Synthesis of the Hemoglobin-Conjugated Polymer Micelles by Thiol Michael Addition Reactions

Article abstract of DOI:10.1002/mabi.201500460

Amphiphilic triblock copolymers mPEG-b-PMAC-b-PCL are synthesized using methoxyl poly(ethylene glycol), cyclic carbonic ester monomer including acryloyl group, and ε-caprolactone. Copolymers are self-assembled into core–shell micelles in aqueous solution. Thiolated hemoglobin (Hb) is conjugated with micelles sufficiently through thiol Michael addition reaction to form hemoglobin nanoparticles (HbNs) with 200 nm in diameter. The conjugation of Hb onto the micelle surface is further confirmed by X-ray photoelectron spectroscopy. Feeding ratio of copolymer micelles to Hb at 1:3 would lead to the highest hemoglobin loading efficiency 36.7 wt%. The UV results demonstrate that the gas transporting capacity of HbNs is well remained after Hb is conjugated with polymeric micelles. Furthermore, the obtained HbNs have no obvious detrimental effects on blood components in vitro. This system may thus have great potential as one of the candidates to be developed as oxygen carriers and provide a reference for the modification of protein drugs. (Figure presented.) .

Full text of DOI:10.1002/mabi.201500460

Macromolecular
Bioscience
Full Paper
Synthesis of the Hemoglobin-Conjugated
Polymer Micelles by Thiol Michael Addition
Reactions
Yanxin Qi, Taihang Li,* Yupeng Wang, Xing Wei, Bin Li, Xuesi Chen,
Zhigang Xie, Xiabin Jing, Yubin Huang*
Amphiphilic triblock copolymers mPEG-b-PMAC-b-PCL are synthesized using meth-
oxyl poly(ethylene glycol), cyclic carbonic ester monomer including acryloyl group, and
ε-caprolactone. Copolymers are self-assembled into core–shell micelles in aqueous solution.
Thiolated hemoglobin (Hb) is conjugated with micelles sufficiently through thiol Michael
addition reaction to form hemoglobin nanoparticles (HbNs) with 200 nm in diameter. The
conjugation of Hb onto the micelle surface is further confirmed by X-ray photoelectron spec-
troscopy. Feeding ratio of copolymer micelles to Hb at 1:3 would lead to the highest hemoglobin
loading efficiency 36.7 wt%. The UV results demonstrate that
the gas transporting capacity of HbNs is well remained after
Hb is conjugated with polymeric micelles. Furthermore, the
obtained HbNs have no obvious detrimental effects on blood
components in vitro. This system may thus have great poten-
tial as one of the candidates to be developed as oxygen carriers
and provide a reference for the modification of protein drugs.
1. Introduction
bioconjugates have vast applications in food, chemical,
and pharmaceutical area.[1] An important require-
ment for protein modification is not to interfere with
the native structure of protein and thus compromise
its biological activity. For example, in the area of blood
Techniques pertaining to the modified proteins have
been developed intensively, since these kinds of
substitutes, modified hemoglobin (Hb) is
a central
Y. Qi, Dr. T. Li, Y. Wang, X. Wei, Prof. Z. Xie,
Prof. X. Jing, Prof. Y. Huang
component in most of the artificial oxygen carrier sys-
tems.[2–6] Application of Hb-based oxygen carriers has
the potential advantage of tackling current transfu-
sion medicine challenges such as blood shortages, red
blood cell storage lesions, and transmission of emerging
pathogens.[7]
Hemoglobin (MW = 64 500) is the dominant protein in
red blood cells (RBCs) and is responsible for the trans-
portation of oxygen. However, cell-free Hb could be
entrapped by the gaseous messenger molecules (NO and
CO) to induce vasoconstriction, hypertension, reduced
blood flow, and tissue oxygenation at microcircula-
tory levels.[8] It could also easily dissociate into dimers
(2αβ) in blood circulation, causing serious renal toxicity.
