Novel nano-materials, RGD-tetrapeptide-modified 17β-amino-11α- hydroxyandrost-1,4-diene-3-one: Synthesis, self-assembly based nano-images and in vivo anti-osteoporosis evaluation

Article abstract of DOI:10.1039/c2jm13983a

To find novel nano-materials with anti-osteoporosis activity and without side effects three novel anti-osteoporosis active amphiphiles, 17β-(RGD-AA-amido)-11α-hydroxyandrost-1,4-diene-3-one (5a: AA = Ser, 5b: AA = Val, 5c: AA = Phe), were constructed by coupling the bone resorption inhibiting active RGD-peptides and anti-osteoporosis active 17β-amino- 11α-hydroxyandrost-1,4-diene-3-one. In the solid state 5a, 5b and 5c exist as dispersed globes of 300 nm-14 μm in diameter, dispersed eggs of 110 nm-19 μm in diameter and beads of 238 nm-22 μm in diameter, respectively. In ultrapure water 1.1 μM of 5a, 5b and 5c form nano-globes of 33-400 nm, 16-278 nm and 54-187 nm in diameter, respectively. At an oral dose of 110 nmol kg ^-15a-c effectively inhibited mice from developing osteoporosis. In contrast to estradiol, 5a-c did not induce mice to develop endometrial hyperplasia and thrombus.

Full text of DOI:10.1039/c2jm13983a

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PAPER
Novel nano-materials, RGD-tetrapeptide-modified
17b-amino-11a-hydroxyandrost-1,4-diene-3-one: synthesis, self-assembly
based nano-images and in vivo anti-osteoporosis evaluation†
*
Yuji Wang, Jianhui Wu, Guifeng Kang, Ming Zhao, Lin Gui, Ning Li, Li Peng, Xiaoyi Zhang, Li Li
*
and Shiqi Peng
Received 16th August 2011, Accepted 13th December 2011
DOI: 10.1039/c2jm13983a
To find novel nano-materials with anti-osteoporosis activity and without side effects three novel anti-
osteoporosis active amphiphiles, 17b-(RGD-AA-amido)-11a-hydroxyandrost-1,4-diene-3-one (5a: AA
¼ Ser, 5b: AA ¼ Val, 5c: AA ¼ Phe), were constructed by coupling the bone resorption inhibiting active
RGD-peptides and anti-osteoporosis active 17b-amino-11a-hydroxyandrost-1,4-diene-3-one. In the
solid state 5a, 5b and 5c exist as dispersed globes of 300 nm–14 mm in diameter, dispersed eggs of 110
nm–19 mm in diameter and beads of 238 nm–22 mm in diameter, respectively. In ultrapure water 1.1 mM
of 5a, 5b and 5c form nano-globes of 33–400 nm, 16–278 nm and 54–187 nm in diameter, respectively.
At an oral dose of 110 nmol kgꢀ1 5a–c effectively inhibited mice from developing osteoporosis. In
contrast to estradiol, 5a–c did not induce mice to develop endometrial hyperplasia and thrombus.
able to improve the anti-osteoporosis potency and reduce
1. Introduction
endometrial hyperplasia of estrogen therapy, but the thrombosis
risk of estrogen therapy was not investigated.23 In contrast
to estrogen, 17b-amino-11a-hydroxyandrost-1,4-diene-3-one,
a novel anti-osteoporosis androgene, possesses higher efficacy,
does not induce endometrial hyperplasia and thrombosis risk.24
To find novel amphiphilic molecules with anti-osteoporosis
activity and without side effects, this paper introduced
RGD-tetrapeptides into 17b-amino-11a-hydroxyandrost-1,4-
diene-3-one to form three 17b-(Arg-Gly-Asp-AA-amido)-11a-
hydroxyandrost-1,4-diene-3-one (5a–c, AA ¼ Ser, Val or Phe),
evaluated their in vivo anti-osteoporosis activities, examined their
endometrial hyperplasia and thrombosis risks, measured their
scanning electron microscopy (SEM) and transmission electron
microscope (TEM) images and discussed the relationship
between their anti-osteoporosis action, side effects and
nanostructure.
