Physical Properties and Stability Mechanisms of

Poly(Ethylene Glycol) Conjugated Liposome

Encapsulated Hemoglobin Dispersions
Dian R. Arifin and Andre F. Palmer
Department of Chemical and Biomolecular Engineering, University of
Notre Dame, Notre Dame, IN, USA
Abstract: Liposomes encapsulating hemoglobin (LEHs) surface-conjugated
with 2000 and 550 Da poly(ethylene glycol) (PEG) were produced via extrusion
through 400, 200 and 100 nm pore diameter membranes in two types of phosphate
buffer with different ionic strengths. The lipid bilayers were composed of
dimyristoyl-phosphatidylcholine (DMPC), cholesterol, dimyristoyl-phosphoetha-
nolamine-PEG (DMPE-PEG), dimyristoyl-phosphatidylglycerol (DMPG), and
a-tocopherol (in a 43:40:10:5:2 mole ratio). N-acetyl-L-cysteine was coencapsu-
lated in order to suppress hemoglobin (Hb) oxidation. Various physical properties
of PEG-LEH dispersions were determined: size distribution, encapsulation
efficiency, P
50
(partial pressure of O
2
where half of the oxygen binding sites are
saturated with O
2
), cooperativity coefficient, and encapsulated methemoglobin
(MetHb) level. In order to study the stabilization mechanism of these dispersions,
the effective bending constant (K
B
) and the spontaneous radius of curvature (R
0
)
of PEG-LEHs were extracted by fitting a mathematical model describing the size
distribution of a liposome dispersion to the experimentally measured size distribu-
tions [1]. We observed that liposome dispersions extruded in phosphate buffer
(PB) were more monodisperse than liposomes extruded in phosphate buffered
saline (PBS), and higher molecular weight PEG promoted the formation of
narrower size distributions. Moreover, extrusion in PB and lipid conjugation with
higher molecular weight PEG imparted higher bilayer rigidity (high K
B
), and sta-
bilized the liposome dispersions by the spontaneous curvature mechanism,
whereas the other liposome dispersions were stabilized by thermal undulations
(low K
B
). The P
50
and cooperativity coefficient of PEG-LEHs extruded in PBS
and PB was comparable to that of human blood, and the encapsulated MetHb
levels were less than 5%. The highest encapsulation efficiencies obtained were
27%36%(82109 mg Hb=mL) for LEH dispersions extruded in PBS and grafted
with 2000 Da PEG. These dispersions yielded K
B
s ranging from 7 kT to 27 kT,
which indicated that these dispersions were stabilized by spontaneous curvature.
Address correspondence to Andre F. Palmer, A51 Fitzpatrick Engineering Hall,
Department of Chemical and Biomolecular Engineering, University of Notre Dame,
Notre Dame, IN 46556, USA. Tel: 1574-631-4776. E-mail: apalmer@nd.edu
Artificial Cells, Blood Substitutes, and Biotechnology, 33: 137162, 2005
Copyright Q Taylor & Francis, Inc.
ISSN: 1073-1199 print/1532-4184 online
DOI: 10.1081/BIO-200055880
137
Whereas the same lipid combination extruded in PBS, however, instead conjugated
with 550 Da PEG resulted in K
B
s ranging from 2 kT to 2.7kT, which indicated that
these dispersions were stabilized by thermal undulations. Thermal undulations per-
mitted Hb leakage through the lipid bilayers, which in turn lowered the encapsula-
tion efficiency to 1%10.7%(332 mg Hb=mL). Taken together, the experimentally
measured size distributions and encapsulation efficiencies of PEG-LEH dispersions
can be readily explained through analysis of the magnitude of K
B
, which dictates the
stability mechanism of the liposome dispersion.
INTRODUCTION
Liposome encapsulated hemoglobin (LEH) is one strategy that is cur-
rently being used to create artificial blood substitutes. This strategy
entails encapsulating potentially toxic stroma-free hemoglobin (Hb)
inside the aqueous core of a lipid membrane shell, which is composed
of phospholipids and cholesterol. LEHs are universal blood substitutes,
and can readily be mass-produced with guaranteed sterility [25]. Their
oxygen binding properties have been shown to be comparable to those
of human red blood cells (P
50
~26 mmHg and cooperativity coefficient
~2.32.4 [3, 4]). Reductants or catalases can be coencapsulated inside
the liposome aqueous core to suppress methemoglobin (MetHb) forma-
tion [3, 6, 7]. However, the circulation half life of LEHs is short
(~1218 hours [8, 9]), and they tend to aggregate and fuse together after
several days of storage [10]. LEHs are primarily eliminated from the
intravascular circulation by the cells of the reticuloendothelial system
(RES) via a complement-mediated phenomenon [11]. The surfaces of
LEHs have been previously modified with ganglioside G
M1
and anionic
lipids in order to increase their circulatory half-life [12, 13]. However, pre-
vious studies have shown that G
M1
surface modification of LEHs did not
increase the intravascular persistence of LEH dispersions [13].
The most promising strategy to improve the circulation halflife and
stability of LEHs is through surface modification with poly(ethylene
glycol) or PEG [3, 14]. PEG is a biologically inert polymer, which has been
used extensively in drug delivery systems and is declared to be clinically
safe by the Food and Drug Administration (FDA) [3, 4, 14]. It was pro-
posed that PEG conjugation creates a steric hydrophilic barrier surround-
ing each LEH, protecting LEHs from opsonizing plasma proteins, and
thus increasing their intravascular persistence [14, 15]. The circulation
half-life of LEH dispersions is dependent on vesicle size, molecular weight
of PEG, and molar percentage of PEG incorporated into the bilayers.
Awasthi et al. [16] studied the size dependence of empty PEG-
conjugated liposomes on their circulation half-life in rabbits. The bilayers
of these PEG-liposomes were composed of phospholipids, cholesterol,
a-tocopherol and 0.025 mol%PEG (molecular weight = 5000 Da). They
138 D. R. Arifin and A. F. Palmer
observed that the optimum diameter of PEG-liposomes corresponding
to maximum liposome circulation was 160220 nm, with circulation
half-lives ranging between 2527 hours. In this size range, liver uptake
was minimized, and spleen uptake was moderate. Beyond this size range,
PEGs ability to camouflage LEHs was compromised, and high accumu-
lation in the RES was observed. Zheng et al. [17] measured the circulation
half-life of LEHs grafted with 5 mol% of 1900 Da PEG in rabbits, and
observed a half-life of 1520 hours, similar to the circulation half-life
of non-modified LEHs [8, 9]. Increasing the PEG mole percentage in lipid
bilayers up to 10 mol% and using 5000 Da PEG, Phillips et al. [18]
demonstrated that PEG-conjugation could indeed extend the circulatory
half-life of LEH dispersions in rabbits up to 65 hours.
The steric barrier created by PEG conjugation also prevents LEH
aggregation and fusion and thus, stabilizes LEH dispersions during
storage [10]. PEG-LEH dispersions stored in a deoxygenated state at 4
and 23

