Article
pubs.acs.org/Langmuir
Preparation of Water-Soluble Hyperbranched Polyester
Nanoparticles with Sulfonic Acid Functional Groups and Their
Micelles Behavior, Anticoagulant Effect and Cytotoxicity
Qiaorong Han,∥ Xiaohan Chen,†,∥ Yanlian Niu,† Bo Zhao,† Bingxiang Wang,† Chun Mao,*,† Libin Chen,†
and Jian Shen*,†,‡
†
Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of
Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P.R. China
‡
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P.R. China
S Supporting Information
*
ABSTRACT: Biocompatibility of nanoparticles has been attracting
great interest in the development of nanoscience and nanotechnology. Herein, the aliphatic water-soluble hyperbranched
polyester nanoparticles with sulfonic acid functional groups
(HBPE-SO3 NPs) were synthesized and characterized. They are
amphiphilic polymeric nanoparticles with hydrophobic hyperbranched polyester (HBPE) core and hydrophilic sulfonic acid
terminal groups. Based on our observations, we believe there are two
forms of HBPE-SO3 NPs in water under different conditions:
unimolecular micelles and large multimolecular micelles. The
biocompatibility and anticoagulant effect of the HBPE-SO3 NPs
were investigated using coagulation tests, hemolysis assay, morphological changes of red blood cells (RBCs), complement and platelet
activation detection, and cytotoxicity (MTT). The results confirmed that the sulfonic acid terminal groups can substantially
enhance the anticoagulant property of HBPE, and the HBPE-SO3 NPs have the potential to be used in nanomedicine due to
their good bioproperties.
■
components of the blood coagulation system are activated.26
The blood compatibility of biomaterials continues need to be
improved and evaluated for further biomedical applications.
Biocompatibility of polymers is directly related to their
architecture, molecular weight, and surface chemistry.27−30 At
present, the original material of hyperbranched polyester
(HBPE) we used only dissolve in organic solvent (e.g.,
dimethyl sulphoxide (DMSO), tetrahydrofuran (THF)) but
not in water. However, it cannot meet the requirement of
biological systems.
In this paper, we synthesized water-soluble nanoparticles by
the chemical modification of aliphatic HBPE with sulfonic acid
functional groups (HBPE-SO3 NPs). The micelles behavior of
HBPE-SO3 NPs in aqueous solution was investigated by
transmission electron microscopy (TEM) and calculator
simulation method. Moreover, a series of specialized experiments were used to assess their blood compatibility and
cytotoxicity.
INTRODUCTION
Hyperbranched polymers have attracted significant interest
because of their unique architecture and novel properties that
include good solubility, special viscosity behavior, and high
density of their functional groups.1−6 Owing to the multifunctionality in hyperbranched polymers, the physical properties can be adjusted to a large extent by the chemical
modification of the end-groups.7,8 The use of hyperbranched
polymers by the chemical modification has attracted increasing
attention in recent years.9−12 These features of hyperbranched
polymers have been used extensively in diverse fields, such as
coatings, additives, blends, nonlinear optics, composites, and
copolymers.13−16 Especially, hyperbranched polymers hold
great potential as drug delivery agents because of their threedimensional shapes and availability of a large number of surface
functional groups amenable to various modification chemistries
for drug conjugation and targeting purposes.17−21
Much attention has been paid to the synthesis methods and
the drug delivery efficiency of hyperbranched polymers, but
there is not enough research works that focus on the blood
compatibility of these materials when they used in the blood
circulation system.22−25 As is well-known, when in contact with
blood, most of conventional and currently used polymers are
still prone to induce clot formation, as platelets and other
© 2013 American Chemical Society
Received: March 5, 2013
Revised: May 23, 2013
Published: May 29, 2013
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Article
spectrometer (EDS) with vantage digital acquisition engine (Thermo
Noran, U.S.). The morphology and structure of the samples were
characterized by TEM which was carried out by HITACHI H-7650
(Hitachi, Japan) and JEOL-2000F (JEOL, Japan). Specimens for
inspection were prepared on a 200 mesh copper grid by slowly
evaporating a drop of prepared solutions covered by a carbonsupported film at room temperature. The Zeta Potential (ζ) of HBPESO3 NPs was detected using a Nano ZS90 Zetasizer (Malvern
Instruments, UK). The measurements were made in automatic mode,
and the data was analyzed using the software supplied by the
manufacturer.
