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Biomaterials 30 (2009) 5077–5085

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Hemoglobin conjugated micelles based on triblock biodegradable polymersas artificial oxygen carriers

Quan Shi a,b, Yubin Huang a, Xuesi Chen a, Meng Wu c, Jing Sun a,b, Xiabin Jing a,*

a State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of Chinab Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of Chinac Department of Materials Science and Engineering of Jilin University, Changchun 130025, People’s Republic of China

a r t i c l e i n f o

Article history:Received 21 March 2009Accepted 21 May 2009Available online 26 June 2009

Keywords:HemoglobinArtificial oxygen carrierRed blood cellsMicelles

* Corresponding author. Tel./fax: þ86 431 8526277E-mail address: xbjing@ciac.jl.cn (X. Jing).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.05.082

a b s t r a c t

An artificial oxygen carrier is constructed by conjugating hemoglobin molecules to biodegradablemicelles. Firstly a series of triblock copolymers (PEG–PMPC–PLA) in which the middle block containspendant propargyl groups were synthesized and characterized. After the amphiphilic copolymer wasself-assembled into core-shell micelles in aqueous solution, azidized hemoglobin molecules protected bycarbon monoxide (CO) were conjugated to the micelles via click reaction between the propargyl andazido groups. The conjugation causes an increase of the micelle’s mean diameter. Maximum conjugationratio is 250 wt% in the hemoglobin-conjugated micelles (HCMs). Oxygen-binding ability of the HCMs wasdemonstrated by converting the CO-binding state of the HCMs into O2-binding state.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Blood, which continuously delivers oxygen to, and takes carbondioxide away from all tissues and cells, is of most essential impor-tance for human lives. The use of blood products in medical practiceoften meets various difficulties, such as short preservation period,immunological responses, potential infections, blood type incom-patibility, and shortage of blood supply. Research of blood substituteshas attracted great attention and rapid progress has been made inthis field [1–4]. Among the blood-cell substitutes developed up todate, hemoglobin vesicles (HbV), prepared by encapsulating hemo-globin (Hb) molecules into lipid or polymeric vesicles, exhibit muchbetter performance than chemically modified Hbs. The HbVs, witha diameter of 100–250 nm, have a layer of membrane whichprevents the Hb molecules from contacting directly with theimmunological system [5] or penetrating vascular wall [6,7]. It isexpected to be the most promising artificial oxygen carrier [8–11],and is referred as ‘‘the third generation of blood-cell substitute’’. Thematrix used for HbVs should combine good biocompatibility, non-toxicity and non-immunogenicity. In 1957, Chang [1] first preparednylon and colloidin microcapsules. Recently, many studies werereported on liposome-encapsulated Hb (LEH) since Djordjevich andMiller established the method in 1977. PEG-modified lipid vesiclesproved to have longer circulation time in vivo [12]. Another HbV

5.

All rights reserved.

system is recombinant human serum albumin (rHSA) and hemo-globin hybrids. They are constructed by non-specific binding forceand have good O2 binding ability [13–15]. In most cases, HbVs needto be modified with PEG to guarantee long storage time, bloodcompatibility and extended circle life [2,16–18].

Recently, a biodegradable polymer, polylactide (PLA), was moreand more studied as the HbV matrix due to its favorable properties.Firstly, it is perfectly biocompatible and biodegradable, it can be finallydegraded into carbon dioxide and water in the body without sideeffects [19,20]. PLA-based materials have been safely used in manybiomedical applications [21,22]. Secondly, the structure of PLA-basedmaterial can be easily tailored to meet demands of different applica-tions. For example, PEG segment can be attached to the end of PLA toform an amphiphilic structure, which can self-assemble into micellesin aqueous solution and widely used in many biomedical fields[23,24]. Functional groups are also introduced into the backbone ofPLA by copolymerization of LA and other functionalized monomers[25–27]. Through these functional groups, many bioactive moleculescan be conjugated to the biodegradable polymer and endow it withcertain bioactivities [28–30]. Thirdly, compared with traditional HbVmatrixes, PLA-based materials are superior in mechanical properties,which lead to easier manufacturing, better stability and less cost [31].Chang’s group first tried to prepare artificial blood cells with PLAmaterial in 1976 [32]. Recently, they reported some approaches tonanoscale Hb encapsulation with PEG–PLA [33–35].

Usually, to achieve encapsulation, organic solvents are used todissolve polymers and then the polymer solution is homogenized withaqueous solution of proteins by vigorous stirring or ultrasonication.

HC

C

CH2

O

O

H3C

H2C

O

H2C

O

O

O

O

CH

CH3

O

O

CH

CH3

*

zy

O

OO

O

yOO

x

O O

O

O

O

CHC

H2C

OO

H3C

H2C

O

H2C

O

O

OHO

x +

+

Et2Zn

OO

x

O

O

O

Scheme 1. Synthesis of the triblock copolymer PEG–PMPC–PLA.