C were stable without observable Hb leakage for one year, and

showed only a slight decrease in pH and P
50
, provided that the P
50
of
LEH dispersions was regulated by coencapsulating the allosteric effector
pyridoxal 5
/
-phosphate (PLP) [10]. PEG-LEH dispersions stored at 40

C
were stable for 6 months, before liposome aggregation and Hb leakage
were observed. MetHb formation of these LEH dispersions was reversed
from an initial MetHb level of 3%to 1%after one month of storage via
coencapsulation of the reductant homocysteine [10].
PEG surface modification improved the rheology and hemodynamic
properties of LEH dispersions, as demonstrated by Sakai et al. [19].
Specifically, it was observed that the viscosity of non-modified LEH dis-
persions suspended in an albumin solution was 8 cP due to LEH aggre-
gation and interaction with albumin. In contrast, the viscosity of PEG-
LEHs suspended in albumin was 3.5 cP, comparable to that of human
blood (~4 cP) [19]. In an additional study using artificial capillaries, it
was shown that both non-modified and PEG-LEH dispersions could eas-
ily permeate through capillaries with diameters of 3 mm and less, while
erythrocytes could hardly traverse these capillaries. It was observed that
PEG-LEH dispersions had a higher permeation rate due to the
suppression of liposomes aggregation [19]. In humans, the capillary dia-
meter ranges from ~3 mm to 15 mm, and during circulatory failure such as
in the case of hemorrhagic shock, the capillary constricts [19]. This study
demonstrates quite convincingly that LEHs can easily permeate through
capillary blockages, and are suitable oxygen carriers in cases of trauma
and routine surgery.
The biocompatibility of PEG-conjugated LEHs was tested in several
studies. Wakamoto et al. [20] studied the effect of PEG-LEHs on human
platelet functions in vitro. Neither platelet aggregation nor platelet acti-
vation was observed, which suggested that PEG-LEHs had no adverse effect
on platelet function in plasma. Sakai et al. [19] showed that erythrocytes
Physical Properties and Stability Mechanisms of PEG-LEHs 139
exhibited no signs of aggregation or deformation in the presence of PEG-
LEHs, indicating that these liposomes did not affect the structural integrity
of erythrocytes. Sherwood et al. [21] infused PEG-LEH and non-modified
LEH dispersions into Listeria monocytogenes-infected mice, and concluded
that PEG-LEHs had significantly less of an adverse impact on host immun-
ity compared to non-modified LEHs. Further animal studies demonstrated
that while infusion of PEG-conjugated and non-modified LEH dispersions
into laboratory animals resulted in normal mean arterial pressure, heart rate
and blood gas parameters, PEG-conjugation prevented vasoconstriction
[22] and significantly improved the hemodynamic state of the laboratory
animals compared to non-modified LEHs [2325].
In this study, PEG-LEH dispersions composed of dimyristoyl-
phosphatidylcholine, cholesterol, a-tocopherol, dimyristoyl-phosphatidyl-
glycerol, and dimyristoyl-phosphoethanolamine-PEG were prepared
via extrusion in phosphate buffered saline (PBS) at pH 7.3 through
100, 200 and 400 nm pore diameter membranes with an initial bovine
Hb concentration of 300 mg=mL. PEG-LEHs were formed with two dif-
ferent PEG molecular weights (550 and 2000 Da). PBS was used as the
extrusion buffer since the allosteric effector of bovine Hb is chloride ions
[2]. A physiological concentration of chloride ions was used to solvate Hb
molecules, in order to obtain P
50
s close to that of human red blood cells.
The effect of using different molecular weight PEGs on LEH: size distri-
bution, encapsulation efficiency, oxygen affinity, and encapsulated
methemoglobin (MetHb) level was measured. The size distribution and
encapsulation efficiency of each PEG-LEH dispersion was measured
using an asymmetric flow field-flow fractionator (AFFF) coupled with
a multi-angle static light scattering (MASLS) photometer, and a differen-
tial interferometric refractometer (DIR) [26]. The oxygen affinity of
PEG-LEHs was determined by evaluating their P
50
s and cooperativity
coefficients. a-tocopherol was incorporated into the lipid bilayers to sup-
press lipid oxidation, and a reductant, N-acetyl-L-cysteine (NAC) was
coencapsulated inside the liposome aqueous core to suppress Hb oxi-
dation [8, 27]. Since it was previously observed that chloride ions can also
increase Hb oxidation [26, 28], LEHs were also extruded in phosphate
buffer (PB, pH 7.3). Moreover, the ionic strength of the extrusion buffers
also affected the physical conformation of the PEG brushes.
Having established that LEH surface modification with PEG could
prolong LEH circulation half-lives [18], the mechanism which stabilized
PEG-conjugated LEHs was investigated. We undertook this effort by
evaluating the effective bending constants, K
B
, and spontaneous radii
of curvatures, R
0
, extracted by fitting Jung et al.s size distribution model
of liposome dispersions [1] to PEG-LEH size distributions measured
using AFFF-DIR-MASLS. The magnitude of K
B
reflected the energy
required to bend a bilayer away from its spontaneous radius of curvature
(R
0
), or it could be thought of as the minimum energy needed to maintain
140 D. R. Arifin and A. F. Palmer
a vesicle of radius R
0
. The mechanism governing LEH stabilization
either by thermal undulations or spontaneous curvature was determined
by examining the magnitude of K
B
[1, 29]. Both mechanisms only permit-
ted formation of unilamellar liposomes. The only known similar study
done on PEG-conjugated vesicles was performed by Kang et al. [30] on
cetyltrimethylammonium bromide=sodium perfluorooctanoate (FC
7
) and
CTAB=sodium perfluorohexanoate (FC
5
) surfactant mixtures. Another
method that is currently being used to probe the stability mechanism
of liposomes is micropipette aspiration, which can only be used to probe
the mechanical properties of giant liposomes (~10 mm in diameter) [31].
However, it is important to note that the size of the liposomes to be
probed will influence [32] the elucidated vesicles stability mechanism,
which can cause discrepancies between the stability mechanism elucidated
for giant liposomes (diameter ~10 mm) and sub-micron sized vesicle
dispersions. In this study, the experimentally measured size distributions
and encapsulation efficiencies of PEG-LEH dispersions will be explained
in light of the magnitude of K
B
, which will determine the stability
mechanism for liposome dispersions. In addition, the effect of PEG mol-
ecular weight and ionic strength of the extrusion buffers on the elucidated
stabilization mechanism of PEG-LEH dispersions will be explored.
MATERIALS AND METHODS
Materials
Dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylgly-
cerol (DMPG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-
N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000), 1,2-
dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-550] (DMPE-PEG550), cholesterol and polycarbonate membranes
were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL).
Phosphate buffered saline (PBS) and phosphate buffer (PB) at physio-
logical pH 7.3, were made using phosphate salts and NaCl obtained from
Sigma-Aldrich (St. Louis, MO). Spectra=Por
1
Cellulose Ester dialysis
bags with 100,000 MWCO were purchased from Spectrum Laboratories,
Inc. (Rancho Dominguez, CA). HEMOX-solution, additive-A and
anti-foaming agent were purchased from TCS Scientific Corp (New
Hope, PA). Octyl-b-D-glucopyranoside (OBG), N-acetyl-L-cysteine, and
a-tocopherol were obtained from Sigma-Aldrich (St. Louis, MO).
Hb Preparation
Tetrameric Hb was extracted from freshly drawn bovine erythrocytes and
assayed by UV visible spectrophotometry (Varian, Inc., Palo Alto, CA)
Physical Properties and Stability Mechanisms of PEG-LEHs 141
according to previous publications [26, 3236]. Bovine Hb has been
shown to function as a compatible and convenient replacement to human
Hb for use in formulating Hb-based blood substitutes [37]. Bovine
erythrocytes were extracted, centrifuged and collected in 3.8% sodium
citrate by Animal Technologies, Inc. (Tyler, Texas). The purity of
extracted tetrameric Hb was established by SDS-PAGE at 99%.
Lipid Composition
A combination of DMPC, cholesterol, DMPE-PEG, DMPG and
a-tocopherol was used in a 43:40:10:5:2 mole ratio to produce LEHs.
Unsaturated phosphatidylcholine (PC) is capable of oxidizing encapsu-
lated Hb within a few days, therefore saturated PC, DMPC in this study,
was found to be suitable in limiting Hb oxidation [8]. Cholesterol was
added to the LEH bilayer to overcome several known problems. Intra-
vascularly, cholesterol in erythrocyte membranes transferred to pure
phospholipid liposome membranes due to the cholesterol concentration
gradient, resulting in osmotically fragile erythrocytes [38]. Addition of
cholesterol to liposomes reduced this concentration gradient. Moreover,
cholesterol enhanced the liposomes resistance to fusion and lysis [8, 39],
and the liposomes impermeability to small ions [40]. DMPG was
included to impart a slight negative charge to the vesicle bilayers, since
neutral liposomes composed of phospholipids and cholesterol displayed
a tendency to aggregate [8]. Also, it was shown [8] that DMPG was safe
when infused into mice, and that addition of DMPG in the amount of
5% mole fraction produced the most optimum encapsulation efficiency.
Incorporation of DMPE-PEG into bilayers membranes is able to increase
the circulation half-life of LEHs [3, 4, 18] by masking the LEHs from
being recognized by the RES. Two molecular weights of PEG were used
in this study: 550 and 2000 Da. Lipid bilayers are susceptible to oxi-
dation, thus a-tocopherol was incorporated in the bilayers to suppress
bilayer oxidation [18]. Hb oxidation was suppressed by inclusion of
200 mg=L of the reductant N-acetyl-L-cysteine (NAC) in the extrusion
buffer [27].
LEH Preparation
Liposomes were extruded through polycarbonate membranes with the
following membrane pore diameters: 400 nm, 200 nm, and 100 nm with
an initial Hb concentration of 300 mg Hb per mL of buffer. Twenty
milligrams of the lipid mixture was dried using a Buchi R-205 rotary
evaporator (Buchi Analytical, Inc., New Castle, DA) for at least 4 hours
at 40