The Self-Assemble Behavior of the HBPE-SO3 NPs Investigated by Calculator Simulation Method. Molecular docking
simulations were performed using the ZDOCK program37,38
integrated into Discovery Studio 2.1 software package,39 The
ZDOCK program is an docking algorithm that provides near-native
structure predictions, where scoring function includes a combination
of shape complementarity. In this study, one HBPE-SO3 unimolecular
micelle was defined as a receptor and the other was defined as a ligand
for their docking. 100 model complexes were generated and the values
of ZDOCK Score were used to choose the optimal complex. The
complex was then subjected to energy minimization for 10 000 steps
by the steepest descents and 10 000 steps by conjugation gradient.
Coagulation Tests. The coagulation assays were performed and
measured by using a Semi automated Coagulometer (RT-2204C,
Rayto, U.S.). The antithrombogenicity of the samples were evaluated
by in vitro coagulative time tests, activated partial thromboplastin time
(APTT), prothrombin time (PT) and thrombin time (TT) tests.
Blood was drawn from healthy New Zealand white rabbits containing
sodium citrate. The platelet-poor plasma (PPP) was obtained by
centrifuging blood at 3000 rpm for 20 min. The final concentrations of
the test samples mixed with PPP were 0.1, 1, 10, and 20 mg/mL.
HBPE-SO3 NPs were dissolved in phosphate buffered saline (PBS),
while HBPE was dissolved in PBS and DMSO mixture solution
(HBPE is not hydrosoluble), and the final DMSO concentration was
1% v/v. PBS was used as a control.
Hemolysis Assay. Red blood cells (RBCs) were separated from
the blood by centrifugation (1500 rpm, 10 min) and washed five times
with PBS. They were used immediately after isolation. Two mL of
diluted 2% RBC suspensions were added to 2 mL sample solutions at
systematically varied concentrations. The final concentrations were 0.1,
1, 10, and 20 mg/mL, HBPE-SO3 NPs were dissolved in PBS, while
HBPE was dissolved in PBS and DMSO mixture solution, and the final
DMSO concentration was 1% v/v). PBS was used as a negative control
whereas DI was used as a positive control. The mixtures were
incubated at 37 °C for 3h then centrifuged at 1500 r/min for 10 min.
The absorbance of the supernatant was measured for release of
hemoglobin at 545 nm.
The percent hemolysis of RBCs was calculated using the following
formula:
EXPERIMENTAL SECTION
Materials. 2,2-Bis(hydroxymethyl)propionic acid (DMPA) was
purchased from Sigma-Aldrich Co. Ltd. and used as received.
Trimethylol propane (TMP), sodium hydride and 1,3-propanesultone
were obtained from Energy Chemical Co. Ltd., China. All solvents are
AR grade and purchased from Sinopharm Chemical Reagent Co. Ltd.,
China. THF was dried by refluxing over sodium and distilled just prior
to use. HUVECs were purchased from Bogoo Biotechnology
Company, China. Human embryonic kidney (HEK 293) cell lines
were purchased from Goybio Biotechnology Company, China. Fetal
bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)
and trypsin/EDTA 0.25% were obtained from Invitrogen (U.S.). All
the other reagents used in the experiments are AR grade. Doubly
distilled deionized (DI) water was obtained from a Milli-Q water
purification system and used throughout the study.
