CN103830205B - A kind of solubilized thrombin nano-particle and its preparation method and application - Google Patents
A kind of solubilized thrombin nano-particle and its preparation method and application Download PDFInfo
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- CN103830205B CN103830205B CN201410072335.4A CN201410072335A CN103830205B CN 103830205 B CN103830205 B CN 103830205B CN 201410072335 A CN201410072335 A CN 201410072335A CN 103830205 B CN103830205 B CN 103830205B
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Abstract
The invention belongs to biomedicine field, disclose a kind of solubilized thrombin nano-particle and its preparation method and application.This solubilized thrombin nano-particle has nucleocapsid structure, and core is thrombin, and shell is by the cationic crosslinked nanoparticle formed of soluble linear starch-polylysine.This nucleocapsid structure is conducive to the biological activity keeping thrombin, at room temperature stable preservation, and have slow-release function, drug treating time is long, enters in body and is not easy to be degraded, economic security.The particle diameter of the preferred nanoparticle of the present invention is 150 ~ 250nm, can keep the biological activity of thrombin better, improves stability and the slow-release function of nano thrombin further.And in order to the stability that improves nano thrombin better and slow-release function, the envelop rate of nano thrombin is preferably 50 ~ 75%.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of solubilized thrombin nano-particle and its preparation method and application.
Background technology
Thrombin (thrombin) is the serine protein hydrolase that a species specificity is very strong, it can be hydrolyzed the peptide bond of fibrinogen, produce insoluble fibrin, make blood become gel and solidify, make blood rapid solidification, filling petechia and reach the object of hemostasis.Because its hemostasis is fast, the advantage such as have no side effect, and be widely used in the hemorrhage and oozing of blood at wound and the position such as clinical operation wound surface and gastrointestinal mucosal, haemostatic effect is good, determined curative effect.But common thrombin is a kind of protease reagent, easily causes biological activity to decline by the pollution factor impact of light, temperature, humidity, particularly microorganism and even lose.At normal temperatures, thrombin aqueous solution preservation after 24 days with regard to complete deactivation.Less stable, makes it apply and receives a definite limitation.
Utilize abundant degradable macromolecule nano material solidification parcel thrombin, prepare thrombin nano-particle, the impact on thrombin such as light, temperature, humidity can be greatly reduced, thus improve the stability of thrombin.Develop some bags at present and carry thrombin medicine, but the carrier that some bags carry thrombin need be dissolved in organic solvent to be prepared bag and carries a thrombin, some bags carry the carrier solution of thrombin in acid, some bags carry thrombin need at high temperature be prepared, and organic solvent, acid solution, high temperature all have considerable influence to the activity of thrombin, thus reduce the drug effect that obtained bag carries thrombin.
Therefore, the activity of a kind of thrombin to package-contained of current necessary exploitation without considerable influence, good stability, safe and efficient, scope of application thrombin novel form widely.
Summary of the invention
Carry the shortcoming that drug effect is low, dissolubility is low, toxicity is large of thrombin medicine with not enough to overcome bag in prior art, primary and foremost purpose of the present invention is the preparation method providing a kind of solubilized thrombin nano-particle;
Another object of the present invention is to the solubilized thrombin nano-particle providing above-mentioned preparation method to obtain;
Another object of the present invention is the application providing above-mentioned solubilized thrombin nano-particle.
Object of the present invention is achieved through the following technical solutions: a kind of preparation method of solubilized thrombin nano-particle, comprises following operating procedure:
(1) amylose-polylysine nano-particle is synthesized by click chemistry;
(2) amylose-polylysine nano-particle is joined in acetic acid aqueous solution, is stirred to and dissolves completely, form amylose-polylysine acetic acid aqueous solution;
(3) thrombin is added in the buffer of pH6.0 ~ 8.0, stirring and dissolving, forms thrombin buffer solution;
(4) thrombin buffer solution is joined in amylose-polylysine acetic acid aqueous solution, adding cross-linking agent to mass concentration is 0.01 ~ 0.10%, be warming up to 30 ~ 50 DEG C, stir 20 ~ 60 minutes with the speed of 100 ~ 800 revs/min, be poured into in liquid paraffin, stir, then to add dispersant to mass concentration be 1 ~ 10%, within 2 ~ 8 hours, carry out emulsifying with the speed high-speed stirred of 300 ~ 1500 revs/min, obtain emulsion;
(5) in step (4) gained emulsion, adding cross-linking agent to cross-linking agent mass concentration is 0.5 ~ 5.0%, stirs 20 ~ 60 minutes, makes it be uniformly dispersed, be placed in 30 ~ 50 DEG C of water-baths and carry out crosslinked 8 ~ 12 hours, product is cooled to room temperature, takes out, filter;
(6) filtrate washing with acetone, makes acetone surface without floating oil mark, obtains the translucent nanoparticle of gel; Again with washing with alcohol dehydration, obtain solid nano microsphere;
(7) by solid nano microsphere vacuum drying at 10 ~ 40 DEG C of temperature, solubilized thrombin nano-particle is obtained.