Self-organization or self-assembly is of practical importance in
the formation of various ordered nanostructures in solution, in
the bulk state, and on a solid surface, such as spherical, lamellar,
honeycomb structures, and so on.1 The advent of nano-materials
opens a door to self-assembly.2 Consequently, numerous self-
assembling molecules, such as highly fluorinated amphiphilic
molecules,3 amphiphilic triblock copolymers with polyrotaxane
as a central block,4 amphiphilic dodecyl ester derivatives from
aromatic amino acids,5 dendritic molecules,6 the shape anisot-
ropy of non-spherical colloidal building blocks,7 alkylated
polycyclic aromatic hydrocarbons,8 porphyrins, graphenes and
fullerenes,9 were designed. Of the self-assembling molecules,
peptides have been considered a kind of special substance,10–17
due to their ability to form nano-structures and to possess
excellent bioactivity.18
It is commonly accepted that bone formation and bone
resorption depend on the activity of osteoblasts and osteoclasts,
respectively. In osteoporosis therapy estrogen has been admin-
istered because it is able to increase the activity of osteoblasts
and decrease the activity of osteoclasts.19–21 By blocking the
adhesiveness of osteoclasts onto the surface of the bone RGD-
tetrapeptides can inhibit bone resorption.22 The conjugates of
RGD-tetrapeptides and estrogen were explored and proved to be
2. Experimental
2.1. General
The protected amino acids with L-configuration were purchased
from Sigma Chemical Co. All coupling and deprotective reac-
tions were carried out under anhydrous conditions. Chroma-
tography was performed on Qingdao silica gel H. The purities of
the intermediates were measured with TLC (Merck silica gel
plates of type 60 F254, 0.25 mm layer thickness) and were more
than 95%. The purities of the products were measured with
HPLC (Waters, C18 column 4.6 ꢁ 150 mm) and were more than
97%. All reagents were of commercial quality and were used as
College of Pharmaceutical Sciences, Capital Medical University, Beijing,
Tel:
100069,
P.
R.
China.
E-mail:
+86-10-8391-1535; sqpeng@bjmu.edu.cn; +86-10-8391-1528
maozhao@126.com;
† Electronic Supplementary Information (ESI) available. See
DOI:10.1039/c2jm13983a
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received. Solvents were dried and purified using standard tech-
niques when it was necessary. Reactions were monitored by thin
layer chromatography, on glass plates coated with silica gel with
a fluorescent indicator. Melting points were measured on a XT5
hot stage microscope (Beijing keyi electro-optic factory) and
were uncorrected. Infrared spectra were recorded on a spectro-
photometer (Perkin-Elmer 983 instrument), and the solid
1.46 (s, 3 H), 1.16 (s, 3 H). 13C NMR (125 MHz, DMSO-d6)
d (ppm) ¼ 187.0, 180.0, 175.2, 170.2, 169.2, 166.0, 144.3, 136.5,
135.7, 123.5, 64.8, 61.2, 60.0, 56.7, 55.2, 50.1, 47.6, 43.0, 42.1,
38.4, 38.0, 37.2, 35.2, 31.7, 27.2, 25.8, 24.9, 19.2, 17,2, 14.9. Anal.
Calcd. for C34H52N8O9: C, 56.97; H, 7.31; N, 15.63. Found: C,
57.18; H, 7.15; N, 15.85.
1
compounds were compressed into KBr pellets. H (500 M Hz)
2.2.2. Synthesis
of
17b-(Arg-Gly-Asp-Val-amido)-11a-
and 13C (125 M Hz) NMR spectra were recorded on a Bruker
Avance II 500 MHz spectrometer by use of DMSO-d6 or CDCl3
as the solvent and tetramethylsilane as the internal standard.
ESI/MS was determined on a ZQ2000 (Waters, US). The content
of calcium was measured on a Varian SpectrAA 220 instrument.
Bone trabecular density was measured on a STRATEC XCT
Research SA+ instrument.
hydroxyandrost-1,4-diene-3-one (5b). Using the procedure of
preparing 5a from 350 mg (0.33 mmol) of 4b 170 mg (72%) of the
title compound was obtained as a colorless powder. Mp 108–109
ꢂC, [a]D ¼ 10.0 (c ¼ 0.13, MeOH), ESI-MS (m/z) 729 [M + H]+.
