polymer/hemoglobin assemblies: biodegradable oxygen carriers for artificial red blood cells

Feature Article

Polymer/Hemoglobin Assemblies:Biodegradable Oxygen Carriers for ArtificialRed Blood Cells

Taihang Li, Xiabin Jing, Yubin Huang*

In routine clinical procedures, blood transfusion is now suffering from the defects of the bloodproducts, like cross-matching, short storage time and virus infection. Various blood substituteshave been designed by researchers through continual efforts. With recent progress innanotechnology, new types of artificial red blood cells with cellular structure are available.This article aims to describe some artificial red blood cellswhich encapsulate or conjugate hemoglobin moleculesthrough various approaches, especially the nanoscaleself-assembly technique, to mitigate the adverse effectsof free hemoglobin molecules. These types of artificialred blood cell systems, which make use of biodegradablepolymers as matrix materials, show advantages over thetraditional types.

Hb

Amphiphilic Copolymers Self-assembly

1

Introduction

As is well known, the blood in our bodies plays an important

role in sustaining our lives, supplying necessary nutrition

and oxygen to the tissues and organs. Blood transfusion has

gradually become a universally practiced, remarkably safe,

routine clinical procedure over the last century. ‘‘Safety’’ is

always of extreme concern; however, transfusion can never

be zero-risk. Many kinds of issues have emerged during the

blood transfusion practice, for example the short shelf life,

cross-matching and virus infection of blood products. These

problems have become the impetus to develop blood

substitutes, along with great challenges and a good future.

T. H. Li, X. B. Jing, Y. B. HuangState Key Laboratory of Polymer Physics and Chemistry,Changchun Institute of Applied Chemistry, Chinese Academy ofSciences, Changchun 130022, ChinaFax: þ86 431 8526 2769; E-mail: [email protected]. H. LiGraduate University of the Chinese Academy of Sciences, Beijing100039, China

Macromol. Biosci. 2011, 11, 865–874

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlin

Ideal blood substitutes, also named artificial oxygen

carriers or artificial red blood cells, refer to synthetic

solutions with the ability to bind, transport and release

oxygen molecules in the body. Ideal blood substitutes

should satisfy some basic requirements: no pathogens; no

blood type; low toxicity for renal corpuscle and easy

clearance; stability at room temperature and longer shelf

life; sufficient half-life circulation; ease of mass production

at low cost. In the 1990 s, concentrated efforts on developing

blood substitutes for public use began seriously,[1–3]

because of the emergence of HIV, hepatitis B and C viruses,

and some new types of infectious diseases in donor blood.

From the development history of blood substitutes, there

are generally two categorized groups: hemoglobin-based

oxygen carriers (HBOCs) and totally synthetic oxygen

carriers (Figure 1). The former is mainly based on human or

bovine hemoglobin (Hb) to obviate renal toxicity from

hemoglobin dissociation. The latter has included perfluor-

ocarbon-based materials[4–8] and totally synthetic heme

hybrids.[9–16]

With the progress of nanotechnology in the fields of

biomaterials and biomedical science, new types of artificial

elibrary.com DOI: 10.1002/mabi.201000469 865

Taihang Li is a Ph.D. candidate at ChangchunInstitute of Applied Chemistry, Chinese Academyof Sciences (CIAC). He obtained his MastersDegree from the School of Biological Engineer-ing, Changchun University of Technology, in2008. Currently, he works in the laboratory ofProf. Yubin Huang at CIAC.

Xiabin Jing is a Professor of Polymer Chemistry atthe State Key Laboratory of Polymer Physics andChemistry, Changchun Institute of AppliedChemistry, Chinese Academy of Sciences (CIAC).He graduated from the University of Science andTechnology of China in Polymer Physics in 1965.His main research involves polymer structureand polymer spectroscopy, conductive polymers

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T. Li, X. Jing, Y. Huang

red blood cells are now a great achievement. Novel

celluliform oxygen carriers with comparatively satisfying

properties are now well recognized by researchers.

