CN102240416A - Heparinizing method and application thereof - Google Patents
Heparinizing method and application thereof Download PDFInfo
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- CN102240416A CN102240416A CN201110103732XA CN201110103732A CN102240416A CN 102240416 A CN102240416 A CN 102240416A CN 201110103732X A CN201110103732X A CN 201110103732XA CN 201110103732 A CN201110103732 A CN 201110103732A CN 102240416 A CN102240416 A CN 102240416A
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Abstract
The invention discloses a heparinizing method and application thereof. The method introduces the confinement effect of a nanopore path of a mesoporous material into a heparin immobilizing method, thereby realizing immobilization and controllable sustained release of heparin in the nanopore path. The heparinizing method is applied to artificial blood vessels. The heparinizing method realizes immobilization and sustained release of the heparin by using the nanopore path of a molecular sieve material, thereby regulating sustained release of the heparin, avoiding reduction of bioactivity caused by a covalent immobilization method and a terminal adhesion immobilization method, and overcoming the uncontrollability of release and generation of degradation products caused by a physisorption blending method and an ionic bond immobilization method. By the method for preparing a novel mesoporous molecular sieve covered artificial blood vessel through growth of the mesoporous material on the surface of an ePTFE (expanded polytetrafluoroethylene) artificial blood vessel, the biological compatibility, long-term antithrombotic property and endothelial cell compatibility of the novel artificial blood vessel can be greatly improved.
Description
Technical field
The present invention relates to a kind of heparin process for fixation and application, is a kind of based on the heparinization method of nano pore and the application in artificial blood vessel's especially diameter＜6mm artificial blood vessel specifically.
Background technology
Heparin is the general designation of cluster acid mucopolysaccharide chemical compound as a kind of important biochemical drug.It is alternately to connect the line style chain macromolecule (structural formula of face heparin as follows) with six sugar or eight sugared units that forms in certain proportion by hexuronic acid (L one iduronic acid, glucuronic acid) and glucosamine sulfate molecule, and molecular weight is between 3000 to 37500.Heparin has very strong anticoagulant property, and since nineteen thirty-seven was used as anticoagulant clinically, it was topmost anticoagulant always.Heparin passes through and Antithrombin III (antithrombin III, AT-III), hepatic binding protein (HBP) (heparin interactingprotein, HIP) and platelet factor (platelet factor, PF) the isoreactivity material has anticoagulant effect in conjunction with bringing into play its physiological function.The practice of many decades has confirmed that the heparinization anticoagulant material is the universal method that improves the biomaterial anticoagulant clinically.But, the storage of heparin and release are restricting the development of biomaterial: reconstructing blood vessel operative treatment angiopathy for example, just be hampered by the unobstructed difficult problem of small-caliber artificial blood vessel (less than 6mm), the new technique of being badly in need of long-term slow release heparin is to prevent forming thrombosis before artificial blood vessel's endothelial cell.Artificial blood vessel's material in clinical mainly contains terylene, expanded PTFE (ePTFE), polyurethane and medical high polymer degradation material or the like, wherein the ePTFE artificial blood vessel is a preferred material, yet therefore its self anticoagulant difference and limited clinical practice need carry out vascular grafts finishing and modification.Handling (surface negative charge, low temperature plasma, nano surface processing etc.) such as physical method, chemical method, biological method (protein modified, polyose modification etc.), in numerous methods such as artificial blood vessel's genetic modification, the material surface fixing heparin is the clearest and the most definite in present effect; But long-term unobstructed but being difficult to so far of small-caliber artificial blood vessel accomplishes.Therefore increase on the ePTFE material heparin supported quantity and prolong action time and become medical science at present, biology, materialogy, one of difficult point of important cross discipline research such as chemistry.