20
1H NMR (500 MHz, DMSO-d6) d (ppm) ¼ 10.62 (w, 1 H), 8.24
(s, 1 H), 8.12 (s, 2 H), 7.91 (s, 1 H), 7.80 (s, 1 H), 6.35 (dd, J ¼ 11.0
Hz, 3.0 Hz, 1 H), 6.27 (d, J ¼ 3.0 Hz,1 H), 6.10 (d, J ¼ 3.0 Hz, 1
H), 4.99 (m, 1 H), 4.78 (m, 1 H), 4.10 (s, 2 H), 3.69 (t, J ¼ 9.5 Hz,
1 H), 3.55 (m, 1 H), 3.16 (m, 1 H), 2.77 (d, J ¼ 11.1 Hz, 2 H), 2.68
(m, 2 H), 2.66 (t, J ¼ 11.2 Hz, 2 H), 2.52 (b, 2 H), 2.50 (b, 1 H),
1.45–1.95 (m, 17 H), 1.96 (t, J ¼ 12.0 Hz, 2 H), 1.46 (s, 3 H), 1.16
(s, 3 H), 1.03 (d, J ¼ 10.1 Hz, 6 H). 13C NMR (125 MHz, DMSO-
d6) d (ppm) ¼ 187.1, 182.1, 175.5, 171.5, 165.3, 144.1, 138.5,
136.0, 132.4, 128.6, 67.8, 62.2, 60.0, 56.8, 56.1, 50.6, 43.5, 43.0,
38.3, 38.0, 37.2, 35.2, 31.7, 29.0, 27.2, 25.8, 24.9, 19.2, 17,2, 16.2,
14.9. Anal. Calcd. for C36H56N8O8: C, 59.32; H, 7.74; N, 15.37.
Found: C, 59.50; H, 7.60; N, 15.14.
2.2. Synthesis of 5a–c
As indicated in Scheme 1, via a five-step-procedure 5a–c were
synthesized. The preparations and the data of intermediates 1,
2a–c, 3a–c and 4a–c are given in item 8 of ESI.†
2.2.1. Synthesis
of
17b-(Arg-Gly-Asp-Ser-amido)-11a-
hydroxyandrost-1,4-diene-3-one (5a). A mixture of 400 mg (0.35
mmol) of 4a, 1.5 ml of dimethyl sulfide, 1.5 ml of phenyl methyl
ether and 3 ml of CF3CO2H/CF3SO3H (4 : 1) was stirred at 0 ꢂC
for 1.5 h. Upon removal of the excess CF3CO2H/CF3SO3H the
residue was triturated with ether and then purified on Sephadex-
G10 and HPLC to provide 190 mg (71%) of the title compound
2.2.3. Synthesis
of
17b-(Arg-Gly-Asp-Phe-amido)-11a-
hydroxyandrost-1,4-diene-3-one (5c). Using the procedure of
preparing 5a from 460 mg (0.41 mmol) of 4c 240 mg (75%) of the
title compound was obtained as a colorless powder. Mp 112–113
20
as a light yellow powder. Mp 114–116 ꢂC, [a]D ¼ 4.0 (c ¼ 0.13,
20
ꢂC, [a]D ¼ 14.0 (c ¼ 0.12, MeOH), ESI-MS (m/z) 777 [M + H]+.
MeOH), ESI-MS (m/z) 717 [M + H]+. 1H NMR (500 MHz,
DMSO-d6) d (ppm) ¼ 10.64 (w, 1 H), 8.20 (s, 1 H), 8.15 (s, 2 H),
7.90 (s,1 H), 6.34 (dd, J ¼ 10.0 Hz, 3.0 Hz, 1 H), 6.28 (d, J ¼ 3.0
Hz,1 H), 6.10 (d, J ¼ 3.2 Hz, 1 H), 4.99 (m, 1 H), 4.78 (m, 1 H),
4.10 (s, 2 H), 3.69 (t, J ¼ 9.5 Hz, 1 H), 3.55 (m, 1 H), 3.16 (m, 1
H), 2.77 (d, J ¼ 11.0 Hz, 2 H), 2.66 (t, J ¼ 11.2 Hz, 2 H), 2.52 (b, 2
H), 2.44 (b, 1 H), 1.45–1.95 (m, 22 H), 1.96 (t, J ¼ 13.1 Hz, 2 H),
1H NMR (500 MHz, DMSO-d6) d (ppm) ¼ 10.77 (w, 1 H), 8.21
(s, 1 H), 8.10 (s, 1 H), 7.93 (s, 1 H), 7.85 (s, 1 H), 7.06–7.22 (m, 5
H), 6.36 (dd, J ¼ 11.1 Hz, 3.0 Hz, 1 H), 6.28 (d, J ¼ 3.0 Hz, 1 H),
6.10 (d, J ¼ 3.1 Hz, 1 H), 4.94 (m, 1 H), 4.74 (m, 1 H), 4.12 (s, 2
H), 3.54 (t, J ¼ 9.5 Hz, 1 H), 3.52 (m, 1 H), 3.16 (m, 1 H), 2.77
(d, J ¼ 11.2 Hz, 2 H), 2.66 (m, 2 H), 2.64 (t, J ¼ 11.2 Hz. 2 H),
Scheme 1 Synthesis route to 17b-(Arg-Gly-Asp-Ser/Val/Phe-amido)-11a-hydroxyandrost-1,4-diene-3-ones. In 2a–4a: AA ¼ Ser(Bzl); in 5a, AA ¼ Ser;
in 2b–5b: AA ¼ Val; in 2c–5c: AA ¼ Phe.