Synthesis of HBPE. All synthetic procedures were carried out
under a dry nitrogen atmosphere. HBPE with TMP as a core was
prepared by a procedure described in the previous literature.31−34 The
schematic diagram illustrating the synthesis route was presented in
Scheme 1. Briefly, esterification reaction was carried out at 140 °C
Scheme 1. Schematic Diagram Illustrating the Synthesis
Route of HBPE-SO3 NPs
with p-toluenesulfonic acid (p-TSA) as an acid catalyst. The chosen
molar ratio of TMP to DMPA was 1:9 corresponding to the
theoretical molecular weight of 1179 g/mol and a HBPE with 12
terminal hydroxyl groups. The crude polymer was precipitated from
acetone in n-hexane and dried under vacuum.
Synthesis of HBPE-SO3 NPs. The HBPE (1.00 g, with 9.6 mM of
−OH groups) was dissolved in THF in a three-necked roundbottomed flask equipped with a magnetic stirrer and a reflux condenser
with a drying tube. Excess sodium hydride (2.0 equiv to −OH groups),
corresponding to the theoretical quantity of hydroxy groups of HBPE,
was then dissolved in THF and added to the polymer solution. The
reaction mixtures were reacted at 70 °C for 12 h with stirring. Then
1,3-propane sulfone was added at 70 °C and the mixture was allowed
to react for more than 12 h . The resulting product was filtered and
dissolved in DMSO, then precipitated in THF and dried in vacuum
oven. The crude product was purified by dialysis through dialysis bag
(MWCO 500) for at least five days. During the dialyzing process, the
fresh DI water was exchanged at appropriate intervals. After dialysis,
the solution was dried under vacuum to give a product HBPE-SO3
NPs that ranged in color from off-white to tan (esterifications
conducted in THF tended to give more colored products).35,36
Characterization. The FTIR spectra were obtained with KBr
pellets on a Bruker Tensor 27 (Bruker, Germany). 1H NMR and 3C
NMR (400 MHz) spectra were recorded on a Bruker Avance 400
spectrometers (Bruker, Germany) at room temperature. The electrospray ionization mass spectrometry (ESI-MS) was obtain from mass
spectrometer (LCQ/M/Z = 50−1850, Finnigan, U.S.). The
compositions of the sample were determined using energy dispersive
%haemolysis = ((sample absorbance
− negative control absorbance)/(positive control absorbance
− negative control absorbance)) × 100%
Morphological Changes of RBCs. For observing morphological
changes of treated RBCs at the early stages of hemolysis, the HBPESO3 NPs were diluted to the required concentrations in RBC
suspensions. The cell pellets obtained after 1.5 h by centrifugation,
were diluted in PBS, and mounted on clean glass slides covered with
coverslips and observed under an Olympus BX41 microscope with a
camera (Olympus E-620, Olympus Ltd., Japan).
Complement Activation. The complement activation of the
samples was determined by turbidimetry method assessing the
depletion of complement C3. Activation studies were performed on
PPP isolated by centrifugation from human whole blood donations.
The sample solutions were incubated for 1 h at 37 °C with PPP, the
final concentration was 1 mg/mL (the final DMSO concentration in
HBPE solution was 1% v/v). The assays were done as per the protocol
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provided by a commercial C3a enzyme immunoassay kit (BD
Biosciences, U.S.). All the complement activation experiments were
done in triplicates.
Platelet Activation Assay. To measure the platelet activation, the
platelet rich plasma (PRP) was incubated at 37 °C with the sample
solution where the final samples concentration was 1 mg/mL (the final
DMSO concentration in HBPE solution was 1% v/v). The incubation
mixture was removed at 30 min to assess the activation state of the
platelets using fluorescence flow cytometry. Expression of the
fluorescently labeled platelet activation marker anti-CD62P and the
platelet pan-marker anti-CD42a was detected using a BD FACSCalibur (BD Biosciences, U.S.). All the platelet activation experiments
were done in triplicates.
