BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556 – 562

Original Article
www.nanomedjournal.com
Poly(citric acid)-block-poly(ethylene glycol) copolymers—new
biocompatible hybrid materials for nanomedicine
Ashkan Tavakoli Naeini, MSa , Mohsen Adeli, PhDb,c,⁎, Manouchehr Vossoughi, PhDa,b
a
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
b
Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran
c
Department of Organic Chemistry, Faculty of Chemistry, Sharif University of Technology, Tehran, Iran
Received 3 June 2009; accepted 24 November 2009

Abstract

Linear-dendritic ABA triblock copolymers containing poly(ethylene glycol) (PEG) as B block and hyperbranched poly(citric acid) (PCA)
as A blocks were synthesized through polycondensation. The molecular self-assembly of synthesized PCA-PEG-PCA copolymers in water
led to formation of nanoparticles and fibers in different sizes and shapes depending on the time and size of PCA blocks. Ten days after
dissolving PCA-PEG-PCA copolymers in water, the size of fibers had reached several millimeters. Mixing a water solution of fluorescein as
a small guest molecule and PCA-PEG-PCA copolymers led to the encapsulation of fluorescein by products of molecular self-assembly. To
investigate their potential application in nanomedicine and to understand the limitations and capabilities of these materials as nanoexcipients
in biological systems, different types of short-term in vitro cytotoxicity experiments on the HT1080 cell line (human fibrosarcoma) and
hemocompatibility tests were performed.

From the Clinical Editor: This manuscript investigates the potentials of linear-dendritic ABA triblock copolymers containing poly(ethylene
glycol) (PEG) as B block and hyperbranched poly(citric acid) (PCA) as A blocks for future applications in nanomedicine.
© 2010 Elsevier Inc. All rights reserved.

Key words: Nanomedicine; Citric acid; Molecular self-assembly; Linear-dendritic; Encapsulation

Preparation of macroscopic objects with a well-defined
structure at the atomic or molecular level continues to be a
very interesting subject, especially when working toward
applications such as nanomedicine that have a critical need for
a well-defined structure. Molecular self-assembly is an approach
to achieve this goal, because in this approach engineered
molecules or macromolecules, as building blocks, self-assemble
in special routes to form different objects such as micelles,1,2
vesicles,3-5 ribbons,6 films,7 fibers,8-10 and tubules.11-13
Supramolecular self-assembly of polymers is an area of
research that has recently attracted much interest. They offer
promise for many applications in different fields ranging from
self-healing polymers and inks to adhesives and personal-care
products. As a result of their intrinsic properties and conforma-
No conflict of interest was reported by the authors of this paper.
⁎Corresponding authors: Sharif University of Technology, Azadi Ave,
Tehran, Iran.
E-mail addresses: adeli@sharif.ir (M. Adeli), vosoughi@sharif.ir
(M. Vossoughi).
1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2009.11.008
tional movements, interaction of polymers with other molecules
or blocks leads to supramolecules, supramolecular polymers,
or nanostructures.14-17
Actually, supramolecules are products of the “bottom-up”
approach in nanotechnology. As a result of the noncovalent
interactions between building blocks in supramolecules, they are
promising multidisciplinary materials for use in nanomedicine,
because they will degrade back into individual molecules that
can be broken down by the body.
Linear-dendritic macromolecules in which linear polymers
are conjugated to dendritic macromolecules are a type of hybrid
materials that have attracted increasing interest in recent
years.18-22 Because of the conjugation of two different types
of macromolecules, linear and dendritic, they are one of the
most important classes of materials for use in supramolecular
self-assembly. Different sizes and shapes of supramolecular
assemblies can be prepared depending on the shape and
properties of the linear and dendritic parts.23-27
Citric acid is a cheap and biocompatible compound that is
used on a large scale in the food and drug industries. Despite the
large-scale production and intrinsic importance of this material,

Please cite this article as: A.T. Naeini, M. Adeli, M. Vossoughi, Poly(citric acid)-block-poly(ethylene glycol) copolymers—new biocompatible hybrid
materials for nanomedicine. Nanomedicine: NBM 2010;6:556-562, doi:10.1016/j.nano.2009.11.008
 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562 557

Figure 1. A schematic representation of poly(citric acid)–poly(ethylene glycol)–poly(citric acid) copolymers.

