Supramolecular prodrug micelles based on the complementary multiple hydrogen bonds as drug delivery platform for thrombosis therapy

Article abstract of DOI:10.1039/c6ra11110f

To improve the effect of thrombosis therapy, an amphiphilic supramolecular prodrug consisting of diosgenin derivative (theophylline-diosgenin) and uracil-terminated poly(ethylene glycol) (PEG-U) was designed and synthesized successfully. The prodrug could self-assemble into micelles which was not only enhanced the drug solubility but also prolonged systemic circulation. Bleeding time assay confirmed that the micelles have a better antithrombotic activity compared to diosgenin in vivo, and platelet aggregation assay in vitro got the same results. The low cytotoxicity of supramolecular copolymer micelles was confirmed by MTT assay against LO2/HK-2 cells and acute toxicity studies in mice. Based on these results, the supramolecular prodrug micelles are very promising candidates for thrombosis therapy.

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ARTICLE
Supramolecular Prodrug micelles Based on the Complementary
Multiple Hydrogen Bonds as Drug Delivery Platform for
Thrombosis Therapy
Received 00th January 20xx,
Accepted 00th January 20xx
Wen’en Li,a,b Haibo Wang,c Zeliang Wei,b Juewei Ning,b Limei Ma ,a,b Huajie Zheng ,b Hai Niu*b,d and Wen
Huang *a, b
DOI: 10.1039/x0xx00000x
www.rsc.org/
To improve the effect of thrombosis therapy, an amphiphilic supramolecular prodrug consisting of diosgenin derivative
(theophyllineꢀdiosgenin) and uracilꢀterminated poly (ethylene glycol) (PEGꢀU) was designed and synthesized successfully.
The prodrug could selfꢀassemble into micelles which was not only enhanced the drug solubility but also prolonged systemic
circulation. Bleeding time assay confirmed that the micells have a better antithrombotic activity compared to diosgenin in
vivo, and platelet aggregation assay in vitro got the same results. The low cytotoxicity of supramolecular copolymer
micelles was confirmed by MTT assay against LO2 / HKꢀ2 cells and acute toxicity studies in mice. Based on these results,
the supramolecular prodrug micelles are very promising candidates for thrombosis therapy.
a new commercial product for thrombosis therapy in China.16ꢀ18 Our
group have been engaged in investigating the extraction and
applications of steroidal saponins from D. zingiberensis for many
Introduction
Thrombosis is one of the most leading causes of morbidity and
mortality throughout the world.1ꢀ6 To prevent and control the
thrombogenic state, some antiꢀthrombotic drugs, such as
anticoagulants, antiꢀplatelet drugs and thrombolytic drugs, have been
used in clinical application. However, most of the drugs exert many
side effects, such as bleeding risk,7 narrow therapeutic window and
incidence of resistance. Therefore, development of new drug
candidates with minimal side effects and high potential for treatment
of ischemic symptoms is urgently needed.8,9
In the process of searching for new antithrombotic drugs,
traditional Chinese medicine have aroused people’s attention.10,11
Dioscorea zingiberensis C.H. Wright (DZW), one of important
Chinese medicinal materials, has been extensively used as a
treatment of various diseases.12,13 More importantly, total steroidal
saponins and diosgenin of Dioscorea zingiberensis C.H. Wright
have been found to exert antithrombotic effect.14,15 Drugs with
steroidal saponins being the main bioactive ingredients, such as
Dioscorea Panthaica Prain et Burkill and Xueꢀsaiꢀtong, have become
years19,20 and have found that diosgenin showed
a better
antithrombotic activity through structural modification.21 Inspired by
these researches, it can be inferred that conjugating diosgenin with
other potential antithrombotic drugs to obtain diosgenin derivatives
will be very promising for thrombosis therapy. However, due to the
strong hydrophobicity of diosgenin, the diosgenin derivatives also
suffer from limitations of poor physicochemical properties.
Therefore, to fully exploit the potential of diosgenin derivatives, it is
essential to address these issues.
