Poly-α,β-aspartyl-Arg-Gly-Asp-Phe: A novel polymeric nanomedicine

Article abstract of DOI:10.1039/c4md00331d

Thrombosis is a pathological condition and has been one of the most prominent causes of morbidity and mortality. Poly-α,β-aspartic acid is a biodegradable polymer, and RGD-tetrapeptides target thrombus. These led to the design of poly-α,β-aspartyl-Arg-Gly

Full text of DOI:10.1039/c4md00331d

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Ming Zhao, Shiqi Peng et al.
Poly-α,β-aspartyl-Arg-Gly-Asp-Phe: a novel polymeric nanomedicine

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Poly-a,b-aspartyl-Arg-Gly-Asp-Phe: a novel
polymeric nanomedicine†
Cite this: Med. Chem. Commun., 2015,
6, 182
a
a
a
b
ac
*
Shuangling Chen, Yuji Wang, Shan Li, Yaonan Wang, Ming Zhao,
Haimei Zhu,a
a
a
*
Jianhui Wu and Shiqi Peng
Thrombosis is a pathological condition and has been one of the most prominent causes of morbidity and
mortality. Poly-a,b-aspartic acid is a biodegradable polymer, and RGD-tetrapeptides target thrombus.
These led to the design of poly-a,b-aspartyl-Arg-Gly-Asp-Phe (PD-RGDF) as
a novel polymeric
Received 31st July 2014
Accepted 13th October 2014
therapeutic drug. In the solid state and ultrapure water PD-RGDF formed nanoparticles of substantially
identical diameter. In vitro the nanoparticles inhibited GPIIb/IIIa expression of activated platelets, thereby
making the in vitro anti-platelet aggregation efficacy greatly increase. In vivo the anti-thrombotic
efficacy of PD-RGDF was 500-fold higher than that of RGDF.
DOI: 10.1039/c4md00331d
www.rsc.org/medchemcomm
Bioactive peptides, including receptor-targeted peptides,
nuclear localization signaling peptides and cell penetrating
Introduction
peptides, function as chemical messengers, nerve transmitters
and ligands of specic receptors capable of regulating physio-
logical and/or pathological processes.24,25 The usual role of
bioactive peptides in polymeric therapeutics is to introduce
therapeutic or targeted functions.26 Of the integrin ligands,
RGD-tetrapeptides plays an important role in a series of physi-
ological and pathological processes. For instance, by inhibiting
the brinogen binding GPIIb/IIIa, RGD-peptides can block the
pathway of platelet aggregation and subsequently inhibit the
formation of thrombus.27–31 The modied RGD-peptides have
even higher anti-thrombotic activity,32,33 and anti-osteoporosis
activity.34–36 Specically, as a nanocarrier RGDV can target the
thrombus,37 a pathological condition and one of the most
prominent causes of morbidity and mortality.38,39
Based on the progress made in polymeric therapeutics, PD,
RGD-tetrapeptides and nanomedicine, as well as those aimed at
thrombus therapy, this work presents a novel nanosized poly-
meric therapeutic, poly-a,b-aspartyl-Arg-Gly-Asp-Phe (PD-
RGDF), evaluated for in vitro anti-platelet aggregation activity
and in vivo anti-thrombotic potency, and discusses their
mechanism of action.
During the past three decades, polymeric therapeutics, i.e.
biologically active polymeric drugs, such as the bioconjugates of
polymer–drugs and polymer–proteins, the block copolymer
micelles covalently bound to drugs and polymeric multilayer
capsules, have been the topic of a numerous excellent reviews.1–7
For polymeric therapeutics, the aspects of nanoscience and
nanobiotechnology have been deeply commented on, in
particular.8–10 So far the polymeric therapeutics in the experi-
mental, preclinical and clinical stages as well as on the market
are mostly anticancer nanomedicines.8,11–15 In respect of the
polymers for constructing nanomedicines, water soluble poly-
mers, reactive polymers and polymer coatings have attracted
special interest.16,17 Of the biodegradable polymers, poly-a,b-
aspartic acid (PD) is highly susceptible to degradation by the
lysosomal enzyme and produces monomeric aspartic acid as
the product.18 The modication of PD with fatty acid side
groups or amino acids have been used to construct polymeric
micelles for encapsulating amphotericin B,19 to treat
thrombus,20,21 and remove Pb(II) in vivo.22,23 These representative
modications ensure the utility of PD in preparing polymeric
therapeutics.
aBeijing Area Major Laboratory of Peptide and Small Molecular Drugs, Beijing
Laboratory of Biomedical Materials, Engineering Research Center of Endogenous
