CN101926984B - Preparation method of artificial nano red blood cells - Google Patents
Preparation method of artificial nano red blood cells Download PDFInfo
- Publication number
- CN101926984B CN101926984B CN2010102462331A CN201010246233A CN101926984B CN 101926984 B CN101926984 B CN 101926984B CN 2010102462331 A CN2010102462331 A CN 2010102462331A CN 201010246233 A CN201010246233 A CN 201010246233A CN 101926984 B CN101926984 B CN 101926984B
- Authority
- CN
- China
- Prior art keywords
- blood cells
- red blood
- starch
- oleic acid
- nano red
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention discloses a preparation method of artificial nano red blood cells. The microencapsulated artificial nano red blood cells with grain diameter of 200-300nm are formed by nano-assembling and coating modified starch with amphiphilicity outside the hemoglobins. The invention prepares the artificial nano red blood cells by taking the hemoglobins as the oxygen-bearing and oxygen-releasing functional molecules and embedding modified starch microcapsules with amphiphilicity outside the hemoglobins by way of nano-assembling. P50 of the prepared artificial nano red blood cells is 28.5mmHg and the artificial nano red blood cells retain good oxygen-bearing and oxygen-releasing capabilities. The prepared artificial nano red blood cells have no obvious activation to the mice complement system in vitro. The thrombin in the blood plasma is not increased and the testing result of the coagulation factors is qualified. After being injected into the bodies of the mice, the artificial nano red blood cells do not have adverse effect on the blood platelets of the mice and do not cause obvious blood platelet aggregation.
Description
Technical field
The invention belongs to HRBC's succedaneum technical field, relate to a kind of method for preparing of artificial nano red blood cells.
Background technology
In recent years, the blood used in clinic amount increases sharply in the worldwide, and the frequent blood famine that occurs makes the blood shortage reach the degree of crisis.In addition, some are under the special environment in burst prominent the Nature disaster, war and terrorist incident etc., and large quantities of wounded will cause these disparities between supply and demand sharply to increase when needing simultaneously treatment of blood transfusion.According to estimates, even peacetime, the whole world needs 1,500 ten thousand liters of fresh bloods every year approximately, but also is the trend of rising appreciably.Nearly 1,300,000 liters of the annual medical blood volume of China, annual also with 7～9% speed increase.Top scientists thinks that all artificial blood is the only way that solves this clinical crisis.
Compare with fresh allosome blood, artificial blood has no surface antigen, does not need cross matching; Avoided transfusion reaction, avoided the pollution and the cross infection of pathogenic microorganism, also do not had in the blood supply of dependence human body; Draw materials conveniently, wide material sources store easily; Long shelf-life, convenient transportation, cheap, but advantages such as sufficient supplies.For this reason, safety, artificial blood effective, that can be mass-produced have wide market application prospect.Also be the research focus of 21st century international concern, the great field of medical product exploitation.
Artificial red cell is also claimed HRBC's succedaneum (human red cell substitutes), is the artificial preparation that has the oxygen propagation function, keeps colloidal osmotic pressure and acid-base balance and expansion capacity.Ideal blood substitute requires to have natural erythrocytic transmission oxygen function, biocompatibility, safety and stability.Be specially: (1) has higher oxygen carrying capacity, and partial pressure of oxygen can be effectively to organizing oxygen supply in the normal physiological scope; (2) has excellent biological compatibility with the human blood all components; (3) can keep colloidal osmotic pressure, acid-base balance, viscosity and blood volume; (4) non-immunogenicity (no sensitization originality), no blood microbiological contamination, no thermal source; (5) body-internal-circulation half life, do not produce side effect such as nephrotoxicity under normal perfusion condition more than 24h; (6) under cryogenic conditions, shelf life of products was greater than 6 months; (7) has the erasability of expection.Simultaneously, as a kind of product, more suitably valency effect ratio, degree of safety and the shelf-life of comparing with blood with existing blood transfusion also depended in its commercial viability.
To so far, blood substitute development both at home and abroad and development experience three generation products.The first generation is representative with the fluorocarbons class mainly; The second filial generation is to be representative with modified hemoglobin (Hb), mainly comprises modifying Hb (crosslinked Hb, poly Hb and macromolecular compound conjugation Hb in being divided into again) and the reorganization Hb that utilizes the generation of molecular genetic engineering; Third generation product is the artificial red blood cells of microencapsulation.Degradable macromolecule hemoglobin microcapsule blood substitute not only can overcome the shortcoming of the acellular envelope barrier of first and second generation DCLHb blood substitute; Having solved the shortcoming of liposome microcapsule blood substitute mechanical property and small-molecule substance permeability difference simultaneously, is a kind of blood substitute with development prospect.
