.4 containing 0.1%
DMSO）were smeared on the belly skin of guinea pigs for seven days consecutively.
After smearing for three days, red and swollen appeared on the area of TP, then after
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six days of TP treatment, scab could be observed. Whereas, no obvious difference can
be observed between TPꢀSꢀSꢀCR7 group and control group (Figure 4). TP treatment
could induce severe damage around corneum and hair follicle. Never the less, there’s
no obvious histopathological changes in TPꢀSꢀSꢀCR7 treated group.
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The in vivo skin retention and penetration experiments were performed to explore
whether TPꢀSꢀSꢀCR7 is suitable for topical therapy by transdermal route. TP 4.3
mg/kg or TPꢀSꢀSꢀCR7 at a dose of 30 mg/kg were transdermal administrated to KM
rats. As shown in Figure 8, TP could be detected at 2 h after administration in both TP
and TPꢀSꢀSꢀCR7 administration groups, the concentrations of TP in epidermis and
dermis in TP administration group and TPꢀSꢀSꢀCR7 administration group are as high
as 6352.22 ng/g and 3647.80 ng/g. Such result demonstrates that even through the
water solubility of TP has been significantly improved, owing to the presence of poly
arginine, the transdermal ability of TP has not been significantly affected. TPꢀSꢀSꢀCR7
also can be detected at 2 h, which could probably indicates TP could release in the
epidermis and dermis in a slow rate. So the effective concentration of TP may last
longer.
3mg/kg TP or 21 mg/kg TPꢀSꢀSꢀCR7 were transdermal administration to SD rats to
detect the concentration of TP in blood. As shown in Figure 8, TP could not be
detected in TP administrated group. In TPꢀSꢀSꢀCR7 administration group, a relatively
low concentration of TP could be detected. The highest concentration is 13.52 ng/ml,
which could cause less systematic toxicity on organs. Such results proves that
TPꢀSꢀSꢀCR7 has the potential for topical therapy by transdermal delivery of TP.
362 DISCUSSION
363 4.1 Design and synthesis of TP-S-S-CR7
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Low molecular weight proteins, which like peptides afford good biodegradability in
vivo, have been employed as drug carriers for transdermal delivery of therapeutic
drugs. Since the guanidine of arginine is proposed to be crucial for achieving
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penetration efficacy30. So in this study, we choose poly arginine to modify the
Cꢀ14ꢀhydroxyl group of TP to investigate whether TPꢀCPP conjugate could be
designed and synthesized, at the same time with the ability to achieve transdermal
delivery, meanwhile, reduce the dermal toxicity of TP. Too much amino acids on the
side chains of poly arginine makes it difficult to conjugate TP and R7 directly. So it is
imperative to induce linkers. As there is a cysteine on the Cꢀterminal of poly arginine,
the most direct linker would be 3ꢀ(pyridineꢀ2ꢀyldisulfancyl) propanoic acid31.
However, it changes into 1,2ꢀdi (pyridineꢀ2ꢀyl) disulfane during the reaction in
CH2Cl2 at room temperature at the presence of 1ꢀ(3ꢀDimethylaminopropyl)ꢀ
3ꢀethylcarbodiimide hydrochloride (EDC) and DMAP. Even modified
3ꢀ(pyridineꢀ2ꢀyldisulfancyl) propanoic acid with benzotriazole, the productivity was
as low as 20%. So in this research, succinic anhydride and pyridyl disulfide
cysteamine hydrochloride are chosen as linkers to conjugate TP and poly arginine.
Because the derivative could be obtained by three steps from the natural product,
scaleꢀup of this method for clinical use of the prodrug does not pose a problem.