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2.52 (b, 2 H), 2.30 (b, 1 H), 1.45–1.96 (m, 19 H), 1.96 (t, J ¼ 13.0
Hz, 2 H), 1.46 (s, 3 H), 1.16 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6) d (ppm) ¼ 187.2, 178.0, 175.2, 171.2, 166.6, 144.3,
140.2, 136.5, 135.7, 132.5, 128.2, 125.8, 123.4, 62.2, 60.0, 56.6,
54.2, 50.2, 43.2, 43.0, 39.2, 38.0, 37.4, 35.2, 30.3, 29.5, 28.1, 25.5,
20.0, 18.4, 17,1, 15.7. Anal. Calcd. for C40H56N8O8: C, 61.84; H,
7.27; N, 14.42. Found: C, 61.62; H, 7.11; N, 14.66.
2.3.3. Testing the bone weight and mineral content of 5a–c
treated mice. After execution the left femurs of the mice were
immediately collected, on complete removal of the muscle their
lengths were measured, and then they were immersed in a 2 : 1
solution of chloroform and methanol twice (each 3 h). After
being defatted the left femurs were heated at 120 ꢂC for 6 h,
cooled and weighed to record their dry weight. The femurs were
calcined in a furnace at 800 ꢂC for 8 h, cooled, weighed to record
the ash weight, and the rate of the ash weight to dry femur weight
(the mineral content of the femur) was calculated. The ashes of
the left femurs were dissolved in 0.5 mL of hydrochloric acid
(6 N) and diluted to 5.00 mL with ultrapure water, from which
0.05 mL of the solution was drawn and diluted to 1.00 mL with
ultrapure water before use. The calcium content of the aqueous
solution was measured with the method of o-methylphenolph-
thalein complexing ketone. The phosphorus content of the
aqueous solution was measured with the method of molybdenum
blue.
2.3. Evaluation of the in vivo anti-osteoporosis and side effects
of 5a–c
2.3.1. Animals and administration. The assessments described
herein were performed based on a protocol reviewed and
approved by the ethics committee of Capital Medical University.
The committee assured that the welfare of the animals was
maintained in accordance to the requirements of the animal
welfare act and according to the guide for care and use of
laboratory animals. ICR mice (weighing 20.7 ꢃ 2.1 g, purchased
from Animal Center of Peking University) were housed under
temperature and humidity controlled conditions at a 12 h light
and 12 h dark (6 pm to 6 am) cycle for one day before being used.
Each of them in drug receiving groups was administered special
food (containing 0.1% of calcium and 0.4% of phosphorus) and
distilled water, while each of them in blank control was admin-
istered normal food (containing 1.76% of calcium and 1.01% of
phosphorus) and running water. Each mouse in the prednisone
control group was injected with 6.3 mg kgꢀ1 of prednisone,
intramuscularly, twice a week and orally administered 0.2 mL of
distilled water once a day. Each mouse in the treated group was
injected with 6.3 mg kgꢀ1 of prednisone, intramuscularly, twice
a week and orally administered 110 nmol kgꢀ1 of 5a–c in 0.2 of
distilled water once a day. All mice were treated in the corre-
sponding way for 4 weeks. On the next day of the last adminis-
tration the mice were weighed, blood was drawn via an eye orbit,
the mice were then sacrificed with pentobarbital sodium (40.0 mg
kgꢀ1, i.p.) anesthesia, and immediately dissected to obtain the
femurs and organs.
2.3.4. Testing the bleeding cease time of 5a–c treated mice.
After the 4 week treatment with estradiol or 5a–c the mice in anti-
osteoporosis assays were weighed to record the body weights.
Thirty and ninety min after the last administration, the mice were
subjected to an in vivo tail bleeding time assay by use of a stan-
dard procedure. Briefly, the mouse was placed in a tube holder
with its tail protruding, and a 2 mm cut was made on the tail.
Flowing blood until it stopped was gently wiped away with
a filter paper every 30 s, yielding the bleeding time.
2.3.5. Testing the uterine weight of 5a–c treated mice. After
the 4 week treatment with estradiol or 5a–c the mice in anti-
osteoporosis assays were sacrificed with pentobarbital sodium
(40.0 mg kgꢀ1, i.p.) anesthesia and immediately dissected to
obtain the uterus to record their weights.