The described amylose of step (1)-polylysine nano-particle molecular weight is between 200-300kDa; Described amylose-polylysine nano-particle obtains according to following preparation process:
(1) by amidation process synthesis first generation dendroid polylysine: by 1.73g L-2,6-bis-tertbutyloxycarbonyl lysine and 5.5mmol propargylamine are dissolved in 20mL dry DMF altogether, at logical stirred under nitrogen 10min, are then cooled to 0 DEG C, add 2.18g BTA-N again, N, N', N'-tetramethylurea hexafluorophosphate and 0.74g1-hydroxy benzo triazole, continue at room temperature to stir 16h, after reaction terminates, add 200mL ethyl acetate, use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification (silica gel, chloroform-methanol=5:1), and then rotary evaporation can obtain the first generation dendroid polylysine of mass fraction 90%;
(2) synthesize second filial generation dendroid polylysine: at room temperature, take 0.96g first generation dendroid polylysine and be dissolved in 15mL trifluoroacetic acid-dichloromethane mixed solution, after reaction 2h, vacuum removes solvent; Add 30mL dry DMF, add 4mL triethylamine and 1.75gL-2 subsequently, 6-bis-tertbutyloxycarbonyl lysine, then add 2.48g BTA-N, N, N', N'-tetramethylurea hexafluorophosphate and 0.86g1-hydroxy benzo triazole, at room temperature stir 24h, after reaction terminates, add 250mL ethyl acetate, use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification, and then rotary evaporation can obtain the second filial generation dendroid polylysine of mass fraction 63%;
(3) third generation dendroid polylysine is synthesized: take 1.12g second filial generation dendroid polylysine, add in 20mL trifluoroacetic acid-dichloromethane mixed solution, L-2 is removed after at room temperature leaving standstill 2h, 6-bis-tertbutyloxycarbonyl, the solid of acquisition is added 40mL dry DMF, adds 6mL triethylamine and 2.08gL-2,6-bis-tertbutyloxycarbonyl lysine subsequently, at logical stirred under nitrogen 10min, be then cooled to 0 DEG C; After reaction terminates, dry by rotary evaporation, the raw product obtained is passed through column chromatography purification, and then rotary evaporation can obtain the third generation dendroid polylysine of mass fraction 60%;
(4) take 0.515g third generation dendroid polylysine, add in 15mL trifluoroacetic acid-dichloromethane mixed solution, after at room temperature leaving standstill 2h, remove residual L-2,6-bis-tertbutyloxycarbonyl; After being evaporated by solvent, add 0.05g amylose and 10mL dimethyl sulfoxide dissolves; Under logical nitrogen, add 18mgCuSO
45H
2o and 65mg sodium ascorbate, and mixed solution is heated to 40 DEG C of constant temperature 48h, after reaction terminates, dialyse product in distilled water 3d, can obtain the amylose-polylysine nano-particle of mass fraction 65% after lyophilizing.
In the described trifluoroacetic acid/dichloromethane mixed solution in step (2) ~ (4), the volume ratio of trifluoroacetic acid and dichloromethane is 1:1.
The mass concentration of step (2) described acetic acid aqueous solution is 0.5 ~ 2.5%; In described amylose-polylysine acetic acid aqueous solution, the mass concentration of amylose-polylysine is 0.5 ~ 8%.
Step (3) described thrombin is human thrombin, pig thrombiase, thrombin of beef or sheep thrombin; In thrombin in step (3) described thrombin buffer solution and step (2) described amylose-polylysine acetic acid aqueous solution, the mass ratio of amylose-polylysine is 4:0.5 ~ 2.
Step (4) is described thrombin buffer solution is joined in amylose-polylysine acetic acid aqueous solution while to stir with the speed of 100 ~ 800 revs/min.
Step (4) and (5) described cross-linking agent are Polyethylene Glycol, sodium alginate or glutaraldehyde; Step (5) described dispersant is sorbester p17, Tween 80 or poloxamer.
The number of times of step (4) described washing with acetone is 2 ~ 5 times.Step (6) described washing with alcohol to be ethanol, the percent by volume being 70% with percent by volume be 80% ethanol, percent by volume be 90% ethanol and dehydrated alcohol dewater successively.