Among the blood substitute of existing two types of microencapsulation being was was being researched and developed: one type was the fluorocarbons carrier of oxygen (PFBOCs), and other one type is hemoglobin-based oxygen carrier (HBOCs).The clinical research in early stage shows that this two series products can be used for tool and partly recovers blood and take O
2Function, the effects such as organ failure that prevent or recover Hypovolemia and cause, but outstanding shortcoming is the release of oxygen, problems such as metabolite toxicity will seriously limit it and further develop.Domesticly start late, and reports such as more representational in the world Chang TMS, adopt the route of polymer embedding hemoglobin in the research aspect the degradable macromolecule hemoglobin microcapsule blood substitute, but the important oxygen property index P that takes
50Be 32mmHg, explain that the affinity of oxygen slightly descends, and envelop rate is also lower, has only 29～47%, the hemoglobin utilization rate is not high.
The synthetic erythrocyte analogies that more adapt to the human body needs of design that develop into of nanotechnology provide strong instrument.Low environmental load, low energy consumption, the low-cost height function nano grain of making are the present focuses of studying; Wherein self-organizing method can finely satisfy this demand: microphase-separated takes place by the amphipathy macromolecule material in this method in aqueous medium; Formation has the nano-micelle of hydrophobic cores and hydrophilic shell, can be used as the carrier of various medicines.At present, can obtain the size homogeneous through self assembly, the perfect nano-micelle of form through amphiphilic polymer.Lapping mainly was the fat-soluble material of high-molecular biologic degradable of synthetic in the past, this type material because of exist toxicity be prone to by phagocyte problems affect such as engulf its further develop.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of method for preparing of artificial nano red blood cells, as lapping, through nanometer self assembly parcel hemoglobin, forms the artificial nano red blood cells of microencapsulation with modified starch.
The present invention realizes through following technical scheme:
A kind of method for preparing of artificial nano red blood cells may further comprise the steps:
1) with behind the starch dissolution, under catalyst, carry out esterification with the oleic acid of 2～8 times of its quality, treat fully reaction after, starch-oleic acid graft polymers that separating reaction generates;
2) starch-oleic acid graft polymers is dissolved in dimethyl sulfoxide, obtains starch-oleic acid graft polymers solution;
The hemoglobin of 1/8～1 times of starch-oleic acid glycerol polymerization amount is scattered in the normal saline; And the tween 20 of adding normal saline volume 1～10%; The concussion mixing under condition of ice bath, splashes into starch-oleic acid graft polymers solution after removing oxygen side by side; Then ultrasonic probe is stretched under the liquid level of mixed solution, under ultrasound condition, make starch-oleic acid graft polymers hemoglobin carried out embedding with the form of nanometer self assembly;
3) after the ultrasonic completion, remove the dimethyl sulfoxide in the reactant mixture, then reactant mixture is used filtering with microporous membrane, the centrifugalize of will filtrating, collecting precipitation obtains the microencapsulation artificial nano red blood cells.
Described starch dissolution is in dimethyl sulfoxide, and wherein the mass concentration of starch is 5～10%, adds the oleic acid of 2～8 times of starch qualities and 0.5～1 times potassium peroxydisulfate then, under 95～105 ℃, carries out esterification; After treating fully reaction, add excess ethanol, starch-oleic acid graft polymers is separated out with precipitation mode, and will precipitate and use the ethanol thorough washing,, obtain starch-oleic acid graft polymers then at 95～105 ℃ of vacuum dryings.
The mass concentration of described starch-oleic acid graft polymers solution is 0.6～4.8%.
After described hemoglobin was scattered in normal saline, the mass concentration of hemoglobin was 0.1～0.8%.
Described starch-oleic acid graft polymers the solution that splashes into is at the uniform velocity splashing into, and the speed of splashing into is 1～2ml/min.
The power setting of described ultrasonic probe is 100～200W, and ultrasonic time is 5～20min.Further the power setting of described ultrasonic probe is 200W, and ultrasonic time is 10min.
Through the dimethyl sulfoxide in the reactant mixture is dialysed to redistilled water, dialysed overnight is to remove the dimethyl sulfoxide in the reactant mixture; In initial 3 hours, per hour change redistilled water one time, changed once in later per 5 hours.
The mass concentration of described normal saline is 0.9%.
The particle diameter of described microencapsulation artificial nano red blood cells is 200～300nm.
Compared with prior art, the present invention has following beneficial technical effects:
The present invention is with the functional molecular of hemoglobin as oxygen carrier, oxygen release, and the amphiphatic modified starch microcapsule of mode embedding in its outside through the nanometer self assembly is with the artificial nano red blood cells of preparation.Such formation had both made the structure of hemoglobin not change; The ability that has kept its oxygen carrier, oxygen release; And starch based natural polysaccharide polymer substance; Good biocompatibility, toxicity are low, and be cheap and have the rhetorical function on nanoparticulate carriers surface, can not produce the antigenicity as the protide material in vivo; Especially have amphiphatic starch after the modification at the ultrasonic spheroid that down can self assembly have formed complete microencapsulation, improved the envelop rate of hemoglobin greatly, reduced percolation ratio, make that artificial nano red blood cells is easy to preserve.