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382 4.2 The toxicity evaluation of TP and TP-S-S-CR7 on HaCaT cell line
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Oxidative stress, which is caused by the overꢀaccumulation of ROS, induces protein
damage and aggregation, leading to impaired cellular homeostasis32. For over a
decade, an increasing number of works have reported that oxidative stress is a
recognized mode of toxic effects of TP exposure which has been observed in vitro and
in vivo. TP exposure has been shown to increase the generation of anion superoxide
and inhibit the activity of such antioxidant enzymes as SOD and subsequently induced
oxidative stress in the liver, kidney and heart tissues33. So in this study, we investigate
whether TPꢀSꢀSꢀCR7 could cause less cytotoxicity on HaCaT cells. The IC50 of
TPꢀSꢀSꢀCR7 is about nineꢀfold higher than that of TP, which indicates that
TPꢀSꢀSꢀCR7 could decrease dermal caused by TP in vitro (Figure 1). Based on such
result, we further investigated whether TPꢀSꢀSꢀCR7 could cause less ROS
accumulation and SOD activity decrease on HaCaT cell line. The results indicated
that there’s obvious increase of intracellular ROS of HaCaT cells at the presence of
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TP (Figure 2). Simultaneously, the activity of SOD significantly decreased.
TPꢀSꢀSꢀCR7 treated group did not display such obvious negative effect.(Figure 3).
Our results prove that the mechanism of dermal toxicity is in accordance with the
toxicity mechanism on other organs.
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400 4.3 In vivo dermal toxicity evaluation of TP and TP-S-S-CR7
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The toxicity of TP has been demonstrated in several experimental animal species. The
tissues and organs affected by toxicity of TP include gastrointestinal tract, liver,
kidney, heart, blood cells, bonemarrow, testes and ovaries. But the toxicity of skin is
rarely investigated34. Obvious toxicities of TP were found toward the kidney and
testicles of mice, and these have even resulted in the death of mice at intraperitoneal
doses of 0.025, 0.05 and 0.1 mg/kg. As previously reported, obvious hepatotoxicity
was also observed at an oral dose of 0.2 mg/kg in mice. It has also been described in
rats with oral dosages as low as 0.1 mg/kg, in dogs at intravenous dosages of 0.04
mg/kg, and in rabbits at an external concentration of 1.11 mmol/L34. Guinea pigs are
commonly used as animal models for skin irritation evaluation35, 34. So in this study,
we choose guinea pigs to conduct the comprehensive dermal toxicity study of TP and
TPꢀSꢀSꢀCR7 at transdermal delivery dosage as low as 0.1 mg/kg/day TP and 722
mg/kg/day TPꢀSꢀSꢀCR7 for 7 days. As shown in Figure 4, TP exposure could cause
significant dermal toxicity on corneum and hair follicle. However, no obvious toxicity
could be observed in TPꢀSꢀSꢀCR7 smeared group, which provide a promising result
for the transdermal delivery of TP.
417 4.4 The degradation of TP-S-S-CR7 in HaCaT cell homogenate, rat
418 skin homogenate, rat plasma and GSH solution
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The results of in vitro and in vivo dermal toxicity experiment prove that TPꢀSꢀSꢀCR7
could cause less dermal toxicity compare with free TP. In order to further discuss the
mechanism of the toxicity reduction, the metabolism activity of TPꢀSꢀSꢀCR7 in
HaCaT cell homogenate, rat skin homogenate and rat plasma was investigated.
In HaCaT cell homogenate, the degradation rate is not so fast, 24 h after TPꢀSꢀSꢀCR7
exposed to the cell homogenate, both TP and TPꢀSꢀSꢀCR7 could be detected. This may
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because the esterases in HaCaT cell is not active.
The existing formation of TPꢀSꢀSꢀCR7 in skin homogenate is very important for us to
understand the transportation form of TPꢀSꢀSꢀCR7 across skin. So in this study, the
degradation of TPꢀSꢀSꢀCR7 in rat skin homogenate was investigated in vitro. After 1h
exposure to rat skin homogenate, about 85% of TPꢀSꢀSꢀCR7 turned into TP, which
was identified by HPLCꢀHRMS with mass tolerance of ±5 ppm. This may because
the activity of esterase is relatively high. What’s more, there’s no other TP related
compounds were detected. As previously reported, human esterases are highly active
and able to hydrolyze substances extensively during permeation through in vitro
human skin, such as 3ꢀalkyl esters of naltrexone and ester derivatives of fluroxypyr36.