Table 1Related data on the copolymerization of MPC and LA initiated by PEG2000.a

Sample MPEG:MPMPC:MPLAb Mw/Mn

d Yield (%) CMC (mg/l)

Feed Productc

Q. Shi et al. / Biomaterials 30 (2009) 5077–50855078

These procedures often cause harmful effects on the activity ofproteins. Therefore, irreversible, quantitative and mild approaches toprotein encapsulation are preferred [36]. Among them, a strategynamed ‘‘click chemistry’’, which means azide/alkyne 1,3-dipolarcycloaddition in aqueous solution under Cu(I) catalysis, is widely usedin bioconjugation [37–39]. Especially in researches based on biode-gradable polymers, the condition is mild enough to keep the polymerfrom degradation while realizing perfect conjugation [40,41].

The purpose of this paper is to construct an oxygen carrier basedon hemoglobin-conjugated biodegradable polymer micelles. Firstlywe designed a PEG–PMPC–PLA triblock copolymer in which themiddle block PMPC contains pendant progargyl groups. After thetriblock copolymer was self-assembled into core-shell micelles inaqueous solution, azidized hemoglobin molecules were conjugatedonto the micelles via click reaction between the propargyl groups inthe micelles and the azido groups in the hemoglobin molecules. Inthe previous papers, we prepared a P(MPC–LA) random polymerand successfully conjugated BSA protein and antibodies on itssurface [42,43]. In those works, click reaction was carried outbetween propargyl groups on the solid surface and azido groups inaqueous solution. Herein, because the propargyl groups areattached to the core surface of the micelles, they are expected toreact with the azido groups without difficulty and the hemoglobinmolecules are hidden in the PEG corona of the micelles, beingprotected against the immunological systems.

PML261 2:0.6:1 2:0.51:1.23 1.67 91 12.1PML263 2:0.6:3 2:0.55:2.87 1.59 90 8.6PML265 2:0.6:5 2:0.58:5.62 1.20 91 3.2

a Molar ratio of Et2Zn to total amount of monomers was 1/200.b Mass ratios.c Determined by 1H NMR spectra.d Determined by GPC (with THF as eluent).

2. Experimental section

2.1. Materials

L-Lactide (LA) was prepared in our own laboratory and recrystallized from ethylacetate for three times prior to use. Cyclic carbonate monomer MPC was synthesized

and purified following the description in our previous paper [42]. Monomethyloxy-poly(ethylene glycol) (PEG) with a molecular weigh of 2000 was purchased fromAldrich. All other organic solvents and reagents were purchased from commercialsources and were used as-received, unless otherwise noted. Bovine hemoglobin waspurchased from Shanghai Kayon Biological Technology Co. Ltd. Carbon monoxidegas was obtained from Dalian Date Gas Co. Ltd. and used without furtherpurification.

2.2. General measurements

1H NMR spectra were measured by a Unity-400 NMR spectrometer at roomtemperature, chemical shifts are given in parts per million with tetramethylsilane asan internal reference. Gel permeation chromatography (GPC) measurements wereconducted with a Waters 410 GPC with CHCl3 as eluent (flow rate: 1 ml/min) at35 �C. The results of molecular weights were calibrated with polystyrene standards.Differential scanning calorimetry (DSC) analyses were carried out at a heating rate of10 �C/min on a Perkin Elmer Pyris 1. UV spectra were obtained on a UV-2401 UV–visspectrophotometer (Shimadzu Scientific Instruments).

2.3. Synthesis of the PEG–PMPC–PLA triblock copolymer (PML)

The PML copolymer was prepared by ring-opening polymerization of MPC andLA using PEG as a macroinitiator. Firstly, calculated amount of PEG2000 was placed

Fig. 1. 1H NMR spectrum of the copolymer PML263 in Table 1.

Q. Shi et al. / Biomaterials 30 (2009) 5077–5085 5079

in a flask and dried via toluene azeotropic distillation for 1 h. Then MPC monomerwas added in the flask followed by addition of calculated amount of ZnEt2 in toluenesolution as catalyst under argon atmosphere. The polymerization system was stirredand heated to 120 �C. After 12 h, LA monomer was added in the flask under argonatmosphere. After another 12 h period the polymerization was ended by cooling theflask to room temperature. The resultant copolymer PEG–PMPC–PLA (PML) waspurified by dissolving in chloroform and precipitating in cold methanol for severaltimes and dried in vacuum.

2.4. Preparation of the PML micelles

PML micelles were prepared as follows: Firstly, 100 mg PML polymer was dis-solved in 10 ml THF. Under vigorous stirring 20 ml double distilled water was

Fig. 2. 13C NMR spectrum of the c

dropped into the solution by a peristaltic pump at a flow rate of 0.1 ml/h. Then thesystem was opened to air overnight, allowing the slow evaporation of THF. Traceamount of THF was removed by rotary evaporation. The resultant aqueous solutionof micelles was lyophilized to obtain powder samples.