The P50 of the artificial nano red blood cells that the present invention is prepared is 28.5mmHg, and the P50 of natural BHb is 26.5mmHg before the embedding, explains that artificial nano red blood cells has kept good oxygen, the oxygen release ability taken.Record that natural BHb Hill coefficient is 2.4 before the embedding, artificial nano red blood cells Hill coefficient is 2.1, and this has explained that BHb still has concertedness preferably behind microencapsulation.
The prepared artificial nano red blood cells of the present invention is external not to have obvious activation to the mice complement system, and thrombin does not have and increases in the blood plasma, and it is qualified that coagulation factors is measured the result, hemolysis rate require 5% in, test passes; And after being expelled to artificial nano red blood cells in the mice body, the platelet of mice is had no adverse effects, do not cause tangible platelet aggregation.This explanation artificial nano red blood cells since embedding the starch microcapsule of modification, its surface does not have the antigenicity of protide molecule, has good biocompatibility and safety.
Description of drawings
Fig. 1 is the comparison diagram of native starch and starch-oleic acid graft polymers infrared absorption spectra, and abscissa is the infrared absorption spectroscopy wave band, and vertical coordinate is a trap;
Fig. 2 is artificial red blood cells's transmission electron microscope photo sketch map.
Fig. 3 is a BHb infrared absorption spectra before and after the modified starch embedding, and abscissa is the infrared absorption spectroscopy wave band, and vertical coordinate is a trap;
Fig. 4 is the oxygen dissociation curve sketch map of BHb before and after the modified starch embedding, and abscissa is a partial pressure of oxygen, and vertical coordinate is an oxygen saturation.
The specific embodiment
The present invention provides a kind of method for preparing of artificial nano red blood cells, and through having amphiphatic modified starch in the hemoglobin outside through nanometer self assembly parcel, forming particle diameter is the artificial nano red blood cells of the microencapsulation of 200～300nm.Prepared artificial nano red blood cells has good oxygen, oxygen release ability and the high envelop rate taken, and platelet is had no adverse effects, and does not cause tangible platelet aggregation.Below in conjunction with accompanying drawing and concrete embodiment the present invention is done to describe in further detail, said is to explanation of the present invention rather than qualification.
1) preparation of starch-oleic acid graft polymers (ST-O1)
The 1g starch dissolution in 10ml DMSO (dimethyl sulfoxide), and is transferred in the round-bottomed flask, add the oleic acid of 5.2g then, and the 0.5g potassium peroxydisulfate is as catalyst; In 95 ℃ of glycerol oil baths, magnetic agitation reaction 8h;
After reaction is accomplished, add the ethanol of 50ml, form starch-oleic acid graft polymers deposition, membrane filtration will precipitate with washing with alcohol 3 times, and drying in 95 ℃ of vacuum drying ovens obtains starch-oleic acid graft polymers then.
Evaluation to starch-oleic acid graft polymers:
The comparison diagram of native starch as shown in Figure 1 and starch-oleic acid graft polymers infrared absorption spectra, the infrared signature absworption peak of native starch is 1157,1080,1016,927cm
-1, and starch-oleic acid graft polymers is at 1740cm
-1Locate the extra characteristic absorption peak that C=O occurs, this shows the esterification completion, amphipathic starch graft modification success; In the spectrum 2926
-1And 2855cm
-1The intensity of absworption peak depends on that methyl and methylene C-H and fatty acid substituents stretch.
2) the nanometer self assembly forms the microencapsulation artificial cell
Starch-oleic acid graft polymers 100mg is dissolved in 3ml DMSO, obtains mass concentration and be starch-oleic acid graft polymers solution of 3.0%;
It is in 0.9% the normal saline that the 20mg BHb is dissolved in the 10ml mass concentration; Obtaining mass concentration is 0.2% BHb solution, adds tween 20 0.3ml then, feeds nitrogen so that oxygen is discharged behind the concussion mixing; Under condition of ice bath; At the uniform velocity splash into starch-oleic acid graft polymers solution with the 2ml syringe with the speed of 1～2ml/min, again ultrasonic probe is stretched under the liquid level of mixed solution the ultrasonic 10min of 200W; In ultransonic process, starch-oleic acid graft polymers carries out embedding with the form of nanometer self assembly to hemoglobin;
3) after the ultrasonic completion, the dialysis remove DMSO: with reactant mixture with MW 12,000gmol
- 1As dialyzer, dialyse to redistilled water dialysed overnight under the magnetic agitation; At initial 3 hours, per hour change a medium (redistilled water), changed once at ensuing 24 hours per 5 hours.After the dialysed overnight; With 0.45 μ m filtering with microporous membrane, filtrating discards supernatant (not residual DMSO in the Hb of embedding assembling and the mixed solution) at 15 ℃ of following 20000r/min high speed centrifugation 30min with reactant mixture; Deposition is again after 0.9% normal saline washing 3 times; Collecting precipitation obtains self assembly and forms the microencapsulation artificial nano red blood cells, and lyophilizing is stored subsequent use.
