Synthesis, Characterization and Evaluation of Triptolide-Cell

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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 Downloaded from http://pubs.acs.org on January 10, 2018

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Molecular Pharmaceutics

4

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

5

Tian Tian†, Yuming Song†, Ke Li†, Yuming Sun , Qing Wang†, §, *

1 2 3

‡

6 7

† School of Pharmaceutical Science and Technology, Dalian University of

8

Technology, No. 2 Linggong Road, Dalian 116024, China

9

§ State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2

10

Linggong Road, Dalian 116024, China

11

‡ Chemical Analysis and Research Center, Dalian University of Technology, No. 2

12

Linggong Road, Dalian 116024, China

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 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

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ABSTRACT

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Triptolide (TP) has been used as one of the most common systemic treatments for

25

various diseases since 1960s. However, TP displays diverse side effects on various

26

organs which limits its clinical application. To overcome this issue, numerous C-14

27

hydroxyl group derivatives of TP have been synthesized. In this research, the C-14

28

hydroxyl group of TP is modified by a cell penetrating peptide poly arginine (R7). The

29

derivative TP-disulfide-CR7 (TP-S-S-CR7) containing a disulfide linkage between TP

30

and R7 possesses less toxicity at various concentrations on immortal human

31

keratinocyte

32

5-diphenyltetrazolium bromide (MTT) assay compared with free TP. Treating HaCaT

33

cells with TP (100 nM) could increase intracellular ROS (reactive oxygen species)

34

and decrease the activity of SOD (Superoxide Dismutase). Meanwhile, treating

35

HaCaT cells with equimolar concentration of TP-S-S-CR7 did not cause both of the

36

above TP-induced alterations. In addition, TP-S-S-CR7 didn’t show significant dermal

37

toxicity on guinea pigs and could efficiently overcome the barrier of corneum then

38

reached epidermis and dermis within 2 h of transdermal administration. In addition,

39

there was a relatively lower concentration of TP in blood indicates a less toxicity on

40

organs. Such results suggest that topical therapy using poly arginine is possible by the

41

transdermal delivery of TP.

42

Key words: Triptolide, Cell penetrating peptide, Toxicity reduction, Drug

43

modification

line

(HaCaT)

cell

line

by

3-(4,

44 45 46 47 48 49


5-dimethylthiazol-2-yl)-2,

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50 51

INTRODUCTION

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Triptolide (TP), an active compound isolated from Tripterygium wilfordii Hook F

53

(TWHF), commonly called lei gong teng or thunder god vine, which displays multiple

54

bioactivities

55

anti-inflammatory, anti-cancer activities1,2. Currently, TP has been used for the

56

treatment of rheumatoid arthritis, psoriasis and leukemia by oral or intravenous route.

57

However, due to its poor water solubility and severe side effects, there are some

58

roadblocks to be applied systemically in clinic. The tissues and organs being inflicted

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mainly include reproductive system3, liver4, kidney5 as well as heart6. Over a long

60

time, the derivatives aimed at the C-14 hydroxyl group of TP were designed and

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synthesized to overcome this issue. For example, TRC4, a TP derivative modified

62

with amine ester is water soluble, which remains potent anticancer activity without

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affecting the growth of normal cells7. Yutaka Aoyagi et al also synthesized a series of

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C-14-hydroxyl group derivatives, which are more effective than free TP against A549

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and HT29 cell lines8. Furthermore, TP-LZM, a C14-hydroxyl group derivative, can

66

solute in water, more importantly, this derivative causes less toxicity in Normal Rat

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Kidney cell line (NRK-52E)9. Moreover, transdermal delivery system can bypass

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hepatic first pass metabolism and reduce the incidence or severity of gastrointestinal

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reactions10. Therefore, transdermal delivery system could also reduce the toxicity of

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TP. Owing to these reasons, there is no doubt that developing the transdermal delivery

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of TP is imperative. Gui Chen et al. used microneedles to break the barrier of skin and

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successfully delivered TP through the skin of rat11. However, microneedles could

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cause micrometer-scale openings on corneum thus causing skin irritation. Based on

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these results, developing a biological enhancement technology for TP is necessary.

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Cell-penetrating peptides (CPPs) such as Antp, TAT, PEP-1 and polyarginine12, is a

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new kind of drug carrier, could enhance the transdermal drug delivery without skin

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irritation. CPPs are first discovered in 198813,

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therapeutic molecules (nucleic acids, drugs, imaging agents) into cells and tissues in a

such

as

immunosuppressive,

anti-fertility,

14

anti-cystogenesis,

and have successfully delivered



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nontoxic manner. Recently, a number of interdisciplinary studies proved that CPPs

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have successfully carried various cargos such as nucleic acids15, polymers16,

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oligonucleotides17, liposomes18, nanoparticles19, and low molecular drugs20 across

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cyto-membrane or other bio-membrane21. Moreover, it is reported that a chain of

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arginines compose one of the most widely used CPPs and more effective than others22.