The design of next generation of artificial oxygen car-
riers is then attempting to prevent the clinical adverse
State Key Laboratory of Polymer Physics and Chemistry
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences
Changchun 130022, P. R. China
E-mail: thli@hisunpharm.com; ybhuang@ciac.ac.cn
Y. Wang, X. Wei
University of Chinese Academy of Sciences
Beijing 100049, P. R. China
Dr. B. Li
Department of Chemistry
SUNY Albany, Albany, NY 12222, USA
Prof. X. Chen
Key Laboratory of Polymer Ecomaterials
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences
Changchun 130022, P. R. China
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DOI: 10.1002/mabi.201500460
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THME (1,1,1-tris(hydroxyl methyl) ethane) was obtained from
Alfa Aesar. Benzaldehyde and Et3N were purchased from Beijing
Chemical Factory. Bovine hemoglobin (MW = 64 500) was pur-
chased from Shanghai Kayon Biological Technology Co. Ltd and
stabilized under CO atmosphere. DL-Dithiothreitol was from Bey-
otime. Other reagents were commercially available and used as
received.
effect of free Hb.[7] Scientists modified Hb through intra
crosslinking of the tetrameric Hb, inter crosslinking of
Hb (polyHb), encapsulation and polymer conjugation
with Hb.[9–12] The common applied modification reac-
tions included amidation reaction, disulfidation reac-
tion, thiol Michael addition, and so on. In a previous
study, we have reported artificial oxygen carriers based
on chemical conjugation of Hb onto micelle-surfaces
through click chemistry and carbonyl-amino condensa-
tion.[4,13,14] Click chemistry is a commonly convenient
tool for modification of bioactive molecules, since this
reaction is tolerable to water, oxygen, and a wide range
of functionalities, and could be preceded with almost
quantitative yields under mild conditions. However,
the Cu catalyst is always of major concern, because the
toxic copper ions are quite difficult to be completely
separated out.[15] Instead, the thiol Michael addition
was not only carried many attributes of click reactions,
but also requiring only small concentrations of rela-
tively benign catalysts and the ready availability of an
enormous range of both thiols and enes.[16] The reaction
is a hydrothiolation reaction and extremely rapid com-
plete in a matter of seconds, which can proceed under
a variety of conditions via catalytic processes mediated
by nucleophiles, acids, or bases. Furthermore, both acti-
vated and nonactivated species of the enes can serve as
suitable substrates.[17]
According to the above-mentioned advantages, thiol
Michael addition was applied to prepare polymer conju-
gated Hb in our work. Amphiphilic triblock copolymers
containing acryloyl groups in the middle blocks were
first synthesized and self-assembled into micelles. Hb
was thiolated using 2-iminothiolate (2-IMT) and then
conjugated onto the surface of the copolymer micelles
through thiol Michael addition to obtain HbNs. X-ray
photoelectron spectroscopy (XPS) was used to address
the conjugation of Hb onto micelles. Moreover, the gas
transporting capacity and blood compatibility of HbNs
were evaluated and confirmed. All these results con-
firmed the reliability, convenience, and high efficiency
of thiol Michael addition reaction for the modification of
functional proteins.
2.2. Characterization
1H Nuclear Magnetic Resonance (NMR) spectrums were recorded
on a Bruker AV 300MHz in CDCl3 at 25 °C. Gel permeation chro-
matography (GPC) measurements were conducted with a Waters
410 GPC equipped with Waters Styragel column (HT3) with CHCl3
as eluent (flow rate: 1 mL min−1, at 35 °C). Transmission electron
microscopy (TEM) measurements were performed on a JEOL JEM-
1011 electron microscope operating at an acceleration voltage of
100 kV. Inverted microscope was performed by Nikon Eclipse Ti.
Steady-state fluorescence excitation spectra of the solution were
monitored by a Perkin Elmer LS50B luminescence spectrometer
(emission wavelength: 390 nm, excitation bandwidth: 5 nm).
Particle size of the micelles and Hb-bearing micelles were deter-
mined by dynamic light scattering (DLS) with a vertically pola-
rized He–Ne laser (DAWN EOS, Wyatt Technology). All sample
solutions were filtered using disposable 0.45 μm Millipore filters
prior to analysis. Thermo Escalab MKII photoelectron spectro-
meter (VG Scientific) equipped with a standard Mg anode X-ray
source (Mg Kα X-rays at 1253.6 eV) and a 150 mm hemispherical
electron energy analyzer were used to analyze surface elemental
compositions. The Bernasek laboratory is equipped with a VG
Scientific ESCALAB MKII spectrometer capable of doing all of the
above with a line spread of as low as 1.1 eV and a spatial reso-
lution of 100 μm. UV–vis spectra were recorded on a UV-2450
UV–vis spectrophotometer (Shimadzu Scientific Instruments) at
room temperature.
2.3. Synthesis of 5-Methyl-5-Acryloyloxymethyl-1,
3-Dioxan-2-One (MAC)
The monomer MAC was synthesized in three steps (Scheme 1).