Figure 2. Matrix-assisted laser diffraction initiation–time of flight mass spectra for PCA-PEG-PCA copolymers synthesized using citric acid/PEG = 2 (A1), 5
(A2), 8 (A3), 10 (A4) molar ratios.

especially in biological systems, there are apparently no reports
of the production of polymeric materials based on this
compound. Herein ABA triblock copolymers of poly(citric
acid) (PCA) and poly(ethylene glycol) (PEG) are synthesized
and characterized. Molecular self-assembly of PCA-PEG-PCA
copolymers in water led to the formation of different objects,
from nanoparticles to fibers that were able to encapsulate
fluorescein as a small guest molecule. Based on their molecular
self-assembly, linear-dendritic PCA-PEG-PCA copolymers are
promising nanomaterials to use in nanomedicine. Hence,
different types of short-term in vitro cytotoxicity experiments
on the HT1080 cell line (human fibrosarcoma) and hemocom-
patibility tests were performed so as to investigate their potential
application in biological systems.
Methods
PEG (molecular weight [Mn] = 1500), citric acid, tetrahy-
drofuran, diethyl ether, and dimethyl sulfoxide (DMSO) were
purchased from Merck. A dialysis bag (Mn cutoff 2000) was
purchased from Sigma-Aldrich (St Louis, Missouri). The cell
line (HT1080, American Type Culture Collection) was obtained
from the Pasteur Institute (Tehran, Iran). MTT (3-(4,5-
558 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562

Figure 3. Gel permeation chromatography diagrams for poly(citric acid)–

poly(ethylene glycol)–poly(citric acid) (PCA-PEG-PCA) copolymers syn-
thesized using CA/PEG molar ratios of 2 (A1), 5 (A2), 8 (A3), and 10 (A4).
Figure 4. Atomic force microscopy image of A2 poly(citric acid)–poly
(ethylene glycol)–poly(citric acid) copolymer in water after 5 days.
Table 1
Some characteristics of PCA-PEG-PCA copolymers synthesized using four using dynamic light scattering (DLS) (Zetasizer ZS; Malvern
different CA/PEG molar ratios* Instruments, Worcestershire, United Kingdom). The molecular
PCA-PEG- Molecular weight Mn PDI %PCA Diameter weight distributions were determined by gel permeation
PCA molar (MALDI-TOF (GPC) (GPC) (TGA) by DLS chromatography (GPC) using a Pump 1000 with a PL
ratio mass) (nm) aquagel-OH mixed-H 8-μm column connected to a differential
A1 1757 2733 1.18 44.38 1.8 refractometer, with water as the mobile phase at 25°C. Pullulan
A2 1889 2877 1.11 55.67 2.0 standard samples were used for calibration. Optical microscopy
A3 3446 2933 1.08 58.41 2.1 images were recorded using an Olympus AHBT3-VANOX
A4 3771 2964 1.09 75.01 2.2 (Tokyo, Japan).
DLS, dynamic light scattering; GPC, gel permeation chromatography; A 2 mg/mL solution of the sample was prepared in 50%
MALDI-TOF, matrix-assisted laser desorption–time of flight; PCA-PEG- acetonitrile in water with 0.1% trifluoroacetic acid (TFA). The
PCA, poly(citric acid)-poly(ethylene glycol)-poly(citric acid)' PDI, polydis- sample was then put into an alpha cyano-4-hydroxycinnamic acid
persity index; TGA, thermogravimetric analysis. (10 mg/mL) matrix, and matrix-assisted laser diffraction
* Molar ratios as follow: A1 (2), A2 (5), A3 (8), and A4 (10).
initiation–time of flight (MALDI-TOF) mass analysis was carried
out in a Voyager-DE STR instrument (Vernon Hills, Illinois).
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) pow- Synthesis of PCA-PEG-PCA copolymers and toxicity tests
der, LDH (lactate dehydrogenase) Kit, crystal violet staining/kit,
RPMI 1640 medium, and fetal calf serum were obtained from Procedures for synthesis of PCA-PEG-PCA copolymers,
Sigma-Aldrich, Roche (Indianapolis, Indiana), Promega (Madi- crystal violet test, MTT assay, LDH cytotoxicity assay, and
son, Wisconsin), Biologos, Illinois, and Biosera (East Sussex, hemolysis assay are reported in the Supplementary Material
United Kingdom), respectively. section (available in the online version of this article).
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded in DMSO-d6 on a Bruker DRX 400 MHz apparatus Molecular self-assembly of PCA-PEG-PCA copolymers
(Vernon Hills, Illinois) (Bruker) with the solvent proton signal
PCA-PEG-PCA copolymers (1 g) were dissolved in deio-
for reference. 13C NMR spectra were recorded on the same
nized water (50 mL) at 25°C and the solution left at this
instrument using the solvent carbon signal as a reference.
temperature without stirring. Then certain volumes of solution
Infrared (IR) measurements were performed using a Nicolet
were withdrawn at set time intervals and used for TEM, atomic
320 FTIR (Nicolet Instrument Corporation, Madison, Wiscon-
force microscopy (AFM), and DLS experiments immediately.
sin). Transmission electron microscopic (TEM) analyses were
performed by a LEO 912 AB electron microscope (JFE Encapsulation of fluorescein as a small guest molecule by
Enterprises, Brookeville, Maryland). Thermogravimetric analy- products of molecular self-assembly
ses (TGA) were carried out in a thermal analyzer (Model DSC
60; Shimadzu, Tokyo, Japan) under a dynamic atmosphere of Water solutions of fluorescein (typically 1 g/100 mL) and
an inert gas (i.e., N2) at 30 mL/min (room temperature [20- PCA-PEG-PCA copolymers (typically 1 g/50 mL) were mixed at
250C]). The particle size and polydispersity were determined room temperature and stirred for 1 hour. The resultant solution,
 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562 559

