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Layer-Controlled Hemoglobin Vesicles by Interaction of Hemoglobin with a Phospholipid Assembly Shinji Takeoka, Takeru Ohgushi, Kouichiro Terase, Taizen Ohmori, and Eishun Tsuchida* Department of Polymer Chemistry, Advanced Research Center for Science & Engineering, Waseda University, Tokyo 169, Japan Received November 28, 1994. In Final Form: November 15, 1995X Hemoglobin vesicles, which encapsulated concentrated hemoglobin (Hb) with a bilayer of dipalmitoylphosphatidylcholine/cholesterol/palmitic acid, were prepared under various preparation conditions in order to decrease the number of bilayers (n) constructing the vesicle and increase the Hb concentration in the interior of the vesicle ([Hb]in). n decreased when the surface potential of the bilayer became more negative because of the electrostatic repulsion between the bilayers, while with a changing ζ-potential of Hb from positive to negative, [Hb]in showed a precipitous fall because of the electrostatic repulsion between Hb and the surface of the bilayer. A temperature decrease leads to a quality increase in the Hb vesicles ([Hb]/[lipid]) in spite of the [Hb]in decrease by the viscosity increase of the Hb solution. This is explained by the effective reduction of n due to the reduction in membrane fluidity and the protonation of Hb.
Introduction Phospholipids spontaneously assemble in aqueous media to form multilamellar vesicles. In the case where the medium is a concentrated hemoglobin (Hb) solution, Hb vesicles, which encapsulate concentrated Hb in the aqueous interior of the vesicle, can be prepared after the removal of Hb from the exterior aqueous phase. Recently, Hb vesicles have been intensively studied in the interdisciplinary fields in order to apply them as red cell substitutes.1-5 The concentration ratio of Hb to total lipid components (phospholipid, cholesterol, and fatty acid) of the vesicles ([Hb]/[lipid]) influences the function of the reticuloendothelial systems (RES) when Hb vesicles are injected so as to retain a sufficient amount of oxygen transport. This leads to the dysfunction of RES, toxicity, or side effects. Because a large amount of lipid means a low-quality oxygen carrier, we have researched a method to reduce the amount of total lipid components, namely, the number of bilayers, thereby retaining the high-oxygencarrying capacity. An ideal structure is a large unilamellar vesicle with a 0.2 µm diameter which was determined for the high encapsulation efficiency and sterilization using a 0.2 µm membrane filter. There are several preparation methods such as reversephase evaporation,6 freeze-thawing,7 extrusion,8 and microfluidization.9 The latter two methods are generally used for a concentrated Hb without denaturation. However, microfluidization does not seem to be useful for * To whom correspondence should be addressed: Tel, +813-32098895; fax, +813-3209-5522. X Abstract published in Advance ACS Abstracts, March 1, 1996. (1) Djordjevich, L; Miller, I. F. Exp. Hematol. 1980, 8, 584. (2) Hunt, C. A.; Burnette, R. R.; MacGregor, R. D.; Strubbe, A. E.; Lau, D. T.; Taylor, N.; Kawada, H. Science 1985, 230, 1165. (3) Farmer, M. C.; Johnson, S. A.; Beissinger, R. L.; Gossage, J. L.; Lynn, A. B.; Carter, R. L. Adv. Exp. Med. Biol. 1988, 238, 161. (4) Rudolph, A. S.; Goins, B.; Ligler, F.; Cliff, R. O.; Spielberg, H.; Hoffman, P.; Phillips, W.; Klipper, R. Prog. Membr. Biotechnol. 1991, 214-216. (5) (a) Tsuchida, E.; Takeoka, S. Artificial Red Cells; Tsuchida, E., Ed.; John Wiley & Sons: Chichester, 1995; p 35. (b)Tsuchida, E. Biomater. Artif. Cells Immobilization Biotechnol. 1992, 20, 337. (6) Szoka, F.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4194. (7) Szoka, F.; Olson, F.; Heath, T.; Vail, W.; Mayhew, E.; Papahadjopoulos, D. Biochim. Biophys. Acta 1980, 601, 559. (8) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9. (9) Sehgal, L. R.; Rosen, A. L. Biomater., Artif. Cells, Artif., Organs 1989, 17, 531.
