Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stability and Blood Compatibility of Polylipid/Hb K. Morizawa, K. Akama, Y. Kawakami & E. Tsuchida To cite this article: K. Morizawa, K. Akama, Y. Kawakami & E. Tsuchida (1992) Stability and Blood Compatibility of Polylipid/Hb, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 641-645, DOI: 10.3109/10731199209119694 To link to this article: http://dx.doi.org/10.3109/10731199209119694
Published online: 11 Jul 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 22 March 2016, At: 15:39
B I O M T . , A R T . CELLS & I H N O B . B I O T E C H . , 20(2-4), 641-645 ( 1 9 9 2 )
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb K.Morizawa,K.Akama,Y.Kawakami
Downloaded by [McMaster University] at 15:39 22 March 2016
Tsukuba Research Lab.,Nippon Oil & Fats Co. Tsukuba 300-26 Japan E.Tsuchida Dept.of Polymer Chemistry,Waseda Univ. Tokyo 169, Japan ABSTRACT The Polylipid/Hb vesicle is a new artificial red cell(ARC) based on liposome-encapsulated Hb. Advantages are derived from the stabilized liposomal bilayer membranes, obtained by polymerization of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phospho choline (DODPC). Furthermore, blood compatibility in vitro are good. 1 INTRODUCTION
One promising method in the development of a suitable blood substitute is the encapsulation of hemoglobin(Hb) in phospholipid vesicles. In our trials, Hb is encapsulated in vesicles, the membranes of which are composed of a special polymerized phospholipid (polylipid/Hb vesicle). Encapsulation of Hb brings many advantages ; such as no need for chemical modification of Hb, the possibility of achieving a red cell substitute with high Hb content, low viscosity, low oncotic pressure, long half life in the blood stream, and also control of oxygen affinity. The vesicles prepared from saturated phospholipids, however, can not solve problems such as instability during storage, aggregation, fusion and leakage of Hb etc. Polylipid/Hb vesicle, a new artificial red cell(ARC), the stability of which was improved significantly by polymerization of unsaturated phospholipid, overcomes all of the problems. In this report, results showing improved stability and sufficient blood compatibility of ARC are described. 2 MATERIALS AND METHODS (1) Preparation of ARC : Stroma-free hemoglobin(SFH) from concentrated human red blood cells according to the general methods, and then stabilized as CO-SFH. The lipid mixture (1,2-bis(2,4octadecadienoyl)-sn-glycero-3-phosphocholine(DODPC)/cholesterol/2,4-octadecadienoicacid:7/7/2) was mixed with CO-SFH solution containing a reductant and allosteric factor. The mixed lipid vesicles were prepared by extrusion method and uncapsulated Hb was removed by passing through a filter(pore size : 0.1 micron), and its lipid membrane was then polymerized by gamma-ray
641
C o p y w t 0 1992 by Marcel Dckker, Inc.
642
MORIZAWA ET AL.
Table 1
Typical Characteristics of ARC
Downloaded by [McMaster University] at 15:39 22 March 2016
Particle Size (nm) Hb Content (g/dl) Osmotic Pressure (mOsm) Oncotic Pressure (torr) (m H g 1 gz?Hb (%) Hill Coefficient (n) viscosity ( cp)
( pH 7.4,
37 OC )
200 5 40 10 280 25 40 < 5 1.8 4
irradiation. Finally CO-SFH in vesicle was dissociated to be free SFH by contacting with oxygen gas and sterilized. Typical characteristics of ARC are summarized in Table 1.
(2) Physical Stability : Polylipid/Hb vesicles were subjected to several times freeze-thawing cycle (freezing : liquid nitrogen, thawing:room temperature), comparing lipid/Hb vesicles which were prepared In the same way as polylipid/Hb without polymerization of its membrane bilayer by gamma-lay irradiation. After each cycle, the solutions were separated in a Sephacryl S-100 (Pharmasia) and the percentage of Hb was determined, and the particle sizes were measured. Polylipid/Hb veaicle(ARC) was rapidly frozen in liquid nitrogen and stored for 1 year at -30 OC. The oxygen binding characteristics ( P50, Hill Coefficient and OTE) before and after storage were compzr
:.
