CN101991595B - Plasma substitute and preparation method thereof - Google Patents
Plasma substitute and preparation method thereof Download PDFInfo
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- CN101991595B CN101991595B CN2009101897437A CN200910189743A CN101991595B CN 101991595 B CN101991595 B CN 101991595B CN 2009101897437 A CN2009101897437 A CN 2009101897437A CN 200910189743 A CN200910189743 A CN 200910189743A CN 101991595 B CN101991595 B CN 101991595B
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Abstract
The invention relates to a plasma substitute and a preparation method thereof. The plasma substitute comprises vesicles, hydroxyethyl starch and an isoosmotic adjusting agent. Blood volume is replenished while the blood oxygen supply is maintained, so that the plasma substitute can be used for treating anaemia, fainting, shock and acute injury.
Description
[technical field]
The invention belongs to formulation art, more particularly, the present invention relates to a kind of method for preparing of blood plasma substitute.
[background technology]
Suitable blood oxygen concentration is the basis of keeping the body normal function.Under the anaerobic environment, can not carry out normal aerobic respiration in the cell, the Organic substance decomposition gives off energy and will reduce, and physiological process is got muddled.Numerous disease such as cerebral infarction; Patient's brain cell can cause permanent damage because of anoxia is downright bad, if certain blood plasma substitute can in time be delivered to brain with oxygen in the prime time of key; Just can make brain cell keep normal operation, extent of injury reduced to minimum.
Existing blood plasma substitute has displacement type blood plasma substitute, platelet succedaneum and red blood cell substitute.Hetastarch has been used to clinical as the displacement type blood plasma substitute, its effect is osmotic pressure, acid-base balance and a blood volume of keeping blood.Patent CN101032512A discloses the method for preparing of hydroxy ethyl starch for injection as blood plasma substitute.But the displacement type blood plasma substitute does not have the delivering oxygen function at present, is not the plasma substitute of real meaning therefore, can not use in a large number.
The platelet succedaneum also is in the research initial stage, does not see the product that success is arranged.Red blood cell substitute it is reported can bring into play erythrocytic oxygen carrier function, therefore is the emphasis of blood plasma blood substitute research.Red blood cell substitute roughly is divided into three kinds, is respectively the hemoglobin and the Optro of fluorocarbons Emulsion, modification, and three's characteristics are following:
(1) fluorocarbons Emulsion is being used oxygen delivery equipment, can let blood carry the oxygen more much more than general case.Fluorocarbons is discharged by lung with original shape after metabolism, and is harmless.But fluorocarbons Emulsion in vivo the half-life shorter; Need the oxygen delivery equipment synchronous applications; And fluorocarbons Emulsion is to remove through reticuloendothelial system; So reticuloendothelial system might can not carry out normal immunity activity because of overload, causes immunity of organisms to descend and causes infection.
The hemoglobin of (2) modifying can play the function of carrying with release of oxygen, if but do not have the protection of cell membrane, the decomposition rate of hemoglobin can be very fast, possibly cause the kidney major injury, so hemoglobin can not be injected directly into human body.Report separation and purification of hemoglobin from multiple materials such as out of date human blood or animal blood is arranged, process much firm modified model hemoglobin after it is modified.But the human hemoglobin polymer PolyHeme of the chemical modification of the expensive 1.72 hundred million dollars of developments of U.S. Northfield company in 2007 is as the clinical research failure of red blood cell substitute, and clinical trial shows that PolyHeme uses the back mortality rate to increase.
(3) Optro utilizes gene recombination technology in escherichia coli, yeast, insecticide, genetically modified animals and plants, to give expression to the hemoglobin of natural human.But this type of technology faces some problems, can not satisfy the large-scale commercial applications production requirement such as the hemoglobin content of producing, and hemoglobin can not be separated etc. with microorganism or animals and plants after expressing fully.
Discover that recently gassiness vesicle suspension has the delivering oxygen function, can be used as novel blood plasma substitute, is used for anoxybiotic treatment.The vesicle of gassiness can absorb oxygen in the environment of oxygen enrichment, atrophy in anoxic environment discharges oxygen, in the environment of oxygen enrichment, is recovered to original shape again.