Biocompatible and biodegradable polymers are commonly

used as the carrier materials,[17–22] and the self-assembly

technique[23,24] has been utilized to easily obtain nano or

mm-sized micelles and vesicles to encapsulate or conjugate

hemoglobin molecules.[25–28] In this article, we present a

brief introduction into how scientists make artificial red

blood cells from using hemoglobin itself to applying the

polymer assembly strategy. For this purpose, totally

synthetic oxygen carriers including perfluorochemicals

(PFCs) and synthetic heme hybrids are not discussed in the

present paper.

and biodegradable polymers and their medicalapplications. He has published over 400 scien-tific papers and applied for more than 80patents.

Yubin Huang, Ph.D., is a Professor of PolymerChemistry at the State Key Laboratory of Poly-mer Physics and Chemistry, Changchun Instituteof Applied Chemistry, Chinese Academy ofSciences (CIAC). He obtained his Ph.D. in PolymerChemistry from CIAC in 1999. He was a post-doctoral researcher and Associate Professor atWaseda University, Japan and collaborated withProf. Eishun Tsuchida. His research involves thedevelopment of high performance PLAmaterials,the synthesis of cell-targeting materials fortumor therapy and artificial red blood cells usingbiodegradable amphiphilic copolymers. He hasover 30 international publications and 20patents. He has been awarded the ‘‘100 TalentsProgram’’ from the Chinese Academy ofSciences.

Modified Hemoglobin

Hemoglobin is the abundant protein in the red blood cells of

mammals and other animals. It contains four sub-units

(tetramer, a2b2; Mw ¼ 64 500; 6.5 nm diameter), and each

sub-unit coordinates with one heme molecule to bind

oxygen. The basic function for hemoglobin is to transport O2

from the lungs to organs and tissues. Hemoglobin is

universally compatible because the antigens responsible

for blood types are not located on hemoglobin itself, but on

the membranes of the red blood cells (RBC). However, cell-

free hemoglobin itself could not be used directly as the

blood substitute, which was first reported by Ambersen in

1930 s.[29] The main reason is that hemoglobin could easily

dissociate into dimers (2ab) in blood circulation, causing

serious renal toxicity.[30,31] Moreover, the free hemoglobin

could be entrapped by the gaseous messenger molecules

(NO and CO) to induce vasoconstriction, hypertension,

reduced blood flow and tissue oxygenation at microcircu-

latory levels.[32,33] Furthermore, the short circulation half-

time (T1/2< 1.5 h), high oncotic pressure and high O2 affinity

(P50, 10–15 mmHg) make the isolated hemoglobin unsui-

table for clinical applications.[30,31]

To obviate the above-mentioned problems from free

hemoglobin molecules, researchers have attempted to

synthesize perfluorocarbon-based materials[4–6] and totally

synthetic heme hybrids[9–16] to transport oxygen. On the

other hand, scientists have made great efforts to prevent

the tetramer of hemoglobin from dissociating into dimers.

These improvements include intra-crosslinking of tetra-

meric hemoglobin, inter-crosslinking of hemoglobin to get

polyhemoglobin and hemoglobin/polymer conjugation.

Bifunctional reagents,[34–38] such as bis(N-maleimido-

methyl) ether[39] or bis(3,5-dibromosalicyl)fumarate,[40]

were often used to crosslink hemoglobin. To obtain

polymerized hemoglobin, utilization of glutaraldehyde

and ring-open saccharides as Hb crosslinking agents has

been extensively investigated in several biological labora-

Macromol. Biosci. 201

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tories. The related works are not included in the present

article and the readers can get detailed information from a

more thorough review by Palmer.[41] However, no matter

whether intra-crosslinked tetrameric hemoglobin or poly-

hemoglobin are used, the vasoconstrictions in microcircu-

lation and hypertension always happen due to the

dissociation of free hemoglobin molecules from these

species.[42,43] These tetrameric hemoglobin molecules could

penetrate across the intercellular junction of the endothe-

lial cells in the vessel walls and react with nitric oxide.[44,45]

Nitric oxide (NO), a so called endothelium-derived relaxing

factor (EDRF), has the main function of maintaining the

relaxation of vessels. Consumption of nitric oxide would

result in systemic vasoconstriction,[46] decreased blood

flow, increased release of proinflammatory mediators and

potent vasoconstrictors, and a loss of platelet inactiva-

tion[47] leading to vascular thrombosis of the heart or other

organs. In addition to the NO-scavenging hypothesis, an

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Figure 1. Main types of artificial oxygen carriers discussed in this paper.