C [41]. The resultant lipid film was rehydrated with one mL of

300 mg=mL Hb solution (diluted in either PB or PBS at pH = 7:3,
142 D. R. Arifin and A. F. Palmer
accordingly). Extrusion was done in steps starting from a larger pore
diameter successively down to the final pore diameter [8]. Ten passes
were executed for each successive step, and 25 passes were completed
at the final step to achieve a homogenous dispersion [26, 32, 4247].
Empty liposomes were extruded in both PB and PBS with 200 mg=L
of NAC to serve as control dispersions. Liposome dispersions were used
immediately after preparation.
Size Distribution and Encapsulation Efficiency
LEH size distributions and encapsulation efficiencies (E%) were mea-
sured simultaneously using an Eclipse asymmetric flow field-flow fractio-
nator coupled in series to a 18-angle Dawn EOS multi-angle static light
scattering photometer equipped with a linearly polarized 30 mW gallium-
arsenide laser operating at 690 nm, and an Optilab DSP differential
interferometric refractometer (Wyatt Technology Corp., Santa Barbara,
CA) [26, 43, 45, 46]. Light scattering spectra were analyzed using the
ASTRA
TM
software (Wyatt Technology Corp.). The mobile phase for
all experiments was PBS at physiological pH = 7:3, filtered through 0.2
micron filters.
Oxygen Affinity
Freshly extruded LEH dispersions were dialyzed overnight in PBS (pH
7.3) at 23

C using dialysis bags with a 100,000 Da MWCO (Spectrum

Labs, Rancho Dominguez, CA) to separate unencapsulated Hb from
LEHs. In preparation for oxygen binding measurements, 0.2 mL of
dialyzed LEHs was lightly mixed with 4.8 mL of HEMOX solution,
20 mL of additive-A and 10 mL of anti-foaming agent. The P
50
and
cooperativity coefficient of LEH dispersions were measured using a
Hemox
TM
-Analyzer from TCS Scientific Corp (New Hope, PA) at
37

C [26, 3236]. The P

50
and cooperativity coefficient were both calcu-
lated by fitting the oxygen binding curve to the Adair equation [48].
MetHb Level
Protein adsorption to phospholipid membranes confers resistance to
detergent lysis. Octyl-b-D-Glucopyranoside (OBG) is a detergent known
to transform LEH dispersions into micelles [8]. To measure the MetHb
level inside the LEH particles, first freshly extruded LEH was dialyzed
overnight in PBS (pH 7.3) at 23