In addition, rat skin also shows relatively high esterase activity, most of the esterases
are located in epidemis and dermis, and the major esterase is carboxylesterase (CES:
EC.3.1.1.1)36. Therefore, the degradation might took place after TPꢀSꢀSꢀCR7
penetrating across the corneum into epidemis and dermis.
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As soon as TPꢀSꢀSꢀCR7 exposed to rat plasma, TP was released. And there’s no other
TP related compounds detected during the experiment. The result of in vivo
penetration experiment showed that TP existed in a relatively low concentration,
which means a relatively low toxicity. Previous research demonstrated that
pꢀNitrophenylacetate, a carboxylic ester containing structure was rapidly hydrolyzed
by rat plasma, and the hydrolase activity was as high as 2540 ± 240 ꢁM/min, which
indicated the esterase activity in rat plasma is relatively high37.
To further investigate whether the esterase is the major factor for the degradation of
TPꢀSꢀSꢀCR7, bisꢀparaꢀnitrophenylphosphate (BNPP; 1 mM), a nonspecific esterase
inhibitor was added to skin homogenate and rat plasma, then incubated for 30 min at
37 ℃before TPꢀSꢀSꢀCR7 was added38. After adding BNPP into skin homogenate, TP
could not be released from TPꢀSꢀSꢀCR7. However, TP still released from TPꢀSꢀSꢀCR7
immediately after TPꢀSꢀSꢀCR7 exposed to rat plasma even when BNPP was added.
There are probably some other components in plasma which could also degrade ester
bond, such as albumin39,40
.
As previously reported, reduced GSH could break disulfide bonds41. So in this study,
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the stability of TPꢀSꢀSꢀCR7 in GSH solution was investigated in vitro. The
degradation product was detected using HPLCꢀHRMS. The concentrations of GSH
used in this experiment are similar to the concentration of GSH in skin homogenate42
(200 ꢁg/ml), HaCaT cell homogenate43 and rat plasma44 (2 ꢁg/ml). The degradation
curve is shown in Figure 5 (A and B), and the degradation product (TPSPꢀD) is
formed by the cleavage of the disulfide bond (Figure 5C). The result of TPꢀSꢀSꢀCR7
stability studies indicated that TPꢀSꢀSꢀCR7 was degraded very quickly in rat plasma,
but it was stable in GSH solution within 1 h, with the GSH concentration level at 2
ꢁg/ml, which was similar with that in rat plasma. This indicated that GSH is not the
main factor for the degradation of TPꢀSꢀSꢀCR7. On the other hand, about 60% of
TPꢀSꢀSꢀCR7 was degraded within 1 h when the GSH was presented at 200 ꢁg/ml,
which was similar to the concentration level of that in skin homogenate. But the
disulfide bond was stable in skin homogenate (as described above). This indicated
that the degradation effect of GSH to TPꢀSꢀSꢀCR7 in skin homogenate was inhibited.
Therefore, though GSH in PBS solution can degrade TPꢀSꢀSꢀCR7 at the concentration
level of 200 ꢁg/ml in vitro and the degradation product was identified by LCꢀHRMS
method as TPSPꢀD (Figure 5), it’s not the main factor for the degradation of
TPꢀSꢀSꢀCR7 in skin homogenate, HaCaT cell homogenate and rat plasma.
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473 4.5 In vivo penetration profile
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Transdermal drug delivery, which was first applied in 1979, has many advantages. For
example, compared with the oral route, transdermal drug delivery can avoid the
firstꢀpass effect of the liver that can prematurely metabolize drugs. What’s more
transdermal drug delivery also has advantages over hypodermic injections, which are
painful, easily generate dangerous medical wastes and pose the risk of disease
transmission by needle reꢀuse45. So it is imperative to develop a transdermal delivery
system for TP. However, there is some difficulties to overcome since TP could cause
severe damage to the corneum (Figure 4). Microinjection, electroporation, and
liposome and viralbased vectors have been used for transdermal delivery. However,
these methods have various drawbacks, such as low efficiency, severe toxicity,
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penurious bioavailability, and poor specificity46. Using cell penetrating peptide poly
arginine to modify the Cꢀ14 hydroxyl group TP could not only avoid some of the
above drawbacks but also can achieve its topical therapy by transdermal route.