First, benzaldehyde (21.4 mL, 0.21 mol) was added dropwisely
to a stirred solution of THME (24.0 g, 0.3 mmol) and TsOH
2. Experimental Section
2.1. Materials
MPEG (MW = 2000 Da, 5000 Da) and 2-IMT (2-iminothiolane
hydrochloride) were from Sigma. ε-Caprolactone (ε-CL) was dis-
tilled over freshly powdered calcium hydride under reduced pres-
sure. Diethylzinc (ZnEt2) was kindly supplied by Prof. Xianhong
Wang from Changchun Institute of Applied Chemistry. Toluene
was dried over Na wire and distilled before use. TsOH (p-tolue-
nesulfonic acid monohydrate) was purchased from Aladdin.
Scheme 1. Synthetic route of monomer MAC.
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(1.2 g, 6.3 mmol) in 300 mL Tetrahydrofuran (THF) at room tem-
perature. The reaction mixture was neutralized with aqueous
ammonia after 24 h. The solvent was evaporated under
reduced pressure and the residues were dissolved in 150 mL of
CH2Cl2 and extracted twice with 150 mL of phosphate buffer
solution (pH 7.4). The organic phase was concentrated to yield
35 g (84%) of white powder.
(2H, s, –CH2–O–), 4.17 (2H, s, –CH2–O–C (O)–), 4.33 (2H, s,
–CH2–O–), 5.91–6.45 (3H, m, –CH=CH2).
2.5. Preparation of PAL Micelles
The acetone solution of PAL copolymer (100 mg of PAL in 5 mL
of acetone) was added dropwisely into PBS solution of (pH = 7.4,
40 mL) under vigorous agitation by a peristaltic pump at a flow
rate of 0.1 mL h−1. The final solution was then opened to air over-
night, allowing slow evaporation of acetone and formation of
micelles.
Secondly, to a stirred solution of the above-obtained white
solid (15 g, 72 mmol) and Et3N (20 mL, 114 mmol) in CH2Cl2
under the ice bath, acyloyl chloride (9.7 g, 108 mmol) was added
dropwisely. After 10 h reaction, the mixture was filtrated and
the filtrate was washed twice with PBS (pH = 7.4). The filtrate
was concentrated and dissolved in 200 mL of CH3OH/1.0 M HCl
(v/v 1:1) and stirred at room temperature for about 2 h. The
solution pH was then adjusted to 7.0 with 2 M NaOH, and then
was concentrated and extracted with ethyl acetate. The organic
phase was concentrated to obtain 9.1 g pale yellow liquid
(yield 73%).
The critical micellar concentration (CMC) of copolymer was
determined by fluorescence measurement using pyrene as a
probe. Pyrene was firstly dissolved in acetone for quantitation.
After acetone was evaporated, PAL triblock copolymer solutions
with different concentrations ranging from 10−4 to 0.1 g L−1 were
added into pyrene. The concentration of pyrene was controlled at
5.94 × 10−7 M. Before the measurements, samples were incubated
overnight at 37 °C. The fluorescence spectra of solution were
recorded on a fluorometer at room temperature. The intensity
ratio of I336/I333 was plotted against the polymer concentration to
determine the CMC.
Last, ethyl chloroformate (11.97 g, 0.11 mol) and 400 mL THF
were charged into the pale yellow liquid. Et3N (16 mL, 91.2 mmol)
was then added dropwisely under ice bath. After 24 h, the reac-
tion mixture was filtrated and the salts of Et3N were removed.
The filtrate was concentrated to yield crude product under
reduced pressure. The residues were crystallized three times
with cold diethyl ether. Yield: 4.8 g (45.9%).
2.6. MTT and Cytocompatibility
The MTT colorimetric assay was performed to detect cells via-
bility of the materials. Briefly, fibroblasts L929 cells (L929 cells)
were plated in 96-well plates (105 cells/well) for 24 h before
added with seven concentrations (0.01–1 mg mL−1) of micelles.
The incubation was continued for 24 h. After that, cells were
treated with 20 μL of 5 mg mL−1 MTT per well and incubated for
4 h at 37 °C.The culture medium was removed and the contained
crystals were dissolved in 150 μL of Dimethyl sulfoxide (DMSO).
The absorbance was measured at 490 nm.