To date, nanocarriers have attracted considerable attention for
their application in drug delivery systems. Especially, polymeric
micelles have emerged as one of the most promising drug delivery
systems22ꢀ27 because of their advantages in increasing drug solubility,
prolonging circulation time28,29 and improving pharmacokinetic
properties.26ꢀ31 In the polymeric micelles systems, hydrophobic drug
is incorporated in a polymeric matrix through chemical conjugation
or physical entrapment, while the hydrophilic shell maintains a
hydration barrier to provide
a stable interface between the
a.Laboratory of Ethnopharmacology, Regenerative Medicine Research Center,
West China Hospita, West China Medical School, Sichuan University, Chengdu
610041, China.
hydrophobic core and the external medium. Therefore, polymer
micelles have great potential to serve as an efficient drug delivery
system.
It is reported that xanthine drugs have potential antithrombotic
effect.30,31 Therefore, in the present work, we designed and
synthesized a novel antithrombotic prodrug of diosgenin and
theophylline which is one kind of xanthine drugs (Scheme 1A).
Furthermore, to enhance the solubility, bioavailability and
pharmacokinetics of the drug, a hydrophilic polymer based on uracil
and PEG was developed in this research. Theophylline and uracil
b.Institute for Nanobiomedical Technology and Membrane Biology, Sichuan
University, Chengdu 610041, China.
C Textile Institute, College of Light Industry, Textile and Food Engineering, Sichuan
University, Chengdu, 610065, China
d College of Mathematics, Sichuan University, Chengdu, Sichuan 610064, China
†Corresponding
author.
WenHuang,
huangwen@scu.edu.cn;
HaiNiu,
niuhai@scu.edu.cn;Tel: +86-028-85164073; Fax: +86-028-85164075.
Wen’en Li and Haibo Wang contributed equally to this work
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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can direct interaction by multiple hydrogen bonds and hydrophilic = 10.5, 5.1 Hz, 1H), 4.31 (dd, J = 13.1, 7.7 Hz, 1H), 3.49 (dd, J
PEGꢀU was employed to construct supramolecular amphiphilic = 10.6, 6.2 Hz, 1H), 3.43 (dd, J = 10.6, 6.2 Hz, 1H), 3.33 (td, J
copolymers (DꢀT:UꢀPEG). Benefiting from their amphiphilicity and = 8.3, 3.8 Hz, 1H), 2.36 – 2.27 (m, 2H), 2.03 (s, 3H), 1.03 (s,
noncovalent connection, DꢀT:UꢀPEG could selfꢀassemble into 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.81 (s,
micelles with hydrophilic PEG shells and hydrophobic DꢀT cores in 3H); 13C NMR (150 MHz, CDCl3) δ 170.54, 139.67, 122.38,
aqueous solution (Scheme 1B). The supramolecular prodrug micelles 90.35, 83.21, 73.89, 68.01, 65.09, 56.90, 50.00, 40.69, 39.40,
could increase the solubility of drug, prolong its systemic circulation 38.09, 37.92, 36.99, 36.70, 35.74, 32.22, 31.98, 31.56, 30.46,
in blood and improve pharmacokinetic properties, which is crucial 30.13, 27.74, 21.42, 20.64, 19.32, 18.92, 16.63, 16.44; HRꢀMS
for improving the drug efficacy in thrombosis therapy.
(m/z) Calcd for C29H46O4: 481.3294 [M+Na]+, found: 481.3283
[M+Na]+.
Synthesis of (25R) furostꢀ5ꢀenꢀ, 3βꢀacetoxy, 26ꢀtheophylline
(DꢀT)
Materials and methods
NꢀIodosuccinimide (NIS, 490 mg, 2.2 mmol) was dissolved in a
10 mL methylene dichloride at room temperature. Then, the
methylene dichloride solution of triphenylphosphine (500 mg,
2.2 mmol) was added dropwise into this solution. After 10
mintes, compound 3 (500 mg, 1.1 mmol) was added into above
mixed solution and stirred for an additional 24 h at room
temperature. The resulting mixture washed with water and
dried over anhydrous sodium sulphate.The organic layer was
removed by vacuum distillation with a rotary evaporator to get
a yellow oily substance, which was purified through column
chromatography over silica gel using petroleum etherꢀethyl
acetate as eluants. compound 4 was obtained with a yield of
95%. After that, compound 4 (1 g, 1.76 mmol), theophylline
(634 mg, 3.52 mmol) and potassium carbonate (486 mg, 3.52
mmol) were dissolved in a 20 mL N,NꢀDimethylformamide and
stirred for an additional 8 h at 80 oC. The resulting mixture was
filtered and filtrate was removed by evaporation to get a yellow
solid substance, which was purified through column
chromatography over silica gel using petroleum etherꢀethyl
acetate as eluants. The desired DꢀT was obtained at 50% ethyl
Materials
N,NꢀDimethylformamide (DMF), azabenzene (C5H5N), and
methylene dichloride (CH2Cl2). Diosgenin (99%, Adams),
acetic anhydride (99%, Adams), triethylamine (TEA, 99%,
Adams),
Sodium
cyanoborohydride
(99%,
Sigma),
triphenylphosphine (99%, Sigma), nꢀiodosuccinimide (99%,
Sigma), theophylline (99%, Sigma), uracil (99%, Sigma) and
acetic acid (99%, J&K) were all used as received.
