CN1153590C - Blood substitute of hemoglobin microcapsule and preparing method thereof - Google Patents
Blood substitute of hemoglobin microcapsule and preparing method thereof Download PDFInfo
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Abstract
The present invention relates to a blood substitute of a haemoglobin microcapsule and a preparation method thereof. The microcapsule contains one or a plurality of monomethoxy-poly(ethylene glycol)-poly(lactic acid) copolymers and haemoglobin, the content of the haemoglobin is high, and the oxygen carrying activity is close to that of the natural red cells. The method comprises the following procedures: (1) the copolymers are dissolved in at least one organic solvent; (2) a haemoglobin solution is added in an organic solution obtained in procedure (1) and homogenized at a high speed, or ultrasonically dispersed to form W1/O emulsion; (3) the emulsion obtained in procedure (2) is put in multiple emulsion and homogenized at a high speed, or ultrasonically dispersed to form W1/O/W2 multiple emulsion; (4) the multiple emulsion obtained in procedure (3) is putt in a solidifying liquid, rotated and evaporated under reduced pressure, or stirred and volatilized at normal temperature and pressure, or dialyzed by cross-flow diffusion to remove the organic solvent; (5) the solidifying liquid obtained in procedure (4) is centrifugally washed, and haemoglobin microcapsules are collected. The preparation method has the advantages of simple process, easy magnification and short time consumption.
Description
Invention field
The invention belongs to biomedical sector, particularly a kind of blood substitute of hemoglobin microcapsule and preparation method thereof.
Background technology
Nowadays, the in short supply day by day and blood cross-infection in clinical blood source has become a worldwide problem.Seeking safely and effectively, blood substitute has become international medical biotechnology area research hot of research and development.
Haemoglobin molecule is by 2 α subunits and 2 tetramers that the β subunit constitutes, diameter 5.5nm, molecular weight 64,500dal.Hemoglobin and O
2In conjunction with being reversible, the affinity for oxygen of hemoglobin refers to that hemoglobin to the bonded firm degree of oxygen, reaches the required partial pressure of oxygen of semi-saturation degree with oxygen in the hemoglobin and represents, note is made P
50Hemoglobin combination and release oxygen are the allosteric processes of a high Collaboration, mainly regulate (this collaborative allosteric degree is represented with the Hill coefficient, normally is 2.8) by allosteric effector.The allosteric effector of human hemoglobin is 2, and 3-DPG is present in the erythrocyte in a large number, and it is by forming salt bridges with two β subunits, thereby reduces the affinity of hemoglobin to oxygen.The P of human hemoglobin in erythrocyte
50Be 26mmHg.
If the highly purified substrate hemoglobin (removing the hemoglobin of impurity such as erythrocyte membrane) of going is directly imported in the body, free haemoglobin molecule dissociates into two dimers rapidly, is leached by kidney, or stops up glomerule generation nephrotoxicity.In addition, free hemoglobin can see through blood vessel in conjunction with the angiotensin NO in the blood, causes hypertension, produces a series of seondary effects.
By embedding, form artificial erythrocyte and be hopeful to solve above-mentioned relevant issues hemoglobin.With the used film assortment of embedding class difference, artificial erythrocyte can be divided into hemoglobin liposome and hemoglobin microcapsule.
Liposome embedded hemoglobin (LEH) is set forth in sixties end as blood substitute.The general composition that constitutes liposome membrane is natural phospholipid and cholesterol.1980, it was the artificial erythrocyte of liposome of 0.2 μ m that Djordjevich and Miller have prepared particle diameter.Many research groups have subsequently also been carried out this research work, such as, Farmer, Hunt, Rudolph, Tsuchida or the like.
At present, existing abroad Experiment Preparation goes out to be suitable for the liposome embedded hemoglobin (LEH) of animal, and has produced in batches.Many research groups have been carried out animal experiment study.A large amount of experiments show that the liposome hemoglobin does not all have toxic and side effects to brain, heart, kidney and the lung of animal.In the exchange transfusion of animal with aspect the massive hemorrhage shock treatment, achieve success, very likely carry out clinical experiment in the recent period.But the hemoglobin liposome still has the following disadvantages: the less stable after in lay up period and the input body; Phospholipid is to reticuloendothelial system tool potential impact; Peroxidating easily takes place in phospholipid; The embedding hemoglobin concentration is on the low side.