To prepare copolymer membranes, 10 mg mL−1 of the
copolymer was added to the coverslip and dried in vacuum oven
for 48 h to form copolymer membranes. The membranes were
subsequently sterilized by UV irradiation for 30 min. L929 cells
were seeded on the membranes at a density of 2.5 × 104 cells/
well in 6-well plate and incubated at 37 °C in a humidified
atmosphere containing 5% CO2. At timed intervals of 24 and 48 h,
2.4. Synthesis of Copolymers Containing
Acryloyl Group
The mPEG-b-PMAC-b-PCL (PAL) triblock copolymer was
obtained through the sequential addition methods. Briefly,
mPEG (MW = 5000 Da) was dried via toluene azeotropic distil-
lation in polymerization vessel for about 2 h. After cooling
to room temperature, MAC monomer was added. Calculated
amount of ZnEt2 (5% mol of MAC and ε-CL) in toluene was
injected under argon atmosphere, and the vessel was sealed.
After reacting at 40 °C for 12 h, ε-CL was added under argon
atmosphere for another 12 h reaction. The reaction was ended
by cooling to room temperature, and the reactant was dissolved
in chloroform. The product PAL was precipitated out in cold die-
thyl ether, washed by methanol, and finally dried in vacuum.
1H NMR (300 Hz, CDCl3, 25 °C): δH 1.09 (3H, s, –CH3), 4.13
Figure 1. A) Typical 1HNMR spectra of triblock copolymers PAL and B) GPC curve of triblock copolymer PAL.
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Table 1. Properties of different PAL copolymers.
Sample Codesa)
MW ratio
In polymers
PDI
CMC [mg L−1
]
Rh [nm]
In feed
2:1:2
PAL
PAL2k-730-1.1k
PAL5k-750-1.49k
2:0.73:1.1
1.14
1.15
3.7
4.9
73
5:1:2
5:0.75:1.49
137
a)The subscript numbers of PAL means the molecular weights of PEG, PMAC, and PCL segments in PAL copolymer, respectively.
samples were rinsed twice with a sterile PBS solution and fixed
by 3% glutaraldehyde for 20 min at room temperature. Glutaral-
dehyde-fixed cells were stained by Giemsa stain for 30 min and
mounted with neutral resin. The samples were observed micro-
scopically using microscope.
2.8. X-Ray Photoelectron Spectroscopy
The surface elemental compositions analysis of Hb, micelles,
and HbNs were performed using photoelectron spectrometer
equipped with a standard Mg anode X-ray source and a 150 mm
hemispherical electron energy analyzer. The spectrums were
obtained for each sample using a takeoff angle of 90°.
The fresh whole blood obtained from Wistar rats were cul-
tivated on the membranes incubated at 37 °C for 12 h. Then,
the coverslips were rinsed by ddH2O and observed using
microscope.
2.9. Oxygen Binding and Dissociation Curves
Different states of Hb would be formed under different atmos-
pheres. The CO stabilized Hb (CO-Hb) could transform to oxy-Hb
or deoxygenated Hb (deoxy-Hb). The oxy-Hb could be gradually
converted to met-HbMs by exposing in air atmosphere at room
temperature. The oxygen binding and dissociation curves of the
HbNs were measured at different atmospheres using a UV–vis
spectrophotometry. The adsorption peaks of different states of
Hb were λmax = 415 nm (oxy-HbNs), λmax = 419 nm (CO-HbNs),
λmax = 430 nm (deoxy-HbNs), and λmax = 405 nm (met-HbNs).
2.7. Preparation of Hemoglobin Nanoparticles (HbNs)
10 mg Hb (under CO atmosphere) was first modified with 2-IMT
(20-fold molar of Hb) in an ice bath under N2 atmosphere gentle
stirring for 1.5 h and 0.007 mg DL-Dithiothreitol (DTT) was added.
After that, the micelle solution was allowed to mix with the modi-
fied Hb in PBS (pH = 7.4) for 6–8 h under CO atmosphere in an ice
bath. HbNs were separated out as the precipitant through ultra-
centrifugation (12 000 rpm 5 min−1) and washed with phosphate
buffer saline (PBS) (pH = 7.4) for three times. The Hb content in
HbNs was determined by UV–vis and the final Hb content in HbNs
was calculated using the following formulation:
2.10. Hemocompatibility of HbNs in vitro
Hb content in HbNs (wt%) = (Total protein in feed − Protein in
the supernatant)/Total amount of the precipitant
The 200 μL fresh whole blood obtained from Wistar rats was
stored in heparinized Eppendorf tubes, and mixed with 300 μL
of HbNs suspended in 0.9% stroke-physiological saline solution.