MTT Assay. The cytotoxicity of HBPE-SO3 NPs as well as HBPE
was assessed by MTT assay that carried out according to the methods
described previously.40−42 Two kinds of cells (HUVECs and HEK 293
cells) were used as the research objects. The cells were cultured in
DMEM medium supplemented with 10% FBS in 96-well culture
plates. The culture was kept in a 5% CO2 atmosphere for 48 h at 37
°C. Then they were detached using 0.25% trypsin-EDTA. Subsequently, the cells were subcultured once again. The media was
changed by fresh ones, and the HBPE (the final DMSO concentration
in HBPE solution was 1% v/v) and HBPE-SO3 NPs samples with
different concentrations were added to the wells at a density of 2 × 104
cells/well (HUVECs) or 1 × 104 cells/well (HEK 293 cells). The cells
of positive control were only incubated with equal Dulbecco’s
modified Eagle’s medium (DMEM) with 10% FBS. All of the cells
were allowed to grow for 72 h before 10 mL MTT (5 mg/mL) was
added to each well. Then, the cells were incubated at 37 °C for an
additional 4 h until purple precipitates were visible. The medium was
replaced by 100 mL DMSO and the cell plate was vibrated for 15 min
at room temperature to dissolve the crystals formed by the living cells.
Finally, the absorption at 540 nm of each well was measured by an
ELISA reader (Behring ELISA Processor, Germany). All of the
samples were assayed in triplicate, and the mean value for each
experiment was calculated. The obtained results are expressed as a
percentage of the control, which is considered to be 100%.
that the original intermolecular hydrogen bonding in HBPE
was broken during sulfation.44 Similarly, the intensity of the
band at 2900 cm−1, attributed to the stretching and/or
deformation vibration of C−O−H bonds, was decreased in
the spectrum of HBPE-SO3 NPs. The new vibration bands at
1195 cm−1 and 1045 cm−1 appeared in HBPE-SO3 NPs, which
were identified as OSO symmetric stretching and SO3−
stretching modes in sulfonic acid groups, respectively.45 The
results indicates that HBPE-SO3 NPs were synthesized based
on a HBPE with terminal hydroxyl groups by modified with
sulfonic acid groups.
The elemental composition of the HBPE-SO3 NPs was
determined using energy dispersive spectrometer (EDS) with
Vantage Digital Acquisition Engine (Thermo NoranA). It was
observed from Figure 2 that the new product HBPE-SO3 NPs
Figure 2. EDS spectrum of HBPE-SO3 NPs.
are composed of the elements C, O, Na, and S. Appearance of
the new element sulfur indicates that the modification is
successful. The result is consistent with FTIR.
The 1H NMR spectrum of HBPE-SO3 NPs with (−SO3)arm=
6 was shown in Figure 3. Comparing the 1H NMR spectrum of
HBPE-SO3 NPs with that of HBPE, new proton signals
appeared at 3.14−3.43, 2.46−2.53, 1.68−1.89 ppm (protons h,
i, and j), which confirmed that 1,3-propane sultone was grafted
successfully through the formation of ester bondings. The
conversion ratio from hydroxyl groups to sulfonic acid groups
was calculated by comparing the integral of peaks f and g with
the integral of peaks a and c (1−5Sf, g /2Sa, c) was about 50%.
(HBPE: 1H NMR (DMSO-d6, ppm): 0.81(t, 3H, CH3CH2−),
1.08(t, 27H, CH3−CR3), 1.33(q, 2H, CH3CH2−), 3.42(t, 24H,
CH2OH), 4.10 (m, 18H, R3C −CH2−OOC), 4.62 (6H,
CH2OH), 4.94 (6H, CH2OH). HBPE-SO3 NPs: 1H NMR
(DMSO-d6, ppm): 0.97(CH3CH2−), 1.08(t, CH3−CR3),
1.21(CH 3 CH 2 −), 1.68−1.89 (m, −OCH 2 −CH 2 −CH 2 −
SO3Na), 2.46−2.53 (t, −OCH2−CH2−CH2−SO3Na), 3.14−
3.43 (m, R3C −CH2−OH, −OCH2−CH2−CH2−SO3Na),
4.03−4.09 (m, R3C −CH2−OOC), 4.50 (CH2OH), 4.71
(CH2OH)).