Polymer/Hemoglobin Assemblies . . .

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over-oxygenation hypothesis is also proposed to explain

the vasoconstriction and development of systemic hyper-

tension.[48,49]

The hypertensive effect can be reduced or diminished by

increasing the molecular volume of the protein.[50–52] In

fact, the retention time in blood circulation could also be

prolonged in this way. A larger particle size would make the

carrier system less easy to be captured by the macrophages

in the liver, which would have some effect in extending the

circulation time in the blood. This approach has been

extended to produce polymer/hemoglobin conjugates, i.e.,

conjugating water-soluble polymers, such as dextran[53] or

poly(ethylene glycol) (PEG)[54–63] onto the crosslinked

hemoglobin molecules. The conjugation of hemoglobin

with PEG chains is known to extend the plasma retention

time.[59] It also increases the viscosity of hemoglobin

solutions, resulting in a vasodilation effect which is also

caused by an enhanced release of nitric oxide from

endothelium.[64,65] However, Mozzarelli et al.[66] found that

PEGylation would promote the hemoglobin dissociation by

comparing PEGylated hemoglobin under anaerobic condi-

tions with that under aerobic conditions. It may be caused

in three ways: i) perturbation of the T and R states;[64] ii)

perturbation of the quaternary transitions;[67] iii) influence

on the tetramer/dimer equilibrium.[68]

In 2009, a paper entitled ‘‘Hemoglobin-based Oxygen

Carriers: Current Status and Future Directions’’ was

simultaneously published in Anesthesiology (111, 946)

and Transfusion (49, 2495). The paper summarized the

scientific presentations and opinions of the speakers at the

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conference, which was sponsored by the Food and Drug

Administration (FDA) and National Institutes of Health

(NIH) on April 29–30, 2008. Many issues about traditional

HBOCs (Table 1) have been raised and discussed. Mean-

while, some novel oxygen carriers have emerged and been

applied to animal experiments.

Since Chang’s group first obtained the polymerized

hemoglobin with glutaraldehyde in 1971,[69] they kept

reporting the synthesis of new kinds of blood substitutes

using nano-biotechnology to assemble hemoglobin

together with enzymes. For example, they used antioxidant

enzymes like superoxide dismutase (SOD) and catalase

(CAT) to form PolyHb-CAT-SOD (1998).[70–73] No evidence of

brain edema[71] was confirmed in animal trials using this

material. They also reported on dual function PolyHb-

tyrosinase (2004) and PolyHb-fibrinogen (2007) in rat

experiments showing superior advantage over simply

crosslinked or polymerized hemoglobin models.[74,75] In

2010, Chang published a review[76] to report on the

development of these products in animal trials.

Liposome/Hemoglobin Assemblies:Hemoglobin Vesicles (HbV)

With a deeper understanding of blood substitutes, the

importance of cellular structures like red blood cells became

apparent. Actually, nature selected this cellular structure

through evolution to give three advantages: i) decrease in

colloidal osmotic pressure of Hb; ii) prevention of Hb

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Table 1. Properties of modified-Hb-based oxygen carriers in development.

Chemical modifiera) Tetramer

[%]

P50[Torr]

Molecular

weight [kDa]

Source/

company

Hemoglobin

brand name

none 100 26.6 64 – native hHb

maleimide PEG, no intra-x,

surface conjugation/

polymerization (PEG¼ 7� 5 kDa)

0 5[59] 95[59] Sangart MP4

(Hemospan)

pyrioxal-phosphate

crosslinking,

a-carboxymethyl,

v-carboymethoxypoly-

oxyethylene/PHP, PEGylation,

16% polymerization between

PEG-Hb tetramers, 90% catalase

and SOD activity of lysed RBCs

0 (<0.5%

unstabilized

tetramer)[117]

20[115] 106 Apex

Biosciences

pyridoxylated

Hb POE

conjugate

(PHP)þcatalase &

SOD

O-raffinose b intra-x or

inter-x

34–42 stabilized

tetramer and

54–62 polymers

(5% unstabilized

tetramer)