The mixture was incubated immediately at 37 °C in a water bath
incubator with constant shaking. At timed intervals of 0, 3, 6,
Following the similar experimental protocol, Hb without
modification with 2-IMT was also reacted with the PAL micelles
(pH = 9) via the Michael addition.
Figure 2. DLS results and TEM image of PAL2k-730-1.1k micelles (bar = 500 nm).
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and 12 h, one drop of the solution was sam-
pled for the RBC morphology observation
using microscope.
3. Results and Discussion
3.1. Synthesis and Characterization
of PAL Copolymers
The triblock PAL copolymers were suc-
cessfully synthesized by sequential
addition of MAC and ε-CL monomers
using mPEG as the microinitiator.
During this polymerization, lower tem-
perature (35 °C) was necessary to pre-
vent acryloyl groups from crosslinking.
Under this situation, acryloyl groups
were settled in the middle segment of
the triblock copolymers and allowed
to be on the surface of the subse-
quently formed micelles for further
modification. In 1H NMR spectrum,
the still remained chemical shifts at
Figure 3. Assessment of growth of L929 cell lines with the PAL2k-730-1.1k.
5.9–6.45 ppm clearly indicated the existence of the acry-
loyl groups in copolymer (Figure 1A). The GPC curve also
showed the high conversion of the PAL copolymers with
narrow Polydispersity Index (PDI) = 1.15 (Figure 1B).
viabilities were obtained, indicating the low cell-toxicity
and good biocompatibility of the PAL micelles (Figure 3).
We also evaluated the cytocompatibility of the
copolymer membrane. After the 24 h and 48 h culture,
seeded L929 cells were able to attach, grow, and multiply
on the surfaces of the copolymer membranes (Figure 4).
Cells grew well, which was in accordance with the close-
packed rule after 48 h. Furthermore, no nonspecific
adsorption was confirmed when the membrane was
cultivated with whole blood for 12 h and rinsed with
water (the date not shown).
3.2. Formation and Characterization of PAL Micelles
The triblock copolymer PAL could aggregate to form micelles
in aqueous media, due to its amphiphilic nature. The PEG
segment would form the hydrophilic shell, and the PCL seg-
ment became the hydrophobic core. Therefore, the acrylic
groups in the middle PMAC segment of the copolymer were
considered to be located on the surface of micelles, which
should be suitable for further Hb conjugation.
3.3. Preparation and Characterization of HbNs
To control the micelle properties, PAL copolymers with
differential block segments were synthesized and sum-
marized in Table 1. The results showed that the CMC
values of copolymer decreased with increasing the ratio of
the hydrophobic block to the hydrophilic block, implying
the increased stability of micelles. Higher molecular
weight of PCL segment would significantly increase the
particle size. In the case of PAL2k-730-1.1k micelles, TEM
images indicated spherical morphology with an average
diameter of 73 nm, which was quite accordant to the DLS
results (Figure 2).
Thiolated Hb was used to bind with PAL micelles via the
thiol Michael Addition. For this purpose, Hb was first thi-
olated by 2-IMT to create active thiol groups on Hb surface.
The in vitro cytotoxicity of PAL
micelles
PAL2k-730-1.1k
was evaluated using MTT assays by mixing the micelles
with L929 cells at different concentrations (0.01 and 1 mg
mL−1) of micelles to confirm the safety of micelles when
contacting with tissues and organs. More than 80% of cell
Figure 4. Micrographs of L929 cells on copolymer PAL2k-730-1.1k
membrane cultured for A) 24 h and B) 48 h.
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Figure 7. XPS spectrum of Hb, blank micelle, and HbNs.
Hb surface, and the exposed amino groups also have the
opportunity to react with the acryloyl groups on micelle
surface via the amino reaction[19] to immobilize Hb. How-
ever, this kind of amino reaction is preferred to take place
at pH = 8–9, and this strong alkaline could easily induce the
rapid hydrolysis of acryloyl group. Based on this considera-
tion, we selected a reaction pH of 7.4. It is obvious that the
amino reaction between Hb and micelles was extremely
restricted under this physiological condition (Figure 5). It is
indicated that thiolation of Hb is necessary and efficient to
obtain higher Hb loading content in HbNs.
After Hb conjugation, HbNs still maintained
nanoscaled spherical morphology with larger particle
size, which were confirmed by TEM and DLS (Figure 6).