Embodiment 2
1) preparation of starch-oleic acid graft polymers (ST-O1)
The 1g starch dissolution in 10ml DMSO (dimethyl sulfoxide), and is transferred in the round-bottomed flask, added the oleic acid of 8g then, the 1g potassium peroxydisulfate is as catalyst; In 105 ℃ of glycerol oil baths, magnetic agitation reaction 8h;
After reaction is accomplished, add the ethanol of 100ml, form starch-oleic acid graft polymers deposition, membrane filtration will precipitate with washing with alcohol 3 times, and drying in 105 ℃ of vacuum drying ovens obtains starch-oleic acid graft polymers then.
2) the nanometer self assembly forms the microencapsulation artificial cell
Starch-oleic acid graft polymers 160mg is dissolved in 3ml DMSO, obtains concentration and be starch-oleic acid graft polymers solution of 4.8%;
The 40mg BHb is dissolved in 10ml 0.9% normal saline; Obtaining concentration is 0.4% BHb solution, tween 20 0.3ml; Feed nitrogen so that oxygen is discharged behind the concussion mixing, under condition of ice bath, using the 2ml syringe is that 1～2ml/min at the uniform velocity splashes into starch-oleic acid graft polymers solution with the speed of splashing into; Again ultrasonic probe is stretched under the liquid level of mixed solution; The ultrasonic 20min of 100W, in ultransonic process, starch-oleic acid graft polymers carries out embedding with the form of nanometer self assembly to hemoglobin;
3) after the ultrasonic completion, the dialysis remove DMSO: with reactant mixture with MW 12,000gmol
- 1As dialyzer, dialyse to redistilled water dialysed overnight under the magnetic agitation; At initial 3 hours, per hour change a medium (redistilled water), changed once at ensuing 24 hours per 5 hours.After the dialysed overnight; With 0.45 μ m filtering with microporous membrane, filtrating discards supernatant (not residual DMSO in the Hb of embedding assembling and the mixed solution) at 15 ℃ of following 20000r/min high speed centrifugation 30min with reactant mixture; Deposition is again after 0.9% normal saline washing 3 times; Collecting precipitation obtains self assembly and forms the microencapsulation artificial nano red blood cells, and lyophilizing is stored subsequent use.
Embodiment 3
1) preparation of starch-oleic acid graft polymers (ST-O1)
The 1g starch dissolution in 10ml DMSO (dimethyl sulfoxide), and is transferred in the round-bottomed flask, added the oleic acid of 2g then, the 0.5g potassium peroxydisulfate is as catalyst; In 100 ℃ of glycerol oil baths, magnetic agitation reaction 8h;
After reaction is accomplished, add the ethanol of 80ml, form starch-oleic acid graft polymers deposition, membrane filtration will precipitate with washing with alcohol 3 times, and drying in 100 ℃ of vacuum drying ovens obtains starch-oleic acid graft polymers then.
2) the nanometer self assembly forms the microencapsulation artificial cell
Starch-oleic acid graft polymers 80mg is dissolved in 3ml DMSO, obtains concentration and be starch-oleic acid graft polymers solution of 2.4%;
The 80mg BHb is dissolved in 10ml 0.9% normal saline; Obtaining concentration is 0.8% BHb solution, tween 20 1ml; Feed nitrogen so that oxygen is discharged behind the concussion mixing, under condition of ice bath, using the 2ml syringe is that 1～2ml/min at the uniform velocity splashes into starch-oleic acid graft polymers solution with the speed of splashing into; Again ultrasonic probe is stretched under the liquid level of mixed solution; The ultrasonic 15min of 150W, in ultransonic process, starch-oleic acid graft polymers carries out embedding with the form of nanometer self assembly to hemoglobin;
3) after the ultrasonic completion, DMSO is removed in dialysis, after the dialysed overnight; With 0.45 μ m filtering with microporous membrane, filtrating discards supernatant (not residual DMSO in the Hb of embedding assembling and the mixed solution) at 15 ℃ of following 20000r/min high speed centrifugation 30min with reactant mixture; Deposition is again after 0.9% normal saline washing 3 times; Collecting precipitation obtains self assembly and forms the microencapsulation artificial nano red blood cells, and lyophilizing is stored subsequent use.
Embodiment 4
1) preparation of starch-oleic acid graft polymers (ST-O1)
The 1g starch dissolution in 10ml DMSO (dimethyl sulfoxide), and is transferred in the round-bottomed flask, added the oleic acid of 4g then, the 0.6g potassium peroxydisulfate is as catalyst; In 98 ℃ of glycerol oil baths, magnetic agitation reaction 8h;
After reaction is accomplished, add the ethanol of 80ml, form starch-oleic acid graft polymers deposition, membrane filtration will precipitate with washing with alcohol 3 times, and drying in 98 ℃ of vacuum drying ovens obtains starch-oleic acid graft polymers then.