The granule of at present suitable particle size range can see through the barrier of pulmonary, controls through the particle diameter to vesicle, and the vesicle of carrier gas is the long period circulation in vivo, has guaranteed that oxygen is transported to the anoxia position continuously.Generally require the particle diameter of vesicle should be limited between the 500nm-8 μ m, necessary oxygen carrier space so both can be provided, can eliminate holding back of pulmonary again.Simultaneously, for improving the oxygen carrier amount of vesicle suspension, vesicle must have suitable concentration, and experiment shows that vesicle concentration is between 10
5Individual/ml～10
10Individual/ml scope is comparatively suitable.
But existing vesicle oxygen therapy carrier circulation time in vivo is shorter, and the persistent period is short, does not possess the characteristics of displacement type blood plasma substitute, can not keep competent blood volume and carry out blood circulation.
[summary of the invention]
The technical problem that the present invention will solve is the weak point to existing plasma substitute, and a kind of novel plasma substitute articles for use and preparation method thereof are provided.
We find in the experiment of the novel plasma substitute of research, and are single with vesicle oxygen therapy carrier or single all undesirable with displacement type blood plasma substitute effect.But vesicle is cured by oxygen therapy the plasma substitute that forms after carrier and the hetastarch organic assembling, and not only physical stability significantly improves, and guarantees that circulation volume can not reduce in the blood, and both bring into play synergism, have complementary advantages.This novel blood plasma substitute more helps solving the low problem of blood oxygen that serial diseases such as anemia, faintness, shock, acute injury cause, replenishes blood oxygen amount in time, efficiently, keeps blood volume simultaneously, recovers the body normal physiological function.
Through test of many times, the technical scheme below the present invention has adopted:
Contain suspension vesicle, hetastarch and isoosmotic adjusting agent in the blood plasma substitute of the present invention, the suspension vesicle has gas core, and vesicle adventitia composition material is lipid, albumen, surfactant or polymer, and the vesicle particle diameter is in micron or nanoscale.
The molecular weight ranges of above-mentioned hetastarch is 9000-600000, and substitution value MS is 0.2-0.7, comprises the high hetastarch that replaces level of Low Moleculor Hydroxyethylstarch, middle molecular ethoxyl starch and HMW.
Above-mentioned isoosmotic adjusting agent is meant the material that is used to regulate colloidal osmotic pressure of generally acknowledging in medical science and the pharmacy, comprises sodium chloride, sulfate, phosphate, citrate, borate, glucose, dextran, mannitol.
Above-mentioned lipid is selected from hydrogenated soya phosphatide HSPC, hydrogenation lecithin HEPC, 1; 2-two palmityls-Sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt DPPG, 1; 2-distearyl acyl group-Sn-glyceryl-3-phosphatidylcholine DSPC, 1; 2-two palmityls-Sn-glyceryl-3-phosphatidylcholine DPPC, 1,2-two palmityls-Sn-glyceryl-3-phosphatidic acid-sodium salt DPPG and their fluoride or carbowax modifier.
Above-mentioned albumen is for the human serum albumin or remove immunogenic animal serum albumin.
Above-mentioned surfactant is the nonionic surfactant of polyoxyethylene groups, comprises tween, span.
Above-mentioned polymer is a biocompatible polymer, comprises polylactic acid, polylactic-co-glycolic acid copolymer, Polyethylene Glycol, poloxamer, chitosan and their Polyethylene Glycol, Polysialic acid or hetastarch modified derivative.
Above-mentioned gas is the acceptable innocuous gas of physiology, comprises fluorine carbon gas, nitrogen, oxygen, carbon dioxide.
Above-mentioned blood plasma substitute is used for that the low patient of blood oxygen amount injects, the additional liquid, the infusion liquid of transplant organ, the oxygen of ishemic part of solution, blood oxygenators and other outer loop of dilute blood provides liquid, the injection solution of liquid ventilation or cultured tissue regenerative cell's culture fluid before the surgical operation.
Above-mentioned blood plasma substitute can adopt the preparation of following method: step (a): vesicle adventitia composition material disperses with organic solvent or aqueous solution, utilizes ultrasonic or mode of heating makes it become a homodisperse mixed system;
Step (b): the mixed system of (a) is removed solution wherein through spray drying or cryodesiccated method, make loose pressed powder.
