Biomaterials, Artificial Cells and Immobilization Biotechnology

ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18

Oxygen-Transport and Solution Properties of Polylipid/HB Vesicles (ARC) Shinji Takeoka, Etsuo Hasegawa, Hiroyuki Nishide, Eishun Tsuchida & Sadayoshi Sekiguchi To cite this article: Shinji Takeoka, Etsuo Hasegawa, Hiroyuki Nishide, Eishun Tsuchida & Sadayoshi Sekiguchi (1992) Oxygen-Transport and Solution Properties of Polylipid/HB Vesicles (ARC), Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 399-404, DOI: 10.3109/10731199209119659 To link to this article: http://dx.doi.org/10.3109/10731199209119659

Published online: 11 Jul 2009.

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Date: 01 May 2016, At: 12:16

BIOMAT., ART. CELLS & IMMOB. BIOTECH.,

20(2-4), 399-404 (1992)

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OXYGEN-TRANSPORT A N D SOLU1'ION PROPEHTIES OF P O L Y L I P I D / H B VESICLES (ARC). Shinji T A K E O K A , Etsuo HASEGAWA, H i r o y u k i NISHIDE, Eishun TSUCHIDA, Departnietir of Polymer Chemisrry. Wuseda University. Tokyo 169, JAPAN Sadayoshi S E K I G U C H I Hokkuido Red Cross Blood Center, Supporo 063, JAPAN Abstract: PolyineriLed phospholipid vesicle encapsulating H b (polylipid/tlb vesicle) was prepared from a niixture o f unsaturated phospholipid, cholesterol and uiisalurated fatty acid and polynierization by y-ray irradiation. The average radius o f resulting vesicles was 203 f 39 nm and concentrated H b (30 wt%) was efficiently encapsulated. y-Ray polymerization proceeds theoretically at low temperature (4 "C).P50 and oxygen transporting efficiency were adjusied to 40 mniHg and 40%, respectively. Oncotic pressure and solution viscosity can be controlled to the same values as blood.

1. Introduction Polymerized phospholipid vesicle encapsulating stroma-free hemoglobin (Polylipid/lib vesicle) i s a new type o f an artificial red cell (ARC) because stabilization o f the vesicle both i t ) v i m and i n vivo can be achieved by using polymerized phospholipid as a niembrane constituent. This paper describes the preparation method of polylipid/Hb vesicle, i t s oxygen-transporting ability, and the control o f solution properties.

2. Materials and Methods 2-1. Materials Lipids to prepare the vesicles encapsulated H b are shown below. Unsaturated phospholipid'-6' was synthesized from 2,4-octadecadienoic acid. This phospholipid is manufactured by NipporiOil and,Fats Co., Japan. Cholesterol and the same fatty acid were used as membrane constiluents. Stioma-free t l b was purified and concenlratetl to be more than 30 wt%. Rate of met-Hb was less than 3% o f total Hh. I t i s noled that stronia-free H b was protected as carboxy-tlb during the preparation in order to prevent met-Hb formation.

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Copyright 0 1992 by Marcel Dekker, Inc.

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2-2. Preparation of polylipidlHb vesicles Large multilainellar vesicles, which were prepared by simply dispersing lipid mixture into concentrated Hb solution, were extruded through membrane filter with penetrated and size-controlled pores at 4 OC7).Vesicles which had smaller size and fewer lamellarily were prepared by mulristzp extrusion with reducing pore size of the filter to 0.2 pm. The polymerization of unsaturated lipid by y-ray was carried out by using 6oCo under an argon atmosphere at 4 OC*~y) 2-3. Measureinent of properties of polylipidlllb vesicle sysleni The oxygen-binding curves were obtained with a Hemox-analyzer (TCSMedical Products) in a Hemox-solution (pH7.4) at 37 'C. Hill coefficient was calculated according to ihe Hill equation from the oxygen-binding percents of Hb and oxygen partial pressures. Oncotic pressure was measured with COP star K A - 1000 (Seiseido Medical Instruments, Co., Ltd) equipped with an ultrafiltration membrane (cut off niolecular weight; 13,000) at 25 OC. Viscoinetric characterization of a polylipid/Hb vesicle dispersion was performed with Biorheolyzer (cone-and-plate type, Tohkisangyo, Co.,) at pH 7.4 and 37 OC. Viscosity at an angular speed of 375 sec- was adopted .

3. Results and Discussion 3-1. Encapsulation of Hb in vesicle Encapsulation aspects of Hb into multilamellar vesicles can be calculated. The structural notation of the vesicle are shown in Fig. 1 , r and d are radius of vesicle and thickness of bilayer, respectively. S and n are the mean area of one lipid and lamellarily, respectively. Calculalion was carried out on the assumplion that there was no space between bilayers. If the volume of inner phase of one vesicle and number of molecules in one vesicle are calculated by fixing r, n, and d i n equations ( I ) and (2), trapping efficiency and [HbMLipid] are represented as the functions of [Lipid] and [Hb] in equations (3) and (4), respectively. I n the experiment, an average diameter of vesicles is adjusted to be 0.2 pm. [Hb]/(L] and trapping efficiency are calculated and shown in Fig 2 (a) and (b), respectively, relating to various lamellarity(n). The experimental data were [Hb]/[Lipid] = 1.2 for the encapsulation of 30 wt% Hb, and 20 % trapping efficiency for the 5 wt% lipid. Therefore, laniellari~yof resulling vcsicles was estimated to be about 2 from those figures. This value was confirmed from measurements where phosplioli ids in outerfacing layer were distinguished from those in inner-facing layer such as PH-NMR and polyrnerizalion behaviors with a waier-soluble radical iniiiatofl). This indicates that Hb is Ihcorelically encapsulaled iiiio vesiclcs by tlic extrusion inelhod.