2) the nanometer self assembly forms the microencapsulation artificial cell
Starch-oleic acid graft polymers 20mg is dissolved in 3ml DMSO, obtains concentration and be starch-oleic acid graft polymers solution of 0.6%;
The 10mg BHb is dissolved in 10ml 0.9% normal saline; Obtaining concentration is 0.1% BHb solution, tween 20 0.1ml; Feed nitrogen so that oxygen is discharged behind the concussion mixing, under condition of ice bath, using the 2ml syringe is that 1～2ml/min at the uniform velocity splashes into starch-oleic acid graft polymers solution with the speed of splashing into; Again ultrasonic probe is stretched under the liquid level of mixed solution; The ultrasonic 12min of 160W, in ultransonic process, starch-oleic acid graft polymers carries out embedding with the form of nanometer self assembly to hemoglobin;
3) after the ultrasonic completion, DMSO is removed in dialysis, after the dialysed overnight; With 0.45 μ m filtering with microporous membrane, filtrating discards supernatant (not residual DMSO in the Hb of embedding assembling and the mixed solution) at 15 ℃ of following 20000r/min high speed centrifugation 30min with reactant mixture; Deposition is again after 0.9% normal saline washing 3 times; Collecting precipitation obtains self assembly and forms the microencapsulation artificial nano red blood cells, and lyophilizing is stored subsequent use.
1) preparation of starch-oleic acid graft polymers (ST-O1)
The 1g starch dissolution in 10ml DMSO (dimethyl sulfoxide), and is transferred in the round-bottomed flask, added the oleic acid of 4.5g then, the 0.6g potassium peroxydisulfate is as catalyst; In 100 ℃ of glycerol oil baths, magnetic agitation reaction 8h;
After reaction is accomplished, add the ethanol of 80ml, form starch-oleic acid graft polymers deposition, membrane filtration will precipitate with washing with alcohol 3 times, and drying in 100 ℃ of vacuum drying ovens obtains starch-oleic acid graft polymers then.
2) the nanometer self assembly forms the microencapsulation artificial cell
Starch-oleic acid graft polymers 60mg is dissolved in 3ml DMSO, obtains concentration and be starch-oleic acid graft polymers solution of 1.8%;
The 20mg BHb is dissolved in 10ml 0.9% normal saline; Obtaining concentration is 0.2% BHb solution, tween 20 0.5ml; Feed nitrogen so that oxygen is discharged behind the concussion mixing, under condition of ice bath, using the 2ml syringe is that 1～2ml/min at the uniform velocity splashes into starch-oleic acid graft polymers solution with the speed of splashing into; Again ultrasonic probe is stretched under the liquid level of mixed solution; The ultrasonic 20min of 120W, in ultransonic process, starch-oleic acid graft polymers carries out embedding with the form of nanometer self assembly to hemoglobin;
3) after the ultrasonic completion, DMSO is removed in dialysis, after the dialysed overnight; With 0.45 μ m filtering with microporous membrane, filtrating discards supernatant (not residual DMSO in the Hb of embedding assembling and the mixed solution) at 15 ℃ of following 20000r/min high speed centrifugation 30min with reactant mixture; Deposition is again after 0.9% normal saline washing 3 times; Collecting precipitation obtains self assembly and forms the microencapsulation artificial nano red blood cells, and lyophilizing is stored subsequent use.
Self assembly forms the evaluation of microencapsulation artificial nano red blood cells
1, the transmission electron microscope observing of outward appearance (TEM)
With artificial nano red blood cells dried frozen aquatic products 10mg, being diluted to mass concentration after the use normal saline redissolves is 0.05～0.1%, behind the 100w ultra-sonic dispersion 5min it is dropped on the copper mesh, and normal temperature drying is placed on observation microsphere form under the transmission electron microscope in the air.
Observed result is as shown in Figure 2, and the artificial nano red blood cells of microencapsulation is spherical in shape mostly, the form rounding; The particle diameter of the formed artificial nano red blood cells of Different Weight proportioning component is 200～300nm.
2, envelop rate detects
After the artificial nano red blood cells preparation of microencapsulation was accomplished, reactant mixture was got the 1ml centrifuged supernatant and is detected wherein free hemoglobin at the centrifugal 30min of 20000r/min, the envelop rate of starch self assembly thereby the calculating hemoglobin is modified.The concentration of the hemoglobin in the middle of the supernatant detects employing standard cyanmethemoglobin method and measures: in the 1ml supernatant, add the hemoglobin conversional solution reagent of 2ml, and on ELIASA, survey the OD value at 540nm place.Ferrum titer, blank sample colorimetric with same processing calculate the free HC in the supernatant, according to the cumulative volume of supernatant and then obtain total free hemoglobin.
Carry out the calculating of envelop rate by following formula:
The envelop rate testing result shows: when modified starch carries out self assembly embedding hemoglobin, when fixing ultransonic power, prolong in time, envelop rate increases gradually; And fixedly when ultrasonic power and time, increase with shell material ratio, envelop rate increases gradually.And be 1: 5 o'clock at the ultrasonic 10min of 200W, hemoglobin and modified starch mass ratio, envelop rate can reach 98.8%.