The heparin structure formula
The utilization ratio that the focus of current heparinization research is to improve heparin be material surface heparinization, avoid the heparin of costliness is buried/be wasted in material internal, because have only the surface of material just to touch blood.Chinese scholars is the main physical absorption blending method that adopts in the technical research of heparin solidified coating, the ionic bond fixation, and the covalent bond fixation, terminal point adheres to means such as fixation.Yet, to use between artificial blood vessel and the heparin after physical absorption blending method, ionic bond fixation are handled to lack stable combining, the controllability that heparin discharges is relatively poor; Covalent bond fixation and terminal point adhere to fixation but owing to too firm combination, limited the native conformation and the biological activity of heparin again, even can cause the loss of bulk material.Therefore, optimize the heparin technique for fixing and need new material badly: make heparin can stably be fixed, improve fixed amount by appropriateness, keep the fixedly natural activity of postheparin to make up a suitable microenvironment; This just provides an opportunity for molecular screen material especially mesoporous material.
Mesoporous material (mesoporous materials) is meant the porous material of aperture between 2～50nm, roughly is divided into " silica-based " and " non-silicon " mesoporous material by its chemical composition, and the former comprises simple silicon oxide again and is doped with two big class materials of other element.Because the transmutability of mesoporous material structure, hydrolysis and polyreaction accurately controlled, roof l network structure stable, surface modificability or the like characteristics, they are studied careful at present.Compare with traditional micro-pore zeolite material, pore passage structure (the aperture of Metaporous silicon dioxide material, duct shape and apparent condition), microscopic appearance and macroscopical body all have successive controllability, widened the application of molecular sieve at aspects such as sensing, nano-device, biomaterials effectively; Moreover they also possess the unexistent advantage of many other materials: physicochemical properties are stable, and certain moisture is arranged in the duct, for protein or enzyme provide a kind of " humidity " environment that their exist that is suitable for; " functionalized " can be realized by various processing in the surface of material.Above-mentioned advantage makes mesoporous material be used to the material of genetic engineering field as biosensor, the conveying of medicine, storage and release, the value preserving phase, drug effect, the minimizing side effect that improve medicine, and fixing protein enzyme.Biomacromolecule in the field of medicaments and its size of most of drug molecule are usually in nanometer range, for example the length of heparin can reach tens nanometers and width is about two nanometers, micro-pore zeolite is difficult to hold and ordered mesoporous material continuously adjustable aperture, no physiology toxicity in 2～50nm scope, special characteristics such as nanometer confinement effect become the effective carrier of medicament slow release: by aperture adjustment and finishing, mesoporous material is expected optionally to adsorb target molecule; Supported in the duct or the bio-pharmaceutical of fixedly embedding also can be improved drug effect by controllable sustained-release.Yet mesoporous material still is in the starting stage in the application of biological field, is applied to the heparin slow release and does not appear in the newspapers to improve the artificial material study on biocompatibility.
Summary of the invention
Goal of the invention:, the purpose of this invention is to provide and a kind ofly can improve the especially heparinization method of the biocompatibility of small-caliber artificial blood vessel of artificial blood vessel in order to overcome the deficiencies in the prior art.
Another object of the present invention is the application of above-mentioned heparinization method in the artificial blood vessel.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of heparinization method, the nano pore confinement effect of introducing mesoporous material is realized the immobilized and controllable sustained-release of heparin in nano pore in the heparin process for fixation.
Described heparin is fixed in the nano pore of mesoporous material and can slowly releases from nano pore.
The pore diameter range of described nano pore is 0.2-50nm, and it is mesoporous wherein to comprise 0.2-2nm micropore and 2-50nm.
Described mesoporous material mainly is mesoporous silicon based material but is not limited to mesoporous silicon based material.
The application of above-mentioned heparinization method in the artificial blood vessel mainly is:
At first mesoporous material is layed onto on the artificial blood vessel, obtains being coated with the artificial blood vessel of mesoporous material, realize the immobilized and controllable sustained-release of heparin in nano pore by the nano pore confinement effect of mesoporous material then.
For supported quantity and the permanently effective slow-release time that improves heparin, in the synthetic process of mesoporous material, need duct shape, pore size, the duct degree of order and the skeleton component modulation of control nano pore.The control of nano pore structure realizes by the various synthesis conditions that change in the preparation process, as changing the proportioning of template, acid source, silicon source, auxiliary agent and each component.Described template can be cetyl ammonium bromide (CTAB), triblock polymer (P123, i.e. EO
20PO
70EO
20) or other synthesising mesoporous employed template; Acid source can be that hydrochloric acid, citric acid or other are used for the employed acid source of synthesize meso-porous material; The silicon source can be that ethyl orthosilicate (TEOS), methyl silicate (TMOS) or other are used for the employed silicon of synthesize meso-porous material source; Auxiliary agent can be expanding agents such as trimethylbenzene (TMB), decane, inorganic additive and other employed additives in the mesoporous material building-up process such as sodium citrate, aluminum nitrate; The proportioning of each component is that the ratio of various synthesis materials can be regulated according to the required nano pore that obtains.