Both TP and TPꢀSꢀSꢀCR7 could overcome the barrier of corneum and accumulate in
epidermis and dermis. Even though TP could not be detected in TP administration
group (Figure 8), which means no toxicity on organs, but TP could cause severe
dermal toxicity limits its application for transdermal administration. Such results
suggest that topical therapy using poly arginine is possible by the transdermal delivery
of TP.
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494 CONCLUSION
495
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502
In this study, we designed and synthesized a water soluble TPꢀCPP derivative
TPꢀSꢀSꢀCR7 which has less dermal toxicity both in vitro and in vivo. Moreover,
TPꢀSꢀSꢀCR7 could cross the barrier of corneum and reach epidermis and dermis at 2 h
after transdermal administration which indicates TPꢀSꢀSꢀCR7 can realize the topical
therapy of TP in a transdermal way. The blood concentration of TP is as low as 13.52
ng/ml, which could cause less toxicity on organs such as liver and heart. In a word,
TPꢀSꢀSꢀCR7 makes topical therapy possible by the transdermal delivery of TP and
poses the potential to cure some disease such as rheumatoid arthritis.
503 NOTES
504
The authors declare no competing financial interest.
505 ACKNOWLEDGEMENT
506
507
508
The study has been financed by Liaoning province natural science funds (2014020015)
and project of outstanding talent support program in universities of Liaoning province
(LR2014002).
509 Associated Content
510
511
Supporting information
Materials; preparation procedures of the solutions of TP and TPꢀSꢀSꢀCR7 used in
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MTT assay, skin irritation test in vivo penetration and the stability of TPꢀSꢀSꢀCR7 in
1
GSH solution experiment; HPLC analysis methods of TPSP and TPꢀSꢀSꢀCR7; H
NMR, 13C NMR and HRMS spectrums of pyridyl disulfide cysteamine hydrochloride,
TPS, TPSP and TPꢀSꢀSꢀCR7; proposed fragmentation pathway of TPꢀSꢀSꢀCR7 are
listed in supporting information.
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517 References
518
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1. Li, X. J.; Jiang, Z. Z.; Zhang, L. Y., Triptolide: progress on research in
pharmacodynamics and toxicology. J Ethnopharmacol.2014, 155 (1), 67ꢀ79.
2. Zhou, Z. L.; Yang, Y. X.; Ding, J.; Li, Y. C.; Miao, Z. H., Triptolide: structural
modifications, structureꢀactivity relationships, bioactivities, clinical development and
mechanisms. Nat Prod Rep.
2012, 29 (4), 457ꢀ75.
3. Hu, J.; Yu, Q.; Zhao, F.; Ji, J.; Jiang, Z.; Chen, X.; Gao, P.; Ren, Y.; Shao, S.; Zhang,
L.; Yan, M., Protection of Quercetin against Triptolideꢀinduced apoptosis by
suppressing oxidative stress in rat Leydig cells. Chem. Biol. Interact. 2015, 240,
38ꢀ46.
4. Jin, J.; Sun, X.; Zhao, Z.; Wang, W.; Qiu, Y.; Fu, X.; Huang, M.; Huang, Z.,
Activation of the farnesoid X receptor attenuates triptolideꢀinduced liver toxicity.
Phytomedicine. 2015, 22 (10), 894ꢀ901.
5. Li, J.; Jin, J.; Li, M.; Guan, C.; Wang, W.; Zhu, S.; Qiu, Y.; Huang, M.; Huang, Z.,
Role of Nrf2 in protection against triptolideꢀinduced toxicity in rat kidney cells.
Toxicol. Lett. 2012, 213 (2), 194ꢀ202.
6. Zhou, J.; Xi, C.; Wang, W.; Yang, Y.; Qiu, Y.; Huang, Z., Autophagy plays an
important role in triptolideꢀinduced apoptosis in cardiomyocytes. Toxicol. Lett. 2015,
236 (3), 168ꢀ183.