Monomethoxy poly (ethylene glycol) (PEG) with Mn = 2000
g/mol was purchased from Fluka and dried by azeotropic
distillation in the presence of dry toluene. Different polystyrene
plates including 6ꢀ and 96ꢀwell ones were provided by Chinese
Sangon Biotech. Male Balb/C mice (four weeks old, 18–22 g,
Chengdu, China) and male Wistar rats (eight weeks old, 200–
250 g, Chengdu, China) were used in this study.
Synthesis of 3βꢀHydroxyꢀ(25R)ꢀSpirostꢀ5ꢀenꢀ3βꢀacetate (2)
Diosgenin (8.3 g, 20 mmol) was taken in pyridine (100 mL) at
room temperature and added acetic anhydride (20 mL, 213
mmol) to it at ice bath, then warmed to 60 oC. After the
reaction, usual workꢀup was done as per reported method.32
Yield: 95%. 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.5 Hz,
1H), 4.78ꢀ4.49 (m, 1H), 4.41 (dd, J = 14.9, 7.5 Hz, 1H), 3.47
(dd, J = 10.0, 3.2 Hz, 1H), 3.37 (t, J = 10.9 Hz, 1H), 2.48ꢀ2.23
(m, 2H), 2.03 (s, 3H), 1.03 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H),
0.79 (d, J = 3.9 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 69.50,
138.67, 121.35, 108.25, 79.79, 72.87, 65.82, 61.07, 55.41,
48.92, 40.59, 39.24, 38.71, 37.07, 35.94, 35.71, 31.02, 30.82,
30.39, 29.28, 27.79, 26.72, 20.41, 19.79, 18.31, 16.12, 15.27,
13.51; ESI mass (MeOH): 457.3 [M+H]+, 479.3[M+Na]+, 495.4
[M+K]+.
1
acetateꢀpetroleum ether as white solid. Yield : 75%, H NMR
(400 MHz, CDCl3) δ 7.52 (1H, ꢀNCHNꢀ), 5.37 (1H, ꢀCCHCH2ꢀ
), 4.60 (1H, ꢀCH2CH(O)CH2ꢀ), 4.29 (1H, ꢀCHCH(CH2)Oꢀ),
4.19 (1H, ꢀCHCH(CH2)Oꢀ), 4.12ꢀ4.01 (2H, ꢀCHCH2Nꢀ), 3.60
(3H, ꢀCH3), 3.41 (3H, ꢀCH3), 3.27 (2H, ꢀCHCH2Cꢀ), 2.03 (3H,
CH3ꢀ), 1.04 (3H, CH3ꢀ), 0.97 (3H, CH3ꢀ), 0.91 (3H, CH3ꢀ), 0.79
(3H, CH3ꢀ); 13C NMR (101 MHz, CDCl3) δ170.54, 155.17,
151.68, 149.12, 148.87, 141.43, 139.69, 122.35, 107.10, 90.06,
83.24, 77.37, 77.26, 77.06, 76.74, 73.87, 64.95, 56.86, 53.16,
49.96, 40.68, 39.37, 38.08, 37.88, 36.98, 36.70, 34.51, 32.19,
31.97, 31.55, 31.01, 30.35, 29.78, 28.00, 27.74, 21.44, 20.63,
19.34, 18.79, 16.88, 16.45. HRꢀMS (m/z) Calcd for
C36H52N4O5: 621.4018 [M+H]+, 643.3836 [M+Na]+, found:
621.4017 [M+H]+, 481.3842 [M+Na]+.