40 1

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POLYLIPID/Hb V E S I C L E S

0

I

0

10

20

30

[Hb] ( w t % )

40

[Lipid]

( wt

%1

Fig. 2 Theoretical calculation of tib encepsulaled vesicles (200 nni d) having various lainellariiy.

[DODPC] (m mol dm-3)

Dose rate (rad hrl)

Fig. 3 Kinetics of ?ray polymerization of unsaturaled vesicles at 4 "C.

TAKEOKA ET AL.

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30

20

10

0

2

4

6

8

[8dditlvesJ(Wm) Fig. 4

Control of oncoiic pressure of polylipidbib vesicles by polymers.

3-2. y R a y polymerizallon of unsaturated lipids in vesicle When y-ray was irradiated to a vesicle dispersion, OH radicals generate from water molecules9). These radicals attack the diene groups of the lipids of vesicle and then polymerization of the unsaturated lipids is initiated. The polymerization rate is proportional to the I .2th power of the unsaturated lipid concentration as shown in Fig. 3(a). This indicates that the polymerization of lipid in vesicle is initiated by OH, not direct excitation of monomer lipids. On the other hand, polymerization rate is proportional to Io.Y2(asshown in Fig. 3(b)). In general, polymerization rate is proportional to lo.' because termination occurs by recombination or disproportionation between two growing polymer chains. In the case of radical polymerization in the bilayer membrane, it is suggested that unimolecular terminalion occurs. The following reaction schemes were, therefore, suggested. H2O

OH' + M Mn' + M M"'

--kd--> OH' + H',

--k'---> M' --kJ-->

Mn+t*

__---__ kc > Pn

Rd = d[OH']/dt

= kdf[H2O]I (6)

(7) (8)

(5)

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POLYLIPID/Hb VESICLES

7 6 human blood

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1

2

3

4

5

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[Lipid] (wt%) Fig. 5 Viscosity of polylipid/llb vesicles 81 k 3 7 5 s (37°C) Oncolic pressure was adjusted 10 25 mmHg.

'

where f, M and Pn mean initiation efficiency, growing polymer chain and a dead polymer chain, respectively. H' does not initiate the polymerization of lipids because of its low reactivity. Polymerization rate(Rp) would be described as equation(9). Rp = -d[ M]/dt = k[ MI1

(9)

where k=kpkdf[HzO]/ki, This shows Ihal R p is proportional to both [ M] and I, satisfying our experimental results. This guarantees good control and reproducibility of polymerization, meaning that complete stabilization of bilayer membrane is always reproduced.

3-3. Oxygen binding Characteristics of polylipid/Hb vesicle system Oxygen binding characteristics could be controlled by adding allosteric effectors such as IHP or 2,3-DPG. PSOof our polylipid/Hb vesicle system was adjusted to 40 mmHg, which meant the lower oxygen-binding affinity than human blood. Hill coefficient was I .8. Oxygen transporting efficiency from lung to terminal tissues was about 40%, which is superior to blood. This suggests that our artificial red cell transports oxygen efficiently in vivo.

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3-4. Solution properly of polylipid/Hb vesicle system Figure 4 shows oncotic pressure of the polylipid/Hb vesicle dispersion. Oncotic pressure increased with adding amount of human albumin or dextran. To adjust the oncotic pressure to that of human blood, 25 niniHg, the 2.2 % solution of dextran with average molecular weight of 40,000 was prepared. The solution was also adjusted by the 5.6 % addition of albumin. It is noted that the oncotic pressure of polylipid/tlb vesicle dispersion in itself is very low, and highly concentrated polylipid/Hb vesicle dispersion is possible. On the other hand, the viscosity of vesicle dispersion with controlled oncotic pressure to human blood (25 mmHg) increases with lipid concentration, especially when dextran was added to polylipid/Hb vesicle dispersion (Fig. 5 ) . This figure indicates that viscosity was optimized by the addition of dextran and/or human albumin up to the lipid concentration of 6.5 wt%, which is sufficient value in our present system. 4. References 1) K. Dorn, R.T. Klingbiel, D. P. Specht. P. N. "yminski, H. Ringsdorf, and D. F. O'Brien, J . Am. Chem. Soc., 106 1627 (1984). 2) H.Ohno, S. Takeoka, H. Iwai, and E. Tsuchida, J . Polym. Sci.. Pur.A, 25,2737 ( 1987). 3 ) H. Ohno, Y. Ogata, and E. Tsuchida, Mucromolecules, 20,929 (1987). 4) H. Ohno, S. Takeoka, and E. Tsuchida, Bull. Chem. SOC.Jpn., 60,2945 (1 987). 5) S. Takeoka, N. Kimura, H. Ohno, and E. Tsuchida, Polymer J., 22,867 (1990). 6) H. Ohno, S. Takeoka, and E. Tsuchida, Macromolecules, 22,61 (1989). 7 ) F. Olson, C.A. Hunt, F.C. Szoka, W.J. Vail, and D. Papahajopoulos, Biochim. Biophys. Acfa., 858, 161 (1986). 8) H. Ohno, Y. Ogata, E. Tsuchida, J. Polym. Sci., Par.A, 24,2959 (1986). 9) E. Hasegawa, N. Kimura, M. Hatashita, C. Makino, E. Tsuchida, Mucromolecules. 24 (I99 I ) in press. 10) C. Makino, N. Kimura, E. Hasegawa, E. Tsuchida, Nihonkugakiiknishi, 8, 1102 ( I99 1 ).