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Poly arginine display many structural and functional advantages over other CPPs may

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because the peptides are positively charged and they interact with critical membrane

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components needed for penetration. Poly arginine (11R) covalently attaching with

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hydroquinone successfully cured UV-induced pigmentation by transdermal delivery.

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Such results demonstrates that topical therapy using the conjugation with poly

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arginine is thought to be possible for the delivery of small molecular drugs by

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transdermal route23.

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The aim of this work is to design and synthesize a TP-poly arginine derivative

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(TP-S-S-CR7)

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hydrochloride as linkers. TP-S-S-CR7 showed lower cell toxicity on immortal human

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keratinocyte (HaCaT) cell line at different concentrations than TP, meanwhile,

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TP-S-S-CR7 could also cause less dermal toxicity on guinea pigs compare to free TP.

using

succinic

anhydride

and

pyridyl

disulfide

cysteamine

96 97

EXPERIMENTAL SECTION

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2.1 Animals

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Male Sprague-Dawley (SD) rats and guinea pigs and Kunming (KM) mice used in all

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experiments were supplied by Dalian Medical University (Dalian, China). The

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animals were kept in an animal room with regulated temperature of 20 ± 2 °C and

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relative humidity of 60 ± 10%. All of them were given free access to food and water.

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2.2 Synthesis of pyridyl disulfide cysteamine hydrochloride

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The synthesis procedure of pyridyl disulfide cysteamine hydrochloride is described as

105

follow. An amount of 10 ml methanol containing 1.04 g cysteamine hydrochloride

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was added to a solution of 2, 2’-dithiodipyridine (4 g) in 16 ml of methanol and 0.6 ml

107

acetic acid dropwisely. The yellow solution was stirred overnight at room temperature. ACS Paragon Plus Environment

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Then the solvent was evaporated off. The product was obtained by precipitation in

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500 ml diethyl ether. After filtration, the residue was dissolved in 15 ml of methanol

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and again precipitated in 500 ml of diethyl ether. The yellow solid was obtained after

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drying under reduced pressure. The structure of pyridyl disulfide cysteamine

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hydrochloride was characterized by 1H-NMR.

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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

115

anhydrous methylene chloride (CH2Cl2) followed by the addition of 1.5 ml CH2Cl2

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containing 4-dimethylaminopyridine (DMAP) 360 mg and trimethylamine 0.5 ml

117

dropwisely. The reaction stirred at room temperature for 24 hours. The reaction was

118

monitored by TLC with the solvent system of ethyl acetate-petroleum ether (1:1, v/v),

119

and the light purple spot was detected by soaking in 2% 3,5-dinitrobenzene in ethanol

120

and 10% potassium hydroxide in methanol9. After evaporating the solvent away, the

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crude product was washed by cold carbon tetrachloride (CCl4) three times. The CCl4

122

was removed and then dried under reduced pressure, yellow powder was obtained.

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The structure of TPS was characterized by 1H-NMR, 13C-NMR and HRMS.

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2.4 Synthesis of TPSP

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Amount of TPS 120 mg, pyridyl disulfide cysteamine hydrochloride 61 mg were

126

dissolved in 0.8 ml tetrahydrofuran containing 0.4 ml triethylamine. Then 54 mg

127

4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

128

was added to the solution. At last, 1.8 ml tetrahydrofuran was added. The reaction

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mixture was stirred at room temperature for 24 hours. After removing the solvent

130

away, the crude product was purified by preparative chromatography (LC-20AP,

131

Shimadzu, Japan) using C18 chromatographic column (ZORBAX SB-C18, 21.2×

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150 mm，Agilent, America, 10-100% B for 30min, A: 0.1% trifluoroacetic acid in

133

water, B: 0.1% trifluoroacetic acid in acetonitrile, flow rate = 7ml/min). After

134

removing the solvent away light yellow spongy solid was obtained. The structure of

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TPSP was characterized by 1H-NMR, 13C-NMR and HRMS. The purity of TPSP was

136

determined by HPLC-UV.


chloride

(DMTMM)


137

2.5 Synthesis of TP-S-S-CR7

138

Amounts of TPSP 60 mg, R7C (adding a cysteine on the N-terminal of R7)·8TFA 203

139

mg were dissolved in 3 ml DMF. Then the reaction solution was stirred for 48 hours at

140

room temperature. After completion of the reaction, the solvent was evaporated off,

141

the crude product was purified by preparative chromatography using SB-C18

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chromatographic column (10-100% solvent B; 30min, flow rate = 7ml/min; solvent A:

143

0.01% trifluoroacetic acid in water, solvent B: 0.01% trifluoroacetic acid in

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acetonitrile). After removing the solvent away, white powder was obtained. The

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structure of TP-S-S-CR7 was characterized by 1H-NMR, HRMS and HRMS2. The

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proposed fragmentation pathway of TP-S-S-CR7 was simulated using Mass Frontier

147

7.0. The purity of TP-S-S-CR7 was determined by HPLC-UV.

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2.6 Cell culture

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HaCaT cell line was kindly offered by Professor Wenli Li (School of Life Science and

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Biotechnology, Dalian University of Technology) was cultured in Dulbecco modified

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Eagle medium (DMEM) high glucose containing 10% fetal bovine serum (FBS) and 1%

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penicillin/streptomycin solution (100 IU/ml penicillin; 100 µg/ml streptomycin) in an

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incubator at 37 °C with 5% CO2, 95% air, and 90% relative humidity and offered

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fresh medium every other day.