39[119] 32 to >500[83,118] Hemosol

(development

terminated)

O-R-PolyHb

(Hemolink)

glutaraldehyde a intra-x

or inter-x

<5% stabilized/

unstabilized

tetramer[83]

40[83,120] 130� 500[83] Biopure bHb PolyBvHb

(Hemopure)

pyridoxal phosphate intra-x,

glutaraldehyde inter-x

<1[83,119] 26� 32[121,122] average 250[123] Nothfield hHb

(development

terminated)

PolyHb

(Polyheme)

a)Intra-X and inter-X denote intramolecular and intermolecular crosslinking, respectively.

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removal from blood circulation; iii) preservation of the

chemical environment in red blood cells. In the cellular

construction, hemoglobin molecules could be well pro-

tected from direct contact with blood, and the leakage of

the hemoglobin molecules from the vessel walls could

be completely eradicated. Furthermore, the appropriate

size[77] of the cellular construction would have great

benefits in realizing long term circulation in body.

HbV is one of the cellular-type hemoglobin-based oxygen

carriers that mimic the red blood cell structure. In 1957,

Chang[78] reported the microencapsulation of Hb and

enzymes using synthetic membranes to form the artificial

red blood cell. He tried nylon, collodion and many other

polymers as the membrane materials. Other scientists and

groups also reported on nano-particles formed by assembling

hemoglobin with gelatine, gum arabic, silicone etc.[79–80]



Unfortunately, particle size regulation was extremely

difficult in order to meet the requirement for blood flow

in capillaries. Furthermore, those particles had insufficient

biocompatibility.

Phospholipid is another important candidate for the

membrane material of the artificial red blood cell, because it

has the capacity to self-assemble into vesicles in aqueous

media and to encapsulate water soluble materials in the

aqueous interior. Considering the necessary stability of

HbV to prevent intervesicular aggregation, fusion and

leakage of the encapsulated Hb, researchers attempted to

stabilize the membrane through the polymerization of

phospholipids containing dienoyl groups[81,82] or through

surface modification with PEG chains.[83–85] Since the

polymerized phospholipids could not be cleared from the

body easily, this method was abandoned. On the other

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hand, the steric barrier created by PEG modification on the

surface of the phospholipid membrane could restrain

the aggregation of HbV, and thus stabilize HbV disper-

sions.[86,87] The methemoglobin (metHb) level of PEGylated

HbV [PEG/ liposome-encapsulated hemoglobin (LEH)]

dispersions could also be suppressed by co-encapsulation

of reductants or catalase, like glutathione (GSH), homo-

cysteine (Hcy) etc.[88–92] The co-encapsulation of GSH in LEH

decreased the rate of metHb formation from 3.8� 10�7 to

1.6� 10�7M � s�1 at 58 Torr.[88] If 5� 10�3

M of Hcy was

encapsulated in HbV, a 40% lower rate of metHb formation

([metHb]¼ 23%) than that of the HbV without any Hcy

([metHb]¼ 35%) was confirmed in vitro atpO2 of 142 Torr at

37 8C for 24 h.[92] The in vivo study also demonstrated the

same results. Tsuchida’s group[93–97] completed a detailed

study on the safety and efficacy of PEG/LEH, finding that

PEG surface modification could improve the rheology,

hemodynamic properties and biocompatibility of the PEG/

LEH dispersions.

Some defects still remained for the liposome assembly

system, such as i) the splenic sequestration of the LEH

particles would saturate the reticuloendothelial system

(RES), which could limit the effectiveness of the immune

system against foreign bodies;[77,98] ii) the lipid may induce

lipid peroxidation during the ischemia reperfusion pro-

cess;[76] iii) the reducing agents (like glucose which is

needed for the metHb reductase system inside red blood

cells) in the circulating blood could not cross the lipid

membrane to depress the rate of metHb formation.[76]

Moreover, HbV also has application limitations[13,15] such

as a shorter functional half-life when compared with RBCs

(2–3 vs. 110 d) and the total amount of the membrane

component of HbV is 2–3 times more than that of RBCs.