The small-caliber artificial blood vessel of artificial blood vessel described in the present invention, especially Φ＜6mm, in the artificial blood vessel, introduce molecular screen material with nano pore immobilized-the slow release heparin.In illustrating, the artificial blood vessel with nano pore is the ePTFE artificial blood vessel of mesopore molecular sieve that has been covered, but is not limited to this, also can be other artificial blood vessel with nano pore.
Lining-grown mesoporous molecular sieve on the artificial blood vessel surface, easier realization in the ePTFE artificial blood vessel: molecular sieve will be overlayed on rough stromal surface, growth gradually again; This breaks away from substrate with regard to requiring the slow hydrolysis-polymerization of synthetic silicon raw material with the molecular sieve particle accumulation phase-splitting that prevents to generate.The present invention prepares molecular sieve-composite artificial blood vessel by the faintly acid synthetic system, easier realization in the ePTFE artificial blood vessel.Specifically, the colloidal sol of the synthesize meso-porous material for preparing is in advance mixed mutually with the ePTFE artificial blood vessel, then the ePTFE artificial blood vessel is joined the mesoporous material synthetic system, after the synthetic step process of mesoporous material, obtain being coated with the artificial blood vessel of mesoporous material.
Among the present invention, the preparation of mesoporous material mainly comprises following two individual system.
One is the P123-CA system, mainly be to adopt P123 to make template, citric acid is made acid source, synthetic aperture is the mesoporous of 7-12nm, concrete preparation method is as follows: under the room temperature, P123 is dissolved in the water, add citric acid then, wherein the mol ratio of citric acid and P123 is 1-180, being stirred to fully, the dissolving back adds the silicon source, gained solution continues stirring reaction and stablizes meso-hole structure to generating, and advances the still ageing then, and cooling, filtration, washing, room temperature are dried the back removed template method and obtained the mesoporous material that the aperture is 7-12nm.The solution stirring reaction generates the preferred 12-48h of response time that stablizes meso-hole structure.
Another is CTAB-CA-CANa
3System mainly is to adopt CTAB to make template, and citric acid is made acid source, and it is the mesoporous of 2-3.4nm that sodium citrate is made the auxiliary agent synthetic aperture; Concrete preparation method is as follows: according to ethyl orthosilicate (TEOS), cetyl ammonium bromide (CTAB), citric acid (CA), sodium citrate (CANa
3) and water between mol ratio be 1: 0.2: 1: 0.5-2.5200-800 reacts; At first with CTAB, CA and CANa
3Be dissolved in the water, add TEOS after the stirring and dissolving and obtain solution mixture, then solution mixture is stablized meso-hole structure 20-50 ℃ of reaction to generating, filtration, washing, room temperature are dried the back removed template method and are obtained the mesoporous material that the aperture is 2-3.4nm; Wherein generate the response time of stablizing meso-hole structure and be preferably 24-72h by solution mixture reaction.
Among the present invention, the colloidal sol of mesoporous material and the mutually blended mode of ePTFE artificial blood vessel comprise direct mixing, ultrasonic auxiliary mixing and perfusion and mix, and the colloidal sol of mesoporous material is contacted the ePTFE artificial blood vessel continuously.
The alcohol extraction method is preferably adopted in the removal of template among the present invention.
Artificial blood vessel's biocompatibility described in the present invention comprises blood compatibility and histocompatibility.
Beneficial effect: compared with prior art, advantage of the present invention is to utilize the nano pore of mesoporous material to realize immobilized, slow release heparin, the slow release of modulation heparin, avoid covalent bond fixation and terminal point to adhere to the biological activity reduction that fixation causes, overcome the caused release uncontrollability of physical absorption blending method and ionic bond fixation with and catabolite generate, be particularly useful for the artificial blood vessel of diameter＜6mm; Thereby obtain the mesopore molecular sieve artificial blood vessel by mesoporous material in artificial blood vessel's superficial growth, can improve artificial blood vessel's biocompatibility, long-term antithrombotic and the endotheliocyte compatibility significantly.