Synthesis of (25R) furostꢀ5ꢀenꢀ,3βꢀacetoxy, 26ꢀol (3)
Compound 2 (2.28 g, 5 mmol ) was dissolved in a mixture
solution of CH3COOH/CH2Cl2 (30 mL:15 mL) at room
temperature, and sodium cyanoborohydride (942 mg, 15 mmol)
was added in portions over a period of 30 min. After 2h, when
the reaction was complete, 10 mL of iceꢀcool water poured the
reaction mixture, extracted with ethyl acetate(3×40 mL),
washed with 5% of sodium chloride solution and dried over
anhydrous sodium sulphate. The organic layer was removed by
vacuum distillation with a rotary evaporator to get a crude
mass, which was purified through column chromatography over
silica gel using petroleum etherꢀethyl acetate as eluants. The
desired alcohol 3 was obtained at 10–12% ethyl acetate–
petroleum ether as white crystalline solid. Yield : 85%, 1H
NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.9 Hz, 1H), 4.60 (tt, J
Preparation of PEGꢀU
PEG (6.0 g, 1.2 mmol) and NaOH (0.14 g, 3.36 mmol) in
anhydrous CH2Cl2 (50 mL) were stirred in an ice water bath.
After stirring for 40 min, a CH2Cl2 solution (20 mL) of pꢀ
toluenesulfonyl chloride (TsCl; 0.41 g, 2.2 mmol) was added
dropwise into the reaction mixture and reacted at 0 oC for 48 h.
After reaction, 10 mL of water was poured into the resulting
mixture and extracted with CH2Cl2 (3×20 mL). The combined
organic extracts were dried with anhydrous sodium sulfate
(Na2SO4), and filtered, then, the filtrate was evaporated. PEGꢀ
sulfanilic acid ester was obtained with a yield of 95%. After
2 | J. Name., 2012, 00, 1-3
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that, uracil (0.36 g, 3.2 mmol), PEGꢀsulfanilic acid ester (8.0 g, five days, rats were anesthetized with i.p. pentobarbital sodium
1.6 mmol), potassium carbonate (K2CO3, 0.44 g, 3.2 mmol) (40 mg/kg), and whole blood was collected by cardiac puncture
were dissolved in 100 mL of DMF and stirred at 90 oC for 24 h. and antiꢀcoagulated with 3.8% trisodium citrate (9:1, v/v).
After reaction, 100 mL of water was poured the reaction Plateletꢀrich plasma (PRP) was obtained by centrifugation at
mixture and extracted with CH2Cl2 (3×50 mL). After drying 100g for 10 min. Plateletꢀpoor plasma (PPP) was prepared by
with anhydrous sodium sulfate (Na2SO4), most of the solvent centrifugation at 1000 g for 10 min.
was removed by evaporation. The residue was precipitated into Platelet aggregation assay in vitro
o
cold ethanol and dried under vacuum to a constant weight. The platelet aggregation assay was performed at 37 C using a
1
Yield: 65%. H NMR (400 MHz, CDCl3, 298 K) δ ppm: 7.7 LBYꢀNJ4 aggregometer (Pulisheng, Beijing, China). PRP were
(1H, ꢀCONHCOꢀ), 7.3 (1H, ꢀNCHCHꢀ), 5.6 (1H, ꢀCHCHCOꢀ), preincubated with the indicated doses of diosgenin and the
3.9 (2H, ꢀCH2CH2Nꢀ), 3.5ꢀ3.7 (PEG, 2H per repeating unit,ꢀ supramolecular prodrug micelles, or control buffer for 40 min
o
OCH2CH2ꢀ, 2H per repeating unit, ꢀOꢀCH2CH2ꢀ), 3.3 (3H, CH3ꢀ at 37 C under static conditions. Aggregation was induced by
OCH2ꢀ). 13C NMR (100 MHz, CDCl3, 298 K) δ: 47.04 (ꢀ 20 uM ADP, and platelet aggregation in vitro was measured.
CH2CH2Nꢀ), 58.0 (CH3Oꢀ), 67.8 (ꢀCH2CH2Nꢀ), 69.7 (ꢀ
CH2CH2Oꢀ), 71.2 (CH3OCH2ꢀ), 100.2 (ꢀCHCHCOꢀ), 146.3 (ꢀ Cell culture
CHCHCOꢀ), 150.9 (ꢀNCONHꢀ), 162.4 (ꢀCHCONHꢀ). HRꢀMS The cultured human hepatocyte LO2 cell line and human
(m/z, Fig.S7): 2131.2563 [M+Na]+.