regulating the size of the vesicles. The extrusion method using polycarbonate membrane filters with a regulated pore size is applicable to prepare Hb vesicles having a 0.2 µm diameter though the production quantity is limited. The increment of encapsulation efficiency of Hb to the vesicles could be possible if negatively charged lipids10 and cholesterol11,12 were incorporated into the bilayer membrane or if the appropriate encapsulating conditions were selected with respect to the nature of the encapsulating molecules. However, there are few reports describing the systematic control of the number of bilayers of the vesicles (n) relating to the preparation conditions.13 Because Hb vesicles are prepared in highly concentrated Hb solutions in order to increase the Hb concentration in the Hb vesicle ([Hb]in), the electrostatic interaction between Hb and the phospholipid vesicles is important and useful for controlling the properties of the resulting Hb vesicles. In this paper, the ζ-potential of the Hb molecules, the surface potential of the phospholipid membrane, the membrane fluidity, and the solution viscosity were studied to clarify their influence on Hb encapsulation into the phospholipid vesicles by controlling n and [Hb]in. Materials and Methods Chemicals. Diacylphosphatidylcholine (PC); and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Nippon Oil & Fats Co., Japan and DPPC was reprecipitated with acetone for purification. Cholesterol was purchased from Kanto Chemical Co., Inc., and was recrystallized from methanol. Fatty acids (FA), myristic acid (MA), palmitic acid (PA), and stearic acid (SA) were also purchased from Kanto Chemical Co., Inc. These lipids (PC/cholesterol/FA) were dissolved in chloroform at a 7/7/2 mole ratio and dried on the inner wall of a flask as a thin solid film using a rotary evaporator. Preparation of an Hb Solution.14 Outdated red cells (RCs) from the Hokkaido Red Cross Blood Center were diluted with an equivalent amount of saline and then shaken under a CO atmosphere to convert HbO2 into HbCO, which was stable toward oxidation (metHb formation) during preparation. After the RCs were washed with saline, the concentrated RC ([Hb] ) 25 g/dL) was hemolyzed14 and then centrifuged (1900g, 15 min). The (10) Hauser, H. Biochim. Biophys. Acta 1984, 772, 37. (11) Inoue, K. Biochim. Biophys. Acta 1974, 339, 390. (12) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839. (13) Takeoka, S.; Terase, K.; Yokohama, H.; Sakai, H.; Nishide, H.; Tsuchida, E. J. Macromol. Sci. Pure Appl. Chem. 1994, A31, 97. (14) Sakai, H.; Takeoka, S.; Yokohama, H.; Seino, Y.; Nishide, H.; Tsuchida, E. Protein Expression Purif. 1993, 4, 563.
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stroma-free Hb solution was heated at 60 °C for 10 h to remove the water-soluble proteins other than Hb. These denaturated proteins were removed by centrifugation (1900g, 20 min). The Hb solution was then dialyzed against pure water to remove any low-molecular-weight electrolytes. This Hb solution contained less than 0.2% of the remaining PC and FA from RC. After the adjustment of pH and ionic strength, the Hb solution was concentrated with ultrafilteration, yielding a highly concentrated Hb solution ([Hb] ∼ 45 g/dL). The Hb concentration was measured using a cyanometHb method. The viscosity of the Hb solution was measured with a rotating viscometer (Shibaura System Co., Vismetron VS-AK) at a rate of 11.5 s-1. Preparation of Vesicles and Hb Vesicles.5 Large multilamellar vesicles were prepared by dispersing the mixed lipid powder into buffer solutions for vesicles or Hb solutions for Hb vesicles. The multilamellar vesicles were extruded through polycarbonate membrane filters with the stepwise reduction of their pore size to 0.2 µm.8 The distribution of the diameter of the vesicles was measured using a light scattering method (Coulter, Particle Analyzer N4-SD). Unencapsulated Hb molecules were removed by gel permeation chromatography (Pharmacia. Co., Sepharose CL-4B). The Structure of Hb Vesicles.13,15 The structure of an Hb vesicle was expressed by several parameters, that is, the radius of the vesicle (r), n, the average molecular area of the lipid molecules (S), the thickness of the bilayer (d), the average distance between bilayers (dw), and [Hb]in. (A) Average Molecular Area of Mixed Lipid, S. A monolayer of mixed lipids (2 mM) was formed on the surface of various kinds of buffer solutions, and then the pressure-area isothermal curves were measured with an HBM-type balancemeter (Kyowa Interface Sci. Co., Ltd.) to obtain the value of S of the mixed lipid at various conditions. (B) The Thickness of Bilayer, d. The density of a bilayer membrane (FL) was measured from the density of a concentrated dispersion of the vesicles and the concentration of lipids in the dispersed system. The thickness of the bilayer was calculated from S and FL using