7. Wang, P. Y.; Zeng, W. J.; Liu, J.; Wu, Y. L.; Ma, Y.; Zeng, Z.; Pang, J. Y.; Zhang, X.
K.; Yan, X.; Wong, A. S.; Zeng, J. Z., TRC4, an improved triptolide derivative,
specifically targets to truncated form of retinoid X receptorꢀalpha in cancer cells.
Biochem. Pharmacol. 2017, 124, 19ꢀ28.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 31
1
2
3
4
5
6
7
8
9
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
8. Aoyagi, Y.; Hitotsuyanagi, Y.; Hasuda, T.; Matsuyama, S.; Fukaya, H.; Takeya, K.;
Aiyama, R.; Matsuzaki, T.; Hashimoto, S., Fluorination of triptolide and its analogues
and their cytotoxicity. Bioorg. Med. Chem. Lett. 2008, 18 (7), 2459ꢀ63.
9. Zheng, Q.; Gong, T.; Sun, X.; Zhang, Z. R., Synthesis, characterization and in vitro
evaluation of triptolideꢀlysozyme conjugate for renal targeting delivery of triptolide.
Arch. Pharm. Res. 2006, 29 (12), 1164ꢀ1170.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10. Sarkar, V.; Yadav, K., Formulation and evaluation of prolonged release
transdermal drug delivery system of atenolol for the treatment of hypertension.
Pharmatutor. 2014, 2 (2), 134ꢀ140.
11. Chen, G.; Hao, B.; Ju, D.; Liu, M.; Zhao, H.; Du, Z.; Xia, J., Pharmacokinetic and
pharmacodynamic study of triptolideꢀloaded liposome hydrogel patch under
microneedles on rats with collagenꢀinduced arthritis. Acta Pharm Sin B. 2015, 5 (6),
569ꢀ76.
12. Chang, M.; Li, X.; Sun, Y.; Cheng, F.; Wang, Q.; Xie, X.; Zhao, W.; Tian, X.,
Effect of cationic cyclopeptides on transdermal and transmembrane delivery of insulin.
Mol. Pharm. 2013, 10 (3), 951ꢀ7.
13. Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A., The third helix of the
Antennapedia homeodomain translocates through biological membranes. J. Biol.
Chem. 1994, 269 (14), 10444ꢀ50.
14. Frankel, A. D.; Pabo, C. O., Cellular uptake of the tat protein from human
immunodeficiency virus. Cell. 1988, 55 (6), 1189ꢀ93.
15. Chen, L.; Fang, S.; Xiao, X.; Zheng, B.; Zhao, M., SingleꢀStranded DNA Assisted
Cell Penetrating PeptideꢀDNA Conjugation Strategy for Intracellular Imaging of
Nucleases. Anal. Chem. 2016, 88 (23), 11306ꢀ11309.
16. Wang, D.; Luo, W.; Wen, G.; Yang, L.; Hong, S.; Zhang, S.; Diao, J.; Wang, J.;
Wei, H.; Li, Y.; Wang, Y., Synergistic effects of negativeꢀcharged nanoparticles
assisted by ultrasound on the reversal multidrug resistance phenotype in breast cancer
cells. Ultrason Sonochem. 2017, 34, 448ꢀ457.
17. Said Hassane, F.; Saleh, A. F.; Abes, R.; Gait, M. J.; Lebleu, B., Cell penetrating
peptides: overview and applications to the delivery of oligonucleotides. Cell. Mol.
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Page 21 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
Life Sci. 2010, 67 (5), 715ꢀ26.
18. Lin, W.; Xie, X.; Yang, Y.; Fu, X.; Liu, H.; Yang, Y.; Deng, J., Thermosensitive
magnetic liposomes with doxorubicin cellꢀpenetrating peptides conjugate for
enhanced and targeted cancer therapy. Drug Deliv. 2016, 23 (9), 3436ꢀ3443.