Prophylactic of Ministry of Education of China, College of Pharmaceutical Sciences
of Capital Medical University, Beijing 100069, P. R. China. E-mail: sqpeng@bjmu.
edu.cn
bEngineering Research Center of Endogenous Prophylactic of Ministry of Education of
China, Medical Experiment and Test Center of Capital Medical University, Beijing
100069, P. R. China
Results and discussion
Preparation of PD-RGDF
The preparation of polysuccimide and PD outlined in Scheme 1
of the ESI† were performed by following the well-known
methods,40,41 in which thermal polycondensation resulted in a
rational polysuccimide used for desirable ring opening by Arg-
Gly-Asp-Phe to keep the PD and the backbone of PD-RGDF
having 119 residues of Asp.20–23 The amino acid analysis of PD-
RGDF gave a ratio of Asp : Phe : Gly : Arg ¼ 2.0 : 1.0 : 0.8 : 0.7,
cFaculty of Biomedical Science and Environmental Biology of Kaohsiung Medical
University, Kaohsiung, Taiwan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00331d
182 | Med. Chem. Commun., 2015, 6, 182–186
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and conrmed that RGDF was successfully introduced into PD.
The HPLC purity of PD-RGDF was 98.4% (retention time
11.5 min).
Nano-images of PD-RGDF
The nano-structure of PD-RGDF in the solid state and in ultra-
pure water was imaged using scanning electron microscopy
(SEM), transmission electron microscopy (TEM) and atomic
force microscopy (AFM), and the images are shown in Fig. 1.
The SEM images show that the powders of PD-RGDF obtained
from the 10ꢀ6 nM aqueous solution are nanoparticles having a
diameter of 66–77 nm (Fig. 1A). The TEM images show that in
ultrapure water, 10ꢀ6 nM PD-RGDF forms nanoparticles having
a diameter of 44–156 nm (Fig. 1B). The AFM images show that in
rat plasma, 10ꢀ6 nM PD-RGDF forms nanoparticles of having a
diameter of 50–152 nm (Fig. 1F). The diameter of 44–156 nm
means that nanoparticles are suitable for escaping the entrap-
ment of macrophages.42
The effect of pH on the particle size was further explained
using the TEM images of PD-RGDF in an aqueous solution (10ꢀ6
M) at pH 1.2 (Fig. 1C), pH 7.4 (Fig. 1D) and pH 7.6 (Fig. 1E). As NS; in the magnified image the red arrowheads point to the pseudo-
Fig. 2 In vitro anti-platelet aggregation activity of PD-RGDF (A) and
RGDF (B), n ¼ 6, SEM image of AA activated rat platelets treated with
pods on the surface of the platelets (C) and SEM image of AA activated
rat platelets treated with PD-RGDF; in the magnified image the
representative nanoparticles on the surface and in the endocytosis
course are labeled with yellow arrowheads and blue arrowheads,
can be demonstrated, in the three pH environments the diam-
eter of the particles ranged from 22 nm to 142 nm, which still
beneted their delivery.
The effect of storage time on the particle size was claried
with 8 day particle size measurements, and the diameter of the
particles ranged from 60 nm to 170 nm, which again beneted
their delivery. The sizes with standard deviation and the zeta
potentials of the particles stored for 12 days in an aqueous
solution are also given in the ESI.†
respectively (D).
activating factor (PAF), adenosine diphosphate (ADP), arach-
idonic acid (AA) and thrombin (TH) were tested. Fig. 2 indicates
that the IC50 value (ꢁ10 nM) of PD-RGDF against PAF induced
platelet aggregation was ꢁ7–35 fold lower than those of it
against ADP, AA and TH induced platelet aggregation, sug-
gesting PD-RGDF selectively blocked the action of PAF in
inducing platelet aggregation. On the other hand, comparing
the IC50 values of RGDF against PAF, ADP, AA and TH induced
platelet aggregation the value of PD-RGDF was ꢁ7–400 fold
lower. To know the reason of PD-RGDF having a greatly higher
inhibition than RGDF, the SEM of 10ꢀ6 nM PD-RGDF treated AA
activated rat platelets and the nanostructure of a 1 mM aqueous
solution RGDF were measured. It was found that in a 1 mM
aqueous solution, RGDF did not form nanoparticles. The SEM
image (Fig. 2C) indicates that in the presence of NS, AA activated
rat platelets have numerous pseudopods on the surface. The
SEM image (Fig. 2D) indicates that in the presence of 10ꢀ6 nM
PD-RGDF the surface of AA activated rat platelets have
numerous nanoparticles, but not pseudopods, some of which
are in the endocytosis course. The nanoparticles capable of
adhering onto the surface and entering into the cytoplasm of