The solubilized thrombin nano-particle prepared by preparation method according to claim 1, the particle diameter of this solubilized thrombin nano-particle is 200 ~ 350nm, and its envelop rate is 50 ~ 75%.This solubilized thrombin nano-particle has nucleocapsid structure, and core is thrombin, and shell is by the cationic crosslinked nanoparticle formed of soluble linear starch-polylysine.This nucleocapsid structure is conducive to the biological activity keeping thrombin, at room temperature stable preservation, and have slow-release function, drug treating time is long, enters in body and is not easy to be degraded, economic security.The particle diameter of the preferred nanoparticle of the present invention is 150 ~ 250nm, can keep the biological activity of thrombin better, improves stability and the slow-release function of nano thrombin further.And in order to the stability that improves nano thrombin better and slow-release function, the envelop rate of nano thrombin is preferably 50 ~ 75%.
Above-mentioned solubilized thrombin nano-particle can be used as the effect of haemostatic medicament or hemostasis spray.
The present invention has following advantage and beneficial effect relative to prior art:
Solubilized thrombin nano-particle of the present invention, owing to adopting nucleocapsid structure, core is thrombin, shell is the nanoparticle of the cationic crosslinked formation of amylose-polylysine, be loaded in the cationic crosslinked nanoparticle formed by amylose-polylysine by thrombin bag, the cation of soluble linear starch-polylysine is reacted by intermolecular or intramolecular crosslinking and formed nanoparticle, it is a kind of electrostatic interaction, do not relate to the high temperature in thermo-crosslinking process and the formation of the covalent bond in chemical crosslinking process, to the activity of the thrombin of package-contained without considerable influence, amylose-polylysine is water miscible, and is avirulence, safe and efficient, good stability, to the activity of the thrombin of package-contained also without considerable influence.Therefore, nano thrombin provided by the invention maintains the biological activity of thrombin.
Solubilized thrombin nano-particle of the present invention, because thrombin is loaded in the nanoparticle of the cationic crosslinked one-tenth of soluble linear starch-polylysine by bag, therefore at room temperature can stablize preservation; The expression activitiy of thrombin is stablized; There is slow-release function, be suitable for intravenous injection; Drug treating time is long, and bioavailability is high; Enter in body and be not easy to be degraded, reduce dosage; Amylose-polylysine can degradation in vivo be aminoacid and glucose, avirulence and bio-compatibility is good, makes nano thrombin safety provided by the invention; By preparing targeted drug preparation to nanoparticle surface modified, nano thrombin provided by the invention has good drug development potentiality, has higher medical value and wide application prospect.
Accompanying drawing explanation
Fig. 1 is amylose-polylysine nano-particle molecular structure.
Fig. 2 is the infrared spectrum analysis chart of amylose-polylysine nano-particle.
Fig. 3 is the scanning electron microscope (SEM) photograph of solubilized thrombin nano-particle of the present invention.
Fig. 4 is the activity change curve chart of solubilized thrombin nano-particle of the present invention.
Fig. 5 is the retention rate of each time point thrombin activity at 25 DEG C.
Fig. 6 is the retention rate of each time point thrombin activity at 0 DEG C.
Fig. 7 is the retention rate of each time point thrombin activity at-20 DEG C.
Fig. 8 is nano thrombin reaction result in fibrinogen solution, and wherein a is nano thrombin solution, and b is that Fibrinogen coagulum is formed, and c is Fibrinogen coagulum deposition.
Fig. 9 is the coagulating effectiveness of variable concentrations nano thrombin to blood, and wherein a is the thrombin nano-particle of variable concentrations gradient, and b is for adding 2ml fresh blood, and c is the Blood Coagulation Process of the nano thrombin of variable concentrations gradient, and d is blood coagulation effect.
Figure 10 is the haemostatic effect of nano thrombin to hepatorrhagia, and wherein a is normal rat liver anatomical, and b is rat liver Hemorrhage Model, and c is the haemostatic effect of nano thrombin to liver Hemorrhage Model.
Figure 11 is the anastalsis of nano thrombin to angiorrbagia, and wherein a is the dissection of Rat Right iliac artery, and b is the Hemorrhage Model of Rat Right iliac artery, and c is the haemostatic effect of the Hemorrhage Model of Rat Right iliac artery.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
The preparation of amylose-polylysine nano-particle:
(1) by amidation process synthesis first generation dendroid polylysine: 1.73g L-2, 6-bis-tertbutyloxycarbonyl lysine (5mmol) and 350 μ L propargylamines (5.5mmol) are dissolved in the anhydrous N of 20mL altogether, in N-dimethylformamide (DMF) solution, at logical stirred under nitrogen 10min, then 0 DEG C is cooled to, 2.18g(5.5mmol is added in solution) BTA-N, N, N', N'-tetramethylurea hexafluorophosphate (HBTU) and 0.74g(5.5mmol) 1-hydroxy benzo triazole (HOBT), continue at room temperature to stir 16h, after reaction terminates, add 200mL ethyl acetate (EtOAc), use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification (silica gel, chloroform-methanol=5:1), and then rotary evaporation can obtain the first generation dendroid polylysine of 90%.