19. Olson, E. S.; Jiang, T.; Aguilera, T. A.; Nguyen, Q. T.; Ellies, L. G.; Scadeng, M.;
Tsien, R. Y., Activatable cell penetrating peptides linked to nanoparticles as dual
probes for in vivo fluorescence and MR imaging of proteases. PNAS. 2010, 107 (9),
4311ꢀ6.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20. Aroui, S.; Brahim, S.; Waard, M. D.; Kenani, A., Cytotoxicity, intracellular
distribution and uptake of doxorubicin and doxorubicin coupled to cellꢀpenetrating
peptides in different cell lines: a comparative study. Biochem. Biophys. Res. Commun.
2010, 391 (1), 419ꢀ25.
21. He, H.; Sun, L.; Ye, J.; Liu, E.; Chen, S.; Liang, Q.; Shin, M. C.; Yang, V. C.,
Enzymeꢀtriggered, cell penetrating peptideꢀmediated delivery of antiꢀtumor agents. J.
Controlled Release. 2016, 240, 67ꢀ76.
22. Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.; Rothbard, J. B.,
Polyarginine enters cells more efficiently than other polycationic homopolymers. J.
Pept. Res. 2000, 56 (5), 318ꢀ325.
23. Candan, G.; Michiue, H.; Ishikawa, S.; Fujimura, A.; Hayashi, K.; Uneda, A.;
Mori, A.; Ohmori, I.; Nishiki, T.; Matsui, H.; Tomizawa, K., Combining polyꢀarginine
with the hydrophobic counterꢀanion 4ꢀ(1ꢀpyrenyl)ꢀbutyric acid for protein
transduction in transdermal delivery. Biomaterials. 2012, 33 (27), 6468ꢀ75.
24. Chen, H.; Chang, X.; Weng, T.; Zhao, X.; Gao, Z.; Yang, Y.; Xu, H.; Yang, X., A
study of microemulsion systems for transdermal delivery of triptolide. J Control
Release 2004, 98 (3), 427ꢀ36.
25. Hao, J.; Sun, Y.; Wang, Q.; Tong, X.; Zhang, H.; Zhang, Q., Effect and mechanism
of penetration enhancement of organic base and alcohol on glycyrrhetinic acid in vitro.
Int J Pharm 2010, 399 (1ꢀ2), 102ꢀ8.
26. Sun, Y.; Yang, Y.; Li, D.; Pu, T.; Xie, X.; Wang, Q., Effect of paeoniflorin on the
pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in rats. Chinese Pharm.
ACS Paragon Plus Environment

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Page 22 of 31
1
2
3
4
5
6
7
8
9
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
J. 2012, 47, 599–603.
27. Lelle, M.; Peneva, K., An amino acidꢀbased heterofunctional crossꢀlinking reagent.
Amino acids 2014, 46 (5), 1243ꢀ51.
28. Åmand, H. L.; Nordén, B.; Fant, K., Functionalization with Cꢀterminal cysteine
enhances transfection efficiency of cellꢀpenetrating peptides through dimer formation.
Biochem. Biophys. Res. Commun. 2012, 418 (3), 469ꢀ474.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29. Schoop, V. M.; Mirancea, N.; Fusenig, N. E., Epidermal organization and
differentiation of HaCaT keratinocytes in organotypic coculture with human dermal
fibroblasts. J. Invest. Dermatol. 1999, 112 (3), 343ꢀ53.
30. Liou, J. T.; Chen, Z. Y.; Ho, L. J.; Yang, S. P.; Chang, D. M.; Liang, C. C.; Lai, J.
H., Differential effects of triptolide and tetrandrine on activation of COXꢀ2,
NFꢀkappaB, and APꢀ1 and virus production in dengue virusꢀinfected human lung cells.
Eur. J. Pharmacol. 2008, 589 (1ꢀ3), 288ꢀ98.