Formation of the supramolecular prodrug micelles
kidney HKꢀ2 cell line were provide by Laboratory of
Transplantation Immunity, SiChuan University and grown in
A typical procedure to obtain the supramolecular prodrug DMEM (HyClone, USA) supplemented with 10% FBS
micelles is as follows: at room temperature, both DꢀT (3.1 mg, (HyClone, USA), 100 units/mL penicillins and 100 ꢁg/mL
0.005 mol) and PEGꢀU (10 mg, 0.005 mol) were dissolved in streptomycin as previously described. Cells were incubated in a
THF (1 mL) . After that, the polymer solution was put dropwise humidified atmosphere of 5% CO2 at 37 °C.
into 10 mL of deionized water and stirred slightly for 40 min.
LO2 cells and HKꢀ2 cells were cultured in complete medium
Then the THF was removed by vacuum distillation. The with 10% FBS to 80% cell confluence and subjected to assays
micelles solution was dialyzed against deionized water for 48 h after overnight serum depletion. the supramolecular prodrug
(MWCO =2000), during which the water was renewed every 4 micelles and diosgenin dissolved in DMSO were added to the
h.
medium. The final concentration of DMSO did not exceed
Dynamic light scattering (DLS) and Transmission electron 0.1%, which did not affect cell viability.
microscopy (TEM)
Cytotoxicity measurements of the supramolecular prodrug
Dynamic light scattering (DLS) measurements were performed micelles and diosgenin in vitro
to determine the micellar size and distribution using a Malvern LO2 cells and HKꢀ2 cells were seeded into 96ꢀwell plates
Zetasizer Nano Series equipped with DTS software and (Corning, USA) at a density of 3ꢀ5×103 cells/well and
operating a 4 mW He
–Ne laser at 633 nm. All samples were incubated overnight, then exposed to various concentrations of
tested at different temperature(from 25 oC to 90 oC) and a micelles and diosgenin dissolved in DMSO and diluted with
scattering angle of 90o. TEM tests were obtained using a JEOL DMEM medium for 24 h. The cytotoxicity of samples in 0.1%
JEMꢀ100CXꢀII instrument at a voltage of 200 kV. Samples DMSO (final DMSO concentration in medium) was tested
were prepared by dropꢀcasting nanoꢀaggregate solutions onto using MTT assay and images were taken by an optical
carbonꢀcoated copper grids and then freezedrying under microscope (Olympus BX51, Japan) after 24 h of incubation. In
vacuum before the measurements.
Bleeding time assay
brief, cells were washed once with PBS carefully, incubated
with 0.2 mL of serumꢀfree DMEM medium containing 0.05%
Male Balb/c mice were randomly divided into three groups of MTT for 4 h. After that, the culture medium was removed and
ten each. Before the bleeding time was assessed, the mice were 0.15 mL of DMSO was added to solubilise the formed
given diosgenin and control buffer orally in a volume of 200 formazan. The absorbance of each well was measured at 490
uL, or other mice were given the supramolecular prodrug nm with a microplate reader (Molecular Devices, USA). We
micelles with intraperitoneal in a volume of 200 uL twice a day compared the absorbance of the treated cells with the control
for five days. The bleeding time was measured by the method cells, which were considered as the 100% viability value.
of Nobukata33 with slight modification. In brief, an incision was Acute toxicity studies in mice
made at the tip of the tail of anesthetized mouse with a scalpel,
The toxicity of diosgenin and the supramolecular prodrug
and the tail was immersed in normal saline (37 oC) micelles to healthy male Kunming mice was conducted by oral
immediately. The time from the incision to the cessation of administration of diosgenin (200 and 400 mg/kg) and the
bleeding was recorded. A bleeding time of 900 s was used as supramolecular prodrug micelles with intraperitoneal injection
the cutꢀoff time for the purpose of statistical analysis. The (50 and 100 mg/kg). Saline was injected as control. After one
measurement of bleeding time was performed in a blinded week, major organs (heart, liver, spleen, lung, kidney and brain)
fashion.
Blood preparation.
and blood samples from each mouse were harvested. Viscera
samples were fixed in formalin, processed routinely into
For in vitro platelet aggregation, Wistar rats (200ꢀ250 g) were paraffin, sectioned, stained with hematoxylin and eosin (H&E)
divided randomly into three groups of ten animals each. After (Aladdin Chemical Co., Ltd., Shanghai, China) and examined
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by an optical microscope (Olympus Corporation, Tokyo, 65%. (B) Schematic illustration of the supramolecular prodrug selfꢀassemble into
Japan). Serum was collected to measure alanine transaminase micelles.