(3) Blood Compatibility: (a) Blood coagulation and platelet aggre ation time : ARC with citrated hole blood was incubat29 at 37 %C for 3 min. Thrombin time(TT)lY, prothr9ybin time( PT) , activated partial t omboand plasma recalcification time(Ca) were plastin time(APTT) measured, according to general test methods. (b) Red cell hemo1ysis:Test solutions with citrated whole blood or 0.6% washed red cells were shaken (at 3Hz) for 2.5 hrs at 37 OC. After incubation, solutions were centrifuged and their supernatants were diluted to be double volume with PBS. 2ml diluted supernatants were passed through Sephacryl 5-300 (Pharmasia) column to separate free Hb. 100% hemolysis was obtained by adding 0.5% Triton X-100 instead of test solution. Degree of hemolysis by ARC was compared with that by lipid/Hb vesicle and also with H-EYPC liposome not containing Hb (hydrogenated egg yolk lecithln/cholesterol/palmiticacid:7/7/2). (c) Red cell aggregation : Test solutions were diluted stepwise. 0.025 ml of washed red cells (dog and rat, 1%) and 0.025 ml of diluted test solutions were added in V-shaped microplates, and then incubated at 37 OC for 1 hr. sequentially incubated at 4 OC for 1 hr. Patterns of washed red cell aggregation were judged under microscope.
2’5
3 RESULTS AND DISCUSSION (a) Physical stability : it was indicated from results in Fig. 1 and Fig. 2 that ARC did not show any change on its particle size and any Hb leakage from vesicles even after 10 times freeze-
Downloaded by [McMaster University] at 15:39 22 March 2016
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb
643
Fig.1 Changes in particle size after freeze-thawing cycle
50
Llpid/Hb
-
PolylipidlHb
v
v
fi
v
A
2
2
3
4
5
J-0 I1
0
1
Freeze-thawing cycle Fig.2 Leakage after freeze-thawing cycle
10
644
MORIZAWA ET AL.
Table 2 Oxygen binding characteristics after subjecting to frozen storage ~
Parameters
Pre storage
Post storage
P ~ (mmHg) O
40.1
40.9
Hill Coefficient
Downloaded by [McMaster University] at 15:39 22 March 2016
OTE (“A)
1.8
1.8
42.0
41.7
Polylipid/Hb vesicles was rapidly frozen in liquid N2 and stored at -30°C for 1 year.
Table 3 Influence on blood coagulation and platelet aggregation
( Rat ) Test solution
TT (sec.)
PT (sec.)
APTT (sec.)
Ca (sec.)
Platelet aggreqation(sec.)
Conlrol(PBS)
14.5
12.3
27.3
201
162
Poiylipid/Hb 1/10
13.8
12.0
23.8
209
172
13.3 13.3 27.6 119 175 Polylipid/Hb Thrombin time TT , gothrombin time(PT), activated panial thromboplastin time(APTT). plasma recalcilicationAm2 ( a)
Table 4 Influence on red cell hemolysis ( Dog )
Test solution
Washed red cells (“A)
Whole blood (%)
Control(PBS)
0.2
3.8
H-EYPC
2.5
79.1
LipidHb
8.0
96.0
Polylipid/Hb 0.2 3.8 A-EVPC: Hydrogenaled egg yolk lecithin/cholesterol/palmiticacid-7/7/2 liposomes.
Table 5 Influence o n red cell aggregation Test solution
Dog red cells
Rat red cells
Control(PBS)
3-
3-
H-EYPC liposomes 1110
f
f
1/40
f
f
1/10
f
1I40
*
f
1/10
3-
3-
Lip‘Wib PolylipidMb
1/40 33-:negative (equal to negative control) f :partial aggregation
+_
3-
Downloaded by [McMaster University] at 15:39 22 March 2016
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb
645
thawing cycles. While, significant change on its particle size and Hb leakage were observed for lipid/Hb vesicles after only one freeze-thawing cycle. All parameters relating to oxygen transporting ability of ARC, shown in Table 2, did not change even after a year storage. As a result, ARC was very stable and this stability was brought by polymerization of unsaturated phospholipids. (b) Blood compatibility : Table 3 shows that ARC did not effect on blood coagulation and also platelet aggregation. Furthermore, Table 4 shows that ARC did not cause any hemolysis, in contrast with great hemolysis in the case of H-EYPC liposomes and lipid/Hb vesicles. In red cell aggregation, ARC had almost the same effect as the negative control.(Table 5 ) In conclusion, it was approved that ARC had good blood compatibility and this is derived from polymerization of unsaturated phospholipid.
REFERENCES 1) Dacie, J. V. and Lewis, S. M.:Practical Haematology, 4th Ed., J. & A. Churchill Led., London, 1970 pp. 312. 2) Quick, A. J. :Hemorrhagic, lst, Ed., Lea and Febiger, Philadelphia, 1957, pp. 379,436. 3) Matchett, M. 0. and Ingram, I. C.:Partial thromboplastin with kaolin normal range and modifications for the diagnosis of hemophilia and Christmas disease, J. Clin. Path., 18:465-471, 1965. 4) Stefanini, M. and Dameshek, W.:The Hemorrhagic Disorders, 2nd Ed., Grune &Stratton, New York, 1962, pp.505.