13
C NMR (in DMSO-d6) spectrum and the ESI-MS of the
HBPE-SO3 NPs were also used to analyze the structure of
samples and precisely calculate the functionalization efficiency
(Supporting Information Figure 1S and 2S).
Micelles Behavior of HBPE-SO3 NPs in Aqueous
Solution. TEM images were performed to estimate the size
and morphology of the HBPE-SO3 NPs. As shown in Figure 4,
the HBPE-SO3 NPs have two forms that include unimolecular
micelles (around 5 nm, Figure 4A) and large multimolecular
micelles (around 50 nm, Figure 4B) in water under different
conditions.46 The amphiphilic architecture of the unimolecular
■. RESULTS AND DISCUSSION
Characterization of HBPE-SO3 NPs. In this case, the
hydroxy-terminated aliphatic HBPE was first activated by NaH
to form HBPE-O-(oxygen anion) and then reacted with 1,3propane sultone to obtain the sulfonic acid functionalized
aliphatic HBPE. The presence of HBPE-SO3 NPs was
confirmed by the FTIR as shown in Figure 1. As exhibited in
the FTIR spectrum of HBPE, the absorptions at 3400 cm−1 and
1735 cm−1 were attributed to the hydroxy stretching and ester
carbonyl asymmetrical.43 Comparing with HBPE, the −OH
stretching vibration bands of HBPE-SO3 NPs at 3400 cm−1
appeared wider and shifted to higher wavenumber, suggesting
Figure 1. FTIR spectra of HBPE and HBPE-SO3 NPs.
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Figure 3. 1H NMR spectra of HBPE and HBPE-SO3 NPs in DMSO-d6.
Figure 4. The TEM images for HBPE-SO3 NPs. (A) unimolecular micelles, the sample aqueous solution was 0.01 mg/mL and treated by ultrasonic
100 Hz for 5 min (The hydrophobic block, HBPE, was expressed by black line, and the hydrophilic block, sulfonic acid functional groups, was
expressed by blue sphere); (B) large multimolecular micelles obtained by the aggregation of unimolecular micelles, the sample was 1 mg/mL at 4 °C
for 3 days.
mV, and this strong negative potential also contributed to the
stability of the large multimolecular micelles.
Anticoagulation Properties. Blood clotting is the result of
a complex process initiated by the intrinsic system or the
extrinsic system and/or a common pathway. As the various
coagulation assays indicate the interactions with different stages
of the coagulation, they provide basic information about the
mode of action of anticoagulants. The APTT, TT, and PT of
control samples for healthy plasma are 13.9 ± 0.46, 16.7 ± 0.47,
and 7.4 ± 0.57 s, respectively. As shown in Figure 5, in the
concentration range of 0.1, 1, 10, 20 mg/mL, the APTT values
of HBPE are slightly lower than the data of control, indicate
that the HBPE itself has coagulative properties to a certain
extent. However, after sulfate modification, the APTT values of
the HBPE-SO3 NPs are higher than control with the
concentration increasement. TT has the same tendency as
APTT. Thus, HBPE-SO3 NPs inhibit both the intrinsic and/or
common pathways of coagulation and thrombin activity or
conversion of fibrinogen to fibrin. On the other hand, PT
values were almost unaffected by the two above samples. The
PT values suggest that HBPE-SO3 NPs almost do not produce
any effects on extrinsic pathway of coagulation. The above
results demonstrate that the HBPE-SO3 NPs have anticoagulation property which attributed to the surface
modification of HBPE with sulfonic acid functional groups.52,53
Complement Activation and Platelet Activation of
HBPE-SO3 NPs. The complement system consists of more
than 20 plasma proteins that function either as enzymes or as
micelles were formed by hydrophobic HBPE core and
hydrophilic sulfonic acid outer shell as shown in Figure
4A.47,48 Typical TEM photos in Figure 4B exhibit the fine
structures in every large multimolecular micelles. The
unimolecular micelles of HBPE-SO3 NPs aggregated into