(ALT), aspartate transaminase (AST).
Result and discussion
Synthesis and characterization of DꢀT and PEGꢀU
The synthetic route of DꢀT and PEGꢀU is outlined in Scheme 1A. Dꢀ
T was synthesized by substitution reaction with a molar feed ratio of
compound 4/theophylline at 1:2. The potassium carbonate catalytic
method was used to make compound 4 react with theophylline to
produce the DꢀT. The chemical structure of DꢀT was confirmed by
1H and 13C nuclear magnetic resonance spectroscopy (1H NMR, 13C
1
NMR). Typical H NMR and 13C NMR spectra of DꢀT is shown in
Fig.
1
1H NMR spectra of diosgenin derivative(DꢀT) and uracilꢀterminated
1
poly(ethylene glycol) (PEGꢀU): (A)DꢀT, (B) PEGꢀU (400 MHz, CDCl3).
Fig. 1A and S4. For the H NMR, the peak at 7.52 (1H, ꢀNCHNꢀ),
3.60 (3H, ꢀCH3) and 3.41 (3H, ꢀCH3) belong to theophylline, and
5.37 (1H, ꢀCCHCH2ꢀ), 4.60 (1H, ꢀCH2CH(O)CH2ꢀ), 4.29 (1H, ꢀ
CHCH(CH2)Oꢀ), 4.19 (1H, ꢀCHCH(CH2)Oꢀ) belong to diosgenin.
The structure of the product was confirmed by these detailed data of
the chemical shifts. The 13C NMR also further demonstrate the
successful conjugating diosgenin with theophylline. The LC and
HRMS techniques were used to characterize the purity and
molecular weight of DꢀT and the results are shown in S5 and S6. The
LC profile gives the retention time of DꢀT as 2.42 min, indicating the
high purity of DꢀT. The HRMS data show that the molecular weight
of DꢀT is (m/z, M+H+,) 621.4017, which is consistent with the
calculated value (m/z, M+H+, 621.4018).
Formation of micelles
The theophylline in DꢀT and uracil in PEGꢀU can direct interaction
by multiple hydrogen bonds to form supramolecular copolymer.
Since the PEGꢀU is a waterꢀsoluble compound, DꢀT is a waterꢀ
insoluble drug. Therefore, the supramolecular copolymer (DꢀT:Uꢀ
PEG) is amphiphilic. Benefiting from this amphiphilic nature, the
supramolecular copolymer could selfꢀassemble to form micelles in
aqueous solution. The supramolecular micelles prodrug were
prepared by dialysis method. After adding the THF solution of DꢀT
and PEGꢀU into deionized water, the THF was removed by dialysis
and a stable nanoparticle solution with a concentration of 1 mg/mL
was obtained. Further, to confirm that the supramolecular prodrug
can form nanoparticles, both DLS and TEM measurements were
used to study the size and morphology of the supramolecular
prodrug micelles. As shown in Fig. 2A, the supramolecular prodrug
micelles aqueous solution forms aggregates and the mean
hydrodynamic diameter of the supramolecular prodrug micelles
aggregates is about 187.2 nm with a unimodal size distribution.
TEM technique was used to further measure and visualize the size
and morphology of the aggregates. The TEM image in Fig. 2B
showed that the supramolecular prodrug micelles aggregateed into
approximate spherical micelles in water and the micelles sizes
determined by TEM were in accordance with the data from DLS in
Fig. 2A. These results demonstrated that the supramolecular prodrug
can selfꢀassemble into stable and wellꢀdefined nanoparticles.
PEGꢀU was synthesized by the reaction of PEGꢀsulfanilic acid
ester with excess uracil. The synthetic route is shown in Scheme 1B.
The chemical structure identification data of PEGꢀU was provided
1
by H NMR and 13C NMR spectroscopy. As shown in Fig. 1B, for
1
the H NMR, The peaks at 3.9 and 7.7 ppm attribute to the protons
of CH2 (ꢀCH2CH2Nꢀ) connected with uracil and NH (ꢀCONHCOꢀ)
on the uracil, which demonstrates the generation of PEGꢀU. The 13
C
NMR (Fig. S8) also further demonstrate the successful grafting of
uracil on PEG.