Description of drawings
Fig. 1 is MVP-1, the curve of MVP-2 and MVP-3 mesopore molecular sieve composite artificial blood vessel slow release heparin;
Fig. 2 be endotheliocyte at mesoporous artificial blood vessel MVP-1, MVP-2 is hatched situation two days later on MVP-3 and the ePTFE artificial blood vessel.
The specific embodiment
Further specify the present invention below by embodiment.
Heparin absorption is by reaching adsorption equilibrium in the buffer that the composite artificial blood vessel material is immersed in heparin.Employed buffer is phosphoric acid buffer physiology salt (phosphate-buffered saline is called for short a PBS) solution, and the pH of this PBS solution is 7.2-7.6, contains 0.02M phosphate and 0.15M NaCl.The heparin that uses is the heparin sodium powder, also can be low molecular weight heparin.
The mensuration of heparin sodium concentration is to use the toluidine blue method of fading.
The following preparation of heparin sodium mother solution: 50mg heparin sodium powder dissolution is at 50ml phosphoric acid buffer physiology salt (phosphate-buffered saline is called for short PBS) solution, and the pH of this PBS solution is 7.2-7.6, contains 0.02M phosphate and 0.15M NaCl.
Mass concentration is 0.005% the following preparation of toluidine blue solution: 25mg o-toluidine blue, 2.5ml 2M HCl and 1g NaCl are dissolved in distilled water and are settled to 500ml.Concentration is respectively 0.5,1,2,4,6,8,10,12,14 μ g ml
-1The heparin sodium standard solution obtain by mother solution dilution.Use 10 test tubes in the mensuration, wherein one is reference.Add the 2.5mL toluidine blue in the every test tube, in nine test tubes, add the heparin sodium standard solution of 2.5ml variable concentrations then successively, in the reference test tube, add 2.5ml PBS solution.All test tubes are thermal agitation 1min all.Add 5ml normal hexane and thermal agitation 1.5min subsequently successively, this is in order to extract the complex except heparin sodium and toluidine blue effect gained in other 9 test tubes of reference test tube.The remaining aqueous solution of n-hexane extraction is surveyed absorbance with visible spectrophotometer in the every test tube under specific wavelength.The concentration mapping of gained absorbance and heparin sodium standard solution can be obtained the standard curve of absorbance-heparin sodium aqua concentration.
The method that the artificial blood vessel who is coated with mesoporous material is adsorbed heparin is: with 5cm
2The artificial blood vessel who is coated with mesoporous material puts into is equipped with the test tube that 5ml concentration is 50mg/ml heparin sodium PBS solution, in 4 ℃ of static adsorption 72h.Be put among the 2.5ml PBS in 37 ℃ of slow release after after the absorption sample being washed 3 times with 10ml PBS solution.At different time sustained-release liquid is taken out the concentration of measuring heparin sodium fully and add the fresh PBS solution of 2.5mL again.Obtain the cumulative release amount according to the burst size of measuring the different time heparin.
Under the room temperature, with 3g P123 (EO
20PO
70EO
20) be dissolved in the 112.5g water, add the citric acid (CA) of amount of calculation then, stir 3h to adding the silicon sources at 40 ℃ after the dissolving fully, continue down to stir 24h at 40 ℃ then, with laggard still ageing, cooling, filtration, washing, room temperature are dried and are obtained former powder sample.Get the former powder sample of 1g and place the Soxhlet extraction device to remove template, obtain the mesoporous molecular sieve SBA-15 sample with 150ml dehydrated alcohol extracting 24h.
According to ethyl orthosilicate (TEOS), cetyl ammonium bromide (CTAB), citric acid (CA), sodium citrate (CANa
3) and water between mol ratio be 1: 0.2: 1: 0.5-2.5: 200-800 reacts, and concrete steps are: at first take by weighing CTAB, CA and CANa
3Be dissolved in the water, after the 303K stirring and dissolving, add TEOS, solution mixture continues to stir 48h at 303K, and filtration, washing, room temperature are dried, and obtain white former powder.The former powder of 0.6g is placed 180ml ethanol, and backflow 24h removed template method obtains mesopore molecular sieve MCM-41 sample in cable-styled extraction tube.