2.4. Identification of the nano-characterization of 5a–c
2.4.1. Measuring the SEM images of 5a–c in solid state. The
shape and size of the nano-species of the lyophilizing powders of
1.1 mM solutions of 5a–c in ultrapure water were measured on
a SEM (JEM-1230, JEOL, Tokyo, Japan) at 50 kV. The lyoph-
ilizing powders were attached on to a copper plate via double-
side tape (Euromedex, France). The specimens were coated with
20 nm gold–palladium using a JEOL JFC-1600 AUTO FINE
COATER. The coater was operated at 15 kV, 30 mA, 200 mTorr
(argon) for 60 s. The shape and size distribution of the nano-
particles were measured from counting over 100 particles in
randomly selected regions on the SEM alloy. All the measure-
ments in triplicate grids were performed. The images were
recorded on an imaging plate (Gatan Bioscan Camera
Model1792) with 20 eV energy windows at 100–10 000ꢁ, and
were digitally enlarged.
2.3.2. Testing the total and trabecula vBMD of the femurs of
5a–c treated mice. Secondary osteoporosis is common in patients
treated with glucocorticoids and in prostate cancer patients
receiving androgen deprivation therapy (ADT) in particular.25–28
Chronic exposure to pharmacological doses of glucocorticoids,
such as prednisone, causes multiple deleterious effects on
osteopenia, osteoporosis and bone fracture in particular.29–31 To
evaluate the efficacy of 5a–c preventing osteoporosis in female
patients treated with glucocorticoids and male patients receiving
ADT,8–10 total volumetric bone mineral density (vBMD) and
trabecula vBMD tests were performed.
Computed tomography (CT) allows the measurement of 3D
bone geometry, and could provide measurements of size-inde-
pendent vBMD. Peripheral quantitative CT (pQCT) allows the
quantitative measurement of 3D bone geometry, and could
provide quantitative measurements of size-independent
vBMD. In the present pQCT tests normal saline (NS) only was
used as the background control and NS plus prednisone was
used as the blank control to measure the total vBMD and
trabecular vBMD of the femurs of 5a–c plus prednisone treated
mice.
2.4.2. Measuring the TEM images of 5a–c in water. The
shape and size of the nano-species of 5a–c were measured on
a TEM (JSM-6360 LV, JEOL, Tokyo, Japan). The solution
(1.1 mM) of 5a–c in ultrapure water was dripped onto a formvar-
coated copper grid and then a drop of anhydrous ethanol was
added to promote the removal of water. At first the grid was
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measurement was repeated for 3 runs per sample, and the data
were calculated automatically using the software from the elec-
trophoretic mobility based on Smoluchowski’s formula.
3. Results and discussion
3.1. Chemical synthesis of 5a–c
As indicated in Scheme 1, via a five-step-procedure 5a–c were
prepared. In brief, the reduction of 11a-hydroxyandrost-1,4-
diene-3,17-dione provided 17b-amino-11a-hydroxyandrost-1,4-
Fig. 1 Total vBMD and images of pQCT scanning at a distance from
the proximal femur growth palate corresponding to <6% of the total
length of the femur of the treated mice.
diene-3,17-dione (1, 90% yield), the coupling of
1 and
Boc-Ser(Bzl), Boc-Val or Boc-Phe provided 17b-[Boc-Ser(Bzl)-
amido]- or 17b-[Boc-Val-amido]- or 17b-[Boc-Phe-amido]-11a-
hydroxyandrost-1,4-diene-3-ones (2a–c, 82–84% yields). After
removing Boc, 2a–c were coupled with Boc-Asp to provide
17b-(Boc-Asp-Ser(Bzl)-amido)- or 17b-(Boc-Asp-Val-amido)- or
17b-(Boc-Asp-Phe-amido)-11a-hydroxyandrost-1,4-diene-3-one
(3a–c, 88–94% yields). After removing Boc, 3a–c were coupled
with Boc-Arg(Tos)-Gly to provide 17b-(Boc-Arg(Tos)-Gly-Asp-
Ser(Bzl)-amido)- or 17b-(Boc-Arg(Tos)-Gly-Asp-Val-amido)- or
17b-(Boc-Arg(Tos)-Gly-Asp-Phe-amido)-11a-hydroxyandrost-
1,4-diene-3-one (3a–c, 88–94% yields). In the presence of
CF3CO2H/CF3SO3H all protective groups were removed and
5a–c were obtained in 72% to 75% yields. Besides, Boc-Arg(Tos)-
Gly was prepared by using the solution method and the yield was
ꢂ
allowed to thoroughly dry in air and then heated at 35 C for
24 h. The sample was viewed under the TEM. The shape and size
of the nano-species on the TEM copper grid were obtained from
counting over 100 species in randomly selected regions. All
determinations were carried out in triplicate grids. TEM was
operated at 80 kV electron beam accelerating voltage. The
images were recorded on an imaging plate (Gatan Bioscan
Camera Model 1792) with 20 eV energy windows at 6000–
400 000ꢁ, and were digitally enlarged.