d)
Vin )
N)
4π S
2MW FLSNA
4 π{r - n(d + dW) + dW}3 3
(1)
(2)
n
∑[{r - (i - 1)(d + d
W)}
2
+ {r - i(d + dW) + dW}2]
i)1
(3)
where MW and NA are the average molecular weight of the mixed lipids and Avogadro’s number, respectively. (C) Concentration of Hb in the Interior of the Vesicle, [Hb]in. The volume of the aqueous interior of one vesicle (Vin) and the number of lipid molecules (phospholipid, cholesterol, and fatty acid) in one vesicle (N) are represented as eqs 2 and 3, respectively, as a function of n. The average distance between bilayers (dw) was measured from the TEM observation of each sample. The weight ratio of Hb to the mixed lipids: [Hb]/[lipid], which is the parameter for evaluating the quality of the resulting Hb encapsulation, is represented by
[Hb]inVin [Hb] ) [lipid] MWN/NA
(4)
The ratio was obtained using a cyanometHb method to measure the concentration of Hb and by Allen’s method16 to measure the phospholipid concentration. [Hb]in was calculated from eq 4. The Number of Bilayers of the Vesicles, n. The total surface area of the vesicles at the same concentration of lipids is proportional to the ratio of the surface area to the number of (15) Takeoka, S.; Sakai, H.; Nishide, H.; Tsuchida E. Jpn. J. Artif. Organs 1993, 22, 566. (16) Cullis, P. R.; Kruijff, B. Biochim. Biophys. Acta 1979, 559, 399.
molecules in one vesicle (N). The relative surface area of the vesicles was measured using a fluorescent probe, N-p-tolyl-2naphthylamine-6-sulfonic acid (TNS, Kanto Chem. Co.). TNS was added to a vesicle dispersion at a concentration of 1.0 × 10-6 M where the lipid concentration was 4.0 × 10-4 M, and then the dispersion was incubated at 25 °C for 8 h. Because the quantum yield of TNS adsorbed on the surface is 720 times that of TNS in bulk water,17 the fluorescence intensity (f) of these samples (vesicles, λex ) 321 nm, λem ) 445 nm; Hb vesicles, λex ) 312 nm, λem ) 456 nm, JASCO FP-770) is proportional to the concentration of lipids in the dispersion.
f ) K[lipid] )
k4πr2NA [lipid] N
fstd ) Kstd[lipid] )
k4πrstd2NA [lipid] Nstd
(5)
(6)
where the fluorescence intensity (fstd) was measured for unilamellar vesicles (n ) 1) extruded through a membrane filter with a 0.05 µm pore size as the standard sample where K and Kstd are constants. The ratio of K to Kstd can afford N, and then n is calculated using eq 3. Regarding the Hb vesicles, the decrease in f due to the quenching effect of Hb was corrected from the degree of quenching of TNS after adding the Hb vesicles to the dispersion of the TNS-introduced vesicles. The Surface Potential of Vesicles.17-21 The surface potential was measured using a fluorescent probe (TNS) which adsorbs on the surface of the vesicles, depending on the electrical double layer near the surface. According to the electrical double layer model of Gouy-Chapman, the fluorescence intensity is represented by
f ) γ[TNS] exp(-eφ0/kT)
(7)
where φ0 is the surface potential of the bilayer, γ is a constant, [TNS] is the concentration of TNS in the bulk water, e is the charge of an electron, k is the Boltzmann constant, and T is the absolute temperature. A zero surface potential was defined for vesicles without fatty acids (only DPPC and cholesterol). The ζ-Potential of Hb. The ζ-potential of Hb was obtained from the migration of Hb measured by electrophoresis. The pH gradient was previously formed on a polyacrylamide gel plate (Pharmacia Co., PhastGel IEF 5-8), and an Hb line was placed on the plate perpendicular to the pH gradient. The ζ-potential of Hb (HbA0) was calculated with
ζ ) uη/E
(8)
where u is the electrophoretic mobility of HbA0, η is the viscosity of the dispersed system, is the permittivity, and E is the electric field applied to the gel. The Membrane Fluidity. The membrane fluidity was measured by the fluorescence depolarization of a rod-type fluorescent probe: 1,6-diphenylhexa-1,3,5-triene (DPH) as the mobility of acyl chain segments in a bilayer membrane.22,23 A THF solution containing 5 mM DPH was added to a vesicle dispersion in order to introduce it into the hydrophobic center of the bilayer membrane at a mole ratio of 1:1000 (DPH:lipid) for an hour at 40 °C in the dark. The fluorescence depolarization was measured (λex ) 357 nm, λem ) 430 nm). The ratio of the anisotropy of polarization (r) to the limiting one (r0), r/r0, is the (17) Eisenberg, M.; Gresalfi, T.; Riccio, T.; McLaughlin, S. Biochemistry 1979, 18, 5213. (18) Tocanne, J. F.; Teissie, J. Biochim. Biophys. Acta 1990, 1031, 111. (19) Ptak, M.; Egret-Charlier, M.; Sanson, A.; Bouloussa, O. Biochim. Biophys. Acta 1980, 600, 387. (20) Cevc, G.; Seddon, J. M.; Hartung, R.; Eggert, W. Biochim. Biophys. Acta 1988, 940, 219. (21) McLaughlin, S. Curr. Top. Membr. Transp. 1977, 9, 71. (22) Shinitzky, M.; Inbra, M. J. Mol. Biol. 1974, 85, 603. (23) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515, 367.