C, using dialysis bags with

100,000 Da MWCO membranes (Spectrum Labs, Rancho Dominguez,
CA) to separate unencapsulated Hb from the LEHs dispersion. LEH
Physical Properties and Stability Mechanisms of PEG-LEHs 143
particles were then lysed with OBG to release the encapsulated Hb into
solution. The MetHb concentration encapsulated inside the LEH disper-
sion was then assayed according to a previously published procedure [26].
THEORETICAL BACKGROUND
Background theories of light scattering, Hb encapsulation efficiency
determination, and oxygen binding equilibria, were described in a pre-
vious publication [26, 43, 45].
Size Distribution Model of Liposome Dispersions
Previous studies [1, 29, 30, 49] have proposed that the polydispersity of
a vesicle dispersion is controlled by the natural fluctuations of the lipid
bilayers curvature around a certain spontaneous curvature, and hence,
the size distribution of this suspension depends on the bilayer fluctuations.
It is possible to apply the thermodynamic theory of aggregation to a lipo-
some dispersion, and calculate the effective bending constant and radius of
curvature of the liposomes bilayers [49, 50]. The size distribution model
presented here is taken from Denkov et al.s derivation [49].
Applying thermodynamic theory, each liposome is considered as
an aggregate composed of N lipid units (assuming that DMPC, DMPG,
DMPE-PEG, a-tocopherol, and cholesterol molecules are uniformly
distributed in the bilayer, we can equally divide the bilayer into N seg-
ments or lipid units consisting of a fixed composition of the 5 molecules).
At equilibrium, the chemical potential of the molecules in all aggregates
must be equal:
l
N
= l
0
N

kT
N
ln
X
N
N

= const (1)
where l
N
is the chemical potential of a lipid unit in a vesicle composed of
N lipid units, l
0
N
is standard chemical potential, which is independent of
the aggregate concentration (however, it depends on the temperature,
pressure and radius of the liposomes), kT is the thermal energy (k is
Boltzmanns constant and T is the absolute temperature of suspension),
and X
N
is the total molar concentration of the lipid units in all aggregates
composed of N lipid units. M is an arbitrary reference state, but in this
case M is taken as the number of lipid units in an aggregate having the
mean size with X
M
as the molar concentration. The probability of finding
an aggregate of N lipid units is [49, 50] determined by the balance
between the entropy and curvature energy:
X
N
N
=
X
M
M
exp
M l
0
M
l
0
N

kT

N
M
(2)
144 D. R. Arifin and A. F. Palmer
Since the temperature and pressure are kept constant throughout the
experiments, the standard chemical potential depends solely on the lipo-
some radius.
For small deviations from the spontaneous curvature, and assuming
that the radius of spontaneous curvature, R
0,
is the mean liposome
radius, the bending energy can be presented as [4951]:
W
B
=
K
2
1
R

1
R
0

2
(3)
where W
B
is the bending elasticity energy per unit area of the bilayer, and
K is the average bending elasticity constant. The total surface area of the
liposomes is proportional to the number of lipid units:
N =
4pR
2
A
0
and M =
4pR
2
0
A
0
(4)
where A
0
is the area of one lipid unit. Combining the previous equations
and substituting C
N
= X
N
=N, the liposome size distribution model can
be derived. Assuming that the liposome suspension is fairly homogeneous
or (1 R=R
0
)
2
<< 1, the model can be simplified by expansion to yield:
C(R) ~ C(R
0
) exp
2pK
kT
R R
0
R
0

2
" #
(5)
where C(R) andC(R
0
) are the molar or number concentrations of liposomes
of radius R and R
0
, respectively. This equation predicts a Gaussian size dis-
tribution around the average radius R
0
. Note that the bending energy
expressed in Eq. 5 is in agreement with Israelachvilis definition [50], which
is four times larger than Helfrichs effective bending energy (K
B
) [51]; hence,
consequently, K is four times larger than Helfrichs effective bending con-
stant [49, 51]. Jung et al. [1] used a similar size distribution model based
on Helfrichs definition of the bending energy (K
B
) [51]:
C(R) ~ C(R
0
) exp
8pK
B
kT
R R
0
R
0

2
" #
(6)
A mathematical tool, Scientist Software (Micromath Scientific Software,
Inc., St. Louis, MO), was used to fit Eq. 6 to the measured differential num-
ber size distribution, toextract K
B
andR
0
as well as their respective standard
deviations [32].
Since Jung et al.s model [1] was developed for spontaneously formed
vesicles at equilibrium, it is pertinent to discuss the validity of applying
this model to our liposome dispersions, which were created via extrusion.
Physical Properties and Stability Mechanisms of PEG-LEHs 145
It is very difficult to prove that liposome dispersions are at equilibrium
[1]. However, if the composition and phase of the dispersion are repro-
ducible regardless of the sample history (i.e. the liposome size distribution
is stable for a long period of time on the order of several months), the
dispersion can be safely assumed to be at equilibrium [1]. Based on our
146 D. R. Arifin and A. F. Palmer
observations, our PEG-liposome size distributions were stable for at least
two months. Sakai et al. have shown that PEG-LEHs created via
extrusion can be stable for as long as one year [10]. In addition, we
observed that when our liposomes were prepared at room temperature,
followed by storage at 23