approximate spherical large multimolecular micelles in water. It
was driven by the intermolecular interactions.49 The amplified
large micelles (arrows) in Figure 4B, clearly indicate that the
large micelles were aggregated and composed of small spherical
building units (unimolecular micelles).50 The self-assemble
behavior and mechanism of the HBPE-SO3 NPs were
investigated by calculator simulation method (Supporting
Information Figure 3S, 4S, and Table 1S). The size of micelles
is a very important parameter for intracellular drug delivery
because the small size (<200 nm) of micelles is beneficial to
maintain lowered level of reticuloendothelial system (RES)
uptake, minimal renal excretion, and effective enhanced
permeability and retention (EPR) effect for passive tumortargeting.51 In this case, the TEM image shows that the HBPESO3 NPs aggregated into approximate spherical micelles in
water, and the diameter was around 50 nm. The result indicates
that the size of HBPE-SO3 NPs is suitable for intracellular drug
delivery. The large multimolecular micelles solution was stored
at 4 °C for one month and it was observed that there was no
change in size and morphology. The results indicate that the
large multimolecular micelles are very stable in aqueous
solution. The zeta potential of the HBPE-SO3 NPs is −22.5
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occur during cardiopulmonary bypass, hemodialysis, as well as
with vascular grafts and catheters.55 Platelet activation upon
interaction with samples is another indication of blood
incompatibility as it could lead to thrombotic complications
under in vivo conditions. The platelet activation was measured
after incubating the samples in PRP for 30 min at 37 °C using
flow cytometry. Platelet activation was expressed as the
percentage of platelets positive for both of the bound
antibodies, anti-CD62P and anti-CD42a. As shown in Figure
6(b), the pristine HBPE caused platelet activation in some
degree. After sulfonic acid modification, the sample exhibits
similar activation behavior to that of the control sample,
indicating that the HBPE-SO3 NPs do not affect platelets
activation.
Hemolysis Assay of HBPE-SO3 NPs. Hemolysis of the
blood is an important problem associated with the
bioincompatibility of materials. Hemolysis causes the release
of hemoglobin and other internal components into the
surrounding fluid, which accelerates the formation of clotting
and thrombus.56 Less than 5% hemolysis is regarded as a
nontoxic effect level.57 During this analysis, PBS was used as a
negative control (0%) and DI as a positive control (100%) of
hemolysis. Figure 7 shows the hemolysis test results of the
Figure 5. APTT, TT, and PT assay of HBPE and HBPE-SO3 NPs (1%
v/v DMSO in PBS did as well, and indicated no statistically different
with the negative control).
binding proteins.54 Opsonization of synthetic carriers with
complement components such as C3a and C5a could
eventually lead to the clearance of such particles by the
reticuloendothelial system, which makes the elucidation of this
interaction significant. In this study, complement activation was
investigated by quantifying the release of C3a, which was shown
in Figure 6(a). PBS was used as a negative control. A significant
Figure 7. Hemolysis (%) after incubation of human erythrocytes with
the samples at different concentrations. DI was used as a positive
control while PBS as a negative control (1% v/v DMSO in PBS did as
well, and indicated no statistically significant different with PBS).
pristine HBPE and HBPE-SO3 NPs at different concentrations.
In this case, all samples have a negligible effect on hemolysis
(<5%) on RBCs comparing with the negative control (PBS).
However, compared with the pristine HBPE, HBPE-SO3 NPs
obviously reduced the hemolysis rate.