Degradable polymer microcapsule embedding hemoglobin is the latest generation blood substitute, have many advantages: the polylactic acid (PLA) that is used for embedding is nontoxic to human body, is through the material of drugs approved by FDA as preparations such as medical operation suture thread and injection microcapsule, microsphere, implants.It has good blood compatibility, and after finishing the oxygen therapy task, degradable is harmless lactic acid, water and CO
2Excrete; Polymeric film porous and mechanical strength are better than the liposome immobilized artificial membrane; This film can also see through the metahemoglobin (Fe in the hemoglobin
2+Be oxidized to Fe
3+, do not have oxygen carrying capability) and reduction needed glucose and other hydrophilic small molecules, the product of reaction also can be spread out by capsule, can not cause refuse concentrating and normally the carrying out of inhibitory reaction in capsule.Degradable polymer microcapsule embedding hemoglobin is the artificial erythrocyte of the complete meaning of tool.
Forming artificial erythrocytic research the earliest about microcapsule embedded hemoglobin is carried out by TMS Chang (1957).He replaces the biomembrane of erythrocyte membrane with the synthetic film of butyl benzoate.By regulating permeability of the membrane, can make 2,3-DPG is embedded in unlikely oozing out in the film, thereby regulate hemoglobin oxygen release ability, make this artificial erythrocytic oxygen decomposition curve and erythrocytic closely similar, in addition, the red blood cell enzyme of embedding, carbonic anhydrase, catalase also can both keep active in film.But this artificial erythrocytic disadvantage is film material butyl benzoate to the human body toxic side effect, and it is bigger to be prepared into the hemoglobin microcapsule particle diameter, is difficult for the blood capillary by pulmonary, and easily by reticuloendothelial system phagocytic.
On JIUYUE 23rd, 1997, Canada scientist Zhang Mingrui of Chinese origin (TMS Chang) professor applies for a patent US5,670,170, respectively with the heavy poly-method of emulsion polymerization, polymer, emulsifying diffusion method, be that to have prepared particle diameter be the hemoglobin Nano capsule of 0.05 μ m-1 μ m to film material embedding hemoglobin with poly-isobutyl group propylene hydrocyanic ester or polylactic acid or polylactic acid poly ethanol copolymer.Describe these three kinds of methods below in detail:
1. emulsion polymerization: 50-100mg isobutyl group propylene hydrocyanic ester, 35-50mg lecithin and 5mg vitamin E are dissolved in 5-10ml ethanol.Then, under stirring condition this solution slowly is injected into 10-25ml and contains 0.1-0.5%Tween20, hemoglobin concentration is in the aqueous solution of 0.5-10%.The polymeric membrane (poly-isobutyl group propylene hydrocyanic ester) that is embedded with hemoglobin forms immediately.Dialysis removes ethanol, and the hemoglobin capsule is collected in the high speed centrifugation washing.
2. the polylactic acid of heavy poly-method: the 150mg of polymer, the phosphatidylcholine of 50mg dissolves in the chloroform that 10ml contains surfactant, 0.5ml go the emulsifying in above-mentioned mixed liquor of substrate hemoglobin solutions, add the 25ml ether again and fully stir, so far can form the polymeric film of embedding hemoglobin.Add cyclohexane extraction again in the solution film is solidified, centrifugalize adds dialysis in phosphate buffer (pH=7.4) under 4 ℃ of the saline that contain surfactant, high speed centrifugation washing, collection hemoglobin capsule at last.
3. emulsifying diffusion method: 100mgPLA and 50mg phospholipid dissolve in 5ml ethanol and the 10ml acetone mixed liquor, under condition of stirring this mixed liquor slowly are injected into 25ml and contain in 5-15% hemoglobin, the 0.4%Tween20 solution.At this moment, ethanol and acetone can dissolve in water and form microcapsule at last, and ethanol in the aqueous solution and acetone can be removed by dialysis in 4 ℃ of saline or phosphoric acid buffer saline solution, and the hemoglobin of embedding is not removed by centrifugal (30000g) or ultrafiltration.