Fig. 2 (A) DLS results for prodrug micelles; (B) TEM photograph of prodrug
micelles.
Scheme 1 (A) Schematic route of diosgenin derivative DꢀT and PEGꢀU. (i) Ac2O,
Pyridine, 60 oC, 2 h, 95%; (ii) AcOH, NaCNBH4, 35 oC, 2h, 85%; (iii) NIS, Ph3P,
CH2Cl2, 25 oC, 24 h, 95%; (iv) Theophylline, K2CO3, DMF, 80 oC, 8 h, 75%. (v)
NaOH, TSCl CH2Cl2, 0 oC, 48 h, 95%; (vi) K2CO3,Uracil, DMF, 90 oC, 24 h,
Acidic and thermal stability of the supramolecular prodrug
micelles
The hydrogen bonding interactions between hydrophobic DꢀT and
hydrophilic PEGꢀU made copolymer micelles unstable at acidic pH.
4 | J. Name., 2012, 00, 1-3
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To study the ability of the micelles in physiological condition, the
micelles were treated with pH 7.4 PBS buffer and pH 5.0 acetate
buffer (50 mM). The particle sizes were measured by DLS at
different time intervals. The result in Fig. 3A showed that the
supramolecular prodrug micelles aggregate rapidly at pH 5.0. The
mean diameter of the copolymer micelles increases from 187.2 nm
to about 520.9 nm in 4 h, while it doesn’t change in PBS buffer
(pH= 7.4). The stability of prepared micelles under physiological pH
condition (pH 7.4) provides the possibility for the introduction to the
body with the potential of therapeutics delivery. The driving force
for the change of micelles size at low pH value is attributed to the
protonation of the nucleobase nitrogen atoms, which leads to the
shedding of hydrophilic PEG shell from the micelles and the
aggregation of hydrophobic DꢀT core.34
The thermal stability of supramolecular copolymers was
investigated at different temperatures. Fig. 3B showed that the effect
of temperature on the supramolecular prodrug micelles was very
slight at the temperature below 60 oC and the mean diameter is
around 187.2 nm. When the temperature was up to 90 oC, the size of
micelles was increased to 217.9 nm because of the dissociation of
complementary hydrogen bonds at high temperature. The stability of
prepared micelles at physiological temperature provided the
possibility for the introduction to the body with the potential of
therapeutics delivery.
Fig. 4 Effect of diosgenin and prodrug micelles on bleeding time. The tail bleeding
time in mice given diosgenin and prodrug micelles, or saline was measured as
described in experiment section.
Platelet aggregation assay in vitro
To investigate whether the supramolecular prodrug micelles have
better antithrombotic effect than diosgenin in vitro. We examined the
prodrug micelles and diosgenin directly to inhibit platelet
aggregation in vitro. As shown in Fig. 5, the results indicated that the
prodrug micelles exhibited more outstanding efficiency in inhibiting
rat platelet aggregation than diosgenin in vitro. At same time, the
results also indicated that the micelles inhibited rat platelet
aggregation in
a doseꢀdependent manner. As known to all,
hemostasis has three major steps: (1) vasoconstriction, (2) formation
of a platelet plug by platelet aggretation and (3) blood coagulation.
Therefore, the measurement of bleeding time and platelet
aggregation can comprehensively reflect the antiꢀthrombotic
activities of drugs. In other words, bleeding time and platelet
aggregation will change if antiꢀplatelet aggregation drugs are taken
up by platelet. As shown in Fig. 4 and in Fig. 5, the results indicated
that the prodrug micelles have better antithrombotic effect than
diosgenin in vivo and in vitro. Videlicet, the results indicated that the
prodrug micelles can be better taken up than diosgenin by platelet.
At the same time, because the prodrug micelles can release the
prodrug in an acidic environment, the prodrug micelles can release
the prodrug in the platelet and exert antithrombotic effect in vivo and
Fig. 3 (A) Size change of prodrug micelles at different pH over time at 25 oC
monitored by DLS. (B) The size of prodrug micelles at different temperatures
determined by DLS. Error bars represent the standard deviation (n = 3).