C for a day and then the temperature was

raised back to room temperature, the size distribution of the originally
extruded liposomes was recovered regardless of temperature cycling.
Hence, we concluded that our liposome dispersions are at equilibrium.
RESULTS AND DISCUSSION
Size Distribution
The size distributions of PEG-LEH dispersions extruded with an initial
Hb concentration of 300 mg=mL and their corresponding controls
(empty liposomes) are displayed in Figures 1 and 2, respectively. Tables
1 and 2 contain the number- (R
n
), weight- (R
w
), z-average radii (R
z
),
and polydispersity indices (R
w
=R
n
and R
z
=R
n
) of PEG-LEH and PEG-
control dispersions. For convenience, PEG-LEHs (PEG molecular
weight = 2000 Da) extruded in PB are referred to as PB-PL2000, and
PEG-LEHs (PEG molecular weight = 550 Da) extruded in PB are
referred to as PB-PL550. Likewise, PEG-LEHs extruded in PBS are
referred to as PBS-PL2000 and PBS-PL550, and the corresponding
PEG-conjugated controls are referred to as PB-C2000, PB-C550,
PBS-C2000, and PBS-C550.
PEG-LEHs and controls extruded through 100 nm pore diameter
membranes exhibited a mean diameter that was larger than the mem-
brane pore diameter, while liposomes extruded through 200 nm pore
diameter membranes exhibited a mean diameter that was close to the
membrane pore diameter, and liposomes extruded through 400 nm mem-
branes exhibited a mean diameter that was smaller than the membrane
pore diameter. Non-modified LEHs (non-PEGylated) and their controls
Figure 1. Size distributions of PEG-conjugated LEH dispersions extruded with
an initial Hb concentration of 300 mg=mL. Differential and cumulative size distri-
butions of 2000 Da PEG-conjugated LEH dispersions extruded in PB are dis-
played in panels A and B respectively; 2000 Da PEG-conjugated LEH
dispersions extruded in PBS are displayed in panels C and D, respectively; 550
Da PEG-conjugated LEH dispersions extruded in PB are displayed in panels E
and F, respectively; and 550 Da PEG-conjugated LEH dispersions extruded in
PBS are displayed in panels G and H, respectively. Dashed lines represent
PEG-LEHs extruded through 400 nm pore diameter membranes, solid lines rep-
resent extrusion through 200 nm pore diameter membranes, and the dotted lines
represent extrusion through 100 nm pore diameter membranes.
3
Physical Properties and Stability Mechanisms of PEG-LEHs 147
size distributions also exhibited a similar trend [26]. Although the poly-
dispersity indices of our dispersions indicated that PEG-LEH and PEG-
control dispersions were monodisperse (indices were close to one in
value), closer inspection revealed that the size distributions of liposomes
extruded in PB were narrower, compared to those of liposomes extruded
148 D. R. Arifin and A. F. Palmer
in PBS, regardless of the PEG molecular weight. The distribution widths
of PB-PL2000 and PB-PL550 dispersions extruded through 100, 200 and
400 pore diameter membranes were approximately 1020 nm and 40 nm,
respectively. In contrast, the distribution widths of PBS-PL2000 and
PBS-PL550 extruded through 100, 200 and 400 pore diameter membranes
were 2060 nm and 3060 nm, respectively. Electrostatic repulsion
between the chloride ions in the buffer, and the negatively charged
DMPG and DMPE-PEG in the bilayers could disrupt the liposomes
dispersion state. Moreover, conjugation with higher molecular weight
Figure 2. Size distributions of PEG-conjugated control (empty liposome) disper-
sions. Differential and cumulative size distributions of 2000 Da PEG-conjugated
control dispersions extruded in PB are displayed in panels A and B, respectively;
2000 Da PEG-conjugated control dispersions extruded in PBS are displayed in
panels C and D, respectively; 550 Da PEG-conjugated control dispersions
extruded in PB are displayed in panels E and F, respectively; and 550 Da PEG-
conjugated control dispersions extruded in PBS are displayed in panels G and
H, respectively. Dashed lines represent PEG-controls extruded through 400 nm
pore diameter membranes, solid lines represent extrusion through 200 nm pore
diameter membranes, and dotted lines represent extrusion through 100 nm pore
diameter membranes.
3
Table 1. Number-, weight-, z-average radii, and polydispersity indices of PEG-
LEH and PEG-control dispersions extruded in PB. Part A corresponds to
PEG-liposomes with a PEG molecular weight = 2000 Da, and part B corresponds
to PEG-liposomes with a PEG molecular weight = 550 Da
PB as the extrusion buffer
[Hb] Pore radius R
n
R
w
R
z
(mg=mL) (nm) (nm) (nm) (nm) R
w
=R
n
R
z
=R
n
A
300 200 118.9 120 120.6 1.01 1.01
100 97.8 98.2 98.1 1 1
50 113.5 113.7 112.4 1 0.99
0 200 100.2 100 99.3 1 0.99
100 93.4 92.7 92.2 0.99 0.99
50 78.1 79.4 79.6 1.02 1.02
B
300 200 89.6 88.3 89.3 0.99 1
100 75.1 78.3 80.4 1.04 1.07
50 72 77.6 80.5 1.08 1.12
0 200 100.2 100 99.3 1 0.99
100 90.7 91 91.1 1 1
50 62.9 65 66.4 1.03 1.06
Physical Properties and Stability Mechanisms of PEG-LEHs 149
PEG resulted in narrower size distributions for PEG-LEH dispersions
extruded in the same buffer. It appeared that higher molecular weight
PEG chains were more effective in suppressing aggregation and fusion
between liposomes, hence creating a more monodisperse suspension.
A similar trend was also observed with PEG-controls. While the
distribution widths of both PB-C2000 and PBS-C2000 extruded through
100, 200 and 400 pore diameter membranes were almost similar, ranging
from 15 nm to 40 nm, those of PB-C550 and PBS-C550 ranged from
35 nm to 50 nm and 40 nm to 150 nm, respectively. In summary, we
observed that PEG-conjugated liposome dispersions extruded in PB were
more monodisperse than those extruded in PBS, and higher molecular
weight PEG promoted the formation of narrower size distributions.
Since this is the first study to measure the absolute size distribution
of PEG-LEHs, we could only compare the average size of PEG-LEH
and PEG-control dispersions with results obtained from other groups.
Extrusion though 220 nm pore diameter membranes yielded 222 62 nm
[10], 230 81 nm[20], 259 82 nm[23], and 250 80 nm [19] PEG-LEHs,
while microfluidization yielded 193 nm diameter PEG-LEHs [18]. The
best comparison was with Awasthi et al. [16] who prepared 136.2, 165.5,
209.2, 275 and 318 nm diameter empty PEG-liposomes via extrusion.
Awasthi [52] experienced difficulties in preparing PEG-liposomes with
Table 2. Number-, weight-, z-average radii, and polydispersity indices of PEG-
LEH and PEG-control dispersions extruded in PBS. Part A corresponds to
PEG-liposomes with a PEG molecular weight = 2000 Da, and part B corresponds
to PEG-liposomes with a PEG molecular weight = 550 Da
PBS as the extrusion buffer
[Hb] Pore Radius R
n
R
w
R
z
(mg=mL) (nm) (nm) (nm) (nm) R
w
=R
n
R
z
=R
n
A
300 200 122.8 131.7 132.3 1.07 1.08
100 121 125.1 124.8 1.03 1.03
50 83.9 86.7 85.6 1.03 1.02
0 200 139.7 139.7 139 1 0.99
100 96 98.1 97.3 1.02 1.01
50 75.2 75.3 75 1 1
B
300 200 120.1 117.2 114.9 0.98 0.96
100 95.3 107.3 109.5 1.13 1.15