Novel Oxygen Carriers from Polymer/Hemoglobin Assemblies

Even if the defects of the lipid membrane could be ignored,

phospholipids are quite expensive. It gives the artificial red

blood cell product a price disadvantage comparing to

donated blood. Instead of using lipid, Chang’s group

reported a novel HbV using biodegradable polylactide

(PLA) polymer to encapsulate hemoglobin and enzymes in

the nano range (80–180 nm).[99–101] They synthesized a

series of copolymers from PLA and PEG.[102] Hemoglobin

with superoxide dismutase catalase and methemoglobin

reductase could be easily encapsulated into nanocapsules

by the self-assembly of the PLA copolymers.[37,42,76,103] They

found that it would take 24.2 h for the systemic Hb level to

reach 1.67 mg �dL�1 when the Hb nanocapsules were

injected into rats, which was much longer than that for the

polyHb (14 h).[102] The metHb level was decreased from 7 to

5.5% in 5 h when the enzyme encapsulated Hb nanocap-




sules were suspended in Ringer lactate (containing

100 mg �dL�1 glucose and 2� 10�5M NADH). They believed

that glucose and NADH could enter the nanocapsules to

start the multi-enzyme reaction. It was also observed that,

unlike HbV, the Hb nanocapsules were permeable to

ascorbic acid, glutathione and other plasma reducing

molecules to maintain the stability of Hb against auto-

oxidation.[75,104,105] Recently, 33% of the blood exchange

infusion of the Hb nanocapsules using rats was reported.

This kind of artificial oxygen carrier did not cause any

vasoconstriction, and no change in biochemistry, enzymes

or histology could be confirmed.[106,107] Their research

brought nanotechnology and the concept of self-assembly

together, leading to a new era for the development of novel

blood substitutes.

As we know, biocompatible and biodegradable amphi-

philic copolymers have been widely investigated and

applied to the biomedical area. These amphiphilic copoly-

mers can not only degrade to removable and harmless

segments in human body fluid, but also form polymer-

somes[108,109] (self-assembled polymer vesicles composed

of amphiphilic diblock copolymers) and micelles in aqueous

solution. With their similar structure to liposomes, poly-

mersomes possess superior properties, such as thickness

and strength of the membrane and ease of adjustment of

the diameter of the particles by changing the diblock

copolymer composition. Some research[77,110] has indicated

that the effect factor on the circulation time in vivo was

principally the diameter of the particle. All of these make

amphiphilic copolymers an excellent platform for scientists

to cultivate novel artificial red blood cells. Up to now, many

achievements for artificial oxygen carriers utilizing biopo-

lymers and hemoglobin as matrix materials have been

obtained through different methods. Some novel oxygen

carriers from biopolymer-hemoglobin assembly are col-

lected in Table 2.

Palmer’s group[98,111] has reported polymersome-encap-

sulated hemoglobin (PEH) dispersions using bioinert PEG/

polybutadiene (PBD) as the membrane material. The sizes of

the polymersomes were controlled to be around 100 or

200 nm by extruding through the pore radii membranes.

The influence of different molecular weights of the diblock

copolymers on the physical properties of PEH was analyzed.

It was confirmed that the hemoglobin molecules could

remain stable in the polymersomes, and the hemoglobin

loading capacities in these polymersomes were higher than

that of liposome particles and nanoscale hydrogel particles.

They also obtained polymersomes from PEG/PLA and PEG/

poly(e-caprolactone) (PCL) to study the size distribution,

hemoglobin encapsulation efficiency, oxygen affinity (P50),

cooperativity coefficient (n) and metHb level. All these

results showed the satisfying relative index for the

hemoglobin encapsulated polymersomes to be the efficient

oxygen carriers in systemic circulation. Furthermore, they

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Figure 2. Hb encapsulated vesicles prepared from PLL-b-PPAsolutions and their SEM images (molecular weightss of PLL andPPA were 8 300 and 880, respectively, bar¼ 20mm).

Table 2. Properties of some oxygen carriers from polymer/hemoglobin assemblies.