The colloidal sol for preparing synthesize meso-porous material by above-mentioned steps earlier, the proportioning of different samples is as shown in table 1, add behind the silicon source to mix with the ePTFE artificial blood vessel and contact, and then proceed according to the method for synthesize meso-porous material among embodiment 1 or the embodiment 2, concrete reaction condition is as shown in table 1.The hydrothermal treatment consists after-filtration, washing.The gained sample dries and is placed in the 180ml ethanol, and backflow 48h removed template method in cable-styled extraction tube obtains containing the artificial blood vessel of mesopore molecular sieve, is labeled as MVP-n.Concrete reaction condition and the mesoporous artificial blood vessel's of gained pore passage structure data are as shown in table 1.The result shows that the method among the embodiment successfully is incorporated into nano pore among the ePTFE artificial blood vessel.
The pore passage structure data of table 1 composite artificial blood vessel series of samples
*Average pore size.
Measure biocompatibility by following means behind the mesopore molecular sieve composite artificial blood vessel absorption heparin:
Embodiment 4 partial thromboplastin times (APTT) and thrombin time (TT) are measured:
Blood picks up from healthy human body, adds citric acid and receives preparation blood plasma, and human blood in this blood plasma: the volume ratio that citric acid is received is 9: 1, and sample is put into sample cell, seals.Behind constant temperature 30min under 37 ℃ of water-baths, test, guarantee no blood seepage, the blood coagulation test is carried out on semi-automatic four-way Blood coagulation instrument, adopts turbidimetry to measure APTT and TT respectively, and specimen detects and repeats more than 5 examples, and each sample repeats 3 times and averages.
Measure the long-term anticoagulant index behind the immobilized heparin of mesoporous artificial blood vessel that gained among the embodiment 3 contains nano pore: the mesoporous artificial blood vessel of gained is placed on slow release heparin in the PBS solution, change PBS solution at set intervals, take out the value of measuring activated partial thromboplastin time (APTT) and thrombin time (TT) simultaneously, as shown in table 2.The result shows the immobilized and slow release heparin of introducing nano pore in the ePTFE artificial blood vessel, has improved long-term anticoagulant.
The mesoporous artificial blood vessel of table 2 anticoagulant index in the slow release process.
* the ePTFE artificial blood vessel is adsorbed behind the heparin sample in contrast.
The experiment of embodiment 5 hemolysis rates
Experimental technique: detected materials is cut into strip, packs in vitro, add mass concentration and be 0.9% NaCl aqueous solution 10ml; Positive control adopts distilled water, and feminine gender is 0.9% NaCl aqueous solution with mass concentration.Adopt the healthy human body anticoagulated blood, blood in this healthy human body anticoagulated blood: the volume ratio of sodium citrate aqueous solution is 4: 1, wherein the mass content of sodium citrate aqueous solution is 3.8%, all test tube is put into 37 ℃ of pre-temperature 30min of water-bath, respectively add the fresh anticoagulant human blood 0.2ml of dilution, this dilutes fresh anticoagulant human blood is by human blood: the volume ratio of normal saline be 4: 5 formulated, continue to be incubated 1h in 37 ℃ of water-baths, centrifugal 5min, rotating speed 2500r/min, get supernatant, measure at spectrophotometer 545nm place and respectively manage absorbance.Positive absorbance is 0.8 ± 0.3, and negative control pipe absorbance should be not more than 0.03.Hemolysis rate is calculated as follows:
Hemolysis rate=(sample absorbance-positive control absorbance)/(negative control absorbance-positive control absorbance) * 100%.