Bleeding time in mice
To investigate the effect of diosgenin and the supramolecular
prodrug micelles on normal thrombosis, we measured bleeding time
one and a halfꢀhour after the final oral administration of diosgenin
(100 mg/kg) and the prodrug micelles with intraperitoneal injection
(50 mg/kg). As shown in Fig 4, both diosgenin and prodrug micelles
showed antithrombotic effects compared to controls, and prodrug
micelles exhibited more outstanding efficiency in prolonging
bleeding time than controls and diosgenin. The mean bleeding time
for the prodrug micelles and diosgenin treatment was 629.4 s and
605.0 s. It can be drawn from the above data that the prodrug
micelles have much better physicochemical properties compared to
diosgenin, and can improve the diosgenin’s drug efficacy in
thrombosis therapy.
in vitro
.
Fig. 5 (A) Effect of diosgenin and (B) effect of prodrug micelles on platelet
aggregation in rats.
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Cytotoxicity of the supramolecular prodrug micelles in vitro
In vitro cell culture systems are a useful tool to rapidly assess the
potential safety or toxicity of chemical constituents. Liver and
kidney, representing the major metabolism, bioactivation and
elimination organ respectively, are the first choice in assessment of
the safety/toxicity of chemical constituents. Here, we investigated
the effect of the prodrug micelles and diosgenin on LO2 cells and
HKꢀ2 cells viability by using MTT assay. As shown in Fig. 6, the
varying concentrations from 2.5 to 50 ꢁM/L of the prodrug micelles
did not influence LO2 and HKꢀ2 cell survival after 24 h exposure.
The 2.5 to 50 ꢁM/L of the prodrug micelles stimulated the cell
proliferation without a significant morphological change (Fig. S9).
However, the cytotoxicity of diosgenin to LO2 and HKꢀ2 cell is
higher than the prodrug micelles at varying concentrations from 2.5
to 50 ꢁM/L. These results suggest thatthe prodrug micelles have low
cytotoxicity and are safe for LO2 and HKꢀ2 cell using concentrations
in the experiment.
Fig. 7 Hematoxylinꢀeosin staining after one week of prodrug micelles given
intraperitoneal injecting and diosgenin given oral administration at different dose
in mice.
Group
AST (U/L)
ALT (U/L)
Control
104.56 ± 0.73
28.37 ± 0.87
200 mg/Kg of
Diosgenin
117.23 ± 6.17
115.60 ± 3.72
109.80 ± 2.34
108.30 ± 1.65
38.53 ± 2.38
30.13 ± 0.61
31.27 ± 0.82
29.63 ± 0.87
400 mg/Kg of
Diosgenin
50 mg/Kg of
Prodrug micelles
100 mg/Kg of
Prodrug micelles
.Table 1 Effects of the diosgenin and prodrug micelles on AST and
ALT levels in the serum of mice
Fig. 6 In vitro toxicity of diosgenin and prodrug micelles to LO2 and HKꢀ2 cells
after 24 h incubation at different concentrations.
Conclusions
Acute toxicity of the supramolecular prodrug micelles studies in
mice
In this study, a novel supramolecular prodrug micelles based on the
complementary multiple hydrogen bonds of nucleobases was
prepared and used for thrombosis therapy. Moreover, the
supramolecular prodrug micelles could selfꢀassemble into micelles,
in which diosgenin derivative was located in the hydrophobic part
and MPEG was the hydrophilic corona. The drug delivery system
increased the solubility and reduced side effects, leading to a better
efficacy of thrombus than the diosgenin. Overall, we believe that the
amphiphilic supramolecular drug micelles may open a new strategy
In the acute toxicity study, no cell death was found in any of the
animals injected through intraperitoneal with the prodrug micelles
(50 and 100 mg/kg body weight) and conducted by oral
administration of diosgenin (200 and 400 mg/kg). As shown in Fig.
7, the histological studies revealed that there was no significant
histological alterations in all major organs (heart, liver, spleen, lung,
kidney and brain) of mice treated with the prodrug micelles and
diosgenin in comparison with control group. These results verified
that no observable toxicity in tissue level of mice was induced by the to improve solubility and bioavailability of drug and are very
prodrug micelles, suggesting good biocompatibility of the prodrug
micelles. In addition, Table 1 shows that no significant difference
was observed in the levels of ALT and AST in the serum of mice
treated with various doses of the prodrug micelles and diosgenin,
further confirming no hepatotoxicity of the prodrug micelles.
promising candidates for improvements in drug efficacy.
Acknowledgements
This study was partly supported by the China National ‘12.5’
Foundation (Grant No. 2011BAJ07B04) and National Natural
Science Foundation of China (Grant No. 20972105, 51503130).
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