Figure 5. Representation of the molecular self assembly of A2 poly(citric acid)–poly(ethylene glycol)–poly(citric acid) copolymer in water by transmission
electron microscopy after (A) 1, (B) 2, (C) 3, (D) 4, (E) 5, (F) 6, (G) 7 days and optical microscopy after (H) 8, (J) 9 days.

containing fluorescein and copolymer, was left at room
temperature. Certain volumes of this solution were withdrawn
at set time intervals and used for different experiments.
Results
Linear-dendritic PCA-PEG-PCA copolymers were synthe-
sized through polycondensation reactions (Figure 1). In this
synthetic route, PEG (Mn = 1500) was used as core and citric
acid was polymerized on it. The molecular weight of copolymers
depended on the molar ratio of citric acid to PEG (CA/PEG). CA/
PEG = 2, 5, 8, and 10 molar ratios were used to synthesize
copolymers (referred to throughout this work as A1, A2, A3, and
A4, respectively).
The IR spectrum of A2 shows absorbance bands of
hydroxyl and carbonyl groups of PCA blocks at 2400–
3600 cm−1 and 1738 cm−1, respectively (see Supplementary
Figure 1, found in the Supplementary Material section in the
online version of this article).
1
H NMR spectra of this copolymer show chemical shifts at
12.5 parts per million (ppm) for acidic hydroxyl groups, 4.2 ppm
for end methylene groups of PEG, 3.6 ppm for PEG methylene
groups, and 2.6–2.8 ppm for methylene groups of PCA (AB
system) (see Supplementary Figure 2, found in the Supplemen-
tary Material section in the online version of this article).
In 13C NMR spectra, chemical shifts at 175, 170, 73, 68, 67,
64, and 40 ppm are assigned to the carbonyl and carbons of PCA
blocks. Chemical shift at 71 ppm is assigned to PEG (see
Supplementary Figure 3, found in the Supplementary Material
section in the online version of this article).
During the last decade, MALDI-TOF mass spectrometry has
become an essential tool for the characterization of dendrimers
and hyperbranched polymers. Figure 2 shows the MALDI-TOF
mass spectra of the synthesized copolymers. According to
MALDI-TOF mass spectrometry results, the molecular weights
of A1, A2, A3, and A4 were 1757, 1889, 3446, and 3771,
respectively. Whereas the molecular weight of PEG used was
1500, it was observed that the molecular weights of PCA blocks
were 257 for A1, 389 for A2, 1946 for A3, and 2271 for A4.
Figure 3 displays the GPC diagrams of A1, A2, A3, and A4.
Molecular weights of copolymers depended on the CA/PEG
molar ratios directly and were between 2730 and 2960. Because
of the self-assembly of copolymers in the solution state, there
560 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562

Figure 6. Optical microscopy images of fibers resulted from water solution of A2, A3, and A4 (from left to right) poly(citric acid)–poly(ethylene glycol)–poly
(citric acid) copolymers, which differ only in the size of the poly(citric acid) block.