HBOC matrix Mw

[kDa]

PEG length

[kDa]

Mean diameter

[nm]

P50[Torr]

na) metHb level EEc)

Hb (native) 64 none �6.5 26.8 2.5 0.5–1 –

PEO/PLA[99–102] 7 or 17 2 80–180 26 2.4–2.9 <6b) 29–47

PEO/PBD[98,111,114] 34.6 12.6 200 22.2 2.4 <1 5.3� 0.4

7.3 2.3 200 28.1 2.1 <1 33.4� 22.6

3.8 1.3 100 30 2.4 <1 30.3� 2.5

2.7 0.9 200 29.8 2.4 <1 22.4� 7.1

PEG/PLA[98,114] 10 5 200 24.2 1.9 <5 <4

2.45 0.55 200 20.81 1.8 <5 <20

PEG/PCL or PCL[115] 42 or 45 6 153 27 1.9 no report 35–87

a)n represents cooperativity coefficients; b)Glucose and NADH were added after 5 h; c)Encapsulation efficiency.

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have made temperature and pH-sensitive hydrogel nano-

particles to entrap hemoglobin as oxygen carriers.[112,113]

On the basis of the aforementioned research, Palmer

et al.[114] recently developed a large scale (50–100 mL)

preparation of PEH dispersions by hollow fiber (0.2mm pore)

extrusion. The membrane of the polymersomes was still

from PEO/PBD with Mn ranging from 1.8 to 10.4 kDa. It is

quite obvious that the preparation method of the PEH

dispersions showed great advantages over the traditional

hydration and manual extrusion method. They have also

proved that various properties (such as P50 ¼12.46, 25.51

and cooperativity coefficients, n¼ 2.74, 2.75) of the human

Hb and bovine Hb encapsulated polymersomes were

comparable to native Hb.

PCL or PEG/PCL were also used as the matrix polymer for

preparation of the hemoglobin-loaded particles (HbP) by

Liu’s group.[115] HbP with a high encapsulation efficiency

(87.35%) and desirable diameter (within 70–200 nm)

was obtained through an improved double emulsion

method.[116] They discussed various effect factors (such

as matrix polymers, solvents and temperature) on the pore-

connecting efficiency, which would take part in the oxygen

delivery through the connected channel. They found that

the pore-connecting efficiency was mainly attributable to

temperature rather than matrix polymer composition.

In addition, hemoglobin molecules were also encapsu-

lated into self-assembled polypeptide vesicles by Jing’s

group.[25] The membrane material was poly(L-lysine)-block-

poly(L-phenylalanine) (PLL-b-PPA) diblock copolymers. They

were self-assembled into hemoglobin vesicles (Figure 2) in

an aqueous medium without using any organic solvents, in

favor of retaining the bioactivity and oxygen transporta-

tion ability of the encapsulated hemoglobin molecules. The

encapsulation process was carried out at pH¼ 5.8, which

was lower than the pKa of PLL (10.0) and the isoelectric point



(pI) of hemoglobin (6.8). Since PLL and hemoglobin possess

the same positive charge at pH¼ 5.8, strong electrostatic

attraction between PLL segments and hemoglobin mole-

cules could be effectively avoided, and the hemoglobin

molecules could be mainly encapsulated into the PLL-b-PPA

vesicles without surface adsorption. The Hb/PLL-b-PPA

vesicles had a spherical shape with a hemoglobin loading

ratio of about 32 wt.-%. The oxygen transportation capacity

was still retained in the Hb/PLL-b-PPA vesicles, as evidenced

by the reversible oxygen binding and release under visible

light irradiation.

More recently, another artificial oxygen carrier was

constructed in our laboratory.[26] Instead of hemoglobin

vesicles, hemoglobin molecules were conjugated on the

surface of the biodegradable micelles, which were made

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Figure 3. (1) Hb is conjugated onto PML micelles via click chemistry. (2) ESEM images of(a) PML micelles and (b) Hb conjugated PML micelles (molecular weights of PEG, PMPCand PLA were 2 000, 600 and 1 000, respectively, bar¼ 500nm).