Step (c): the pressed powder of step (b) quantitatively is sub-packed in the cillin bottle, in bottle, charges into gas, sealing.
Step (d): the pressed powder of step (c) is added in the clinical injection solvent commonly used, form gassiness vesicle aaerosol solution.
Wherein aqueous solution in the step (a) or the injection solvent in the step (d) are dissolved with hetastarch and isoosmotic adjusting agent in advance.
[specific embodiment]
Below further specify the present invention through several embodiment.
Embodiment 1: the preparation of lipid carrier vesicle suspension in the oxygen carrier blood plasma substitute
Take by weighing hydrogenated soy phosphatidyl choline (HSPC) 40mg, polyethylene glycol 1500 (PEG1500) 1000mg, tween 100mg in the beaker of 50ml, add tert-butyl alcohol 10ml (analytical pure), 60 ℃ of ultrasonic dissolutions of water-bath.Treat that above-mentioned substance dissolves fully, after the solution becomes clarification, be sub-packed in the cillin bottle of 10ml, every bottled amount 2ml, in 0-4 ℃ of cold putting more than 30 minutes, liquid is creamy white solidifies shape, lyophilization 40 hours.The sample that lyophilizing is good fills the air in the perfluoropropane displacement bottle, close plug.
Take by weighing 300mg hetastarch 130/0.4 and be dissolved in the 5ml distilled water, be injected in the above-mentioned cillin bottle,, obtain the lipid vesicle suspension with lyophilizing sample mix, jolting at a distance from plug with 45mg sodium chloride.Sampling is observed, and the vesicle particle size distribution is at 3-6 μ m, and vesicle concentration is 3 * 10
8Individual/ml.
Embodiment 2: the preparation of albumin carrier vesicle float in the oxygen carrier blood plasma substitute
Take by weighing 600mg hetastarch 130/0.4 and be dissolved in the 10ml distilled water, add 500mg bovine serum albumin mixing, be mixed with 5% bovine serum albumin solution with 90mg sodium chloride; Under the output of 120W, continuous ultrasound vibration 60s feeds a large amount of oxygen from container bottom simultaneously with this solution; After supersound process finishes; The microvesicle suspension that obtains in 0 ℃ of standing demix, is got upper strata milky microvesicle suspension and inserted in the cillin bottle, with the gas in the replacement of oxygen bottle; Close plug is preserved in 4 ℃ of refrigerators.Microscopically is observed microbubble concentration greater than 2.5 * 10
8Individual/ml, the microvesicle diameter is between 2-4 μ m, and the half-life is 120min in the body.
Embodiment 3: spray drying method for preparation oxygen carrier blood plasma substitute carrier vesicle float
Take by weighing Semen Myristicae phosphatidylcholine (DMPC) 0.3g, Semen Myristicae PHOSPHATIDYL ETHANOLAMINE (DMPE) 0.02g and poloxamer (188) 0.05g are dissolved in the 50ml phosphate buffer, and heating makes it to become clear solutions.Add perflexane liquid 20ml under the 800rpm mixing speed, form the micron and even the nano level colostrum that comprise perflexane.The colostrum that forms adds the aqueous solution of isopyknic 5% hetastarch 40, and 800rpm at the uniform velocity stirs, and makes its abundant mixing, utilizes spray dryer to carry out drying (temperature is controlled at 35 ℃), makes blank microvesicle powder.
Get the 100mg spray drying and make blank microvesicle powder and be sub-packed in the 5ml cillin bottle, the air in the displacement bottle that fills oxygen, close plug.Use is injected the 2ml normal saline after plug, and jolting gets the lipid vesicle suspension.Microscopically is observed microbubble concentration greater than 2.5 * 10
8Individual/ml, the microvesicle diameter is between 2-4 μ m, and the half-life is 120min in the body.
Embodiment 4: the research of vesicle suspension oxygen carrier amount
Take by weighing hydrogenated soy phosphatidyl choline (HEPC) 40mg, DSPC-PEG20001mg, poloxamer 750mg, polysorbas20 mg in the beaker of 80ml, add tert-butyl alcohol 10ml (analytical pure), 60 ℃ of ultrasonic dissolutions of water-bath.Treat that above-mentioned substance dissolves fully, after the solution becomes clarification, be sub-packed in the cillin bottle of 10ml every bottled amount 2ml.Divide the solution install rapidly in 0-4 ℃ of cold putting more than 30 minutes, liquid is creamy white solidifies shape, lyophilization 45 hours.The sample that lyophilizing is good fills the air in the perfluoropropane displacement bottle, close plug.