3, FTIR spectrum analysis
(Nicolet5SXC, whether Germany) BHb of modified starch embedding front and back carries out infrared analysis respectively, change to detect its structure to adopt infrared spectrum analyser.
The infrared analysis result is as shown in Figure 3, and natural BHb detection curve Hb compares before the BHb detection curve SEHb after the embedding and the embedding: the infrared signature of natural Hb absorbs 1540cm
-1Be the bending vibration of N-H, and 1647cm
-1The absorption at place then is the absorbed oscillation of C=O; Curve S EHb compares absorption peak with curve Hb and does not change basically; This explanation BHb by embedding after its feature structure obtained good reservation, and the embedding of modified starch self assembly is little to the influence of the chemical constitution of hemoglobin.
4, P
50Mensuration with the Hill coefficient
P
50Be partial pressure of oxygen corresponding when the Hb oxygen saturation is 50% among the artificial red blood cells, this is an important indicator of expression artificial red blood cells oxygen carrying capacity.The Hill coefficient is the synergitic important indicator that haemachrome combines oxygen on each subunit of reflection Hb.
With blood oxygen analysis appearance Hemox
TMAnalyzer (U.S. TCS Scientific Corp product) is at 37 ℃ of P that measure the BHb of modified starch embedding front and back
50With the Hill coefficient: the blood oxygen analysis appearance is measured partial pressure of oxygen with Clark type oxygen electrode (U.S. Yellow Spring company), and uses the oxygen saturation of the spectrophotometric determination hemoglobin of dual wavelength simultaneously.The oxygen balance curve with method of least square and Adair equation model, can be calculated various oxygen-balance parameters.The logarithm through calculating 40～75% oxygen saturations (Y) ratio and the logarithm (logPO of partial pressure of oxygen
2) ratio, can obtain Hill coefficient (n).
The computing formula of Hill coefficient (n) is following:
Testing result shows: the P50 of natural BHb is 26.5mmHg before the embedding, and the artificial nano red blood cells P after the modified starch embedding
50Be 28.5mmHg, explain that artificial nano red blood cells has kept good oxygen, the oxygen release ability taken.Record that natural BHb Hill coefficient is 2.4 before the embedding, artificial nano red blood cells Hill coefficient is 2.1, slightly descend, and or not very big but change, this has explained that BHb still has concertedness preferably behind microencapsulation.
And the oxygen dissociation curve of BHb before and after the embedding as shown in Figure 4; Wherein, Abscissa is a partial pressure of oxygen; Vertical coordinate is an oxygen saturation, and natural BHb detection curve Hb compares before the BHb detection curve Nano-Hb after the embedding and the embedding: it is suitable with natural BHb ability to depress entrained oxygen at the same oxygen branch, has kept good oxygen, the oxygen release ability taken.
5, external complement activation and blood coagulation, hemolysis test
The testing in vitro artificial nano red blood cells adopts mice serum, erythrocyte or blood plasma to carry out respectively to complement activation and blood coagulation, hemolytic detection.During mensuration, with the artificial nano red blood cells suspension (to include the amount of Hb, 150g/L) respectively with mice serum, red blood cell suspension 150g/L, the mixed of blood plasma with 1: 10 (volume ratio) after, measure.Complement C3 content detects with instrument BehringNephelometer 100Analyzer, and as positive control, 0.9% normal saline is as negative control with zymosan (0.25mg/ μ l) simultaneously; Hemolysis rate with distilled water as positive control, with 0.9% normal saline as negative control; As positive control, 0.9% normal saline is as negative control with heparin sodium 15U/ml for setting time.Each test point is got 3 parallel appearance, results averaged.Testing result is as shown in table 1:
The external complement activation of table 1, hemolytic and thrombotest
| The artificial red blood cells | Positive control | Negative control | |
| C3 content (g/L) | 0.293±0.004 | 0.009±0.002 | 0.282±0.005 |
| Hemolysis rate (%) | 1.8 | 100 | 0 |
| Setting time (s) | 15.9±0.5 | - | 15.0±0.6 |
Testing result shows: artificial nano red blood cells is with after mice serum mixes, and C3 content is 0.293 ± 0.004g/L, and hemolysis rate is 1.8%; Setting time 15.9 ± 0.5s with matched group SC amount no difference of science of statistics, explains that the artificial red blood cells does not have obvious activation to the mice complement system; Thrombin does not have and increases in the blood plasma; It is qualified that coagulation factors is measured the result, hemolysis rate require 5% in, test passes.