Measure the hemolysis rate behind the immobilized heparin of mesoporous artificial blood vessel that contains nano pore among the embodiment 3, MVP-1, MVP-2, MVP-3, MVP-4, MVP-5, MVP-6, MVP-7, MVP-8, the hemolysis rate of MVP-9 and MVP-10 sample is respectively: 1.2%, 1.5%, 2.6%, 3.6%, 4.6%, 3.8%, 3.7%, 4.3%, 4.5% and 2.3%.As reference, ePTFE artificial blood vessel's hemolysis rate is 4.7%.The result shows the immobilized and slow release heparin of introducing nano pore in the ePTFE artificial blood vessel, has improved blood compatibility.
The experiment of embodiment 6 water contact angles
Experimental technique: testing sample is cut into lamellar, uses contact angle tester CAM200 to measure water contact angle.Each sample is measured five times in zones of different, tries to achieve meansigma methods.
Measure the water contact angle that contains the mesoporous artificial blood vessel of nano pore among the embodiment 3, MVP-1, MVP-2, MVP-3, the water contact angle of MVP-9 and MVP-10 sample is respectively 72 °, and 88 °, 88 °, 106 ° and 109 °.As reference, ePTFE artificial blood vessel's water contact angle is 112 °.The result shows that introducing behind the nano pore PTFE artificial blood vessel goes up immobilized and slow release heparin, has improved artificial blood vessel's hydrophilic.
Embodiment 7
Measure the ability that the mesoporous artificial blood vessel that contains nano pore among the embodiment 3 is adsorbed the slow release heparin, as table 3 and shown in Figure 1.The result shows the immobilized and slow release heparin of introducing nano pore in the ePTFE artificial blood vessel, has improved the ability of the long-term slow release heparin of artificial blood vessel.
Table 3MVP-1, the ability of the mesoporous artificial blood vessel's absorption/slow release of MVP-2 and MVP-3 heparin.
aUnit is μ g cm
-2h
-1 bUnit is ng cm
-2h
-1
The experiment of embodiment 8 recalcification times
Experimental technique: extract the blood 5ml of fresh and healthy human body, add the anticoagulant of 0.13mol/L liquor sodii citratis with mass ratio 9: l, get whole blood 3ml, it is standby that the centrifugal 15min of 5000r/min takes out supernatant blood plasma.Detected materials is cut into little of 0.5cm * 0.5cm, puts into 0.9%NaCl solution and soak 24h, move to the test tube bottom then, add the 0.2ml supernatant, add 0.2ml 0.025mol/L CaCl again
2Solution, and pick up counting, jiggle test tube and make CaCl
2Solution and supernatant are evenly mixed, and experiment is carried out 37 ℃ of waters bath with thermostatic control.When the fine shape of white of appearance precipitates in the test tube, writing time.
Measure the recalcification time behind the immobilized heparin of mesoporous artificial blood vessel that contains nano pore among the embodiment 3, MVP-1, the recalcification time of MVP-2 and MVP-3 sample is respectively 12～24h, 8h and 4 minutes and 36 seconds.As reference, ePTFE artificial blood vessel's recalcification time is 112 seconds.The result shows the immobilized and slow release heparin of introducing nano pore in the ePTFE artificial blood vessel, has improved anticoagulant property.
Embodiment 9
Measure behind the immobilized heparin of mesoporous artificial blood vessel that contains nano pore among the embodiment 3 activity of surperficial heparin in PBS solution slow release process, as shown in table 4.The result shows the immobilized and slow release heparin of introducing nano pore in the ePTFE artificial blood vessel, and the artificial blood vessel surface has very high heparin activity, and can keep for a long time.
Mesoporous artificial blood vessel is at surperficial heparin activity of the difference moment in the table 4 slow release process.