50 63.9 67.2 69 1.05 1.08
0 200 174.7 171.2 165 0.98 0.94
100 96.8 106.8 109.7 1.1 1.13
50 70.1 70 71.1 1 1.01
150 D. R. Arifin and A. F. Palmer
diameters less than 100 nm via extrusion, while microfluidization yielded
liposomes with sizes ranging from 80 nm to 120 nm. If anionic lipids
were used (in our case, DMPG is negatively charged), liposomes with
diameters larger than 320 nm could not be prepared via extrusion [52].
This observation confirmed our finding that only PEG-liposomes with
diameters ranging from 160 nm to 240 nm could easily be prepared by
extrusion.
Encapsulation Efficiency and Oxygen Affinity
Hb Encapsulation efficiencies, estimated entrapped Hb concentrations,
P
50
s and Hill coefficients (n) of PEG-LEHs are displayed in Tables 3
and 4. The encapsulated MetHb levels for all samples were less than
5%. Although, it was reported that chloride ions promoted Hb oxidation
[26, 28], we did not observe MetHb level differences in PEG-LEHs
extruded in PB and those extruded in PBS. The P
50
s of PEG-LEHs
extruded in PBS were higher than those of PEG-LEHs extruded in PB,
which was expected since chloride ions are an allosteric effector of bovine
Hb [2]. Regardless of the extrusion buffer, PEG molecular weight, and
extrusion membrane pore size, all PEG-LEHs exhibited P
50
s and coop-
erativity coefficients close to the measured P
50
and cooperativity coef-
ficient of bovine Hb (28 torr and 2.42.5, respectively), and were
comparable to that of human blood (26 torr and 2.32.4, respectively)
[4]. Surface modification with PEG clearly did not compromise the oxy-
gen binding properties of PEG-LEHs. However, we found that the
encapsulation efficiencies of PB-PL2000, PB-PL550 and PBS-PL550 were
Table 3. Hb encapsulation efficiencies, estimated entrapped Hb concentrations,
P
50
s and Hill coefficients (n) of PEG-LEHs extruded in PB. Part A corresponds
to PEG-liposomes with a PEG molecular weight = 2000 Da, and part B corre-
sponds to PEG-liposomes with a molecular weight = 550 Da
PB as the extrusion buffer
Pore radius [Hb entrapped] P
50
(nm) E% (mg=mL) (mmHg) Hill no.
A
200 4.27% 12.81 24.72 2.29
100 10.54% 31.62 22.53 2.67
50 7.46% 22.38 21.35 2.67
B
200 5.18% 15.54 30.67 2.23
100 3.89% 11.67 27.56 2.25
50 6.02% 18.06 37.57 1.92
Physical Properties and Stability Mechanisms of PEG-LEHs 151
very low (less than 11%) and the estimated entrapped Hb concentrations
were significantly less than the Hb concentration of human erythrocytes
(150 mg Hb=mL of blood) [4]. Note that in our PEG-LEHs preparation
scheme, PEG chains were incorporated into both the inner and outer
leaflets of the liposomes; and consequently, PEG brushes in the inner
leaflet reduced the core volume available for Hb encapsulation. Strangely,
the encapsulation efficiencies of PBS-PL2000 were high (2736%), where
the highest encapsulation efficiency (36.31%) was obtained by extruding
PEG-LEHs with the largest pore diameter membrane (400 nm). However,
we did not expect this result, since incorporation of lower molecular
weight PEG should have increased the entrapped Hb concentration.
Moreover, we observed that lowering the ionic strength of the extrusion
buffer generally lowered the Hb encapsulation efficiency.
Phillips et al. [18] reported that PEG-LEHs with an average diameter
of 193 nm could encapsulate 1.2 g=dL or 12 mg=mL of Hb (with a lipid
bilayer composed of 10 mol% of DSPE-conjugated 5000 Da PEG). Note
that in these dispersions [18], PEG brushes were also incorporated into the
inner leaflet of the liposomes, thus, a low encapsulated Hb concentration
was expected. Our estimated entrapped Hb concentrations of PEG-LEHs
extruded through 200 nm pore diameter membranes in PB and PBS were
31.62 and 92.70 mg=mL, respectively, for LEHs conjugated with 2000 Da
PEG, and 11.67 and 32.19 mg=mL, respectively, for LEHs conjugated with
550 Da PEG. We expected to obtain higher Hb encapsulation efficiencies
than Phillips et al. [18] since we employed shorter PEG chains. Hence, it
was surprising to observe that PB-PL550 (extruded through 200 nm pore
diameter membranes) exhibited an encapsulated Hb concentration close
to the value obtained by Phillips et al. [18]. Sakai et al. [10, 19, 23] managed
Table 4. Hb encapsulation efficiencies, estimated entrapped Hb concentrations,
P
50
s and Hill coefficients (n) of PEG-LEHs extruded in PBS. Part A corresponds
to PEG-liposomes with a PEG molecular weight = 2000 Da, and part B corre-
sponds to PEG-liposomes with a molecular weight = 550 Da
PBS as the extrusion buffer
Pore radius [Hb entrapped] P
50
(nm) E% (mg=mL) (mmHg) Hill no.
A
200 36.31% 108.93 25.05 2.51
100 30.90% 92.7 38.25 2.06
50 27.35% 82.05 37.86 2.13
B
200 1.43% 4.29 33.15 2.2
100 10.73% 32.19 24.99 2.57
50 1.00% 3 17.1 2.16
152 D. R. Arifin and A. F. Palmer
to increase the entrapped Hb concentration to 10 g=dL or 100 mg=mL by
exclusively incorporating 5000 Da PEG on the outer surface of the PEG-
LEHs (220250 nm in diameter). Note that although the lower limit of Hb
concentration in a Hb-based blood substitute has never been established,
for acellular, Hb-based artificial blood substitutes, 5 g=dL (50 mg=mL)
of Hb is theoretically sufficient to deliver adequate amounts of oxygen
at rest without taxing cardiac output, provided that vasoconstriction does
not occur [53]. For cellular-based blood substitutes, the required Hb con-
centration should be higher because oxygen has to first diffuse out of the
artificial membranes, before it is transported to tissues.
Stability Mechanisms
In order to understand the stabilization mechanism of our dispersions, we
extracted effective bending constants, K
B
, and spontaneous radius of cur-
vatures, R
0
, by fitting Jung et al.s liposome size distribution model [1] to
our differential number size distributions measured by AFFF-MASLS-
DIR. The results of these curve fittings and their corresponding standard
deviations are displayed in Tables 5 and 6. Note that K
B
and its standard
deviation, D
K
, are presented in units of kT, where k is the Bolztmanns
constant and T is the absolute temperature. The magnitude of K
B
dictates
the stabilization mechanism of liposome dispersion. If K
B
~kT then the
liposome dispersion is stabilized by thermal undulations, otherwise, if
K
B
>> kT then the stabilization mechanism is spontaneous curvature,
and the bilayers are more rigid [54, 55].
PB-PL2000 dispersions were stabilized by spontaneous curvature,
regardless of the membranes pore diameters used for extrusion. By chan-
ging the extrusion buffer to PBS, the bilayers rigidity decreased (K
B
s
decreased), but the PBS-PL2000 dispersions were still stabilized by
spontaneous curvature, except for PEG-LEHs extruded through
400 nm pore diameter membrane. Conjugation with lower molecular
weight PEG (PB-PL550) decreased the bilayers rigidities, but, changing
the extrusion buffer to PBS, decreased K
B
further and shifted the stabili-
zation mechanism of PBS-PL550 dispersions from spontaneous curvature