Take by weighing 300mg hetastarch 130/0.4 and be dissolved in the 5ml distilled water with 45mg sodium chloride, be injected in the above-mentioned cillin bottle that contains dried frozen aquatic products at a distance from plug, jolting obtains the lipid vesicle suspension.Sampling is observed, vesicle mean diameter 6 μ m, and vesicle concentration is 4.2 * 10
9Individual/ml.
Get lipid vesicle suspension 2ml in the cillin bottle of 10ml, close plug.Charge into the pure oxygen of different pressures respectively at a distance from plug, the air in the displacement bottle.Behind the shake well 10min, adopt top oxygen content method to measure the bonded amount of oxygen of lipid vesicle.
Table 1 pure oxygen branch is pressed on the relation of vesicle oxygen carrier amount
Experimental result is seen table 1, shows that discovery vesicle when pure oxygen is breathed can carry 0.24pl oxygen.Vesicle concentration reaches 4.5 * 10 in blood
8Individual/as during ml, enough arteriovenous oxygen difference can be provided.When pressure breathing oxygen, a vesicle can be carried 0.46pl oxygen, and the material that this vesicle only needs in blood plasma, to inject seldom just can provide oxygen delivery.
Embodiment 5: blood plasma substitute is used for the treatment of anemia
10 of anemia rats, auricular vein injection mean diameter is 6 μ m, concentration is 4.2 * 10
9The blood plasma substitute of individual/ml (method for preparing is seen embodiment 4) lipid vesicle suspension 1ml once a day, injected 15 days continuously, was index evaluation treatment situation with packed cell volume and hemoglobin content.
Table 2 packed cell volume and hemoglobin content situation of change
Experimental result is seen table 2; Through the anemia rat is injected blood plasma substitute; The packed cell volume of evaluation rat anemia index and hemoglobin numerical quantity all in normal range, show that gassiness vesicle suspension as blood plasma substitute, can prolong the life cycle of anemia rat.
Embodiment 6: the treatment of shock due to blood plasma substitute is used to suffocate
This blood plasma substitute is supply oxygen rapidly, improves oxygen content fast, can also improve hemorheology properties simultaneously, and expanding blood volume makes the disorderly normalization of recovering of the body physiological function that is caused by anoxia.
The equal particle diameter of making even is 6 μ m, and concentration is 1.2 * 10
9Albumin vesicle suspension (method for preparing the is seen embodiment 2) 2ml of individual/ml in the cillin bottle of 10ml, close plug.Charge into 2 * 10 at a distance from plug
6The pure oxygen of KPa pressure, the air in the displacement bottle.Shake well 10min makes the abundant saturated oxygen of vesicle.
10 of asphyxial rats, auricular vein injection blood plasma substitute saturated oxygen suspension 2ml after 10 minutes, surveys matched group and the art pO2, oxygen capacity, arterial oxygen saturation, the arterial oxygen content that give the blood plasma substitute group respectively.
Table 3 asphyxial rat gives blood plasma substitute of the present invention each item index situation of change after 10 minutes
Experimental result is seen table 3, and contrast shock matched group, art pO2, oxygen capacity, arterial oxygen saturation, the arterial oxygen content that gives the experimental group of blood plasma substitute all have increasing in various degree.Inject after five minutes asphyxial rat consciously, it is clear-headed to begin recovery about eight minutes, can be free movable about ten minutes.
Embodiment 7: blood plasma substitute is used for the infusion liquid of transplant organ
This blood plasma substitute can be used as the infusion liquid of organ transplantation with normal saline, glucose injection, cell-free protein solution etc.
The equal particle diameter of making even is 6 μ m, and concentration is 4.2 * 10
9The lipid vesicle suspension 10ml that the pure oxygen of individual/ml is saturated adds normal saline 50ml, glucose injection 50ml, and cell-free protein solution 50ml is made into infusion liquid.Be connected with the new zealand white rabbit kidney of transplanting through perfusion unit, carry out circumfusion, infusion liquid consumption 80-120ml.Through detection, after the perfusion, same state when the kidney organ of transplanting keeps with the body intracellular metabolic.