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Figure 1. Surface potential of vesicles as a function of the content of palmitic acid (PA) in mixed lipids of the membranes.
Figure 2. The number of bilayers of vesicles (n) as a function of surface potential of vesicles with various compositions of palmitic acid at pH ) 7.0 (O), and of vesicles (DPPC:cholesterol: PA ) 7:7:2) at various pHs and ionic strengths (b). order parameter of DPH in the bilayer membrane, which expresses the membrane fluidity.
Results and Discussion Influence of the Surface Potential on the Number of Bilayers (n).10 Vesicles, which had no Hb but a buffer solution (150 mM HEPES, pH ) 7.0) in the interior of the vesicle, were used to eliminate the effect of Hb during the experiments, especially spectral analyses.24 The diameter of the vesicles was controlled to 0.20 ( 0.04 µm by extrusion through a polycarbonate membrane filter of which the pore size was strictly regulated to 0.2 µm. The surface potential proportionally and negatively increased with introducing fatty acid in the bilayer membrane as shown in Figure 1. This proportional relation suggests the homogeneous distribution of fatty acid in the membrane and electrostatic interaction among the negative charges of the fatty acid which influences the degree of dissociation of the fatty acid do not exist in this range of fatty acid content. Thus, the surface potential of the bilayer was confirmed to be controllable by adjusting the fatty acid content in the membrane.25 Open plots in Figure 2 show the relationship between the number of bilayers (n) and the surface potential which were set by adjusting the fatty acid content. Vesicles, of which the average n was 2, were prepared in the range of the surface potential from 0 to -20 mV, but upon further (24) Shviro, Y.; Zilber, I.; Shaklai, N. Biochim. Biophys. Acta 1982, 687, 63. (25) Djordjevich, L.; Ivankovich, A. D. Liposomes as Drug Carriers; Gregoriadis, G., Ed.; John Wiley & Sons Ltd.: New York, 1988; 551567.
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Figure 3. Influence of ionic strength of the vesicle dispersion on the number of bilayers of vesicles (n) at various pHs. pH ) 9.0 (O), 7.2 (Y), 6.8 (y), and 5.0 (b).
decreasing the potential to -42 mV, n decreased to unity. These results suggested that the processes to convert vesicles into smaller ones with extrusion, including the deformation of the vesicle, the separation of bilayers from the lamellar structure, the destruction of parts of the membrane, and the reconstruction of the vesicles would accompany the invasion of water into the space between the separated membranes following the dissociation of the fatty acid. Thus, electrostatic repulsion between the negatively charged bilayer membranes should accelerate the separation of the membrane from the lamellar structure. This can be supported by the result that the dissociation of fatty acids upon extrusion was observed as a pH decrease in the buffer-free disperse system (data are not shown here). In order to confirm this electrostatic effect, vesicles (at a mole ratio of DPPC:cholesterol:PA ) 7:7:2) were prepared by extrusion with variation of the solution pH and the ionic strength, and n values are summarized in Figure 3 relating to these factors. n decreased with increasing pH, and unilamellar vesicles with a 0.2 µm diameter were formed at pH 9.0. The surface potential of the vesicles at this pH was calculated to be -40 mV, which agreed well with the results shown in Figure 2 as described below. Moreover, n increased with the ionic strength. It is suggested that the shielding effect of electrostatic repulsion between the bilayer membranes by added ions inhibits the separation of the bilayer membranes from the lamellar structure, resulting in the higher n. The surface potential of the vesicles with different solution pH and ionic strength (µ) could be calculated using the Gouy-Chapman theory of the diffusion of electrical double layers.21 It increases negatively with a pH increase and a µ decrease. Because the charge on the surface of the bilayer membrane originated from the dissociated fatty acid in the bilayer membrane, the degree of dissociation was calculated from the Henderson-Hasselbach equation, where pKa and n were 7.4 and 1.2, respectively.18-20 pKa was corrected for µ of the disperse system.19 All the plots in Figure 3 were replotted against the calculated surface potential as closed circles in Figure 2, and some plots with similar surface potentials were averaged. The dependence of n on the surface potential calculated for the vesicles with various pHs and µ shows good agreement with that for the surface potential varied with the incorporation of fatty acid. Therefore, it is reasonable to conclude that the effective methods to decrease n are to enhance the electrostatic repulsion between the membranes, e.g., the introduction of negative charges on the vesicular surface, the increase in solution pH, and the decrease in µ. ζ-Potential of Hb and [Hb]in of Hb Vesicles. In the case of the preparation of Hb vesicles, an Hb solution is
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Figure 4. ζ-potentials of Hb molecules as a function of pH. The elliptical marks are pH markers.