1. and 3. method uses a large amount of ethanol as film material solvent, easily makes degeneration, loses the oxygen carrier function.In addition, method 1. in, during isobutyl group propylene hydrocyanic ester polymerization film formation, the amino on haemoglobin molecule surface also is easy to participate in reaction as initiator, by and make the hemoglobin degeneration.Though the capsule grain diameter that 3. method forms is less, about 0.1 μ m, it can not form effective embedding (embedding rate is too low, does not appear in the newspapers) to hemoglobin.
2. method has used a large amount of organic solvents, and apparent, in this process, hemoglobin is easy to take place degeneration.In the method, embedding 0.5ml hemoglobin need be used 10ml chloroform, 25ml ether and 100ml cyclohexane extraction, and preceding two kinds of organic solvents are poisonous, and last a kind of inflammable and explosive dangerous goods especially obviously is not suitable for as hemoglobin microcapsule preparation technology.
On the whole, there is the hemoglobin changeableness in three kinds of methods of this of TMS Chang, and embedding rate is lower, and preparation technology need use a large amount of organic solvents, are difficult for shortcomings such as technology amplification.
1994, Frenchman N.Cedrati was the film material with polylactic acid (PLA), and with multi-emulsion method embedding hemoglobin, making particle diameter at last is the hemoglobin microcapsule of 10 μ m-500 μ m.(N.Cedrati(1994)Art.Cells，Blood?Subs.and?Immob.Biotech.22(3)，867-873)
1997, N.Cedrati used similar approach again, was that the film material makes the hemoglobin microcapsule of particle diameter less than 200 μ m with polylactic acid and ethyl cellulose (146000-166000).(N.Cedrati(1997)Art.Cells，BloodSubs.and?Immob.Biotech.25(5)，457-462)
Concrete preparation process is as follows, at first the mixture with 0.5g polylactic acid (PLA) or polylactic acid and ethyl cellulose is dissolved in the 20g dichloromethane, the hemoglobin solutions 5ml that adds 8-30% under the magnetic agitation condition pours 1.5L polyvinyl alcohol (PVA) concentration then into and is in 1% the aqueous solution under magnetic agitation (1500rpm).Stirred 2 hours under the room temperature, last centrifuge washing is collected the hemoglobin microcapsule precipitation.
The preparation method of N.Cedrati report is simple relatively, and the hemoglobin activity is preserved.His embedding be commercially available human hemoglobin because 2, the P50 before the loss of 3-DPG, this hemoglobin embedding is 13.8mmHg, almost remains unchanged after the embedding, is 13.9mmHg.But his particle diameter of preparation is bigger than normal a bit, 200 μ m, and the fine, soft fur tubule diameter of human body only is 4 μ m.
Summary of the invention
The objective of the invention is to overcome many deficiencies of above-mentioned blood substitute of hemoglobin microcapsule, and a kind of blood substitute of hemoglobin microcapsule with the Biodegradable high-molecular embedding is provided;
Another object of the present invention is to provide the preparation method of this blood substitute of hemoglobin microcapsule.
Embodiment of the present invention are as follows:
Blood substitute of hemoglobin microcapsule provided by the invention, it is characterized in that, this blood substitute of hemoglobin microcapsule contains one or more polylactic acid mono methoxy polyethylene glycol copolymer and pure hemoglobins, and the weight portion proportioning of described polylactic acid mono methoxy polyethylene glycol copolymer and pure hemoglobin is: 1: 2-5; Described polylactic acid mono methoxy polyethylene glycol molecular weight of copolymer is 20000-120000, and wherein the molecular weight of mono methoxy polyethylene glycol is 1000-10000; Described pure hemoglobin is pure bovine hemoglobin, the white or pure human hemoglobin of pure Sanguis caprae seu ovis red eggs.