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2.7 Cytotoxicity assay of TP and TP-S-S-CR7 on HaCaT cell line

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The effect of TP-S-S-CR7 and TP on HaCaT cell viability was measured at different

157

concentrations using MTT assay. HaCaT cells (7×103 cells/well) were seeded in

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96-well tissue culture plates. After 24 h, the medium was replaced with fresh medium

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containing various concentrations of TP, TP-S-S-CR7 (10, 1, 0.1, 0.01, 0.001 µM) or

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medium containing 0.5% dimethyl sulfoxide (DMSO). After incubation for 24 h, 110

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µL of 500 µg/ml MTT was added, and the plates were incubated for another 4 hours.

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Thereafter, the medium was removed and replaced with 150 µL of DMSO, the optical

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density (OD) was measured at 490 nm using a microplate reader. The cytotoxic

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effects of tested agents were expressed as the 50% inhibiting concentration (IC50), and

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the IC50 values were calculated using SPSS. ACS Paragon Plus Environment

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2.8 The stability of TP-S-S-CR7 in HaCaT cell homogenate, rat skin

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homogenate and rat plasma

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The HaCaT cells were seeded in the culture plate (diameter was 10 cm), after 80% -

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90% confluent, cells and cell culture medium were then separated. Cells were then

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washed with cold PBS 3 times and collected using cell scraper with 1 ml cold PBS in

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a 10 ml Eppendorf tube. Then 500 µl (1 mg/ml) TP-S-S-CR7 was added to 500 µl of

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the cell homogenate and mixed then incubated at 37 ℃. After 24 h, an aliquote of the

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sample (100 µl) was withdrawn from the solution and equal volume acetonitrile was

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added to stop the reaction. The samples were analyzed by HPLC-HRMS. (n=3)

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The abdominal skin was obtained soon after the rat was executed. Then the skin

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samples (200 mg) were cut into small pieces in a 10 ml centrifuge tube, 2.0 ml of PBS

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was added. The samples were homogenized at 18000 rpm for about 1 min, and the

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supernatant was obtained by centrifugation at 6×103 g for 5 min. Then 1 ml

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TP-S-S-CR7 (1 ml) was added to 1ml of the skin homogenate and mixed then

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incubated at 37℃. Then after 1h, an aliquote of the sample (100 µl) was withdrawn

181

from the solution and equal volume acetonitrile was added to stop the reaction. The

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amount of TP-S-S-CR7 was detected by HPLC-HRMS. (n=3)

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Blood was collected from adult male SD rats from orbital plexus to heparinized tubes,

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then the plasma was separated immediately by centrifugation (6×103 g, 10 minutes).

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Immediately after the collection of plasma, 1 ml TP-S-S-CR7 (1 mg/ml) was added to

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1ml plasma. Then an aliquote of the sample (100 µl) was withdrew from the solution

187

and equal volume acetonitrile was added to stop the reaction. The amount of

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TP-S-S-CR7 was detected by HPLC-HRMS. (n=3)

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2.9 The stability of TP-S-S-CR7 in glutathione (GSH) solution

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1 ml TP-S-S-CR7 (1 mg/ml) was mixed with 1 ml (200 µg/ml or 2 µg/ml) GSH in

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PBS and incubated at 37°C for 24 h. Aliquots were withdrawn from the solution at

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specified time points (0, 1, 3, 6, 12 and 24 h) and analyzed by HPLC-UV method, and

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the corresponding degradation products were identified by HPLC-HRMS method.

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(n=3) ACS Paragon Plus Environment


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2.10 Intracellular ROS determination

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DCFH-DA, a lipophilic dye was used to determine the intracellular accumulation of

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ROS. HaCaT cells were seeded on 6-well plate until about 80%-90% confluent.

198

Subsequently, the cells were treated with 100 nM TP, TP-S-S-CR7 or fresh medium

199

for 12 h respectively. After that, the medium was removed; the cells were washed

200

twice with PBS. Then, cells were incubated with DCFH-DA (20 µM) for about half an

201

hour at 37 ℃ in dark. Then washed with serum free fresh DMEM ３ times.

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Fluorescence was detected with flow cytometry. Excitation: λ 488 nm and Emission:

203

λ 520 nm. (n=3)

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2.11 SOD activity determination

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HaCaT cells were seeded in a 6-well plate until about 80% - 90% confluent. Then

206

cells were exposed to TP, TP-S-S-CR7 or fresh medium (as negative control) for 12h.

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After that, the medium was removed, and then the cells were washed twice with cold

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PBS (pH = 7.4, NaCl: 8 g/L, Na2HPO4: 2.86 g/L, KH2PO4: 0.2 g/L, KCl: 0.19 g/L).