Figure 6. Influence of preparation temperature on (a) [Hb]/ [lipid] ratio (Y) and (b) [Hb]in (O) and n (b). Table 1. The Number of Bilayers (n) of Vesicles Consisting of Several Lipids mixed lipid
Figure 5. [Hb]in/[Hb]o ratio as a function of ζ-potential of Hb: [Hb]in, the Hb concentration in the interior of Hb vesicles; [Hb]o, the Hb concentration at the preparation of Hb vesicles.
introduced into the space formed between the membranes of the lamellar structure. On comparison of [Hb]in to that at the preparation of Hb vesicles, the former is always lower than the latter, suggesting the difficulty of invasion of Hb molecules into the aqueous interior newly formed by extrusion. Hb solutions used in the experiments are a mixture of Hb variants, though proteins other than Hb were removed during the purification of the Hb solutions. The main component is HbA0, of which the isoelectric point (pI) is 7.02 at 25 °C.26 Thus, the ζ-potential of Hb can be controlled by adjusting the pH of the solution as shown in Figure 4. For example, when the pH of the Hb solution is higher than pI, the ζ-potential of Hb becomes negative. [Hb]in was measured at various pHs during preparation at a constant temperature and ionic strength. Figure 5 shows the relationship between the ζ-potential of Hb and [Hb]in to the concentration of Hb used for the preparation of the Hb-vesicles ([Hb]o), ([Hb]in/[Hb]o). When the pH of the Hb solutions is high, namely, the ζ-potential of Hb is negative, the ratio of [Hb]in/[Hb]o fell significantly. This ineffective introduction of negatively charged Hb molecules into the interior of the vesicle should be due to the electrostatic repulsion between Hb molecules and the surface of the bilayer membrane, while the positively charged Hb molecules can easily approach the vicinity of the bilayer membranes. Therefore, the pH of the Hb solution for the preparation of Hb vesicles should be lower than the pI of Hb. Influence of the Membrane Fluidity on the Number of Bilayer Membranes. Vesicles with various (26) Basset, P. J. Chromatogr. 1982, 227, 267.
r/r0
pH
n
DMPC/Chol/MA DPPC/Chol/PA DSPC/Chol/SA
Tc of PC (°C) 24 41 54
0.70 0.72 0.74
5.0 5.0 5.0
3.8 2.3 2.3
EYPC/Chol/SA HSPC/EYPC/Chol/SA HSPC/Chol/SA
-5 -5, 55 55
0.57 0.70 0.79
7.0 7.0 7.0
2.1 1.6 1.2
DMPC/Chol/MA DPPC/Chol/PA DSPC/Chol/SA
24 41 54
0.71 0.73 0.73
9.0 9.0 9.0
1.5 1.2 1.1
membrane fluidities at a constant temperature (25 °C) were prepared with different diacylphosphatidylcholines whose phase transition temperature as a pure bilayer membrane was different. Table 1 shows the order parameters (r/r0) of the probe molecule (DPH) in the bilayer membrane and n of the resulting vesicles without Hb. The unity value of r/r0 indicates that the anisotropy of fluorescence does not change within its lifetime, and the parameter becomes smaller with increasing membrane fluidity. The influence of membrane fluidity on the n was evaluated at three pH values. As mentioned above, the high pH leads to the lower n. At each pH, the bilayer membrane with a higher r/r0, namely, a lower membrane fluidity, leads to a smaller n. It is suggested that the multilamellar membranes with high membrane fluidity can deform their shape and cleave when the vesicles are passing through the pores of the membrane filters, leading to the smaller vesicles with a relatively high n. When the fluidity of the multilamellar membranes is low, the vesicles cannot pass through the pores because deformation of the curvature of the multilamellar membrane is difficult. Therefore, the multilamellar vesicles would be forced to reduce the number of bilayers in order to pass through the pores, leading to the smaller vesicles with a lower n. Preparation Temperature and the Structure of Hb Vesicles. Figure 6 shows the influence of the preparation temperature on the structure of the resulting Hb vesicles. The [Hb]/[lipid] increases nonlinearly with
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Figure 7. (a) Influence of temperature on pH of Hb solutions (O) and vesicle dispersions (b). (b) The viscosity of Hb solution as a function of temperature (y). [Hb] ) 38 g/dL, shear rate ) 11.5 s-1.