2.5. Micelle characterization

Critical micelle concentration was measured by fluorescence spectroscopy usingpyrene as a hydrophobic probe. A series of aqueous micelle solutions withconcentrations from 10�4 to 10�1 g/ml were prepared with saturated pyreneaqueous solution (6�10�7 mol/l). The flasks were kept at 25 �C overnight toequilibrate the pyrene partition between water and micelles. Then steady statefluorescence excitation spectra of the solution series were monitored by a Perkin

opolymer PML263 in Table 1.

Fig. 3. DSC curves of (1) PML261, (2) PML263, and (3) PML265 in Table 1 (secondheating run).

300 310 320 330 340 350 360

0

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In

te

ns

ity

(a

rb

itra

ry

u

nits

)

Wavelength(nm)

1

23456

Fig. 4. Excitation spectra of pyrene in aqueous solution of various PML copolymerconcentrations (lem¼ 390 nm).

Q. Shi et al. / Biomaterials 30 (2009) 5077–50855080

Elmer LS50B luminescence spectrometer (emission wavelength: 390 nm, excitationbandwidth: 5 nm).

Particle size and size distribution of the micelles were determined by dynamiclight scattering (DLS) with a vertically polarized He–Ne laser (DAWN EOS, WyattTechnology). The scattering angle was fixed at 90� and the measurement was carriedout at 25 �C. The sample solutions were filtered using disposable 0.45 mm Milliporefilters prior to analysis. The morphology of the micelles was examined by environ-mental scanning electron microscopy (ESEM) performed on an XL 30 ESEM FEGscanning electron microscope (Micrion FEI Philips).

2.6. Preparation of carbonylhemoglobin (CO–Hb)

Hb (500 mg) was dissolved in 250 ml phosphate buffer solution (PBS, pH¼ 7.4).Into 100 ml of Hb solution was added 25 mmol sodium ascorbate. Then with CO gasflowing over the fluid surface, the solution was stirred by a magnetic stirring bar tillthe UV spectrum of the solution remained constant. The resultant solution was thenlyophilized to obtain blood-red powders (CO–Hb). A standard curve of absorbance at419 nm vs. CO–Hb concentration was prepared.

2.7. Conjugation of CO–Hb onto PML micelles

CO–Hb was firstly azidized with NaN3 [42]. Then calculated amounts of theazidized CO–Hb and PML micelles were dissolved in PBS solution (pH¼ 7.4). CuSO4,sodium ascorbate and histidine were sequentially added to make their concentra-tion 6�10�5 mol/l in the mixed system. The mixture was then protected by N2

atmosphere and shaken at 37 �C. After 24 h, the mixture was separated by ultra-centrifugation at 15,000 g on a Sigma 1-14 benchtop centrifuge (Sartorius StedimBiotech S. A.). Hemoglobin-conjugated micelles (HCMs) from the ultracentrifugation

PLA-PMPC

= PEG

= Progargyl

Scheme 2. A micelle formed from PML copolymers.

was washed with PBS solution (pH¼ 7.4) for several times. Complete elution of thefree Hb molecules can be indicated by the disappearance of adsorption peak at 400–600 nm in the spectra of the eluate. All the eluted solutions were collected in a 50 mlvolumetric flask and diluted with PBS solution (pH¼ 7.4) to the calibration mark.After treated with CO gas for 2 h, absorbance value of the solution at 419 nm was

Fig. 5. ESEM photographs of (a) PML261 micelles and (b) CO–Hb conjugated PML261micelles.

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Fig. 6. Size distributions of PML261 (a), PML263 (b), and PML265 (c) micelles determined by DLS.

Q. Shi et al. / Biomaterials 30 (2009) 5077–5085 5081

measured. Quantity of the eluted CO–Hb can thus be calculated by virtue of thestandard curve.

2.8. O2 binding ability of HCM

HCM solution was decarbonylated by exposing it to an O2 atmosphere underirradiation of a visible light. The process was monitored by the 400–420 nmadsorption peak of the UV spectra.

350 400 450 500 550 600 6500.0

0.5

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1.5

2.00 min40 min80 min120 min160 min

Ab

so

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ce

Wavelength (nm)

Fig. 7. UV spectra changes of O2–Hb when treating with CO gas (scanning was per-formed at 40 min intervals, immediately after aerating of CO gas).