The preparation method of blood substitute of hemoglobin microcapsule provided by the invention is characterized in that, its step is as follows:
1. one or more polylactic acid mono methoxy polyethylene glycol copolymers are dissolved in and make organic solution O at least a organic solvent, described organic solvent is dichloromethane, ethyl acetate, ethyl propionate or propyl acetate;
2. with hemoglobin solutions W
1Among the organic solution O that 1. the adding step forms,, make W through high speed homogenize or ultra-sonic dispersion
1/ O colostric fluid;
3. with 2. gained W of step
1/ O colostric fluid is poured double emulsion W into
2In, make W through high speed homogenize or ultra-sonic dispersion
1/ O/W
2Double emulsion; Described double emulsion W
2Be the mixed liquor of polyvinyl alcohol water solution and common salt aqueous solution, they at the content of mixed liquor are: contain polyvinyl alcohol 0.1-6g in every 100ml mixed liquor, contain Sal 0.4-3.0g; Described consolidation liquid is that concentration is that the common salt aqueous solution of 0.4-3.0g/100ml, the D/W that concentration is 0.4-3.0g/100ml, glycerine water solution or the concentration that concentration is 0.4-3.0g/100ml are the glycol water of 0.4-3.0g/100ml;
4. with 3. gained W of step
1/ O/W
2Double emulsion is poured in the consolidation liquid, stirs volatilization or cross-flow diffusion dialysis through decompression rotary evaporation, normal temperature and pressure and removes wherein organic solvent;
5. the centrifuge washing step is 4. resulting has removed organic solvent and has contained W
1/ O/W
2The consolidation liquid of double emulsion promptly obtains hemoglobin microcapsule of the present invention, lyophilization, and collect;
Described organic solvent is dichloromethane, ethyl acetate, ethyl propionate or propyl acetate; Described organic solvent is any two or more the blended mixed organic solvents in dichloromethane, ethyl acetate, ethyl propionate, propyl acetate and the acetone; The molecular weight of described one or more polylactic acid mono methoxy polyethylene glycol copolymers is 20000-120000, and wherein the molecular weight of mono methoxy polyethylene glycol is 1000-10000; Described hemoglobin solutions is bovine hemoglobin solution, sheep hemoglobin solutions or human hemoglobin solution; Described hemoglobin solutions is the described double emulsion W of the broken centrifuged supernatant of cattle, sheep or human red blood cell
2Be the mixed liquor of polyvinyl alcohol water solution and common salt aqueous solution, they at the content of mixed liquor are: contain polyvinyl alcohol 0.1-6g in every 100ml mixed liquor, contain Sal 0.4-3.0g; Described consolidation liquid is that concentration is that the common salt aqueous solution of 0.4-3.0g/100ml, the D/W that concentration is 0.4-3.0g/100ml, glycerine water solution or the concentration that concentration is 0.4-3.0g/100ml are the glycol water of 0.4-3.0g/100ml.
In the hemoglobin microcapsule of the present invention's preparation, pure hemoglobin content height, the weight portion proportioning of polylactic acid mono methoxy polyethylene glycol copolymer and pure hemoglobin is up to 1: 2-5, and the oxygen carrier activity of product is very near neutral red cell (P50=26mmHg, Hill coefficient=2.4);
Blood substitute of hemoglobin microcapsule preparation method provided by the invention, technology is simple, amplifies easily, and weak point consuming time.
Used PLA/MPEG molecular weight of copolymer is 20000-120000 among the present invention, and wherein MPEG (mono methoxy polyethylene glycol) molecular weight is 1000-10000.Embedding rate is improved greatly, reach more than 90%.This is owing to MPEG block hydrophilic in this polymer is stronger, is forming W
1After/O the emulsion, the MPEG segment extends in the water droplet in the copolymer, and the PLA segment only is dissolved in organic facies O, by and form a polymeric membrane at oil-water interfaces, when next step emulsion, be difficult for making hemoglobin to leak out to the second water W
2, by and obtain higher embedding rate.
The used organic solvent of the present invention is dichloromethane, chloroform, ethyl acetate, propyl acetate, ethyl propionate, acetone or their mixed solvent, and concrete kind or volume need be decided on used film material.