a drop in temperature as shown in Figure 6a. This indicates that Hb vesicles with high quality can be prepared at low temperature, though extrusion becomes somewhat difficult because of the high viscosity of the Hb solution and the lipid membrane. Figure 6b shows that n decreases with a drop in temperature, especially below 25 °C. While [Hb]in shows a maximum at 25 °C. These profiles were considered in relation to temperature, electrostatic interactions, or dynamic properties. The dissociation of Hb or fatty acid in a bilayer membrane would be influenced by temperature and also change the pH of an Hb solution, affecting the n and [Hb]in of the resulting Hb vesicles after extrusion. As shown in Figure 7a, the pH increases with a drop in temperature of the Hb solution (40 g/dL) due to the protonation of the proteins.27,28 This protonation was also supported by an increase in the pI of HbA0 with a temperature decrease (pI ) 7.02 at 25 °C, 7.39 at 4 °C). The protonation makes (27) Radola, B. J.; Graesslin, D.; Walter de G. Electrofocusing and Isotachophoresis; Berin: New York, 1977; 71-83. (28) Fredriksson, S. J. Chromatogr. 1978, 151, 347.
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the ζ-potential of Hb positive and the solution pH higher. On the other hand, the aqueous disperse system of vesicles without Hb at a mole ratio of DPPC:cholesterol:PA ) 7:7:2 showed no change in pH. In these situations, the promoted dissociation of fatty acids by a pH increase would result in a smaller n, and the less electrostatic repulsion between Hb molecules and negatively charged bilayers is considered to increase [Hb]in. The second point is the membrane fluidity which decreases with a drop in temperature. As mentioned above, the low fluidity decreases n. The third point is the viscosity of Hb solution. The introduction of Hb molecules into the space formed between the separated bilayers during extrusion is considered to be influenced by the viscosity of the Hb solution during preparation. The viscosity of the Hb solution becomes high with a drop in temperature as shown in Figure 7b. Hb molecules form flocks (aggregations),29 which cause the increase in the solution viscosity. It should be difficult for Hb to enter the space between the separated bilayers in comparison with free Hb molecules, leading to a reduction in [Hb]in with a drop in temperature. Accompanied by the electrostatic influence of temperature on the increment of [Hb]in, [Hb]in show a maximum as shown in Figure 6b. However, the decrease in n with a drop in temperature contributes to the improved quality of the Hb vesicles, [Hb]/[lipid], exceeding the negative influence of the decrease in [Hb]in (Figure 6a). Conclusions The factors, which influence the structure of Hb vesicles, were studied based on the interaction among Hb molecules and lipid membranes. The number of bilayers decreased by accelerating the electrostatic repulsion between the bilayers or by reducing the membrane fluidity. The concentration of Hb in the interior of the vesicles increases when the ζ-potential of Hb becomes positive and decreases with increasing the solution viscosity. These profiles can be used to explain the temperature dependence of the structure of the Hb vesicles. The Hb vesicles of which [Hb]/[lipid] was near 1.7, the highest value reported so far, was prepared. Acknowledgment. This work has been partly supported by a Grant-in-Aid for Scientific Research No. 5650930 and 07508005 from the Ministry of Education and Culture and a Iwaki research grant. The authors also thank Kyowa Interface Science Co., Ltd., for surface pressure measurements. LA940936J (29) De Young, L. R.; Fink, A. L.; Dill, K. A. Acc. Chem. Res. 1993, 26, 614.