3. Results and discussion

3.1. Synthesis of copolymer PML

LA and cyclic carbonate monomers have been reported tocopolymerize by the initiation of poly(ethylene glycol) to form di-or triblock copolymers under the catalysis of ZnEt2. But to ourknowledge, a triblock copolymer containing PEG, PC (poly-carbonate), and PLA has never been studied. So in this paper wefirst examined the sequential copolymerization of MPC and LAusing PEG as macromolecular initiator to form a triblock copolymer.Scheme 1 shows the synthetic procedure. Firstly, polymerization ofMPC monomer was initiated by PEG. Because of the pseudo-activenature of the polymerization system, the active center on thepropagating PEG–PMPC chain remains effective to initiate the ring-opening polymerization of LA to achieve a triblock copolymer PEG–PMPC–PLA. Table 1 summarizes related data of the polymerizationreactions. From Table 1, the yield of copolymerization is quite high,and the weight ratios of different blocks in the products are similarto those in feed. Every product shows a unimodal peak in GPC curve(data not shown), implying that the copolymerization is completedsuccessfully and no homopolymerization of LA or MPC occurs.These data suggest that composition of the copolymer can be easilycontrolled in the synthesis process.

1H NMR and 13C NMR were used to characterize the structure ofthe PML copolymers. Fig. 1 displays the 1H NMR spectra of copoly-mer PML263. Every proton of the polymer can find its characteristicsignal in the spectrum. Typically, the signal at 3.6 ppm is due to theCH2–CH2 protons in the PEG block. The signal at 4.7 ppm is attrib-uted to the O–CH2–C protons in MPC units. The signal at 5.2 ppm isattributed to the O–CH–CH3 proton in LA units. Relative proportionsof the three blocks can be then calculated from their intensities.Fig. 2 displayed 13C NMR spectrum of the copolymer PML263. Asshown in the figure, every carbon atom can find its characteristicpeak. Especially, signal d at 169.7 ppm is attributed to the methinecarbon atom in LA unit. It is sensitive to the sequence distribution of

Click

= CO-Hb

= PEG

= Progargyl

PLA-PMPC PLA-PMPC

Scheme 3. Conjugation of CO–Hb to the core of PML micelle via click chemistry.

Q. Shi et al. / Biomaterials 30 (2009) 5077–50855082

the copolymer. As reported previously [44–47], if LA and carbonateunits polymerize randomly, the signal at this position will split intoseveral peaks due to the coupling effect of neighboring units inpossible sequence distributions. In Fig. 2, the 169.7 ppm peak onlyshows a little shoulder that can be assigned to the LA unit at theboundary between the LA and MPC blocks. This providesconvincing evidence that the copolymer has a triblock structure.

Thermal properties of the copolymers were tested by DSCmeasurements. Fig. 3 shows the second heating run in DSC test ofdifferent copolymers in Table 1. All copolymers have no distinct Tg.PML261 and PML263 both show a melting peak of PEG at 50 �C andmultiple melting peaks of PLA at 110–150 �C, while PML265 only

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Micelle-HbCOMicelle

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Fig. 8. (a) UV spectra of pure PML261 micelles and CO–Hb-conjugated PML261micelles; (b) difference spectrum between the two spectra in (a).

displays the melting peaks of PLA. The co-existence of the PEG andPLA melting peaks in PML261 and PML263 is owing to the blockstructure of the copolymers. Disappearance of the melting peak ofPEG blocks from PML265 can be ascribed to the long chain length ofthe PLA blocks. Actually, PEG segments crystallize under a con-strained condition caused by crystallization of the PLA segments.When the PLA length is long enough, the PEG segments are difficultto crystallize. Stronger melting peak of PLA was observed when PLAcontent increases.

3.2. Micelle formation

The amphiphilic nature of the triblock copolymer PML providesit with an opportunity to self-assemble into shell-core micelles inaqueous solution. Assumed structure of such formed micelles isshown in Scheme 2. It consists of three layers: hydrophilic PEGsegments serve as the outermost layer to stabilize the nano-structure in aqueous solution; hydrophobic MPC and PLA segmentstogether constitute the core of the micelle, with MPC segments asthe outer layer and PLA segments as the inner core because of thetriblock structure of the copolymer. For such a structure, the reac-tive alkynyl groups may reside in between the PMPC layer and thePEG layer and thus are available for later conjugation process. Theformation of micelles was confirmed by fluorescence probingtechnique. Fig. 4 illustrates excitation spectra of pyrene in thepresence of various concentrations of PML copolymer. An obviousred-shift from 332.5 to 334.5 nm can be observed when concen-tration of PML copolymer increases. This photophysical propertychange of pyrene indicates the formation of micelles because in theformation process of micelles, hydrophobic pyrene molecules arepreferentially packed into the hydrophobic core of the micelles.Moreover, the critical micelle concentration (CMC) can be obtainedby plotting pyrene fluorescent intensity ratio I334.5/I332.5 versuslogarithm of PML concentration and by taking the tangents of thecurve. The CMC values of the three PML copolymers examined arelisted in Table 1. As expected, the PML with a longer PLA block hasa lower CMC.