Used hemoglobin solutions (W among the present invention
1) be the broken liquid of ORBC, concrete preparation process is as follows.Get fresh Sanguis Bovis seu Bubali 100ml, the centrifugal 40min of 4000g gets ORBC 45ml, add 1.6% saline 50ml washing, the centrifugal 30min of 4000g adds normal saline 50ml washing and the centrifugal 30min of 4000g then, repeat twice of aforesaid operations, get ORBC 35ml, pour in the 35ml double distilled water magnetic agitation 20min into, last 30000g is centrifugal, gets its supernatant and carries out embedding operation (hemoglobin concentration is about 17%).More than all washing fragmentation procedures all in aseptic 4 ℃ of down operations.The aforesaid operations object is a Sanguis Bovis seu Bubali, also can wash broken Sanguis caprae seu ovis, expired human blood with method, obtains sheep, human hemoglobin solution.
The used stabilizing agent of second water is the polyvinyl alcohol of molecular weight 20000-100000 degree of hydrolysis 85-95% among the present invention, or molecular weight is the Polyethylene Glycol of 4000-20000.In addition, this second aqueous phase also need add micromolecule such as a certain amount of Sal, glucose or glycerol, is made into certain density aqueous solution, to improve the osmotic pressure of second water, guarantees to obtain higher embedding rate.
The emulsion step is used the ultrasonic or homogenize emulsion process of homogenize speed more than 8000rpm of 30-80w among the present invention, makes the hemoglobin microcapsule particle diameter at last below 3 μ m, less than the fine, soft fur tubule internal diameter 4 μ m of human body.
The present invention forms hemoglobin microcapsule with multi-emulsion method embedding hemoglobin, makes that the hemoglobin activity is kept, and has the similar oxygen carrier activity of neutral red cell (P
50Be 28mmHg, the Hill coefficient is 2.4).Selecting PLA/MPEG for use is the film material, adds certain density Sal at second water, has improved embedding rate, and has reduced particle diameter.
Embodiment
Embodiment 1:
(the PLA/MPEG molecular weight is 30,000 with 50,mg3 ten thousand (5000), wherein the MPEG molecular weight is 5000, as follows) be dissolved in the 3ml dichloromethane, add the 600ul bovine hemoglobin, 10000rpm is emulsifying 30s just, pours 100mlNaCl0.9% into, in the double emulsion of PVA6%, with 8000rpm emulsion 1min, to pour into again in the 200ml normal saline, the 1000rpm magnetic agitation is 1.5 hours under the normal temperature and pressure.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 2 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 2:
40,mg6 ten thousand (5000) is dissolved in the mixed solvent of 2ml dichloromethane and 1ml ethyl acetate, add the 800ul hemoglobin, 10000rpm is emulsifying 30s just, pour 100ml1% glycerol into, in the double emulsion of PVA6%, with 8000rpm emulsion 1min, to pour into again in the 200ml normal saline, the 1000rpm magnetic agitation is 1.5 hours under the normal temperature and pressure.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 3:
20,mg2 ten thousand (1000) and 30,mg6 ten thousand (10000) are dissolved in the 3ml dichloromethane, add the 600ul hemoglobin, 12000rpm is emulsifying 30s just, pour the 100ml2% glucose into, in the double emulsion of PVA4%, with 10000rpm emulsion 1min, to pour into again in the 200ml2% D/W, the 1000rpm magnetic agitation is 1.5 hours under the normal temperature and pressure.With normal saline 12000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 2 in the microcapsule, and particle diameter is less than 3 μ m).