to thermal undulations. The size distributions of liposome dispersions
with high K
B
s are expected to be stabilized by spontaneous curvature
and are expected to be narrower [1, 55], which was the trend observed
with the measured size distributions in this study. Moreover, the decrease
in bilayer rigidity with decreasing PEG molecular weight explained
the trend previously observed with the measured Hb encapsulation
efficiencies. Highly flexible (less rigid) bilayers permitted Hb leakage
[56]. However, it did not explain why the presence of salt in the extrusion
buffer increased Hb encapsulation, while decreasing bilayer rigidity. We
propose that extrusion in PBS increased the ionic strength inside the
Physical Properties and Stability Mechanisms of PEG-LEHs 153
liposomes. Consequently, these ions screened the charges on the PEG
molecules, which caused the PEG tails to collapse into a globular confor-
mation to shield the hydrophobic layers of the membranes from the
highly charged aqueous core [56], and hence permitting more Hb to be
encapsulated, such as the case with PBS-PL2000. For PBS-PL550, the
fluctuating bilayers (stabilized by thermal undulations) caused extensive
Hb leakage, and consequently, exhibited low Hb encapsulation efficien-
cies, despite the favorable extrusion buffer. Comparing the effective
bending constants of PEG-LEHs and non-modified LEHs (data was
taken from a previous study [32], refer to Table 7), we found that conju-
gation with 2000 Da and 550 Da PEG increased the bilayers rigidity.
A more significant increase in bilayer rigidity was observed with
2000 Da PEG-conjugation compared to 500 Da PEG-conjugation.
The incorporation of wedge-shaped PEG molecules into bilayers
Table 5. Effective bending constants, K
B
, and spontaneous radii of curvature, R
0
,
and their corresponding standard deviations, D
K
and D
Ro
of PEG-LEH and PEG-
control dispersions extruded in PB. Note that K
B
and its standard deviation, D
K
,
are presented in units of kT, where k is Bolztmanns constant and T is the absolute
temperature. Part A corresponds to PEG-LEHs with a PEG molecular
weight = 2000 Da, part B corresponds to PEG-LEHs with a PEG molecular
weight = 550 Da, part C corresponds to PEG-controls with a PEG molecular
weight = 2000 Da, and part D corresponds to PEG-controls with a PEG
molecular weight = 550 Da
Extrusion buffer: PB
Pore radius K
B
D
K
R
0
D
Ro
(nm) (kT) (kT) (nm) (nm)
A
200 19.86 0.413 120.41 0.0386
100 29.89 0.178 97.73 0.0072
50 23.41 0.856 109.83 0.057
B
200 5.74 0.072 88.03 0.0324
100 6.15 0.083 79.72 0.0302
50 2.67 0.044 78.35 0.0542
C
200 1.69 0.021 94.35 0.061
100 21.78 0.168 91.45 0.01
50 2.82 0.03 76.88 0.0326
D
200 1.55 0.031 113.46 0.1118
100 2.13 0.036 88.12 0.0682
50 0.78 0.015 61.71 0.0866
154 D. R. Arifin and A. F. Palmer
Table 6. Effective bending constants, K
B
, and spontaneous radii of curvature, R
0
,
and their corresponding standard deviations, D
K
and D
Ro
of PEG-LEH and PEG-
control dispersions extruded in PBS. Note that K
B
and its standard deviation, D
K
,
are presented in units of kT, where k is the Bolztmanns constant and T is the
absolute temperature. Part A corresponds to PEG-LEHs with a PEG molecular
weight = 2000 Da, part B corresponds to PEG-LEHs with a PEG molecular
weight = 550 Da, part C corresponds to PEG-controls with a PEG molecular
weight = 2000 Da, and part D corresponds to PEG-controls with a PEG molecu-
lar weight = 550 Da
Extrusion buffer: PBS
Pore radius K
B
D
K
R
0
D
Ro
(nm) (kT) (kT) (nm) (nm)
A
200 1.41 0.005 125.97 0.0242
100 26.98 0.498 122.97 0.0309
50 6.98 0.098 83.24 0.0312
B
200 2.66 0.039 110.13 0.0677
100 2.05 0.013 105.93 0.0335
50 2.45 0.038 67.08 0.046
C
200 127.6 2.8 138.34 0.0188
100 58.82 1.05 96.59 0.0158
50 88.17 1.78 74.53 0.0112
D
200 0.52 0.005 148.16 0.1355
100 0.17 0.002 79.47 0.1053
50 4.18 0.091 70.24 0.052
Table 7. Effective bending constants, K
B
, and corresponding standard deviations,
D
K
of non-modified LEHs dispersions, extruded with an initial Hb concentration
of 300 mg=mL (data taken from a previous study [32]). Note that K
B
and its
standard deviation, D
K
, are presented in units of kT, where k is the Bolztmanns
constant and T is the absolute temperature
Extrusion buffer: PB Extrusion buffer: PBS
Pore radius K
B
D
K
K
B
D
K
(nm) (kT) (kT) (kT) (kT)
200 9.16 0.082 7.98 0.096
100 1.31 0.008 2.87 0.023
50 0.17 0.003 3.48 0.07
Physical Properties and Stability Mechanisms of PEG-LEHs 155
composed of tubular-shaped phospholipid molecules induced surface
lateral expansion in the bilayers, and as a result rigidified the bilayers
[57]. Moreover, the extrusion buffer had a prominent effect on the
stabilization mechanism, specifically PEG-LEH bilayers were more rigid
(and the stabilization mechanism was via spontaneous curvature) when
the extrusion buffer was PB.
There was a mixture of both types of stabilization mechanisms for
PB-C2000 dispersions. The PEG-control dispersion extruded through
200 nm pore diameter membranes was stabilized by spontaneous curva-
ture, while the other dispersions were stabilized by thermal undulations.
Opposite to the trend observed with PEG-LEH dispersions, the bilayer
rigidity increased with extrusion in PBS, and the PBS-C2000 dispersions
were stabilized by spontaneous curvature, regardless of the vesicles size.
However, when the PEG molecular weight was decreased to 550 Da, K
B
s
decreased and the stabilization mechanisms of PEG-control dispersions
extruded in both PB and PBS shifted to thermal undulations. Based on
the theory presented earlier for the stabilization mechanism of a liposome
dispersion, the magnitude of K
B
determines the polydispersity of the lipo-
some dispersion. In our study, we observed that K
B
for PEG-control dis-
persions increased with increasing PEG molecular weight. This correlated
with a narrowing of the size distribution as K
B
increased. However, we
observed that if the molecular weight of the PEG-control dispersion
was held constant and the ionic strength of the extrusion buffer varied,
no difference was detected in the widths of the PEG-control size distribu-
tions. Taken together, these results showed that although the stability
mechanism (determined from the magnitude of K
B
) can explain the mea-
sured size distribution and encapsulation efficiency of PEG-LEH disper-
sions, it could not predict the behavior of the PEG-control dispersions.
Overall, it appeared that incorporation of higher molecular weight
PEG molecules into the bilayer imparted higher bilayer rigidity, which
agreed with reported observations that PEG-LEH circulation half-life
increased with increasing PEG molecular weight [18].
To examine the effect of Hb encapsulation on the liposome disper-
sion stabilization mechanism, we compared K
B
values between PEG-
LEH and PEG-control dispersions. For liposomes conjugated with
2000 Da PEG and extruded in PB, K
B
s increased regardless of the vesi-
cles size with Hb encapsulation, and the stabilization mechanism shifted