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from the triblock amphiphilic copolymer

PEG-b-PMPC-b-PLA or simply PML, in

which PMPC¼poly-5-methyl-5-propar-

gyloxycarbonyl-1,3-dioxan-2-one. In this

micellar structure, hydrophilic PEG seg-

ments serve as the outermost layer to

stabilize the micellar structure of PML in

aqueous solution; PLA segments form the

hydrophobic core; PMPC segments with

pendant propargyl groups constitute the

middle layer between the PEG and PLA

parts. The reactive propargyl groups

might reside in between the PMPC layer

and the PEG layer making them available

for a later conjugation process. As a

matter of fact, after mixing with azided

hemoglobin, the hemoglobin could be

conjugated onto the micellar surface via a

click reaction between the propargyl and

azido groups with a hemoglobin loading

ratio of about 70 wt.-%, which was

confirmed by the typical absorbance of

hemoglobin in UV, and the enlarged

micelle size was confirmed by DLS and

350 400 450 500 550 6000.0

0.5

1.0

1.5

2.0

2.5

Abs

orba

nce

Wavelength (nm)

PO2 Increase

600550500

Figure 4. UV spectra of Hb-conjugated micelle solutions underdifferent O2 partial pressures.

ESEM (Figure 3) measurements. The hemoglobin conju-

gated PML micelles could bind and release oxygen

reversibly with a P50 of 30 Torr, which was proved by the

changes in UV absorbance under different oxygen partial

pressures (Figure 4). Triblock copolymers containing other

groups (for example carboxyl groups) were also synthe-

sized, and the related characterization is still ongoing. The

advantage of these hemoglobin conjugated biodegradable

micelles is obvious, because conjugation between hemo-

globin and the micelles results in immobilization of

hemoglobin molecules inside the micelles. The hemoglobin

molecules are protected by the PEG corona from the attack

of the immunological systems on one hand, and they keep

in touch with the aqueous medium in blood in favor of

oxygen binding and release on the other hand. Moreover,

the PEG segments can stabilize the micelle particles in

aqueous media and prolong the systematic circulation of

the micelles.

Future Aspects

The development of artificial red blood cells is a complicated

and tough process. It is still hard now to predict which type

of the blood substitutes mentioned above could be applied

in clinical trials and become a kind of commodity in the near

future. Up to now, the efforts of Baxter and Biopure to

develop HBOCs have not met with success. Worse still, it is a

heavy blow that Northfield went out of business in 2009,

since its artificial blood product ‘‘PolyHeme’’ did not win

approval from the FDA.




Nevertheless, the merit of artificial red blood cells is

always a great driving force for researchers. Although it is a

time consuming and high cost task, more and more

researchers are attracted into this field because of the huge

social demands for artificial blood, especially during wars

and natural disasters, or in regions of viral disease

prevalence. At the same time, the introduction of nano-

technology and self-assembly techniques into this area

provides a great opportunity to make a breakthrough in

artificial red blood cells. It is becoming common knowledge

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that artificial red blood cells should have a cellular form and

the membrane should be made of biocompatible and

biodegradable materials. Crosslinking of the membrane

may be necessary to improve the stability of the artificial

cells, but penetration of oxygen through the membrane

should not be weakened. A high loading ratio of hemoglo-

bin molecules is preferred to have sufficient oxygen supply.

Finally, the polymer/hemoglobin complex should have

enough water solubility for a longer circulation time in

blood. At the present time, a few artificial red blood cell

products in the cellular form are undergoing animal

experiments and even phase-II or III clinical trials.

Achievements have also been reported all along which

show significant advantages compared to traditional

methods. We have full confidence that new generations

of artificial red blood cells with novel structures and

excellent performance will be created and finally accepted

in the near future.

Acknowledgements: The authors acknowledge financial supportfrom the National Natural Science Foundation of China (No.50733003, 51021003 and 20874097), the Ministry of Science andTechnology of China (973 Project, No. 2009CB930102; 863 Project,No. 2007AA03Z535), the ‘‘100 Talents Program’’ of the ChineseAcademy of Sciences (No.KGCX2-YW-802) and the Jilin ProvincialScience and Technology Department (No. 20082104, 20100588).

Received: November 29, 2010; Published online: February 10,2011; DOI: 10.1002/mabi.201000469

Keywords: biocompatibility; biodegradable; biological applica-tions of polymers; self-assembly

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polymer/hemoglobin assemblies: biodegradable oxygen carriers for artificial red blood cells

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