the platelets should be responsible for the excellent in vitro anti-
platelet aggregation activity.
PD-RGDF selectively inhibiting PAF-induced platelet
aggregation
Using an in vitro platelet aggregation model, the IC50 values of
PD-RGDF inhibition of platelet aggregation induced by platelet
Fig. 1 The nanoimages of PD-RGDF. (A) SEM image of PD-RGDF in
the solid state; (B) TEM image of PD-RGDF in ultrapure water; (C) TEM
image of PD-RGDF in an aqueous solution of pH 1.2; (d) TEM image of PD-RGDF inhibiting rats to develop thrombus
PD-RGDF in an aqueous solution of pH 7.4; (E) TEM image of PD-
Using a rat model, the anti-thrombotic activity of PD-RGDF was
RGDF in an aqueous solution of pH 7.6; (F) AFM image of PD-RGDF in
rat plasma (final concentration 10ꢀ6 nM); (G) AFM image of rat plasma
alone.
evaluated using doses of 0.1, 1 and 10 nmol kgꢀ1 and repre-
sented with thrombus weight. Fig. 3 indicates that PD-RGDF
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of GPIIb/IIIa expressed by 10 mM RGDF and NS treated platelets,
suggesting that at 10 mM, RGDF does not block the expression of
GPIIb/IIIa. However, 1 mM RGDF effectively inhibits the
expression of GPIIb/IIIa, and its potency equals that of 1 mM PD-
RGDF. Therefore, in respect of inhibiting GPIIb/IIIa expression,
the potency of PD-RGDF is 1000 fold higher than that of RGDF.
The advantage of PD-RGDF to RGDF in inhibiting GPIIb/IIIa
expression could also be attributed to PD-RGDF being able to
form nanoparticles, which are capable of adhering onto the
surface and entering into the cytoplasm of the platelets.
Experimental
Fig. 3 Effect of PD-RGDF and RGDF on rats developing thrombus, The chemicals, animals and equipment used herein, the
n ¼ 10.
synthetic route, the preparations and characteristics of poly-
succimide, PD and PD-RGDF, the measurements of the TEM,
SEM and AFM images, the in vitro anti-platelet aggregation
assay, the in vivo anti-thrombotic assay, the SEM images of rat
platelets, the GPIIb/IIIa expression assay, the stability of the
nanoparticles and the zeta potentials are described in the ESI.†
inhibits rats to develop thrombus in a dose dependent manner,
and the effective dose is 0.1 nmol kgꢀ1. Moreover, the anti-
thrombotic efficacy of 1 nmol kgꢀ1 and 10 nmol kgꢀ1 PD-RGDF
equal those of 167 mmol kgꢀ1 aspirin and 5 mmol kgꢀ1 RGDF,
respectively. This implies that the effective dose of PD-RGDF is
167 000 and 500 fold lower than those of aspirin and RGDF,
respectively. At a dose of 1 nmol kgꢀ1 both PD and a mixture of
PD and RGDF exhibit no anti-thrombotic activity, suggesting
that the excellent anti-thrombotic activity of 1 nmol kgꢀ1 PD-
RGDF is a result of it being a polymeric therapeutic.
Conclusions
Poly-a,b-aspartic acid, consisting of 119 Asp residues is highly
susceptible to degradation by lysosomal enzymes and therefore
could be used as a biodegradable polymer. To develop a poly-
meric therapeutic drug, Arg-Gly-Asp-Phe was covalently conju-
gated with poly-a,b-aspartic acid and gave poly-a,b-aspartyl-Arg-
Gly-Asp-Phe as a novel polymeric therapeutic, capable of form-
ing substantially identical nanoparticles in the solid state,
ultrapure water and in rat plasma, effectively inhibiting the
expression of GPIIb/IIIa and dramatically enhancing the anti-
thrombotic efficacy in vivo.
Effect of PD-RGDF on GPIIb/IIIa expression
To clarify the interaction of PD-RGDF with GPIIb/IIIa, the
expression of GPIIb/IIIa on PD-RGDF and RGDF treated plate-
lets activated by AA was examined using an ELISA experiment
according to the procedure for the Rat Platelet Membrane
Glycoprotein IIb/IIIa ELISA Kit (Shanghai Yuanye Biotechnology
Co. China). Fig. 4 indicates that the level of GPIIb/IIIa expressed
by 1 mM PD-RGDF treated platelets is signicantly lower than
that of GPIIb/IIIa expressed by NS treated platelets, suggesting
that at 1 mM, PD-RGDF blocks the expression of GPIIb/IIIa.
Moreover, no statistical difference was found between the levels
Acknowledgements
This work was supported by Beijing Municipal Science &
Technology Commission (Z141100002114049), the Project of
Construction of Innovative Teams and Teacher Career Devel-
opment for Universities and Colleges Under Beijing Munici-
pality, the NSFC (81172930, 81273379, 81202412, 81373264),
Beijing NOVA Programme (XX2013039) and the Beijing Natural
Science Foundation (7122023).
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