from thermal undulations to spontaneous curvature for liposomes
extruded through 400 and 100 nm pore diameter membranes. The
opposite trend occurred with PEG 2000-liposomes extruded in PBS,
K
B
s decreased with Hb encapsulation, but spontaneous curvature was
still maintained as the preferred stabilization mechanism. For liposomes
conjugated with 550 Da PEG and extruded in PB, K
B
s also increased
with Hb encapsulation and shifted the stabilization mechanism from
thermal undulations to spontaneous curvature. K
B
s slightly increased
156 D. R. Arifin and A. F. Palmer
with Hb encapsulation for PEG 550-liposomes extruded in PBS, but the
stabilization mechanisms of these dispersions were predominantly ther-
mal undulations. In conclusion, K
B
s generally increased with Hb encap-
sulation. It was previously observed that lipid membranes and Hb
molecules could initiate complex formation, followed by intercalation
of Hb into the bilayers [58]. Farmer et al. [8] previously observed that this
interaction manifested itself as an increase in the bilayers mechanical
strength.
Using a similar method as ours to measure the stability mechanism,
Kang et al. [30] reported that when DMPE-PEG (PEGM
w
= 5000 Da)
was mixed with spontaneously formed vesicle dispersions composed of
cetyltrimethylammonium bromide=sodium perfluorooctanoate (FC
7
)
and CTAB=sodium perfluorohexanoate (FC
5
) mixtures, the DMPE-
PEG did not insert into the vesicle bilayers, leading to macro-phase sep-
aration. Incorporation of lower molecular weight PEG (2000 Da) into
CTAB=FC
7
did not induce this phenomenon, but shifted the stabilization
mechanism from spontaneous curvature to thermal undulations (K
B
decreased from 6 kT to 0.3 kT with incorporation of PEG). As for the
CTAB=FC
5
system, the vesicles were already stabilized by thermal undu-
lations, however, addition of PEG favored the formation of a narrower
size distribution. Although the values of K
B
measured did not match
our values, the trend exhibited by these vesicle dispersions agreed with
our observations. Taken together, these results show that the influence
of PEG on the stability of vesicle dispersions depends on the molecular
weight of PEG and the composition of the vesicle bilayers.
The effective bending constants of liposomes was shown to vary as
DMPE-PEG of different degrees of polymerization were inserted into
the bilayers. K
B
s for DMPE-PEG molecules of polymerization degree
114, 45, 17 and 8 were about ~113 kT, 30 kT, 25 kT and 25 kT, respect-
ively, for 0.1 mole fraction of PEG-lipids incorporated into the bilayers
[57]. DMPE-PEG used in our study has a polymerization degree of 45
for 2000 Da PEG and 12 for 550 Da PEG. K
B
s for PB- and PBS-
PL2000, and PB- and PBS-C2000 dispersions extruded through 200 nm
pore diameter membranes were close to values obtained by Marsh [57],
while the values for liposomes grafted with 550 Da PEG were lower
than those obtained by Marsh [57]. These differences could be attributed
to the different extrusion buffers and liposome sizes employed. Never-
theless, the trend that K
B
decreased with decreasing degree of PEG
polymerization was similar to what we observed. In addition, it was
observed that short polymers (degree of polymerization _ 8) contri-
bution to effective bending constants never exceeded the K
B
for non-
modified liposomes [59]. For long polymers (2000 Da or 5000 Da
PEG), the polymer contribution could exceed that of non-modified
liposomes because of the cubic dependence of K
B
on the degree of poly-
merization [59].
Physical Properties and Stability Mechanisms of PEG-LEHs 157
Optimized Design Criteria for LEHs
In summary, extrusion in PB imparted higher bilayer rigidity (high K
B
)
and promoted the formation of narrower PEG-LEH size distributions.
Taken together, we recommend preparing PEG-LEHs in PB to create
stronger and more homogeneous artificial Hb-based blood substitutes,
which correlates directly with their circulation half-life. However, the
encapsulation efficiencies of PEG-LEHs extruded in PB were very low
(less than 11%) and their estimated entrapped Hb concentrations (less
than 33 mg=mL of buffer) were less than that of human erythrocytes
(150 mg=ml of blood [4]). This can be solved by exclusively conjugating
PEG onto the outer surface of LEHs, as demonstrated by Sakai et al.
[10, 19, 60]. Using this preparation method, even PEG brushes longer
than 2000 Da can be incorporated into the outer leaflet of the bilayers.
Another method to solve this problem is to increase the total lipid con-
centration used in the PEG-LEH dispersions. While we employed a total
lipid concentration of 20 mg=mL, Sakai et al. used a total lipid concen-
tration of 62 mg=mL [60] or 57 mg=mL [19, 23] to produce PEG-LEH dis-
persions with encapsulated Hb concentrations of 100 mg=mL. If
sufficient encapsulated Hb concentrations cannot be achieved with either
of these methods, PBS can be used as an extrusion buffer, however, the
size distributions of PEG-LEH dispersions extruded in PBS are expected
to be more polydisperse, and their bilayers are expected to be less rigid.
CONCLUSIONS
We observed that PEG-conjugated liposome dispersions extruded in PB
were more monodisperse than those extruded in PBS, and higher molecu-
lar weight PEG promoted the formation of narrower size distributions.
Moreover, extrusion in PB and conjugation with higher molecular weight
PEG imparted higher bilayer rigidity and stabilized liposome dispersions
by the spontaneous curvature mechanism. The experimentally measured
size distributions and encapsulation efficiencies of PEG-LEH dispersions
can be readily explained through analysis of the magnitude of the effective
bending constant, which dictates the stability mechanism of the liposome
dispersion. Despite previous findings that sodium chloride promotes Hb
oxidation [26], the oxygen binding properties of PBS- and PB-PEG-LEHs
were not significantly different. The P
50
s and cooperativity coefficients of
both PBS- and PB-PEG-LEHs were comparable to that of human blood,
indicating that PEG-LEHs display good potential as an artificial blood
substitute. Taking these results together, we recommend preparing PEG-
LEHs in PB to create stronger and more homogeneous artificial Hb-
based blood substitutes, which correlates directly with their circulation
half-life. However, the encapsulation efficiencies of PEG-LEHs extruded
158 D. R. Arifin and A. F. Palmer
in PB were very low (less than 11%). This can be solved by exclusively
conjugating PEG onto the outer surface of LEHs or by increasing the
total lipid concentration used in the PEG-LEH dispersions.
ACKNOWLEDGEMENTS
We acknowledge support from the National Science Foundation BES-
0196432 (Arlington, VA). We would also like to thank Dennis Birdsell
and Rian Galloway from the Center for Environmental Science and
Technology (University of Notre Dame, South Bend, IN) for the use
of the Centers facilities.
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