209

Through tissue homogenized on ice, centrifuged at 1×104 g for 15min at 4℃, the

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total intracellular protein could be obtained. Total protein of each sample was

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determined by the BCA Protein Assay Kit. SOD activity was determined using SOD

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Detection Kit. (n=3)

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2.12 Skin irritation test in guinea pigs

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6 guinea pigs (200-300 g) were used for this skin irritation test. Three days prior to

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application, the hair on the dorsal area was removed using clipper and shaver, then the

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belly skin of each guinea pig was divided into three areas (3 × 3 cm2). Then 100

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µg/kg/day TP or 722 µg/kg/day TP-S-S-CR7 (equimolar concentration) and equal

218

volume of PBS were smeared on each depilated skin areas for seven days

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successively24. 24 h after the last administration, collect each area then fixed in 4%

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paraformaldehyde for at least 24 h. Tissue sections (5 µm) were prepared by mounted

221

on common slides, and then stained with hematoxylin and eosin.

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2.13 In vivo penetration experiment


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Six male SD rats (weighing 150-200 g) were randomly divided into two groups. 24

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hours prior to the experiment, the hair on the dorsal area was removed using clipper

225

and shaver. Then TP at a dose of 3 mg/kg or TP-S-S-CR7 at a dose of 21 mg/kg

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(equimolar concentration) were smeared onto the dorsed areas of rats (3×3 cm2).

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Blood samples were collected from the retro-orbital plexus into heparinized tubes at

228

designated time intervals (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 hours) post

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administration then plasma was separated immediately by centrifugation (6×103 g, 10

230

minutes). The plasma samples were stored at -40℃ until analysis. Plasma sample 50

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µL was mixed with 50 µL of internal standard solution (200 µg/ml triamcinolone

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acetonide in methanol), then 50 µL of methanol was added and vortexed for 1 minute.

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After centrifugation at 6×103 g for 5 min, the supernatant was transferred to a new

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tube. 20 µL of each sample was injected into the LC-MS system for quantitative

235

analysis.

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Separation was performed on an Agilent Zorbax SB-C18 column (4.6×150 mm, 5

237

µm, Agilent Technologies, USA). The mobile phase was composed of 40-80% B for 6

238

min, then 80-100% B for 1min, then 100% B for 1min (A: water, B: acetonitrile),

239

delivered at a flow rate of 0.5 ml/min.

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Thermo LTQ orbitrapXL mass spectrometer with high resolution was operated in ESI

241

positive mode. The parameters were as follows: vaperizer temperature 350 ℃; sheath

242

gas flow rate 30 Arb; auxiliary gas flow rate 10 Arb; ion spray voltage 3.5 kV;

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capillary temperature 350 ℃; capillary voltage 39 V. The resolution was set at 30000

244

with mass error tolerance of ±5 ppm. The retention times of TP and internal standard

245

(triamcinolone acetonide) were 7.01 and 9.69 minutes. Over the range of 1.25-200

246

ng/ml, TP concentrations were linearly proportional to the area ratio of TP/internal

247

standard. Calibration curves were constructed for TP is y=-0.000136353 +

248

0.00144784x (R=0.999).

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2.14 In vivo skin retention experiment

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Twenty-four KM male mice weighing 20-25 g were randomly divided into two groups.

251

Sodium sulfide 4 g, Amidulin 3.5 g, sucrose 2 g, Sodium tetraborate 0.5 g, Glycerin



252

2.5 g were carefully weighed and dissolved in 50 ml water and stirred to obtain a

253

homogeneous solution. The hairs on the back of the mice were lightly rubbed with a

254

pledget pre-wetted in the above solution. After all the hairs were removed, the

255

remaining solution on the back of the animal was washed away with warm water. The

256

treated animal was kept for at least 48 h to ensure that no visible defect was present25.

257

Then TP at a dose of 4.3 mg/kg or TP-S-S-CR7 at a dose of 30 mg/kg (equimolar

258

concentration) were smeared onto the dorsed areas of mice (2 cm2). After 2, 6, 12

259

hours of administration, the animals were executed and the administration areas of

260

skin were collected. (n=3)

261

The corneum of the skin samples were removed and then the skin samples (100 mg)

262

were cut into small pieces in a 10 ml centrifuge tube, 1.0 ml of methanol was added.

263

The samples were homogenized at 18000 rpm for about 1 min, and the supernatant

264

was obtained by centrifugation at 6×103 g for 5 min26. The supernatant was treated

265

using the protocol used for the plasma samples and analyzed by HPLC-MS method

266

which was the same as that for plasma samples.

267 268

RESULTS

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3.1 Synthesis and characterization of pyridyl disulfide cysteamine

270

hydrochloride

271

To make the amino acid cysteine at the N-terminal of poly arginine accessible for TP,

272

the corresponding sulfydryl-reactive functionalities had to be introduced. In this

273

research, we choose pyridyl disulfide cysteamine hydrochloride because it is widely

274

used and easy to be synthesized27. The synthesis procedure is described on Scheme 1.