6, platelet count detects in the body
(concentration to be including the amount of Hb, and ratio 150g/L) behind mouse tail vein injection artificial nano red blood cells suspension, is investigated behind the infusion situation of change of mouse platelets quantity in the 24h by the injection 10ml artificial nano red blood cells suspension of every kg mice body weight.Each test point is got 3 mices, results averaged.Testing result is as shown in table 2, and be 1.57 * 10 the normal range of mouse platelets counting
5～2.60 * 10
5Individual/μ l, behind the infusion artificial red blood cells suspension, the platelet of mice is had no adverse effects, do not cause tangible platelet aggregation.
Table 2 platelet count result (Table 3 Results of platelet counts)
| Sampling time point (h) | Artificial red blood cells's group (* 10 5·μl -1) | Matched group (* 10 5·μl -1) |
| ?12 | 1.812±0.164 | 1.798±0.133 |
| ?24 | 1.955±0.173 | 1.812±0.156 |
7, different preservation condition artificial nano red blood cells leak test
The 50mg artificial red blood cells is added 10ml 0.9% normal saline fully to be disperseed; Mixing is divided into two groups, and one group is kept under 4 ℃ of conditions, and another group is-20 ℃ of freezing preservations; Respectively at 1,5,30,60d gets the supernatant of two groups of goods, measures the supernatant content of hemoglobin.The result is as shown in table 3 ,-20 ℃ freezing 30 days, artificial red blood cells's percolation ratio is 5.4%, 60 day to be 5.9%, 4 ℃ to preserve 30 days that percolation ratio 6.7%, 60 day was 7.0%, this explanation the present invention prepares artificial nano red blood cells and is easy to preserve.
The different preservation condition artificial red blood cells's percolation ratios of table 3 (%)
| Holding time | -20 ℃ freezing group | 4 ℃ of cold preservation groups |
| 1d | 4.2 | 4.9 |
| 5d | 4.8 | 5.3 |
| 30d | 5.4 | 6.7 |
| 60d | 5.9 | 7.0 |
Claims (8)
1. the method for preparing of an artificial nano red blood cells is characterized in that, may further comprise the steps:
1) with starch dissolution in dimethyl sulfoxide, wherein the mass concentration of starch is 5～10%, adds the oleic acid of 2～8 times of starch qualities and 0.5～1 times potassium peroxydisulfate then, under 95～105 ℃, carries out esterification; After treating fully reaction, add excess ethanol, starch-oleic acid graft polymers is separated out with precipitation mode, and will precipitate and use the ethanol thorough washing,, obtain starch-oleic acid graft polymers then at 95～105 ℃ of vacuum dryings;
2) starch-oleic acid graft polymers is dissolved in dimethyl sulfoxide, obtains starch-oleic acid graft polymers solution;
The hemoglobin of 1/8～1 times of starch-oleic acid glycerol polymerization amount is scattered in the normal saline; And the tween 20 of adding normal saline volume 1～10%; The concussion mixing under condition of ice bath, splashes into starch-oleic acid graft polymers solution after removing oxygen side by side; Then ultrasonic probe is stretched under the liquid level of mixed solution, under ultrasound condition, make starch-oleic acid graft polymers hemoglobin carried out embedding with the form of nanometer self assembly;
3) after the ultrasonic completion, remove the dimethyl sulfoxide in the reactant mixture, then reactant mixture is used filtering with microporous membrane, the centrifugalize of will filtrating, collecting precipitation obtains the microencapsulation artificial nano red blood cells.
2. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, the mass concentration of described starch-oleic acid graft polymers solution is 0.6～4.8%.
3. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, after described hemoglobin was scattered in normal saline, the mass concentration of hemoglobin was 0.1～0.8%.
4. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, the described starch-oleic acid graft polymers solution that splashes into is at the uniform velocity splashing into, and the speed of splashing into is 1～2ml/min.
5. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, the power setting of described ultrasonic probe is 100～200W, and ultrasonic time is 5～20min.
6. the method for preparing of artificial nano red blood cells as claimed in claim 5 is characterized in that, the power setting of described ultrasonic probe is 200W, and ultrasonic time is 10min.
7. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, the mass concentration of described normal saline is 0.9%.