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stability and Blood Compatibility of Polylipid/Hb K. Morizawa, K. Akama, Y. Kawakami & E. Tsuchida To cite this article: K. Morizawa, K. Akama, Y. Kawakami & E. Tsuchida (1992) Stability and Blood Compatibility of Polylipid/Hb, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 641-645, DOI: 10.3109/10731199209119694 To link to this article: http://dx.doi.org/10.3109/10731199209119694
Published online: 11 Jul 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 22 March 2016, At: 15:39
B I O M T . , A R T . CELLS & I H N O B . B I O T E C H . , 20(2-4), 641-645 ( 1 9 9 2 )
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb K.Morizawa,K.Akama,Y.Kawakami
Downloaded by [McMaster University] at 15:39 22 March 2016
Tsukuba Research Lab.,Nippon Oil & Fats Co. Tsukuba 300-26 Japan E.Tsuchida Dept.of Polymer Chemistry,Waseda Univ. Tokyo 169, Japan ABSTRACT The Polylipid/Hb vesicle is a new artificial red cell(ARC) based on liposome-encapsulated Hb. Advantages are derived from the stabilized liposomal bilayer membranes, obtained by polymerization of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phospho choline (DODPC). Furthermore, blood compatibility in vitro are good. 1 INTRODUCTION
One promising method in the development of a suitable blood substitute is the encapsulation of hemoglobin(Hb) in phospholipid vesicles. In our trials, Hb is encapsulated in vesicles, the membranes of which are composed of a special polymerized phospholipid (polylipid/Hb vesicle). Encapsulation of Hb brings many advantages ; such as no need for chemical modification of Hb, the possibility of achieving a red cell substitute with high Hb content, low viscosity, low oncotic pressure, long half life in the blood stream, and also control of oxygen affinity. The vesicles prepared from saturated phospholipids, however, can not solve problems such as instability during storage, aggregation, fusion and leakage of Hb etc. Polylipid/Hb vesicle, a new artificial red cell(ARC), the stability of which was improved significantly by polymerization of unsaturated phospholipid, overcomes all of the problems. In this report, results showing improved stability and sufficient blood compatibility of ARC are described. 2 MATERIALS AND METHODS (1) Preparation of ARC : Stroma-free hemoglobin(SFH) from concentrated human red blood cells according to the general methods, and then stabilized as CO-SFH. The lipid mixture (1,2-bis(2,4octadecadienoyl)-sn-glycero-3-phosphocholine(DODPC)/cholesterol/2,4-octadecadienoicacid:7/7/2) was mixed with CO-SFH solution containing a reductant and allosteric factor. The mixed lipid vesicles were prepared by extrusion method and uncapsulated Hb was removed by passing through a filter(pore size : 0.1 micron), and its lipid membrane was then polymerized by gamma-ray
641
C o p y w t 0 1992 by Marcel Dckker, Inc.
642
MORIZAWA ET AL.
Table 1
Typical Characteristics of ARC
Downloaded by [McMaster University] at 15:39 22 March 2016
Particle Size (nm) Hb Content (g/dl) Osmotic Pressure (mOsm) Oncotic Pressure (torr) (m H g 1 gz?Hb (%) Hill Coefficient (n) viscosity ( cp)
( pH 7.4,
37 OC )
200 5 40 10 280 25 40 < 5 1.8 4
irradiation. Finally CO-SFH in vesicle was dissociated to be free SFH by contacting with oxygen gas and sterilized. Typical characteristics of ARC are summarized in Table 1.
(2) Physical Stability : Polylipid/Hb vesicles were subjected to several times freeze-thawing cycle (freezing : liquid nitrogen, thawing:room temperature), comparing lipid/Hb vesicles which were prepared In the same way as polylipid/Hb without polymerization of its membrane bilayer by gamma-lay irradiation. After each cycle, the solutions were separated in a Sephacryl S-100 (Pharmasia) and the percentage of Hb was determined, and the particle sizes were measured. Polylipid/Hb veaicle(ARC) was rapidly frozen in liquid nitrogen and stored for 1 year at -30 OC. The oxygen binding characteristics ( P50, Hill Coefficient and OTE) before and after storage were compzr
:.