31. Chuan, X.; Song, Q.; Lin, J.; Chen, X.; Zhang, H.; Dai, W.; He, B.; Wang, X.;
Zhang, Q., Novel freeꢀpaclitaxelꢀloaded redoxꢀresponsive nanoparticles based on a
disulfideꢀlinked poly(ethylene glycol)ꢀdrug conjugate for intracellular drug delivery:
synthesis, characterization, and antitumor activity in vitro and in vivo. Mol. Pharm.
2014, 11 (10), 3656ꢀ70.
32. Prakash, C.; Soni, M.; Kumar, V., Mitochondrial oxidative stress and dysfunction
in arsenic neurotoxicity: A review. J Appl Toxicol. 2016, 36 (2), 179ꢀ88.
33. Xi, C.; Peng, S.; Wu, Z.; Zhou, Q.; Zhou, J., Toxicity of triptolide and the
molecular mechanisms involved. Biomed. Pharmacother. 2017, 90, 531ꢀ541.
34. Xu, L.; Pan, J.; Chen, Q.; Yu, Q.; Chen, H.; Xu, H.; Qiu, Y.; Yang, X., In vivo
evaluation of the safety of triptolideꢀloaded hydrogelꢀthickened microemulsion. Food
Chem. Toxicol. 2008, 46 (12), 3792ꢀ9.
35. Frosch, P. J.; Schulzedirks, A.; Hoffmann, M.; Axthelm, I., Efficacy of skin barrier
creams (II). Ineffectiveness of a popular "skin protector" against various irritants in
the repetitive irritation test in the guinea pig. Contact Derm. 1993, 29 (2), 74ꢀ77.
36.Imai, T.; Ariyoshi, S.; Ohura, K.; Sawada, T.; Nakada, Y., Expression of
Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug
ACS Paragon Plus Environment

Page 23 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
in Rat Skin. J Pharm Sci. 2016, 105 (2), 714ꢀ721.
37. Bahar, F. G.; Ohura, K.; Ogihara, T.; Imai, T., Species difference of esterase
expression and hydrolase activity in plasma. J Pharm Sci. 2012, 101 (10), 3979ꢀ88.
38. Fu, J.; Sadgrove, M.; Marson, L.; Jay, M., Biotransformation Capacity of
Carboxylesterase in Skin and Keratinocytes for the PentaꢀEthyl Ester Prodrug of
DTPA. Drug Metab Dispos. 2016, 44 (8), 1313ꢀ1318.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
39. Jin, Q.; Feng, L.; Zhang, S. J.; Wang, D. D.; Wang, F. J.; Zhang, Y.; Cui, J. N.;
Guo, W. Z.; Ge, G. B.; Yang, L., RealꢀTime Tracking the Synthesis and Degradation
of Albumin in Complex Biological Systems with a nearꢀInfrared Fluorescent Probe.
Anal Chem. 2017, 89 (18), 9884ꢀ9891.
40. Ge, G. B.; Feng, L.; Jin, Q.; Wang, Y. R.; Liu, Z. M.; Zhu, X. Y.; Wang, P.; Hou, J.;
Cui, J. N.; Yang, L., A novel substrateꢀinspired fluorescent probe to monitor native
albumin in human plasma and living cells. Anal Chim Acta. 2017, 989, 71ꢀ79.
41. Pillow, T. H.; Sadowsky, J. D.; Zhang, D.; Yu, S. F.; Del Rosario, G.; Xu, K.; He,
J.; Bhakta, S.; Ohri, R.; Kozak, K. R.; Ha, E.; Junutula, J. R.; Flygare, J. A.,
Decoupling stability and release in disulfide bonds with antibodyꢀsmall molecule
conjugates. Chem Sci. 2017, 8 (1), 366ꢀ370.
42. TunaliꢀAkbay, T.; Alev, B.; Tunali, S.; Oktay, S.; EmekliꢀAlturfan, E.; Ipekci, H.;
Ozturk, L. K.; Yanardag, R.; Yarat, A., Protective role of edaravone against valproic
acid induced changes in skin. Indian J Exp Biol. 2017, 55 (5), 286ꢀ291
43. Inbaraj, J. J.; Chignell, C. F.; Cytotoxic action of juglone and plumbagin: a
mechanistic study using HaCaT keratinocytes. Chem Res Toxicol, 2004, 17(1): 55ꢀ62.