The experiment of embodiment 10 thrombin inactivations
Experimental technique: blood vessel sample (0.5 * 0.5cm
2) be put among the PBS of 3ml, 37 ℃ of immersions (every 2-3 days sustained-release liquid is taken out fully, adds the fresh PBS of equivalent).Took out the blood vessel sample respectively at the 5th day with Ahau and carry out the thrombin experiment in immersion process, concrete experimental procedure is as follows.The blood vessel sample take out put into a HEPES buffer that contains 350 μ l (the HEPES buffer is: 25mmol/L HEPES, 190mmol/L NaCl, the 0.5mg/mL bovine serum albumin soaks 10min at 37 ℃ in EP pipe pH=7.5).Add 50ul Antithrombin III (concentration of Antithrombin III is 1IU/mL) then, soak 5min in 37 ℃ subsequently, add 100ul thrombin normal saline solution (thrombin is dissolved in 0.9% the normal saline, concentration 10IU/mL) subsequently again.Take out behind 37 ℃ of concussion 3min that (Tris-ethylenediaminetetraacetic acid buffer is: 50mmol/L Tris basal liquid in Tris-ethylenediaminetetraacetic acid buffer that 20ul solution joins 380ul, 175mmol/L NaCl, 7.5mmol/L ethylenediaminetetraacetic acid, pH=8.4).Adding 100ul concentration then is the thrombin luminous substrate (s2238) of 0.2mmol/L, 37 ℃ leave standstill 3min after, adding 100ul volume fraction is 30% glacial acetic acid solution, gets 200ul solution behind the mix homogeneously and is put on the microtitration orifice plate, is determined at the absorbance at 405nm place with microplate reader.Prepare the heparin solution of various concentration (0.005-0.02IU/mL), the solution 20ul that gets every kind of concentration measures according to the method described above, tries to achieve the standard curve of heparin.
Measure behind the immobilized heparin of mesoporous artificial blood vessel that contains nano pore among the embodiment 3 in PBS solution slow release process thrombin inactivation situation, as shown in table 5.The result shows in the ePTFE artificial blood vessel and to introduce the immobilized and slow release heparin of nano pore, has improved the deactivation of artificial blood vessel to thrombin, and after one period long period, can also keep stronger thrombin deactivation at slow release.
The mesoporous artificial blood vessel of table 5 joins in the thrombin in difference constantly in PBS solution slow release process, changes the amount (nmol) of residual thrombin in time.
Embodiment 11 endotheliocytes adhere to experiment
Extract lymphocyte from the blood of healthy human body, endotheliocyte is cultivated in differentiation.In the culture plate of one 6 lattice, every lattice add the 2ml cell suspension, and cell concentration is 5 * 10 in this cell suspension
6Cells mL
-1, then the artificial blood vessel is put into, with 37 ℃ under at 5%CO
2Hatch under the atmosphere after 2 days and fix with volume content 2.5% glutaraldehyde water solution, spending the night at 4 ℃. sample is ethanol/aqueous solution gradient of 50% → 70% → 80% → 90% → 100% each 15min that dewaters with volume content, detects blood vessel surface cell adhesion situation with scanning electron microscope behind the last vacuum drying.
Measure the growing state on endotheliocyte contains nano pore in embodiment 3 the mesoporous artificial blood vessel, as shown in Figure 2.The result shows in the ePTFE artificial blood vessel and to introduce the immobilized and slow release heparin of nano pore, has promoted artificial blood vessel surface endothelial cell growth, the ePTFE artificial blood vessel surface of the not containing nano pore endotheliocyte of then almost can not growing.
The experiment of embodiment 12 platelet adhesion reactions
In the silication system, (rotating speed: (Platelet-rich-plasma PRP), places clean silication test tube to get supernatant platelet rich plasma 2000rpm) at 20 ℃ of centrifugal 10min to gather healthy human body anticoagulated blood 10ml.Blood vessel sample to be measured (is put into normal saline after the washing of 1cm * 1cm) and soaked 24h, put into fresh PRP again, hatch 1h for 37 ℃, taking-up is washed with phosphate buffer, volume content is to be the ethanol water of 50% → 75% → 95% → 100% gradient each 10～15min that dewaters in succession with volume content behind the 12h fixedly in 2% the glutaraldehyde water solution, and volume content is isoamyl acetate-each 10～15min of alcoholic solution dehydration of 50% → 75% → 95% → 100% gradient; Take out sample, CO
2Gold-plated behind the critical point drying, on the scanning electron microscope instrument, observe platelet form and number at last.The positive control material of unmodified.Experimental result shows that all MVP-n blood vessel surface do not adhere to platelet, and there are a lot of platelet on the ePTFE artificial blood vessel surface of not containing nano pore, immobilized and the slow release heparin of nano pore is introduced in explanation in the ePTFE artificial blood vessel, improved the adherent ability of blood vessel antiplatelet.