275

The productivity is 74%. 1H NMR (400 MHz D2O): δ = 3.15-3.08 (2H, t), 3.40-3.33

276

(2H, t), 7.37-7.31 (1H, m), 7.77-7.72 (1H, d), 7.87-7.80 (1H, t-d), 8.49-8.44 (1H, m).

277

The spectrum of pyridyl disulfide cysteamine hydrochloride is shown in supporting

278

information.

279

3.2 Synthesis and characterization of TPS


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280

TP has an active hydroxyl group at C-14 position, which can be conjugated to poly

281

arginine via spacers. In this research, we choose succinic anhydride as the first spacer.

282

The schematic synthetic route is shown in Scheme 2. The productivity is 94%.

283

1

284

3.54-3.51 (1H, d), 3.47-3.42 (1H, d), 2.84-2.62 (5H, m), 2.34-2.25 (1H, d), 2.21-2.04

285

(2H, m), 1.95-1.82 (2H, m), 1.61-1.47 (1H, dd), 1.30-1.13 (1H, m), 1.05-1.01 (3H, s),

286

0.95-0.90 (3H, d), 0.85-0.79 (3H, d). HRMS m/z 461.1807 [M+H]+. The spectrums of

287

TPS are shown in supporting information.

288

3.3 Synthesis and characterization of TPSP

289

In order to modify TPS with 2-(methyldisulfanyl) pyridine residue, pyridyl disulfide

290

cysteamine hydrochloride is chosen as the second spacer. DMTMM is chosen as

291

catalyst, because it is insoluble in THF, thus easy to remove after the reaction. The

292

reaction procedure was described in scheme 3. The productivity is 50%. 1H-NMR

293

(400 MHz, CDCl3): 8.56-8.48 (1H, s), 7.67-7.58 (1H, s)7.58-7.50 (1H, d), 7.21-7.07

294

(2H, s), 5.11-5.04 (1H, s), 4.71-4.62 (2H, s), 3.88-3.78(1H, s), 3.62-3.43 (4H, t),

295

2.96-2.52 (8H, m), 2.39-2.23 (1H, m), 1.97-1.79 (2H, d), 1.56-1.50 (1H, m), 1.34-1.19

296

(1H, m), 1.06-1.00 (3H, s), 0.96-0.91 (3H, d), 0.84-0.79 (3H, d). HRMS m/z 629.1984

297

[M+H]+. The spectrums of TPSP are shown in supporting information.

298

3.4 Synthesis and characterization of TP-S-S-CR7

299

As previously studied, adding a cysteine to the C-terminal of CPPs could not affect

300

their activities28. So, in this study, we add a cysteine to the N-terminal of poly arginine

301

to offer a sulfydryl in order to react with TPSP. The reaction procedure is described on

302

scheme 4. The productivity is 60% and the purity is 98%. HRMS m/z 289.4912 (z=6),

303

347.1882 (z=5), 433.7333 (z=4), 577.9754 (z=3), 866.4596 (z=2). The structure of

304

TP-S-S-CR7 is shown in Scheme 5. The spectrums of HRMS2, the proposed

305

fragmentation pathways and 1H-NMR of TP-S-S-CR7 are shown in supporting

306

information.

307

3.5 Cytotoxicity of TP and TP-S-S-CR7 on HaCaT cell line

308

HaCaT cell line is frequently used as a paradigm for skin29, so in this study, we

H-NMR (400 MHz, CDCl3): 5.06-5.05 (1H, s), 4.72-4.61 (2H, s), 3.83-3.80 (1H, d),



309

choose HaCaT cell line as a cellular model to investigate the dermal toxicity of TP

310

and TP-S-S-CR7 in vitro using MTT assay. The cells were treated with

311

various concentrations of TP and TP-S-S-C-R7 for 24 h. A dose-dependent effect of

312

TP and TP-S-S-CR7 could be observed on HaCaT cell line (Figure 1). The IC50 value

313

of TP on HaCaT cell line for 24 h was 1.00 µM, while the IC50 value of TP-S-S-CR7

314

was 9.06 µM indicating that TP-S-S-CR7 can reduce dermal toxicity in vitro,

315

especially at 10, 1 and 0.1 µM cell viability can be significantly improved. Moreover,

316

poly-arginine has no cell toxicity (data not shown). Moreover, from the result of the

317

stability of TP-S-S-CR7 in HaCaT cell homogenate, TP-S-S-CR7 was found to

318

degrade into TP. But only part of TP-S-S-CR7 was degraded. This perhaps was

319

because the lower esterase concentration in HaCaT cell line and further led to the

320

result of lower cytotoxicity.

321

3.6 Induction of oxidative stress in HaCaT cells by TP and

322

TP-S-S-CR7

323

As previously reported, TP could induce intracellular ROS accumulation and then

324

lead to tissue damage. So in this research, we attempt to investigate whether

325

TP-S-S-CR7 could cause oxidative damage on HaCaT cells as TP does. The cells were

326

treated with 100 nM TP, TP-S-S-CR7 and fresh medium for 12 h. Then, intracellular

327

ROS level as well as SOD activity were measured. As shown in Figure 2, compared

328

with the control group, TP exposure increased the accumulation of intracellular ROS.