Morphological Changes of RBCs. Erythrocyte interaction
with samples is particularly important in the use of polymers for
in vivo applications. Aggregation, crenation and hemolysis are
indicators of interaction and incompatibility of samples with
red blood cells.58,59 In general, the untreated RBCs in PBS
appear in a normal biconcave shape (Figure 8a). As we know,
exposure to materials with the bad blood compatibility will
induce appearance of morphological aberrant forms for RBCs
such as echinocyte-like forms with numerous surface spikes,
swollen RBCs, and the phenomena of ghost cells (lysed
RBCs).60 Figure 8 shows the images of RBCs incubated with
HBPE-SO3 NPs. From Figure 8 we can conclude that the RBCs
do not show any morphological changes even at high
concentration (20 mg/mL) of HBPE-SO3 NPs. This result is
in agreement with the above hemolysis analysis.
Cytocompatibility. MTT assays, considered as the “gold
standard” for cytotoxicity, is a colorimetric assay that measures
the enzymatic activity of cellular mitochondria.61 In this case,
MTT assays were performed to test the effects of HBPE and
HBPE-SO3 NPs samples on the metabolic activity of cells. As
Figure 6. (a) Complement activation; and (b) platelet activation assay
of HBPE and HBPE-SO3 NPs. PBS was used as a negative control (1%
v/v DMSO in PBS did as well, and indicated no statistically different
with the negative control).
difference was observed between the PBS and pristine HBPE.
On the other hand, the HBPE-SO3 NPs were found to be
neutral to the complement system in the amount of C3a
produced even at high concentration (1 mg/mL).
Platelet activation (platelet release, PMP formation, Pselectin expression, aggregation) and adhesion are known to
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Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The work is supported by NSFJS (BK2011781, BE2011196),
the Priority Academic Program Development of Jiangsu Higher
Education Institutions, Major Program for the Natural Science
Fundamental Research of the Higher Education Institutions of
Jiangsu Province (12KJA150006), Key Scientific and Technological Project of Shanghai Municipal Science and Technology
Commission (10391901800), and Base of production,
education & research of prospective joint research project of
Jiangsu Province (BY2011109).
Figure 8. Optical images of RBCs treated by HBPE-SO3 NPs (a:
negative control (PBS); b: positive control (DI); c: 0.1 mg/mL; d: 1
mg/mL; e: 10 mg/mL; f: 20 mg/mL).
■
depicted in Figure 9A, all samples did not exerted any
noticeable cytotoxicity in HUVECs at concentration below 1
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hyperbranched polymers. Eur. Polym. J. 2004, 40, 1257−1281.
Figure 9. Cytocompatibility of HBPE and HBPE-SO3 NPs to (A)
HUVECs and (B) HEK 293 cells (The final concentration of samples
in the media: a: 0.01 mg/mL; b: 0.1 mg/mL; and c: 1 mg/mL).
mg/mL. Similarly, the samples did not destory the HEK 293
cell viability under experimental conditions (Figure 9B).
Moreover, the HBPE-SO3 NPs samples exhibited better
noncytotoxicity than the pristine HBPE samples. The reason
for the increase of cell viability in the presence of HBPE-SO3
NPs will be investigated in our further research. These in vitro
evaluations suggest that HBPE-SO3 NPs could be further used
as biomaterials.
■
CONCLUSION
In this paper, the water-soluble HBPE-SO3 NPs were
synthesized successfully and their micelles behavior, anticoagulant effect and cytotoxicity were also investigated. The results
showed that HBPE-SO3 NPs exhibit good anticoagulant effect
that attributed to the sulfonic acid functional groups that
grafting on the surface of HBPE. The strategies open up more
exciting possibilities of using HBPE materials in the blood
medium, and provide a promising platform for biomedical
applications in future.
■
ASSOCIATED CONTENT
S Supporting Information
*
Additional information as noted in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected] (C. M.); shenjian@nju.
edu.cn (J. S.).
Author Contributions
∥
REFERENCES
These authors contributed equally to this work.
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