Morphology of the micelles formed was observed by ESEM. Asshown in Fig. 5(a), micelles formed from copolymer PML displayshomogeneous spherical structure. Their diameter is about 40–60 nm. Their size distribution is unimodal (Fig. 6), and their meandiameter increases with increasing PLA block length in thecopolymer.

3.3. Preparation of CO–Hb

As is well known, Hb in ferrous state can be slowly auto-oxidized into nonfunctional ferric state and therefore loses itsoxygen carrying ability [48]. So usually the Hb (O2–Hb) moleculesare transformed into CO binding state (CO–Hb) to get stabilized

a b

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Fig. 9. Size distributions of PML261-CO–Hb (a), PML263-CO–Hb (b), and PML265-CO–Hb (c) determined by DLS.

Q. Shi et al. / Biomaterials 30 (2009) 5077–5085 5083

before performing any treatment or processing [49, 50]. In thepresent study, tiny excessive ascorbic acid was used to reduce thepossible ferric-state Hb molecules, and then the O2–Hb form wastransferred into CO–Hb form. The state transformation can bemonitored by the absorbance peak in UV spectra. As shown in Fig. 7,in the original status, O2–Hb shows a typical absorbance peak at405 nm. As the CO gas flows over the liquid surface under stirring,the O2 bonded on the Hb molecule is substituted by CO gradually.The absorbance peak at 405 nm disappears and a new peak cor-responding to the CO–Hb formed appears at 419 nm. The completedisappearance of the 405 nm peak indicates complete trans-formation of O2–Hb into CO–Hb.

0 1 2 3 4 5 6 7 8 90

50

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Co

nju

gatio

n R

atio

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MHbCO

:Mmicelle

Fig. 10. Conjugation ratio as a function of feed mass ratio of CO–Hb to PML261micelles.

3.4. Preparation of hemoglobin-conjugated micelles (HCM)

To obtain oxygen carrier based on hemoglobin and biodegrad-able block copolymer, azidized CO–Hb molecules were conjugatedwith PML micelles. In an aqueous solution, water-soluble PEGsegments on the micellar corona are flexible and extensible freely,allowing azido groups on the azidized CO–Hb to ‘‘click’’ withalkynyl groups on the PMPC layer of the micellar cores under thecatalysis of Cu(I). As shown in Scheme 3, because of the watersolubility of Hb molecules and their chemical binding with the MPCsegments, they are located closely on the surface of the PMPC layerand the whole HCM formed still assumes a spherical shape asoriginal PML micelles (Scheme 2). The difference between the twois that the outer layer of the HCMs is composed of two components:conjugated CO–Hb molecules and freely movable PEG segments.The former keeps in contact with the aqueous medium and mayexhibit oxygen-carrier ability and the later not only stabilizes thewhole micelle in aqueous medium but also protects the conjugated

CO–Hb molecules from the attack of the immunological systems. Asa whole, each micelle may serve as an oxygen carrier.

The conjugation between CO–Hb and PML micelles was moni-tored by UV spectroscopy. As shown in Fig. 8(a), pure PML micellesshow a declining UV spectrum without fine structure in the wave-length range examined because of the scattering effect of nanoscalemicelles. After conjugation with CO–Hb molecules, a new absor-bance peak appears at 419 nm over a declining background. Whenthe spectrum of the pure PML micelles is subtracted from that ofHCMs, Fig. 8(b) is obtained. Obviously, it is analogous to the spec-trum of CO–Hb (Fig. 7, 160 min). This similarity provides a powerful

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Fig. 11. UV spectra of HCM solution before (a) and after (b) O2 feeding for 2 h.

Q. Shi et al. / Biomaterials 30 (2009) 5077–50855084

support to the successful conjugation of CO–Hb molecules onto themicelles. Of course, there were residual CO–Hbs in the reactionsystem. In order to completely remove them, the reaction productswere ultracentrifuged [51], and HCMs obtained were washed withPBS solution and ultracentrifuged several times till the absorbancepeak of the elute near 419 nm diminished to zero.

DLS measurement was performed on HCMs. As shown in Fig. 9,the three samples all show unimodal distributions as their parentmicelles (Fig. 6). But their mean diameters are larger than those ofcorresponding PML micelles (70, 100, and 110 nm vs. 45, 50 and60 nm, respectively). Direct measurement by ESEM was performedon HCMs from PML261 (Fig. 5(b)). It can be seen clearly that theHCMs are also spherical and have a diameter of 150–200 nm.Meanwhile, some smaller particles with diameter of about 40–60 nm can also be seen in the view. It seems that they are thePML261 micelles that have not conjugated with CO–Hb molecules.All these facts demonstrated that the diameter increase is mainlydue to the CO–Hb conjugation.