329

Meanwhile, the intracellular SOD activity decreased significantly. However,

330

TP-S-S-CR7 treated cells have approximated the same intracellular ROS level and

331

SOD activity as the control group (Figure 2 and 3). Such results indicated that

332

TP-S-S-CR7 didn’t show significant oxidative damage on HaCaT cell line.

333

3.7 Skin irritation test in guinea pigs

334

To further investigate whether TP-S-S-CR7 has less skin irritation, 0.1 mg/kg/day TP,

335

0.722 mg/kg/day TP-S-S-CR7 and equal volume of PBS（pH=7.4 containing 0.1%

336

DMSO）were smeared on the belly skin of guinea pigs for seven days consecutively.

337

After smearing for three days, red and swollen appeared on the area of TP, then after ACS Paragon Plus Environment

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338

six days of TP treatment, scab could be observed. Whereas, no obvious difference can

339

be observed between TP-S-S-CR7 group and control group (Figure 4). TP treatment

340

could induce severe damage around corneum and hair follicle. Never the less, there’s

341

no obvious histopathological changes in TP-S-S-CR7 treated group.

342

3.8 In vivo skin retention and penetration analysis

343

The in vivo skin retention and penetration experiments were performed to explore

344

whether TP-S-S-CR7 is suitable for topical therapy by transdermal route. TP 4.3

345

mg/kg or TP-S-S-CR7 at a dose of 30 mg/kg were transdermal administrated to KM

346

rats. As shown in Figure 8, TP could be detected at 2 h after administration in both TP

347

and TP-S-S-CR7 administration groups, the concentrations of TP in epidermis and

348

dermis in TP administration group and TP-S-S-CR7 administration group are as high

349

as 6352.22 ng/g and 3647.80 ng/g. Such result demonstrates that even through the

350

water solubility of TP has been significantly improved, owing to the presence of poly

351

arginine, the transdermal ability of TP has not been significantly affected. TP-S-S-CR7

352

also can be detected at 2 h, which could probably indicates TP could release in the

353

epidermis and dermis in a slow rate. So the effective concentration of TP may last

354

longer.

355

3mg/kg TP or 21 mg/kg TP-S-S-CR7 were transdermal administration to SD rats to

356

detect the concentration of TP in blood. As shown in Figure 8, TP could not be

357

detected in TP administrated group. In TP-S-S-CR7 administration group, a relatively

358

low concentration of TP could be detected. The highest concentration is 13.52 ng/ml,

359

which could cause less systematic toxicity on organs. Such results proves that

360

TP-S-S-CR7 has the potential for topical therapy by transdermal delivery of TP.

361 362

DISCUSSION

363

4.1 Design and synthesis of TP-S-S-CR7

364

Low molecular weight proteins, which like peptides afford good biodegradability in

365

vivo, have been employed as drug carriers for transdermal delivery of therapeutic

366

drugs. Since the guanidine of arginine is proposed to be crucial for achieving ACS Paragon Plus Environment


Page 14 of 31

367

penetration efficacy30. So in this study, we choose poly arginine to modify the

368

C-14-hydroxyl group of TP to investigate whether TP-CPP conjugate could be

369

designed and synthesized, at the same time with the ability to achieve transdermal

370

delivery, meanwhile, reduce the dermal toxicity of TP. Too much amino acids on the

371

side chains of poly arginine makes it difficult to conjugate TP and R7 directly. So it is

372

imperative to induce linkers. As there is a cysteine on the C-terminal of poly arginine,

373

the most direct linker would be 3-(pyridine-2-yldisulfancyl) propanoic acid31.

374

However, it changes into 1,2-di (pyridine-2-yl) disulfane during the reaction in

375

CH2Cl2 at room temperature at the presence of 1-(3-Dimethylaminopropyl)-

376

3-ethylcarbodiimide

377

3-(pyridine-2-yldisulfancyl) propanoic acid with benzotriazole, the productivity was

378

as low as 20%. So in this research, succinic anhydride and pyridyl disulfide

379

cysteamine hydrochloride are chosen as linkers to conjugate TP and poly arginine.

380

Because the derivative could be obtained by three steps from the natural product,

381

scale-up of this method for clinical use of the prodrug does not pose a problem.

382

4.2 The toxicity evaluation of TP and TP-S-S-CR7 on HaCaT cell line

383

Oxidative stress, which is caused by the over-accumulation of ROS, induces protein

384

damage and aggregation, leading to impaired cellular homeostasis32. For over a

385

decade, an increasing number of works have reported that oxidative stress is a

386

recognized mode of toxic effects of TP exposure which has been observed in vitro and

387

in vivo. TP exposure has been shown to increase the generation of anion superoxide

388

and inhibit the activity of such antioxidant enzymes as SOD and subsequently induced

389

oxidative stress in the liver, kidney and heart tissues33. So in this study, we investigate

390

whether TP-S-S-CR7 could cause less cytotoxicity on HaCaT cells. The IC50 of

391

TP-S-S-CR7 is about nine-fold higher than that of TP, which indicates that

392

TP-S-S-CR7 could decrease dermal caused by TP in vitro (Figure 1). Based on such

393

result, we further investigated whether TP-S-S-CR7 could cause less ROS

394

accumulation and SOD activity decrease on HaCaT cell line. The results indicated

395

that there’s obvious increase of intracellular ROS of HaCaT cells at the presence of

hydrochloride

(EDC)

and


DMAP.