Kidney after the perfusion is transplanted; After two weeks, White Rabbit each item physical signs of transplanting is all normal, and immunological rejection does not take place; The kidney of transplanting can carry out normal physiological function, shows that the oxygen carrier blood plasma substitute has better protect and repair ability as infusion liquid to transplant organ.
Claims (3)
1. the method for preparing of a blood plasma substitute is characterized in that: take by weighing 600mg hetastarch 130/0.4 and be dissolved in the 10ml distilled water with 90mg sodium chloride, add 500mg bovine serum albumin mixing; Be mixed with 5% bovine serum albumin solution, with this solution under the output of 120W, continuous ultrasound vibration 60s; Feed a large amount of oxygen from container bottom simultaneously, after supersound process finishes, with the microvesicle suspension that obtains in 0 ℃ of standing demix; Get upper strata milky microvesicle suspension and insert in the cillin bottle, with the gas in the replacement of oxygen bottle, close plug; In 4 ℃ of refrigerators, preserve, obtain blood plasma substitute.
2. the method for preparing of a blood plasma substitute, it is characterized in that: take by weighing hydrogenated soy phosphatidyl choline 40mg, DSPC-PEG2000 1mg, poloxamer 750mg, polysorbas20 mg add tert-butyl alcohol 10ml in the beaker of 80ml; 60 ℃ of ultrasonic dissolutions of water-bath treat that above-mentioned substance dissolves fully, after the solution becomes clarification, are sub-packed in the cillin bottle of l0ml; Every bottled amount 2ml divides the solution install rapidly in 0-4 ℃ of cold putting more than 30 minutes, and liquid is creamy white solidifies shape; Lyophilization 45 hours, the sample that lyophilizing is good fills the air in the perfluoropropane displacement bottle; Close plug takes by weighing 300mg hetastarch 130/0.4 and is dissolved in the 5m1 distilled water with 45mg sodium chloride, is injected in the above-mentioned cillin bottle that contains dried frozen aquatic products at a distance from plug; Jolting obtains the lipid vesicle suspension, i.e. blood plasma substitute.
3. blood plasma substitute according to claim 1 and 2 is used to prepare the injection liquid that is applied to the low patient of blood oxygen amount, preparation and is applied to solution, the preparation of dilute blood before the surgical operation and is applied to the culture fluid that infusion liquid that blood oxygenators, preparation be applied to transplant organ, oxygen that preparation is applied to ishemic part provide injection solution that liquid, preparation be applied to the liquid ventilation or preparation to be applied to the cultured tissue regenerative cell.
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| CN101991595B true CN101991595B (en) | 2012-11-28 |
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| CN106092848B (en) * | 2016-03-22 | 2018-07-06 | 威海威高血液净化制品有限公司 | Dialyzer ultrafiltration performance test fluid |
| CN106749579A (en) * | 2017-02-05 | 2017-05-31 | 河南康元生物科技股份有限公司 | A kind of wheat germ globulin applied in blood substitutes |
| CN108760233A (en) * | 2018-05-30 | 2018-11-06 | 威海威高血液净化制品有限公司 | dialyzer pressure drop performance test liquid |
| CN108849861A (en) * | 2018-09-30 | 2018-11-23 | 南京麦荻泉生物科技有限公司 | One kind containing fluorocarbon emulsion type organ preservative fluid |
| CN109486907B (en) * | 2018-12-21 | 2022-02-11 | 陕西师范大学 | Loop-mediated isothermal amplification reagent capable of being transported at normal temperature, preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1613504A (en) * | 2004-07-22 | 2005-05-11 | 周翔 | Method and use of closed hydrophilic and lipophilic liquid-phase hollow capsules with cores |
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|---|---|---|---|---|
| CN1613504A (en) * | 2004-07-22 | 2005-05-11 | 周翔 | Method and use of closed hydrophilic and lipophilic liquid-phase hollow capsules with cores |
Non-Patent Citations (1)
| Title |
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| 陈新等.中分子羟乙基淀粉.《新编药物学》.人民卫生出版社,2007,第583-584页. * |
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