Furthermore, efficiency of the conjugation was investigated.Mass of PML micelles was kept constant, and reaction was per-formed with different amounts of CO–Hb. Conjugation ratio wasexpressed as the quotient of mass of CO–Hb conjugated on themicelles divided by mass of the original micelles. Fig. 10 shows theconjugation ratio as a function of mass ratio of CO–Hb to PMLmicelles in the reaction feed. It is seen that the conjugation ratio isalmost proportional to the mass ratio of CO–Hb to micelles beforethe later ratio is below 6. Beyond 6, the conjugation ratio does notincrease any longer. This result can be well explained with thechemical reaction between CO–Hb and the micelles. The clickreaction takes place between the propargyl groups on the micellesand the azido groups on CO–Hbs. Available amount of the azidogroups will determine the amount of CO–Hbs needed. When theazido groups are not completely assumed, the reaction continueswith the addition of CO–Hbs. When all the active sites are coveredby CO–Hbs, the reaction stops and the conjugation ratio does notincrease any longer. The maximum conjugation ratio obtained is250%. It implies that the CO–Hb content in the HCMs may reach ashigh as 70 wt%.

3.5. O2 binding ability of HCMs

It is well known that free hemoglobin molecules in CO-bindingstate can be transformed into O2-binding state by contacting withO2 under visible light irradiation [3]. The same procedure was usedto examine the O2-binding ability of the HCMs prepared. Fig. 11shows the UV spectra of the HCM solution before and after O2

binding process. It is seen that after O2 gas was fed for 2 h, typical

absorbance peak of hemoglobin changed obviously from 419 nm toabout 409 nm, indicating the state of hemoglobin was successfullytransformed to O2-binding state. It implies that the Hb-conjugatedmicelles have oxygen-carrying ability. Further quantitative evalu-ation and improvement of this ability are undertaken, and theresult will be published elsewhere.

4. Conclusion

An amphiphilic PEG–PMPC–PLA triblock biodegradable copoly-mer was synthesized. The copolymer formed three-layer micellesin aqueous solution with PEG as shell, PMPC as outer core and PLAas inner core. Through the propargyl groups on the PMPC layer,hemoglobin molecules were conjugated to the micelles via clickchemistry and the micellar diameter increased to about 150–200 nm. The Hb content in the HCMs could reach as high as 70 wt%and the conjugated hemoglobins retained their O2-binding ability.Therefore the HCMs prepared may be considered as artificialoxygen carriers.

Acknowledgement

This project was financially supported by the National NaturalScience Foundation of China (Project Nos. 20674084 and20774093), by the National Fund for Distinguished Young Scholars(No. 50425309) and by the Ministry of Science and Technology ofChina (‘‘973 Project’’, No. 2009CB930102).

Appendix

Figures with essential colour discrimination. Certain figures inthis article, in particular Figures 3, 4, 6, 7, 9 and 11 are difficult tointerpret in black and white. The full colour images can be found inthe on-line version, at doi:10.1016/j.biomaterials.2009.05.082.

References

[1] Chang TMS. Blood substitutes: principles, methods, products and clinicaltrials, vol. 1. Karger Landes Systems; 1997.

[2] Sakai H, Sou K, Horinouchi H, Kobayashi K, Tsuchida E. Haemoglobin-vesiclesas artificial oxygen carriers: present situation and future visions. J Intern Med2008;263:4–15.

[3] Winslow RM. Blood substitutes. Adv Drug Deliver Rev 2000;40:131–42.[4] Saxena R, Wijnhoud AD, Carton H, Hacke W, Kaste M, Przybelski RJ, et al.

Controlled safety study of a hemoglobin-based oxygen carrier, DCLHb, in acuteischemic stroke. Stroke 1999:993–6.

[5] Chang TMS. Blood substitutes based on nanobiotechnology. Trends Biotechnol2006;24:372–7.

Q. Shi et al. / Biomaterials 30 (2009) 5077–5085 5085

[6] Goda N, Suzuki K, Naito M, Takeoka S, Tsuchida E, Ishimura Y, et al. Distri-bution of heme oxygenase isoforms in rat liver. Topographic basis for carbonmonoxide-mediated microvascular relaxation. J Clin Invest 1998;101:604–12.

[7] Kyokane T, Norimizu S, Taniai H, Yamaguchi T, Takeoka S, Tsuchida E, et al. Carbonmonoxide from heme catabolism protects against hepatobiliary dysfunction inendotoxin-treated rat liver. Gastroenterology 2001;120:1227–40.

[8] Sakai H, Yuasa M, Onuma H, Takeoka S, Tsuchida E. Synthesis and physico-chemical characterization of a series of hemoglobin-based oxygen carriers:objective comparison between cellular and acellular types. Bioconjugate Chem2000;11:56–64.