2.4.3. Measuring the nano-size of 5a–c in NS. The size of the
nano-particles of 5a–c in NS was measured on a Malvern’s Zeta
Sizer (Nano-ZS90) with DTS (Nano) Program. The concentra-
tion of the solution of 5a–c in NS was 1.1 mM, the testing
temperature was 25 ꢂC, the time interval was 30 s, the mean size
and half-peak width of ten times were recorded.
2.4.4. Measuring the zeta-potential of 5a–c in NS. The zeta-
potential of the surface of nano-particles of 5a–c in NS was
measured on a ZetaPlus Potential Analyzer (ZetaPlus S/N 21394,
Brookhaven Instruments Coorporation) with BIC Zeta Potential
Analyzer. The concentration of the solution of 5a–c in NS was
1.1 mM and the testing temperature was 25 ꢂC. The zeta potential
Fig. 3 Dry weights, ash weights, Ca2+ and BMC of the femurs of 5a–c
treated mice. a) Weights of dry femur and femur ash are represented with
mean ꢃ SD mg, femur Ca2+ and BMC is represented with mean ꢃ SD %,
n ¼ 12, PDN ¼ prednisone, dose of 5a–c ¼ 110 nmol kgꢀ1. For dry femur
weight: b) compared to NS + PDN and 5a + PDN p < 0.01, to 5b + PDN
p < 0.05; c) compared to NS + PDN p < 0.01, to 5a + PDN p < 0.05; d)
compared to NS + PDN p < 0.01. For femur ash weight: b) compared to
NS + PDN, 5b + PDN and 5a + PDN p < 0.01; c) compared to NS +
PDN p < 0.01, to 5a + PDN p < 0.05; d) compared to NS + PDN p < 0.01.
For femur BMC: b) compared to NS + PDN and 5c + PDN p < 0.01, to 5b
+ PDN p < 0.05; c) compared to NS + PDN p < 0.01, to 5a + PDN p <
0.05; d) compared to NS + PDN p < 0.01. For femur Ca2+ : b) compared
to NS + PDN and 5a + PDN p < 0.01, to 5b + PDN p < 0.05; c) compared
to NS + PDN p < 0.01, to 5a + PDN p < 0.05; d) compared to NS + PDN
p < 0.01.
Fig. 2 Trabecular vBMD of the femurs of the treated mice at a distance
from the proximal femur growth palate corresponding to <6% of the total
length of the femurs of the treated mice. a) Trabecular vBMD is repre-
sented with mean ꢃ SD mg cmꢀ3, n ¼ 12, PDN ¼ prednisone, dose of 5a–
c ¼ 110 nmol kgꢀ1. b) Compared to NS + PDN, 5a + PDN and 5b + PDN
p < 0.01. c) Compared to NS + PDN and 5a + PDN p < 0.01. d)
Compared to NS + PDN p < 0.01.
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3.3. Dry weights, ash weights, Ca2+ and BMC of the femurs of
5a–c treated mice
Physical and chemical studies were generally used in the in vivo
anti-osteoporosis assay. In the present tests of the dry weights,
ash weights, Ca2+ and bone mineral content (BMC) of the femurs
of 5a–c treated mice NS alone was used as the background
control, NS plus prednisone was used as the blank control and
the data are shown in Fig. 3.
Fig. 3 indicates that the dry weight of the femur of NS plus
prednisone treated mice is significantly lower than that of the
femur of NS alone treated mice. This means that prednisone
effectively induces the mice to lose the femur. The dry weights of
the femur of 5a–c plus prednisone treated mice are significantly
higher than those of the femur of NS plus prednisone treated
mice. This means that 5a–c effectively inhibit prednisone treated
mice to lose femur. Similar effects of 5a–c on femur ash weights,
femur Ca+2 and femur BMC of the treated mice were also
observed from Fig. 3. Besides, the activity order of 5a–c inhib-
iting prednisone treated mice to lose femur’s dry weights, femur’s
ash weights, femur’s Ca2+ and femur’s BMC is also 5c > 5b > 5a.
Fig. 4 Uterine weights of 5a–c treated mice. Uterine weight is repre-
sented by mean ꢃ SD mg, n ¼ 12, PDN ¼ prednisone, E2 ¼ estradiol,
dose of E2 and 5a–c ¼ 110 nmol kgꢀ1. a) Compared to NS alone p < 0.01.
at the same level as that reported previously.23,24 This five-step-
procedure is suitable for preparing 5a–c.