Claims (10)
1. heparinization method is characterized in that: the nano pore confinement effect of introducing mesoporous material is realized the immobilized and controllable sustained-release of heparin in nano pore in the heparin process for fixation.
2. heparinization method according to claim 1 is characterized in that: described heparin is fixed in the nano pore of mesoporous material and can slowly releases from nano pore.
3. heparinization method according to claim 1 is characterized in that: the pore diameter range of described nano pore is 0.2-50nm, and it is mesoporous wherein to comprise 0.2-2nm micropore and 2-50nm.
4. the application of the described heparinization method of claim 1 in the artificial blood vessel.
5. application according to claim 4, it is characterized in that: at first mesoporous material is layed onto on the artificial blood vessel, obtain being coated with the artificial blood vessel of mesoporous material, realize the immobilized and controllable sustained-release of heparin in nano pore by the nano pore confinement effect of mesoporous material then.
6. application according to claim 5 is characterized in that: described mesoporous material is overlayed on the rough stromal surface of described artificial blood vessel.
7. according to claim 5 or 6 described application, it is characterized in that: the method that described mesoporous material is layed onto on the artificial blood vessel is: preparation mesoporous material colloidal sol in the mesoporous material synthetic system, then the mesoporous material colloidal sol for preparing is mixed mutually with the artificial blood vessel, again the artificial blood vessel is joined in the mesoporous material synthetic system, according to the artificial blood vessel who obtains being coated with mesoporous material after the synthetic step process of mesoporous material.
8. application according to claim 7 is characterized in that: described mesoporous material synthetic system comprises triblock polymer-citric acid system and cetyl ammonium bromide-citric acid-sodium citrate system.
9. application according to claim 8, it is characterized in that: described triblock polymer-citric acid system is that the employing triblock polymer is a template, it is the mesoporous of 7-12nm that citric acid is made the acid source synthetic aperture, concrete preparation method is as follows: under the room temperature, triblock polymer is dissolved in the water, add citric acid then, stirring fully, the dissolving back adds the silicon source, gained solution continues stirring reaction and stablizes meso-hole structure to generating, advance the still ageing then, cooling, filtration, washing, room temperature are dried and are obtained former powder sample; The template that removes in the former powder sample promptly obtains the mesoporous material that the aperture is 7-12nm; Wherein the mol ratio of citric acid and triblock polymer is 1-180.
10. application according to claim 8 is characterized in that: described cetyl ammonium bromide-citric acid-sodium citrate system is to adopt the cetyl ammonium bromide to make template, and citric acid is made acid source, and it is the mesoporous of 2-3.4nm that sodium citrate is made the auxiliary agent synthetic aperture; Concrete preparation method is as follows: according to the mol ratio between ethyl orthosilicate, cetyl ammonium bromide, citric acid, sodium citrate and the water is 1: 0.2: 1: 0.5-2.5: 200-800 takes by weighing; Then cetyl ammonium bromide, citric acid and sodium citrate are dissolved in the water, add ethyl orthosilicate after the stirring and dissolving and obtain solution mixture, again solution mixture is stablized meso-hole structure 20-50 ℃ of reaction to generating, filtration, washing, room temperature are dried, and obtain white former powder; The template that removes in the white former powder promptly obtains the mesoporous material that the aperture is 2-3.4nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110103732XA CN102240416A (en) | 2011-04-25 | 2011-04-25 | Heparinizing method and application thereof |
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| CN108992711A (en) * | 2018-07-06 | 2018-12-14 | 东华大学 | A kind of internal layer passes through the preparation method for the double-layer artificial small-caliber vascular modified |
| CN110859995A (en) * | 2019-11-14 | 2020-03-06 | 浙江大学 | Drug sustained-release coating based on double-layer heterogeneous structure and preparation method and application thereof |
| CN113694255A (en) * | 2021-09-02 | 2021-11-26 | 南昌大学附属口腔医院(江西省口腔医院) | Organic-inorganic antibacterial composite coating on surface of medical implant and preparation method thereof |
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| CN113694255A (en) * | 2021-09-02 | 2021-11-26 | 南昌大学附属口腔医院(江西省口腔医院) | Organic-inorganic antibacterial composite coating on surface of medical implant and preparation method thereof |
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