[9] Chang TMS. Future generations of red blood cell substitutes. J Intern Med2003;253:527–35.

[10] Chang TMS. Hemoglobin-based red blood cell substitutes. Artif Organs2004;28:789–94.

[11] Lowe KC, Ferguson E. Benefit and risk perceptions in transfusion medicine:blood and blood substitutes. J Intern Med 2003;253:498–507.

[12] Phillips WT, Klipper RW, Awasthi VD, Rudolph AS, Cliff R, Kwasiborski V, et al.Polyethylene glycol-modified liposome-encapsulated hemoglobin: a longcirculating red cell substitute. J Pharmacol Exp Ther 1999;288:665–70.

[13] Tsuchida E, Komatsu T, Yanagimoto T, Sakai H. Preservation stability and invivo administration of albumin-heme hybrid solution as an entirely syntheticO2-carrier. Polym Adv Technol 2002;13:845–50.

[14] Curry S, Tsuchida E, Zunszain PA, Ohmichi N, Komatsu T, Nakagawa A. O2 andCO binding properties of artificial hemoproteins formed by complexing ironprotoporphyrin IX with human serum albumin mutants. J Am Chem Soc2005;127:15933–42.

[15] Komatsu T, Nakagawa A, Zunszain PA, Curry S, Tsuchida E. Genetic engineeringof the heme pocket in human serum albumin: modulation of O2 binding ofiron protoporphyrin IX by variation of distal amino acids. J Am Chem Soc2007;129:11286–95.

[16] Sou K, Klipper R, Goins B, Tsuchida E, Phillips WT. Circulation kinetics andorgan distribution of Hb-vesicles developed as a red blood cell substitute.J Pharmacol Exp Ther 2005;312:702–9.

[17] Nakai K, Usuba A, Ohta T, Kuwabara M, Nakazato Y, Motoki R, et al. Coronaryvascular bed perfusion with a polyethylene glycol-modified hemoglobin-encapsulated liposome, neo red cell, in rats. Artif Organs 1998;22:320–5.

[18] Wakamoto S, Fujihara M, Abe H, Yamaguchi M, Azuma H, Ikeda H, et al. Effectsof hemoglobin vesicles on resting and agonist-stimulated human platelets invitro. Artif Cell Blood Sub 2005;33:101–11.

[19] Kissel T, Brich Z, Bantle S, Lancranjan I, Nimmerfall F, Vit P. Parenteral depot-systems on the basis of biodegradable polyesters. J Control Release 1991;16:27–41.

[20] Shirahama H, Kanetani A, Yasuda H. Synthesis and biodegradability ofcopolymers of ethylene carbonate with lactones. Polym J 2000;32:280–6.

[21] Kulkarni RK, Pani KC, Neuman C, Leonard F. Polylactic acid for surgicalimplants. Plast Reconstr Surg 1967;39:430.

[22] Langer R. Biomaterials in drug delivery and tissue engineering: one labo-ratory’s experience. Accounts Chem Res 2000;33:94–101.

[23] Nasongkla N, Shuai X, Ai H, Weinberg BD, Pink J, Boothman DA, et al. cRGD-functionalized polymer micelles for targeted doxorubicin delivery. AngewChem Int Ed 2004;43:6323–7.

[24] Rosler A, Vandermeulen GWM, Klok HA. Advanced drug delivery devices viaself-assembly of amphiphilic block copolymers. Adv Drug Deliver Rev 2001;53:95–108.

[25] Lee RS, Yang JM, Lin TF. Novel, biodegradable, functional poly(ester-carbonate)sby copolymerization of trans-4-hydroxy-L-proline with cyclic carbonatebearing a pendent carboxylic group. J Polym Sci Polym Chem 2004;42:2303–12.

[26] Al-Azemi TF, Harmon JP, Bisht KS. Enzyme-catalyzed ring-opening copoly-merization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MBC) withtrimethylene carbonate (TMC): synthesis and characterization. Bio-macromolecules 2000;1:493–500.

[27] Lecomte P, Riva R, Schmeits S, Rieger J, Van Butsele K, Jerome C, et al. Newprospects for the grafting of functional groups onto aliphatic polyesters. Ring-opening polymerization of a- or d-substituted-caprolactone followed by chem-ical derivatization of the substituents. John Wiley & Sons, Ltd; 2006. p. 157.

[28] Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, et al. Targetednanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. ProcNatl Acad Sci U S A 2006;103:6315–20.

[29] Choi SW, Kim WS, Kim JH. Surface modification of functional nanoparticles forcontrolled drug delivery. J Dispersion Sci Technol 2003;24:475–87.

[30] Cheng J, Teply BA, Sherifi I, Sung J, Luther G, Gu FX, et al. Formulation offunctionalized PLGA–PEG nanoparticles for in vivo targeted drug delivery.Biomaterials 2007;28:869–76.