Embodiment 4:
40,mg6 ten thousand (5000) is dissolved in the mixed solvent of 2ml dichloromethane and 1ml ethyl propionate, it is white to add 800ul Sanguis caprae seu ovis red eggs, 10000rpm is emulsifying 30s just, pour 100ml1% glycerol into, in the double emulsion of PVA6%, with 8000rpm emulsion 1min, to pour into again in the 200ml normal saline, the 1000rpm magnetic agitation is 1.5 hours under the normal temperature and pressure.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 5:
The hybrid films material of 50,mg6 ten thousand (5000) and 30,mg2 ten thousand (2000) is dissolved in 2ml dichloromethane and the 2ml ethyl acetate, add the 1500ul haemoglobin aqueous solution, 10000rpm is emulsifying 30s just, pour 100mlNaCl0.9% into, in the double emulsion of PVA4%, with 8000rpm emulsion 1min, pour into again in the 200ml normal saline.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 6:
The hybrid films material of 40,mg9 ten thousand (5000) and 20,mg3 ten thousand (2000) is dissolved in the 4ml ethyl acetate, add the 1500ul human hemoglobin, 10000rpm is emulsifying 30s just, pour 100ml1% glycerol into, in the double emulsion of PVA6%, with 8000rpm emulsion 1min, pour in the 200ml1% glycerine water solution 40 ℃ of rotary evaporations 1 hour again into.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 4.2 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 7:
50,mg3 ten thousand (2000) is dissolved in the 3ml ethyl propionate, add the 1000ul hemoglobin, the ultrasonic 30s of 80w under the ice-water bath, pour 100mlNaCl0.9% into, in the double emulsion of PVA4%,, pour 200ml again into 10000rpm emulsion 1min, in the normal saline of PVA0.4%, 40 ℃ of rotary evaporations 1 hour.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3.2 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 8:
30m,g12 ten thousand (5000), 20,mg3 ten thousand (1000) and 10,mg2 ten thousand (2000) are dissolved in the 3ml dichloromethane, add the 1200ul hemoglobin, 10000rpm is emulsifying 30s just, pour 100mlNaCl0.9% into, in the double emulsion of 4%PEG6000, with 8000rpm emulsion 1min, to pour into again in the normal saline of 200ml0.4%PEG6000, the 1000rpm magnetic agitation is 1.5 hours under the normal temperature and pressure.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3.2 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 9:
20,mg3 ten thousand (2000) is dissolved in the 3ml ethyl acetate, it is white to add 600ul Sanguis caprae seu ovis red eggs, the ultrasonic 30s of 80w under the ice-water bath, pour 100mlNaCl0.9% into, in the double emulsion of PVA4%, the ultrasonic 1min of 50w under the ice-water bath pours in the normal saline of 200mlPVA0.4% again, 40 ℃ of rotary evaporations 1 hour.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 4.8 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 10:
20m,g12 ten thousand (10000), 20,mg3 ten thousand (1500) and 10,mg2 ten thousand (1000) are dissolved in the 4ml dichloromethane, add the 1500ul bovine hemoglobin, the ultrasonic 30s of 80w under the ice-water bath, pour 100mlNaCl0.9% into, in the double emulsion of PVA4%, the ultrasonic 1min of 50w under the ice-water bath pours in the normal saline of 200mlPVA0.4% again, 40 ℃ of rotary evaporations 1 hour.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 4.5 in the microcapsule, and particle diameter is less than 4 μ m).
Embodiment 11:
30,mg6 ten thousand (5000) is dissolved in the 2ml ethyl acetate, add the 800ul human hemoglobin, the ultrasonic 30s of 80w under the ice-water bath, pour 100mlNaCl1.2% into, in the double emulsion of PVA2%, the ultrasonic 1min of 50w under the ice-water bath pours in the normal saline of 200ml0.4%PEG12000 again, 40 ℃ of rotary evaporations 1 hour.With normal saline 10000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 4.2 in the microcapsule, and particle diameter is less than 3 μ m).
Embodiment 12:
60,mg6 ten thousand (5000) is dissolved in 1ml ethyl acetate and the 3ml chloroform, add the 1200ul hemoglobin, the ultrasonic 40s of 80w under the ice-water bath, pour 100mlNaCl2% into, in the double emulsion of PVA2%, the ultrasonic 2min of 50w pours 200ml0.4%PEG10000 again under the ice-water bath, in the aqueous solution of NaCl1.5%, 40 ℃ of rotary evaporations 1 hour.With normal saline 12000g centrifuge washing four times, collect the hemoglobin microcapsule precipitation at last.(copolymer and hemoglobin weight proportion are 1: 3.2 in the microcapsule, and particle diameter is less than 3 μ m).
Claims (8)
1, a kind of blood substitute of hemoglobin microcapsule, it is characterized in that, this blood substitute of hemoglobin microcapsule contains one or more polylactic acid mono methoxy polyethylene glycol copolymer and pure hemoglobins, and the weight portion proportioning of described polylactic acid mono methoxy polyethylene glycol copolymer and pure hemoglobin is: 1: 2-5.