Figure 8. Influence of concentration of A1 (gray bars) and A3 (white bars)

copolymers on viability of HT1080 cell line after 24 hours' incubation time
Figure 7. Encapsulation of fluorescein by fibers resulted from molecular self- (MTT assay). The red bars show the result for untreated control cultures.
assembly of A2.

DLS experiments showed that the diameter of synthesized

was a small difference between their molecular weights
compared to the MALDI-TOF mass spectrometry results
(Table 1). However, in low CA/PEG molar ratios a shoulder at
higher molecular weights can be seen. It seems that in low CA/
PEG molar ratios homogeneity of reaction decreases, and the
proportion of citric acid is not sufficient to react with all end
hydroxyl functional groups of PEG. Polydispersity indexes of
molecular weights of copolymers were relatively narrow—
between 1.08 and 1.18 (Table 1).
TGA experiments confirmed the MALDI-TOF mass
spectrometry and GPC results. According to these experiments,
the PCA content of PCA-PEG-PCA copolymers directly
depended on the CA/PEG molar ratios. There are two main
weight-loss temperatures for synthesized copolymers; the first
(at 150–210°C) is assigned to the decomposition of PCA
blocks, and the second (at 380–410°C) is related to the
decomposition of the PEG block (see Supplementary Figure 4
in the Supplementary Material section in the online version of
this article). According to the TGA diagrams, the PCA
contents for A1, A2, A3, and A4 are 44%, 55%, 68%, and
75%, respectively.
PCA-PEG-PCA copolymers in a fresh solution (1 g/100 mL) is
smaller than 5 nm and that there is a direct relationship between
size of PCA-PEG-PCA copolymers and CA/PEG molar ratios
(see Supplementary Figure 5 in the Supplementary Material
section in the online version of this article; and Table 1).
Molecular self-assembly was observed for PCA-PEG-PCA
copolymers in aqueous solutions. When a water solution of
copolymers was left at room temperature, supramolecular
assemblies appeared gradually, so that after several days they
were observed visually.
Figure 4 shows the AFM image of molecular assemblies
of A2 produced in water after 5 days. As can be seen, after
this time point the size of molecular self-assemblies had reached
40–50 nm. In this figure each particle is coupled with other
particles, precluding detection of a single particle; in other
words, particles are self-assembling.
The molecular self-assembly of copolymers was investigated
using TEM experiments. In these experiments water solutions of
copolymers (typically 1 g/50 mL) were prepared and their self-
assembly followed day by day. Figure 5 shows the molecular self-
assembly of A2 during 9 days. During the first day aggregations
 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562
Figure 9. Effect of concentration of A1 (gray bars) and A3 (white bars)
copolymers on functionality of HT1080 cell line after 24 hours' incubation
(crystal violet staining assay). The red bars show the result for untreated
negative control cultures.
appeared as small particles with an average size around 3 nm. On
days 2, 3, and 4 particle sizes increased gradually, reaching 5, 7,
and 12 nm, respectively. On day 5 the size of particles increased
steeply—probably as the result of self-assembly of produced
particles (see also Figure 4). After 6 days the size of particles had
reached 150 nm (Figure 5, F). On day 7 rodlike particles, which
are the precursors of the fibers, had appeared (Figure 5, G). Self-
assembly of rodlike aggregations finally led to the long fibers.
After day 7 the size of aggregations had increased sufficiently to
investigate by optical microscopy. On days 8 and 9 the fiber-like
objects were observed by optical microscopy.
DLS experiments were used to investigate the self-assembly
of PCA-PEG-PCA copolymers in water. For this reason DLS
diagrams of A2 (1 g/50 mL) in water during 4 days were
recorded (see Supplementary Figure 6 in the Supplementary
Material section in the online version of this article). On day 1
the diameter of aggregations was around 1 nm. On days 2, 3,
and 4 not only had the diameter and size distribution of
aggregations increased, but multiple peaks had also appeared.
These results confirmed the TEM observations and self-
assembly of copolymers.
Size and shape of supramolecular assemblies depended on the
structure of copolymers. Figure 6 shows the optical microscopy
images of fibers produced by molecular self-assembly of
copolymers, which differ only in the size of PCA blocks. This
figure shows that the size of PCA blocks directly affects the
diameter of the fibers produced, so that copolymers with larger
PCA blocks produce fibers with greater diameters.
The diameter of fibers resulting from molecular self-assembly
of A2 after 10 days was 1.2 μm, whereas for A3 and A4