Even

modified

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396

TP (Figure 2). Simultaneously, the activity of SOD significantly decreased.

397

TP-S-S-CR7 treated group did not display such obvious negative effect.(Figure 3).

398

Our results prove that the mechanism of dermal toxicity is in accordance with the

399

toxicity mechanism on other organs.

400

4.3 In vivo dermal toxicity evaluation of TP and TP-S-S-CR7

401

The toxicity of TP has been demonstrated in several experimental animal species. The

402

tissues and organs affected by toxicity of TP include gastrointestinal tract, liver,

403

kidney, heart, blood cells, bonemarrow, testes and ovaries. But the toxicity of skin is

404

rarely investigated34. Obvious toxicities of TP were found toward the kidney and

405

testicles of mice, and these have even resulted in the death of mice at intraperitoneal

406

doses of 0.025, 0.05 and 0.1 mg/kg. As previously reported, obvious hepatotoxicity

407

was also observed at an oral dose of 0.2 mg/kg in mice. It has also been described in

408

rats with oral dosages as low as 0.1 mg/kg, in dogs at intravenous dosages of 0.04

409

mg/kg, and in rabbits at an external concentration of 1.11 mmol/L34. Guinea pigs are

410

commonly used as animal models for skin irritation evaluation35, 34. So in this study,

411

we choose guinea pigs to conduct the comprehensive dermal toxicity study of TP and

412

TP-S-S-CR7 at transdermal delivery dosage as low as 0.1 mg/kg/day TP and 722

413

mg/kg/day TP-S-S-CR7 for 7 days. As shown in Figure 4, TP exposure could cause

414

significant dermal toxicity on corneum and hair follicle. However, no obvious toxicity

415

could be observed in TP-S-S-CR7 smeared group, which provide a promising result

416

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

419

The results of in vitro and in vivo dermal toxicity experiment prove that TP-S-S-CR7

420

could cause less dermal toxicity compare with free TP. In order to further discuss the

421

mechanism of the toxicity reduction, the metabolism activity of TP-S-S-CR7 in

422

HaCaT cell homogenate, rat skin homogenate and rat plasma was investigated.

423

In HaCaT cell homogenate, the degradation rate is not so fast, 24 h after TP-S-S-CR7

424

exposed to the cell homogenate, both TP and TP-S-S-CR7 could be detected. This may ACS Paragon Plus Environment


425

because the esterases in HaCaT cell is not active.

426

The existing formation of TP-S-S-CR7 in skin homogenate is very important for us to

427

understand the transportation form of TP-S-S-CR7 across skin. So in this study, the

428

degradation of TP-S-S-CR7 in rat skin homogenate was investigated in vitro. After 1h

429

exposure to rat skin homogenate, about 85% of TP-S-S-CR7 turned into TP, which

430

was identified by HPLC-HRMS with mass tolerance of ±5 ppm. This may because

431

the activity of esterase is relatively high. What’s more, there’s no other TP related

432

compounds were detected. As previously reported, human esterases are highly active

433

and able to hydrolyze substances extensively during permeation through in vitro

434

human skin, such as 3-alkyl esters of naltrexone and ester derivatives of fluroxypyr36.

435

In addition, rat skin also shows relatively high esterase activity, most of the esterases

436

are located in epidemis and dermis, and the major esterase is carboxylesterase (CES:

437

EC.3.1.1.1)36. Therefore, the degradation might took place after TP-S-S-CR7

438

penetrating across the corneum into epidemis and dermis.

439

As soon as TP-S-S-CR7 exposed to rat plasma, TP was released. And there’s no other

440

TP related compounds detected during the experiment. The result of in vivo

441

penetration experiment showed that TP existed in a relatively low concentration,

442

which means a relatively low toxicity. Previous research demonstrated that

443

p-Nitrophenylacetate, a carboxylic ester containing structure was rapidly hydrolyzed

444

by rat plasma, and the hydrolase activity was as high as 2540 ± 240 µM/min, which

445

indicated the esterase activity in rat plasma is relatively high37.

446

To further investigate whether the esterase is the major factor for the degradation of

447

TP-S-S-CR7, bis-para-nitrophenylphosphate (BNPP; 1 mM), a nonspecific esterase

448

inhibitor was added to skin homogenate and rat plasma, then incubated for 30 min at

449

37 ℃ before TP-S-S-CR7 was added38. After adding BNPP into skin homogenate, TP

450

could not be released from TP-S-S-CR7. However, TP still released from TP-S-S-CR7

451

immediately after TP-S-S-CR7 exposed to rat plasma even when BNPP was added.