[31] Yu WP, Chang TMS. Submicron polymer membrane hemoglobin nanocapsulesas potential blood substitutes: preparation and characterization. Artif CellBlood Sub 1996;24:169–83.

[32] Chang TMS. Biodegradable semipermeable microcapsules containingenzymes, hormones, vaccines, and other biologicals. J Bioeng 1976;1:25–32.

[33] Chang TMS, Powanda D, Yu WP. Analysis of polyethylene-glycol-polylactidenano-dimension artificial red blood cells in maintaining systemic hemoglobinlevels and prevention of methemoglobin formation. Artif Cell Blood Sub2003;31:231–47.

[34] Chang TMS. Evolution of artificial cells using nanobiotechnology of hemo-globin based RBC blood substitute as an example. Artif Cell Blood Sub2006;34:551–66.

[35] Chang TMS, Powanda D, Yu WP. Ultrathin polyethylene-glycol-polylactidecopolymer membrane nanocapsules containing polymerized Hb and enzymesas nanodimension red blood cell substitutes. Artif Cell Blood Sub2003;31:231–48.

[36] Rusmini F, Zhong Z, Feijen J. Protein immobilization strategies for proteinbiochips. Biomacromolecules 2007;8:1775–89.

[37] Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical functionfrom a few good reactions. Angew Chem Int Ed 2001;40:2004–21.

[38] Bock VD, Speijer D, Hiemstra H, Maarseveen JH. 1,2,3-Triazoles as peptidebond isosteres: synthesis and biological evaluation of cyclotetrapeptidemimics. Org Biomol Chem 2007;5:971–5.

[39] Lewis WG, Green LG, Grynszpan F, Radic Z, Carlier PR, Taylor P, et al. Clickchemistry in situ: acetylcholinesterase as a reaction vessel for the selectiveassembly of a femtomolar inhibitor from an array of building blocks. AngewChem Int Ed 2002;41:1053–7.

[40] Riva R, Schmeits S, Stoffelbach F, Jerome C, Jerome R, Lecomte P. Combinationof ring-opening polymerization and click chemistry towards functionalizationof aliphatic polyesters. Chem Commun 2005;42:5334–6.

[41] Parrish B, Breitenkamp RB, Emrick T. PEG-and peptide-grafted aliphaticpolyesters by click chemistry. J Am Chem Soc 2005;127:7404–10.

[42] Shi Q, Chen X, Lu T, Jing X. The immobilization of proteins on biodegradablepolymer fibers via click chemistry. Biomaterials 2008;29:1118–26.

[43] Han Y, Shi Q, Hu J, Du Q, Chen X, Jing X. Grafting BSA onto poly(L-lactide-co-carbonate) microspheres by click chemistry. Macromol Biosci2008;8:638–44.

[44] Ray WC, Grinstaff MW. Polycarbonate and poly(carbonate-ester)s synthesizedfrom biocompatible building blocks of glycerol and lactic acid. Macromole-cules 2003;36(10):3557–62.

[45] Guan HL, Xie ZG, Zhang PB, Wang X, Chen XS, Wang XH, et al. Synthesis andcharacterization of novel biodegradable block copolymer poly(ethyleneglycol)-block-poly(L-lactide-co-2-methyl-2-carboxyl-propylene carbonate).J Polym Sci Polym Chem 2005;43:4771–80.

[46] Xie Z, Hu X, Chen X, Sun J, Shi Q, Jing X. Synthesis and characterization ofnovel biodegradable poly(carbonate ester)s with photolabile protectinggroups. Biomacromolecules 2008;9:376–80.

[47] Hu X, Liu S, Chen X, Mo G, Xie Z, Jing X. Biodegradable amphiphilic blockcopolymers bearing protected hydroxyl groups: synthesis and characteriza-tion. Biomacromolecules 2008;9:553–60.

[48] Sakai H, Masada Y, Onuma H, Takeoka S, Tsuchida E. Reduction of methe-moglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipidvesicles. Bioconjugate Chem 2004;15:1037–45.

[49] Takeoka S, Sakai H, Kose T, Mano Y, Seino Y, Nishide H, et al. Methemoglobinformation in hemoglobin vesicles and reduction by encapsulated thiols. Bio-conjugate Chem 1997;8:539–44.

[50] Atoji T, Aihara M, Sakai H, Tsuchida E, Takeoka S. Hemoglobin vesicles con-taining methemoglobin and L-tyrosine to suppress methemoglobin formationin vitro and in vivo. Bioconjugate Chem 2006;17:1241–5.

[51] Sakai H, Hamada K, Takeoka S, Nishide H, Tsuchida E. Physical properties ofhemoglobin vesicles as red cell substitutes. Biotechnol Progr 1996;12:119–25.