44. Semercioz, A.; Baltaci, A. K.; Mogulkoc, R.; Avunduk, M. C., Effect of Zinc and
Melatonin on Oxidative Stress and Serum InhibinꢀB Levels in a Rat Testicular
TorsionꢀDetorsion Model. Biochem genet. 2017, 55 (5ꢀ6), 395ꢀ409.
45. Prausnitz, M. R.; Langer, R., Transdermal drug delivery. Nat. Biotechnol. 2008, 26
(11), 1261ꢀ8.
46. Copolovici, D. M.; Langel, K.; Eriste, E.; Langel, Ü., Cellꢀpenetrating peptides:
design, synthesis, and applications. Acs Nano. 2014, 8 (3), 1972ꢀ94.
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Figure 1 Cell viability of HaCaT cell line at the presentence of different
concentrations of TP, TPꢀSꢀSꢀCꢀR7 for 24 hours (n=3) (*P<0.05 and **P<0.01 vs.
control).
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Figure 2 Flow cytometry analysis of DCFHꢀDA density on HaCaT cells. The cells
were treated with 100 nm TP, TPꢀSꢀSꢀCR7 or fresh medium for 12 h (n=3, Mean±
S.D.).
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Figure 3 The activity of intracellular SOD on HaCaT cells were measured after the
treatment of triptolide, TPꢀSꢀSꢀCR7 or fresh medium for 12h (n=3) (*P<0.05 and
**P<0.01 vs. control).
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Figure 4 Histopathological features of the belly skin of guinea pigs after treating with
100 ꢁg/ml triptolide, 722 ꢁg/ml TPꢀSꢀSꢀCR7 and equal volume of PBS for 7 days
successively. The signals were observed by confocal microscopy. Scale Bars = 500
ꢁm.
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Figure 5 The degradation curves and the degradation product spectrogram of
TPꢀSꢀSꢀCR7 in different concentrations of GSH solutions for 24 h. (A represents the
degradation curve in 200 ꢁg/ml GSH solution; B represents the degradation curve in
2ꢁg/ml GSH solution; C represents the HPLC chromato of the reaction mixture aftre
24 h.HPLC mobile phase: 10%ꢀ100% solvent A for 25 min, A: 0.3% acetic acid in
acetonitrile; B: 0.3% acetic in water.)
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Figure 6 The stability of TPꢀSꢀSꢀCR7 in rat plasma. A represents 1 mg/ml
TPꢀSꢀSꢀCR7 in PBS, B represents 1 mg/ml TPꢀSꢀSꢀCR7 in 1 ml rat plasma. C also
represtets 1 mg/ml TPꢀSꢀSꢀCR7 in 1 ml rat plasma. D represents TP dissolved in
methanol (A and B are detected using the following method, 5%ꢀ100% solvent A for
15 min; then 100% A for 5min. A: 0.3% acetic acid in acetonitrile; B: 0.5% acetic in
water. C and D are detected using the following method, 40ꢀ80% B for 6 min, then
80ꢀ100% B for 1min, then 100% B for 1min. A: water, B: acetonitrile)
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Figure 7 Mean Plasma Concentration ꢀ Time profiles of TP in SD rats after
transdermal administration of TP and TPꢀSꢀSꢀCR7 at a dose of 3mg/kg and 21 mg/kg,
respectively (n=3, Mean±S.D.)
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Figure 8 Mean Concentration in Skin Homogenate of Mice ꢀ Time profiles of TP after
transdermal administration of TP and TPꢀSꢀSꢀCR7 at a dose of 4.3 mg/kg and 30
mg/kg respectively (n=3, Mean±S.D.)
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Scheme 1 Synthesis procedure of pyridyl disulfide cysteamine hydrochloride
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Scheme 2 Synthesis procedure of TPS
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Scheme 3 Synthesis procedure of TPSP
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Scheme 4 Synthesis procedure of T PꢀSꢀSꢀCR7 (
represents poly argninie).
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