8. the method for preparing of artificial nano red blood cells as claimed in claim 1 is characterized in that, the particle diameter of described microencapsulation artificial nano red blood cells is 200～300nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102462331A CN101926984B (en) | 2010-08-05 | 2010-08-05 | Preparation method of artificial nano red blood cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102462331A CN101926984B (en) | 2010-08-05 | 2010-08-05 | Preparation method of artificial nano red blood cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101926984A CN101926984A (en) | 2010-12-29 |
| CN101926984B true CN101926984B (en) | 2012-09-05 |
Family
ID=43366585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102462331A Expired - Fee Related CN101926984B (en) | 2010-08-05 | 2010-08-05 | Preparation method of artificial nano red blood cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101926984B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK2714017T3 (en) * | 2011-06-02 | 2018-09-03 | Univ California | MEMBRANE-WRAPPED NANOPARTICLES AND METHOD OF USE |
| CN103091149B (en) * | 2013-01-10 | 2015-08-05 | 济南金域医学检验中心有限公司 | The preparation method of HE liquid |
| CN107677658A (en) * | 2017-10-10 | 2018-02-09 | 广西师范学院 | Utilize the method for hemoglobin concentration in nitrogen-doped carbon quantum dots characterization solution |
| CN110437377B (en) * | 2019-06-28 | 2021-02-05 | 江南大学 | Bio-based core-shell particle and preparation method thereof |
-
2010
- 2010-08-05 CN CN2010102462331A patent/CN101926984B/en not_active Expired - Fee Related
Non-Patent Citations (10)
| Title |
|---|
| F.T.Meng等.Microencapsulation of bovine hemoglobin with high bio-activity and high entrapment efficiency using a W/O/W double emulsion technique.《Colloids and Surfaces B:Biointerfaces》.2004,第33卷177-183. |
| Improvement of the encapsulation efficiency of oligonucleotide-containing biodegradable microspheres;T.Freytag等;《Journal of controlled release》;20001231;第69卷;197-207 * |
| Jian Zhao等.Preparation of hemoglobin-loaded nano-sized particles with porous structure as oxygen carriers.《Biomaterials》.2006,第28卷第1414-1422页. |
| Microencapsulation of bovine hemoglobin with high bio-activity and high entrapment efficiency using a W/O/W double emulsion technique;F.T.Meng等;《Colloids and Surfaces B:Biointerfaces》;20041231;第33卷;177-183 * |
| Preparation of hemoglobin-loaded nano-sized particles with porous structure as oxygen carriers;Jian Zhao等;《Biomaterials》;20061128;第28卷;第1414-1422页 * |
| T.Freytag等.Improvement of the encapsulation efficiency of oligonucleotide-containing biodegradable microspheres.《Journal of controlled release》.2000,第69卷197-207. |
| 人工红细胞及微囊包被血红蛋白的制备与鉴定;樊晶等;《中国输血杂志》;20041231;第17卷(第2期);75-78 * |
| 樊晶等.人工红细胞及微囊包被血红蛋白的制备与鉴定.《中国输血杂志》.2004,第17卷(第2期),75-78. |
| 王岱娟等.血红蛋白氧载体研究进展及应用现状.《中国生化药物杂志》.2005,第26卷(第6期),379-382. |
| 血红蛋白氧载体研究进展及应用现状;王岱娟等;《中国生化药物杂志》;20051231;第26卷(第6期);379-382 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101926984A (en) | 2010-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101926984B (en) | Preparation method of artificial nano red blood cells | |
| CN102533650B (en) | Cell separation medium and cell separation method | |
| CN105028389A (en) | Erythrocyte preservative solution | |
| CN101926983B (en) | Artificial nano red blood cells | |
| CN101785764A (en) | Method for improving blood compatibility of microcapsule | |
| CN102507961B (en) | Newborn ABO/Rh blood grouping reagent card and preparation method thereof | |
| Hortin et al. | Progress toward preparation of universal donor red cells | |
| CN106635769B (en) | Cell separation apparatus and cell isolation method | |
| CN104865232A (en) | Method for selectively detecting ascorbic acid by utilizing metal-organic framework material | |
| CN111713487B (en) | Reagent erythrocyte preservation system and preparation method thereof | |
| CN107873696B (en) | Preserving fluid for red blood cells of animals in family of bear and application thereof | |
| CN106750429B (en) | Preparation process of onion polysaccharide-gelatin composite membrane | |
| CN103675298B (en) | Immune hemolytic anemia detection kit and detection method thereof | |
| CN102113999A (en) | Novel multi-layer polysaccharide structure-modified liposome-encapsulated hemoglobin nano-capsule blood substitute | |
| CN112778421A (en) | Polydopamine hemoglobin-loaded micro-nano particle and preparation method and application thereof | |
| Mostaghni et al. | Haematology and serum biochemistry of captive wild sheep (Ovis orientalis esphahanica) in Iran | |
| CN202086790U (en) | Anticoagulation blood collection tube | |
| Nimaev et al. | New Carbon-Mineral Sorbent Based on Aluminum Oxide, Polydimethylsiloxane, and Single-Wall Carbon Nanotubes: Assessment of the Effect on Erythrocytes In Vitro | |
| CN109432012B (en) | Preparation method of baicalein drug-loaded nanorod | |
| CN103667174B (en) | A kind of composition for the treatment of biological specimen | |
| CN110568184A (en) | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier | |
| CN104739853A (en) | Method for establishing iron-overload mouse model corresponding to thalassemia Pesaro risk classification | |
| Zhang et al. | Effects of different bicarbonate on spirulina in CO2 absorption and microalgae conversion hybrid system | |
| CN1763102A (en) | Cell membrane antigen preservation method and magnetic particle envelope human cell membrane antigen | |
| Compagnoni et al. | Influence of parenteral nutrition on blood rheology and platelet aggregation in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 Termination date: 20140805 |
|
| EXPY | Termination of patent right or utility model |