2, by the described blood substitute of hemoglobin microcapsule of claim 1, it is characterized in that described polylactic acid mono methoxy polyethylene glycol molecular weight of copolymer is 20000-120000, wherein the molecular weight of mono methoxy polyethylene glycol is 1000-10000.
3, by the described blood substitute of hemoglobin microcapsule of claim 1, it is characterized in that described pure hemoglobin is pure bovine hemoglobin, the white or pure human hemoglobin of pure Sanguis caprae seu ovis red eggs.
4. the preparation method of the blood substitute of hemoglobin microcapsule of a claim 1 is characterized in that, its step is as follows:
1. one or more polylactic acid mono methoxy polyethylene glycol copolymers are dissolved in and make organic solution O at least a organic solvent, described organic solvent is dichloromethane, ethyl acetate, ethyl propionate or propyl acetate;
2. with hemoglobin solutions W
1Among the organic solution O that 1. the adding step forms,, make W through high speed homogenize or ultra-sonic dispersion
1/ O colostric fluid;
3. with 2. gained W of step
1/ O colostric fluid is poured double emulsion W into
2In, make W through high speed homogenize or ultra-sonic dispersion
1/ O/W
2Double emulsion; Described double emulsion W
2Be the mixed liquor of polyvinyl alcohol water solution and common salt aqueous solution, they at the content of mixed liquor are: contain polyvinyl alcohol 0.1-6g in every 100ml mixed liquor, contain Sal 0.4-3.0g; Described consolidation liquid is that concentration is that the common salt aqueous solution of 0.4-3.0g/100ml, the D/W that concentration is 0.4-3.0g/100ml, glycerine water solution or the concentration that concentration is 0.4-3.0g/100ml are the glycol water of 0.4-3.0g/100ml.
4. with 3. gained W of step
1/ O/W
2Double emulsion is poured in the consolidation liquid, stirs volatilization or cross-flow diffusion dialysis through decompression rotary evaporation, normal temperature and pressure and removes wherein organic solvent;
5. the centrifuge washing step is 4. resulting has removed organic solvent and has contained W
1/ O/W
2The consolidation liquid of double emulsion promptly obtains hemoglobin microcapsule of the present invention, lyophilization, and collect.
5. press the preparation method of the described blood substitute of hemoglobin microcapsule of claim 4, it is characterized in that described organic solvent is any two or more the blended mixed organic solvents in dichloromethane, ethyl acetate, ethyl propionate, propyl acetate and the acetone.
6. press the preparation method of the described blood substitute of hemoglobin microcapsule of claim 4, it is characterized in that, the molecular weight of described one or more polylactic acid mono methoxy polyethylene glycol copolymers is 20000-120000, and wherein the molecular weight of mono methoxy polyethylene glycol is 1000-10000.
7. by the preparation method of the described blood substitute of hemoglobin microcapsule of claim 4, it is characterized in that described hemoglobin solutions is bovine hemoglobin solution, sheep hemoglobin solutions or human hemoglobin solution.
8. by the preparation method of the described blood substitute of hemoglobin microcapsule of claim 4, it is characterized in that described hemoglobin solutions is the broken centrifuged supernatant of cattle, sheep or human red blood cell.
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| CN1733298B (en) * | 2005-08-16 | 2011-08-24 | 华东理工大学 | Microcapsule type blood substitute based on hemoglobin and process for preparing the same |
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| CN102091043B (en) * | 2011-01-25 | 2012-09-26 | 中国人民解放军第三军医大学第二附属医院 | Growth factor slow release microballoon and preparation method thereof |
| CN102113999B (en) * | 2011-01-30 | 2012-06-27 | 中国人民解放军第四军医大学 | Novel multi-layer polysaccharide structure-modified liposome-encapsulated hemoglobin nano-capsule blood substitute |
| CN102861322A (en) * | 2012-08-09 | 2013-01-09 | 四川大学华西医院 | Hemoglobin oxygen carrier and preparation method thereof |
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| CN1419936A (en) | 2003-05-28 |
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