452

There are probably some other components in plasma which could also degrade ester

453

bond, such as albumin39,40.

454

As previously reported, reduced GSH could break disulfide bonds41. So in this study, ACS Paragon Plus Environment

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455

the stability of TP-S-S-CR7 in GSH solution was investigated in vitro. The

456

degradation product was detected using HPLC-HRMS. The concentrations of GSH

457

used in this experiment are similar to the concentration of GSH in skin homogenate42

458

(200 µg/ml), HaCaT cell homogenate43 and rat plasma44 (2 µg/ml). The degradation

459

curve is shown in Figure 5 (A and B), and the degradation product (TPSP-D) is

460

formed by the cleavage of the disulfide bond (Figure 5C). The result of TP-S-S-CR7

461

stability studies indicated that TP-S-S-CR7 was degraded very quickly in rat plasma,

462

but it was stable in GSH solution within 1 h, with the GSH concentration level at 2

463

µg/ml, which was similar with that in rat plasma. This indicated that GSH is not the

464

main factor for the degradation of TP-S-S-CR7. On the other hand, about 60% of

465

TP-S-S-CR7 was degraded within 1 h when the GSH was presented at 200 µg/ml,

466

which was similar to the concentration level of that in skin homogenate. But the

467

disulfide bond was stable in skin homogenate (as described above). This indicated

468

that the degradation effect of GSH to TP-S-S-CR7 in skin homogenate was inhibited.

469

Therefore, though GSH in PBS solution can degrade TP-S-S-CR7 at the concentration

470

level of 200 µg/ml in vitro and the degradation product was identified by LC-HRMS

471

method as TPSP-D (Figure 5), it’s not the main factor for the degradation of

472

TP-S-S-CR7 in skin homogenate, HaCaT cell homogenate and rat plasma.

473

4.5 In vivo penetration profile

474

Transdermal drug delivery, which was first applied in 1979, has many advantages. For

475

example, compared with the oral route, transdermal drug delivery can avoid the

476

first-pass effect of the liver that can prematurely metabolize drugs. What’s more

477

transdermal drug delivery also has advantages over hypodermic injections, which are

478

painful, easily generate dangerous medical wastes and pose the risk of disease

479

transmission by needle re-use45. So it is imperative to develop a transdermal delivery

480

system for TP. However, there is some difficulties to overcome since TP could cause

481

severe damage to the corneum (Figure 4). Microinjection, electroporation, and

482

liposome and viralbased vectors have been used for transdermal delivery. However,

483

these methods have various drawbacks, such as low efficiency, severe toxicity,



484

penurious bioavailability, and poor specificity46. Using cell penetrating peptide poly

485

arginine to modify the C-14 hydroxyl group TP could not only avoid some of the

486

above drawbacks but also can achieve its topical therapy by transdermal route.

487

Both TP and TP-S-S-CR7 could overcome the barrier of corneum and accumulate in

488

epidermis and dermis. Even though TP could not be detected in TP administration

489

group (Figure 8), which means no toxicity on organs, but TP could cause severe

490

dermal toxicity limits its application for transdermal administration. Such results

491

suggest that topical therapy using poly arginine is possible by the transdermal delivery

492

of TP.

493 494

CONCLUSION

495

In this study, we designed and synthesized a water soluble TP-CPP derivative

496

TP-S-S-CR7 which has less dermal toxicity both in vitro and in vivo. Moreover,

497

TP-S-S-CR7 could cross the barrier of corneum and reach epidermis and dermis at 2 h

498

after transdermal administration which indicates TP-S-S-CR7 can realize the topical

499

therapy of TP in a transdermal way. The blood concentration of TP is as low as 13.52

500

ng/ml, which could cause less toxicity on organs such as liver and heart. In a word,

501

TP-S-S-CR7 makes topical therapy possible by the transdermal delivery of TP and

502

poses the potential to cure some disease such as rheumatoid arthritis.

503

NOTES

504

The authors declare no competing financial interest.

505

ACKNOWLEDGEMENT

506

The study has been financed by Liaoning province natural science funds (2014020015)

507

and project of outstanding talent support program in universities of Liaoning province

508

(LR2014002).

509

Associated Content

510

Supporting information

511

Materials; preparation procedures of the solutions of TP and TP-S-S-CR7 used in


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512

MTT assay, skin irritation test in vivo penetration and the stability of TP-S-S-CR7 in

513

GSH solution experiment; HPLC analysis methods of TPSP and TP-S-S-CR7; 1H

514

NMR, 13C NMR and HRMS spectrums of pyridyl disulfide cysteamine hydrochloride,

515

TPS, TPSP and TP-S-S-CR7; proposed fragmentation pathway of TP-S-S-CR7 are

516

listed in supporting information.

517

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518

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Figure 1 Cell viability of HaCaT cell line at the presentence of different

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concentrations of TP, TP-S-S-C-R7 for 24 hours (n=3) (*P

Synthesis, Characterization and Evaluation of Triptolide-Cell

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