3.2. Total and trabecula vBMD of the femurs of 5a–c treated
mice
3.4. Side effects of 5a–c in vivo
CT measured 3D bone geometry and the size-independent
vBMD, as well as pQCT quantitatively measured 3D bone
geometry and size-independent vBMD were used to represent
the anti-osteoporosis efficacy of 5a–c and are shown in Fig. 1
and 2.
Endometrial hyperplasia and the thrombosis risk are common
side effects in estrogen therapy. The endometrial hyperplasia risk
of 5a–c therapy is represented with the uterine weights of the
treated mice and the data are shown in Fig. 4. The thrombosis
risk of 5a–c therapy is represented with the tail bleeding time of
the treated mice and the data are shown in Fig. 5.
Fig. 1 indicates that the total vBMD of the femurs of NS plus
prednisone treated mice is significantly lower than that of the
femurs of NS alone treated mice. This means that prednisone
effectively induces the mice to decrease the total vBMD. The
total vBMDs of the femurs of 5a–c plus prednisone treated mice
are significantly higher than those of the femurs of NS plus
prednisone treated mice. This means that 5a–c effectively inhibit
prednisone treated mice to decrease the total vBMD. Besides, the
total vBMD of the femurs of 5a plus prednisone treated mice is
significantly lower than those of the femurs of 5b plus prednisone
treated mice, while the total vBMD of the femurs of 5b plus
prednisone treated mice are significantly lower than those of the
femurs of 5c plus prednisone treated mice. This means that the
activity order of 5a–c inhibiting prednisone treated mice to
develop osteoporosis is 5c > 5b > 5a.
Fig. 4 indicates that the uterine weights of the mice receiving
estradiol plus prednisone are significantly higher than those of
the mice receiving NS alone. This means that estradiol therapy
induces the mice to develop endometrial hyperplasia. In contrast
to estradiol, the uterine weights of the mice receiving 5a–c plus
prednisone are equal to those of the mice receiving NS alone.
This means that 5a–c have no endometrial hyperplasia risk.
Fig. 5 indicates that 30 min and 90 min after of the last oral
administration the tail bleeding times of the mice receiving
estradiol plus predsone are significantly shorter than those of the
mice receiving NS plus predsone. This means that estradiol
therapy induces thrombosis risk. However 30 min and 90 min
after of the last oral administration the tail bleeding times of the
Fig. 2 indicates that the trabecular vBMD of the femurs of NS
plus prednisone treated mice is significantly lower than that of
the femurs of NS alone treated mice. This means that prednisone
effectively induces the mice to decrease trabecular vBMD. The
total trabecular vBMDs of the femurs of 5a–c plus prednisone
treated mice are significantly higher than those of the femurs of
NS plus prednisone treated mice. This means that 5a–c effec-
tively inhibit prednisone treated mice to decrease trabecular
vBMD. Besides, the trabecular vBMD of the femurs of 5a plus
prednisone treated mice is significantly lower than that of the
femurs of 5b plus prednisone treated mice, while the trabecular
vBMD of the femurs of 5b plus prednisone treated mice are
significantly lower than that of the femurs of 5c plus prednisone
treated mice. This again means that the activity order of 5a–c
inhibiting prednisone treated mice to develop osteoporosis is
5c > 5b > 5a.
Fig. 5 Effect of 5a–c on the tail bleeding time of the treated mice. Tail
bleeding time is represented by mean ꢃ SD s, n ¼ 12, PDN ¼ prednisone,
E2 ¼ estradiol. a) Compared to NS + PDN, p < 0.01; b) compared to NS
+ PDN, p > 0.05.
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Fig. 6 SEM images of 5a in solid state. Dispersing globes of 300 nm–14 mm in diameter (5a–A and 5a–B), and fusion globes (5a–C).
Fig. 7 SEM images of 5b in solid state. Dispersing eggs of 110 nm–19 mm in diameter, a number of small eggs are on the faces of the four bigger eggs
(5b–A, 5b–B and 5b–C), one egg remains with its tail incomplete, as well (5b–C).
Fig. 8 SEM images of 5c in solid state. Beads of 238 nm–22 mm in diameter, and some small beads are on the faces of the bigger beads (5c–A and 5c–B),
one side of a bead remains incomplete (5c–B and 5c–C).
Fig. 9 TEM images of 1.1 mM of 5a in ultrapure water. The nano-globe of 400 nm in diameter consists of numerous smaller globes (5a–A), the
dispersing globes of 55–200 nm in diameter and some fusion globes (5a–B), as well as the dispersing globes of 18–146 nm in diameter (5a–C).