diameters were 2 and 3.5 μm, respectively.
Interestingly, the products of molecular self-assembly of
PCA-PEG-PCA copolymers were able to encapsulate fluores-
cein as a small guest molecule. Figure 7 shows a fiber resulting
from molecular self-assembly of A2 in the presence of
fluorescein. As can be clearly seen, fluorescein has been
encapsulated in certain segments of the fiber. Probably these are
the fiber segments containing assembled PCA blocks, because
these blocks contain a large number of functional groups that
can interact with fluorescein.
561
Figure 10. Effect of concentration of A1 (gray bars) and A3 (white bars)
copolymers on cell membrane integrity after 24 hours' incubation. The red
bars show the result for dimethyl sulfoxide (0.1% vol/vol)–treated negative
control cells.
Because molecular self-assemblies of PCA-PEG-PCA
copolymers can encapsulate fluorescein, they can be used to
transport drugs and other molecules in biological systems. One
of the most interesting topics to investigate in the use of these
copolymers in biological systems is the effect of their
molecular self-assembly on their toxicity, transport capacity,
reorganization by the immunogenic system, and internalization
into the tissues and cells. Thus, to investigate their potential
application in biological systems (especially for drug delivery)
and to understand the limitation and capability of these
materials as nanoexcipients in such systems, we performed
different types of short-term in vitro cytotoxicity experiments
on the HT1080 (human fibrosarcoma) cell line, as well as
hemocompatibility tests.
The results of the MTT assay, as an indicator of
mitochondrial function, showed negligible toxicity compared
with the control group up to 1 mg/mL concentration after
24 hours' incubation (Figure 8).
The crystal violet staining assay is a colorimetric method
based on counting the adherent and almost functional cells that
can take up the dye. The results of the crystal violet staining
assay for A1 and A3 after 24 hours' incubation are shown in
Figure 9. According to these results, negligible toxicity
compared with the control group up to 0.5 mg/mL concentration
was observed after 24 hours' incubation, whereas 1 μg/mL
concentration showed toxicity against the controlled medium
after this time.
Figure 10 shows the results of the LDH assay for treated cells
with A1 and A3 copolymers against 0.1% (vol/vol) DMSO as
negative control, after 24 hours' incubation. According to these
results, toxicity was observed only for 2 mg/mL concentration.
Hemolysis assay showed no sign of harsh hemolysis (b9%)
for A1 and A3 copolymers up to 0.5 mg/mL concentration
within 2 hours of incubation (see Supplementary Figure 7 in
the Supplementary Material section in the online version of
this article).
Discussion
Results of the crystal violet staining test and of the LDH and
hemolysis assays showed a dose toxicity profile that can be
ascribed to a variety of reasons including chemical and physical
562 A.T. Naeini et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 556–562
interactions between copolymers and cellular components or
cell membranes.28-30
In this work fresh water solutions of copolymers were used for
toxicity tests. As mentioned above, as time passed copolymers
tended to occur in increasingly aggregated forms; thus, the
toxicity profile of copolymers should be assigned to both
nonaggregated (in the preliminary stages) and aggregated
forms. In other words, molecular self-assembly of copolymers
is a dynamic process, and its products grow during toxicity
studies; hence their interactions with the cell membrane can
change and induce heterogeneous interactions with it. As can be
seen from this explanation, the molecular self-assembly of
synthesized copolymers is important in the case of toxicity and
biological applications.
However, many points must be clarified to understand the
role of molecular self-assembly of copolymers in their interac-
tions with biological media and with cell membranes and
organelles. To achieve a comprehensive understanding, the
effect of all factors such as size of PCA blocks and concentration
of copolymers on the molecular self-assemblies and their
interactions with biological media in vitro and in vivo conditions
should be investigated.
According to the biocompatibility tests (crystal violet,
hemolysis, LDH, and MTT assays), the toxicity of synthesized
copolymers in 1 mg/mL or higher concentrations was consid-
erable. Because self-assembly depends directly on the concen-
tration of copolymers, it is probable that in high concentrations
self-assembly is the main reason for toxicity of the materials.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.nano.2009.11.008.
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