(3) Blood Compatibility: (a) Blood coagulation and platelet aggre ation time : ARC with citrated hole blood was incubat29 at 37 %C for 3 min. Thrombin time(TT)lY, prothr9ybin time( PT) , activated partial t omboand plasma recalcification time(Ca) were plastin time(APTT) measured, according to general test methods. (b) Red cell hemo1ysis:Test solutions with citrated whole blood or 0.6% washed red cells were shaken (at 3Hz) for 2.5 hrs at 37 OC. After incubation, solutions were centrifuged and their supernatants were diluted to be double volume with PBS. 2ml diluted supernatants were passed through Sephacryl 5-300 (Pharmasia) column to separate free Hb. 100% hemolysis was obtained by adding 0.5% Triton X-100 instead of test solution. Degree of hemolysis by ARC was compared with that by lipid/Hb vesicle and also with H-EYPC liposome not containing Hb (hydrogenated egg yolk lecithln/cholesterol/palmiticacid:7/7/2). (c) Red cell aggregation : Test solutions were diluted stepwise. 0.025 ml of washed red cells (dog and rat, 1%) and 0.025 ml of diluted test solutions were added in V-shaped microplates, and then incubated at 37 OC for 1 hr. sequentially incubated at 4 OC for 1 hr. Patterns of washed red cell aggregation were judged under microscope.
2’5
3 RESULTS AND DISCUSSION (a) Physical stability : it was indicated from results in Fig. 1 and Fig. 2 that ARC did not show any change on its particle size and any Hb leakage from vesicles even after 10 times freeze-
Downloaded by [McMaster University] at 15:39 22 March 2016
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb
643
Fig.1 Changes in particle size after freeze-thawing cycle
50
Llpid/Hb
-
PolylipidlHb
v
v
fi
v
A
2
2
3
4
5
J-0 I1
0
1
Freeze-thawing cycle Fig.2 Leakage after freeze-thawing cycle
10
644
MORIZAWA ET AL.
Table 2 Oxygen binding characteristics after subjecting to frozen storage ~
Parameters
Pre storage
Post storage
P ~ (mmHg) O
40.1
40.9
Hill Coefficient
Downloaded by [McMaster University] at 15:39 22 March 2016
OTE (“A)
1.8
1.8
42.0
41.7
Polylipid/Hb vesicles was rapidly frozen in liquid N2 and stored at -30°C for 1 year.
Table 3 Influence on blood coagulation and platelet aggregation
( Rat ) Test solution
TT (sec.)
PT (sec.)
APTT (sec.)
Ca (sec.)
Platelet aggreqation(sec.)
Conlrol(PBS)
14.5
12.3
27.3
201
162
Poiylipid/Hb 1/10
13.8
12.0
23.8
209
172
13.3 13.3 27.6 119 175 Polylipid/Hb Thrombin time TT , gothrombin time(PT), activated panial thromboplastin time(APTT). plasma recalcilicationAm2 ( a)
Table 4 Influence on red cell hemolysis ( Dog )
Test solution
Washed red cells (“A)
Whole blood (%)
Control(PBS)
0.2
3.8
H-EYPC
2.5
79.1
LipidHb
8.0
96.0
Polylipid/Hb 0.2 3.8 A-EVPC: Hydrogenaled egg yolk lecithin/cholesterol/palmiticacid-7/7/2 liposomes.
Table 5 Influence o n red cell aggregation Test solution
Dog red cells
Rat red cells
Control(PBS)
3-
3-
H-EYPC liposomes 1110
f
f
1/40
f
f
1/10
f
1I40
*
f
1/10
3-
3-
Lip‘Wib PolylipidMb
1/40 33-:negative (equal to negative control) f :partial aggregation
+_
3-
Downloaded by [McMaster University] at 15:39 22 March 2016
STABILITY AND BLOOD COMPATIBILITY OF POLYLIPID/Hb
645
thawing cycles. While, significant change on its particle size and Hb leakage were observed for lipid/Hb vesicles after only one freeze-thawing cycle. All parameters relating to oxygen transporting ability of ARC, shown in Table 2, did not change even after a year storage. As a result, ARC was very stable and this stability was brought by polymerization of unsaturated phospholipids. (b) Blood compatibility : Table 3 shows that ARC did not effect on blood coagulation and also platelet aggregation. Furthermore, Table 4 shows that ARC did not cause any hemolysis, in contrast with great hemolysis in the case of H-EYPC liposomes and lipid/Hb vesicles. In red cell aggregation, ARC had almost the same effect as the negative control.(Table 5 ) In conclusion, it was approved that ARC had good blood compatibility and this is derived from polymerization of unsaturated phospholipid.
REFERENCES 1) Dacie, J. V. and Lewis, S. M.:Practical Haematology, 4th Ed., J. & A. Churchill Led., London, 1970 pp. 312. 2) Quick, A. J. :Hemorrhagic, lst, Ed., Lea and Febiger, Philadelphia, 1957, pp. 379,436. 3) Matchett, M. 0. and Ingram, I. C.:Partial thromboplastin with kaolin normal range and modifications for the diagnosis of hemophilia and Christmas disease, J. Clin. Path., 18:465-471, 1965. 4) Stefanini, M. and Dameshek, W.:The Hemorrhagic Disorders, 2nd Ed., Grune &Stratton, New York, 1962, pp.505.