Fig. 10 TEM images of 1.1 mM of 5b in ultrapure water. The smaller globes, blocks and awls are on the face of the larger globe in a fusion manner (5b–
A), most of the nano-globes exist as separate objects, but four are fusion globes (5b–B) and similarly most of the nano-globes exist as separate objects,
some are fusion globes (5b–C).
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Fig. 11 TEM images of 1.1 mM of 5c in ultrapure water. Numerous nano-particles and their aggregators spread around the nano-globe of 183 nm in
diameter and a number of nano-particles are on its face in a fusion manner (5c–A), a hemisphere of 275 nm in diameter and some smaller globes adhere
on its incomplete face (5c–B), five globes of 48–188 nm in diameter and one fusion globe of 130 ꢁ 145 nm (5c–C).
mice receiving 5a–c plus predsone are equal to those of the mice
receiving NS plus predsone. This means that 5a–c therapy has no
thrombosis risk.
nano-globes or similar nano-species including nano-eggs and
nano-beads. The similar characterization was observed in the
TEM images of 5a–c. This implies that it is the common chemical
structure of 17b-(Arg-Gly-Asp-AA-amido)-11a-hydroxyan-
drost-1,4-diene-3-ones that determines the similarity of the nano-
image characterization of 5a–c in the solid state and in aqueous
solution. On the other hand, the moiety 17b-amino-11a-
hydroxyandrost-1,4-diene-3-one determines the size of the
nano-globes and the amino acid residue of the C-terminus of the
RGD-tetrapeptide moiety determines the feature of the nano-
globes.
3.5. Comparing in vivo activities and side effects of 5a–c with
estradiol
The in vivo anti-osteoporosis activities, the endometrial hyper-
plasia risk and the thrombosis risk of 5a–c were also compared
with those of estradiol and the data are shown in Fig. 2, 3, 4, 5
and 7 of ESI.† The figures indicate that at the dose of 110 nmol
kgꢀ1 the in vivo anti-osteoporosis activities of oral 5a–c are
significantly higher than that of estradiol. Besides, in contrast to
estradiol, 5a–c do not induce the mice to develop endometrial
hyperplasia and thrombus.
3.8. Sizes and zeta-potential of the nano-particles of 5a–c
As seen in Fig. 12, the mean diameters of the particles of 5a–c
are in a range of 128–266 nm during the monitored 10 days. In
the first 5 days the diameters decrease gradually and in the
second 5 days they have a relatively stable value. The mean
diameters less than 270 nm could be attributed to the hydro-
phobicity of the 17b-amino-11a-hydroxyandrost-1,4-diene-3,17-
dione moiety of 5a–c, which leads to stronger hydrophobic
interaction and the formation of aggregators with closer packing
density. On the other hand, the order of the mean diameters is
5a > 5b > 5c.
3.6. Comparing in vivo activities of 5a–c with 17b-amino-11a-
hydroxyandrost-1,4-diene-3-one
The in vivo anti-osteoporosis activities of 5a–c were further
compared with that of the unmodified 17b-amino-11a-hydrox-
yandrost-1,4-diene-3-one, and the data are shown in Fig. 2, 3 and
4 of ESI.† The figures indicate that at the dose of 110 nmol kgꢀ1
the in vivo anti-osteoporosis activities of oral 5a–c are signifi-
cantly higher than that of 17b-amino-11a-hydroxyandrost-1,4-
diene-3-one. This means that the introduction of RGD-tetra-
peptides enhances the in vivo anti-osteoporosis activity. Due to
RGD-tetrapeptides being able to block the adhesiveness of
osteoclasts onto the surface of the bone and lead 5a–c to form
nano-structures the enhanced in vivo anti-osteoporosis activities
should be attributed to both the blockage of the adhesiveness of
osteoclasts onto the surface of the bone and the formation of the
nano-structures.
The zeta-potentials of the nano-globes of 5a–c are in a range of
73.6–126.0 mV during the monitored 10 days, and the order of
the zeta-potentials is 5a < 5b < 5c (Fig. 13). This order matches
the order of the mean diameters, i.e., 5a > 5b > 5c. The lower
diameters and the higher zeta-potentials imply that the formed
3.7. Nano-images of 5a–c
SEM and TEM have been widely used for the investigation
of the three-dimensional structure of hydrogels, cells entrapped
in nanofiber matrices or the individual nanostructures of
peptides. To visualize the size and morphology of 5a–c in solid
state and in solution their SEM images and TEM images
were measured and are shown in Fig. 6 to 8 and Fig. 9 to 11,
respectively.
The SEM images demonstrate that in the solid state these
amphiphilic molecules (5a–c) are able to self-assemble and form
Fig. 12 Mean sizes of the particles of 5a–c within 10 days.
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