The conjugation of Hb onto micelles was further inves-
tigated and confirmed by XPS. Characteristic peaks for
nitrogen around 399 eV could be observed from both Hb
and HbNs samples (Figure 7). Since blank micelles did
not contain any nitrogen element, and the only nitrogen
resource was from the amino acid residue in Hb, Hb mole-
cules could be confirmed to be located on the surface of
micelles in HbNs.
Figure 5. Relationship between the Hb content in micelles with
the Hb feed ratios under different reactions.
2-IMT (also called as Traut's reagent) has the specific char-
acter to selectively and spontaneously modify the primary
amines (like lysine residue on Hb). This modification not
only introduced SH groups to protein without producing
any toxic side product, but also preserved the positive
charge similar to the original amino groups to maintain
protein solubility. In our case, 20-fold molar excess of
2-IMT was allowed to react with Hb to generate enough
SH groups on Hb surface, making Hb to have more oppor-
tunity to bind with micelles. There were 5.9 free thiols per
Hb that were formed on average upon 2-IMT treatment.[18]
By increasing the thiolated Hb feeding ratio to micelles,
the Hb content in HbNs gradually increased to about
36.7 wt%, and then kept constant (Figure 5). The final
feeding ratio of thiolated Hb to micelles was decided to be
3:1 (w/w) for further application. It is needed to be men-
tioned that, there are many lysine residues located on
Figure 6. DLS results and TEM image of HbNs. HbNs were prepared from PAL2k-730-1.1k micelles at pH = 7.4 (bar = 500 nm).
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Figure 8. UV–vis spectra of HbNs and Hb under various atmosphere.
3.4. Gas Transporting Capacity of the HbNs
analysis of Hb conveniently. According to these, the gas
binding ability of HbNs was tested to determine whether
Hb conjugated micelles still remained the oxygen trans-
portation capacity. Figure 8 showed the typical UV–vis
characteristic absorbance peaks of HbNs, and the conver-
sion between different gas-binding states (CO-HbNs, oxy-
HbNs, and deoxy-HbNs) could be carried out reversibly as
the atmosphere changed in comparison with Hb. These
results indicated the excellent gas transporting capacity
and demonstrated that the biological activity of Hb was
not damaged after Hb was conjugated onto polymeric
micelles.
Hb is commonly known as the oxygen carrier since the
heme units inside Hb could bind and release O2 reversibly.
In fact, Hb has several different gas-binding states while
exposing to different atmospheres. For example, Hb could
rapidly bind with CO gas to form the very stable state
CO-Hb. And CO-Hb could transform to oxy-Hb or deoxy-
Hb under O2 or N2 atmosphere, respectively, under visible
light irradiation. If further exposed to air at room temper-
ature, oxy-Hb could be gradually oxidized to met-Hb and
lose the capacity to carry oxygen. All these states of Hb
have the specific adsorption peaks in UV–vis spectrum,
and could be used for the qualitative and quantitative
3.5. Blood Compatibility
The biocompatibility test was evaluated
utilizing the in vitro RBC morphology
with the sample by microscope.[20]
When blood was mixed with 60% PBS for
12 h, the number of RBC was decreased
significantly and the RBC morphology
changed because of hemolysis (Figure 9).
While HbNs were applied to mix with
blood, the RBC morphology remained
stable, indicating the satisfying blood
compatibility of HbNs system.
4. Conclusion
In summary, micelles formed by
amphiphilic triblock copolymer PAL
could successfully bind with thiolated
Hb molecules via thiol Michael addi-
Figure 9. Morphology of RBC mixed with different solutions observed by microscope
(bar = 20 μm).
tion with significantly improved
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morphology with particles size around 200 nm. HbNs
system had comparable gas transporting capacity
and had no obvious detrimental effects on blood com-
ponents. The thiol Michael addition provides a new
strategy for the synthesis of reliable and efficient Hb-
based artificial oxygen carrier, which could have the
potential to be applied in clinical applications such as
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Acknowledgements: Financial support was provided by the
Ministry of Science and Technology of China (863 Project, No.
SS2012AA020603) and National Natural Science Foundation of
China (Grant Nos. 51321062 and 21174143).
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Received: December 21, 2015; Revised: January 25, 2016;
Published online: March 3, 2016; DOI: 10.1002/mabi.201500460
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Keywords: hemoglobin; oxygen carriers; protein delivery; thiol
Michael addition
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