(2) second filial generation dendroid polylysine is synthesized: first use trifluoroacetic acid (TFA) to cut first generation dendroid polylysine; At room temperature, taking 0.96g(2.5mmol) first generation dendroid polylysine is dissolved in 15mL trifluoroacetic acid-dichloromethane (v/v=1:1) mixed solution, after reaction 2h, vacuum removes solvent, add 30mL dry DMF, add 4mL triethylamine and 1.75g(5.1mmol subsequently) L-2,6-bis-tertbutyloxycarbonyl lysine, then, add 2.48g(6.2mmol) HBTU and 0.86g(6.2mmol) HOBt, at room temperature stirs 24h, after reaction terminates, add 250mL ethyl acetate, use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification (silica gel, chloroform-methanol=10:1), and then rotary evaporation can obtain the second filial generation dendroid polylysine of mass fraction 63%.
(3) third generation dendroid polylysine is synthesized: take 1.12g(1.30mmol) second filial generation dendroid polylysine, add 20mL trifluoroacetic acid-dichloromethane (v/v=1:1), L-2 is removed after at room temperature leaving standstill 2h, 6-bis-tertbutyloxycarbonyl, the solid of acquisition is added 40mL dry DMF, adds 6mL triethylamine and 2.08g(5.5mmol subsequently) L-2,6-bis-tertbutyloxycarbonyl lysine, at logical stirred under nitrogen 10min, be then cooled to 0 DEG C; After reaction terminates, dry by rotary evaporation afterwards, the raw product obtained is passed through column chromatography purification (silica gel, chloroform-methanol=15:1), and then rotary evaporation can obtain the third generation dendroid polylysine of mass fraction 60%;
(4) solubilized thrombin nano-particle is prepared: by 0.515g(0.29mmol) third generation dendroid polylysine, add 15mL trifluoroacetic acid-dichloromethane (v/v=1:1), after at room temperature leaving standstill 2h, remove residual L-2,6-bis-tertbutyloxycarbonyl; After being evaporated by solvent, add 0.05g amylose (Amy-N
3, buy in sigma company of the U.S.) and 10mL dimethyl sulfoxide, limpid solution can be obtained after dissolving; Then, under logical nitrogen, 18mg CuSO is taken
45H
2o and 65mg sodium ascorbate adds solution, and mixed solution is heated to 40 DEG C of constant temperature 48h, after reaction terminates, dialyse product in distilled water 3d(MWCO=14000), can obtain tree solubilized thrombin nano-particle (Amy-g-PLLD) of mass fraction 65% after lyophilizing, its building-up process as shown in Figure 1.According to infrared spectrum analysis, polylysine connects amylose, as shown in Figure 2.
Embodiment 2
Example 1 gained amylose-polylysine nano-particle 20mg, inserts in three-neck flask, adds the acetic acid aqueous solution 500ml of mass percent 2%, stirs and makes it dissolve completely; Separately take thrombin (being purchased from sigma company of the U.S.) 3000u, add phosphate buffer (pH7.4) 100ml, stirring and dissolving, form thrombin buffer solution; At room temperature stir with the speed of 100 ~ 800 revs/min, thrombin buffer solution is added drop-wise in above-mentioned amylose-polylysine acetic acid aqueous solution, time for adding is 30min, is warming up to 40 DEG C, drips mass fraction 0.1% glutaraldehyde solution 30ml, with the speed continuous stirring 30min of 200 revs/min, stop stirring, be poured into in 500ml liquid paraffin, stir 25min, add the Tween 80 of 50ml, within 4 hours, carry out emulsifying with the speed high-speed stirred of 500 revs/min; Get the glutaraldehyde solution 100ml that emulsion adds mass fraction 25% again, stirring 30min makes it be uniformly dispersed, be placed in 40 DEG C of water-baths and carry out crosslinked self assembly 8h, product is cooled to room temperature, take out, filter, obtain product washing with acetone 3 times, each 300ml, obtains the translucent nanoparticle of gel; After more successively with volume fraction 70% ethanol, volume fraction 80% ethanol, volume fraction 90% ethanol and each 200ml washing dehydration of anhydrous alcohol, obtain solid nano microsphere; Obtained solid nano microsphere is placed in vacuum desiccator 30 DEG C of vacuum dryings, obtains solubilized thrombin nano-particle, the envelop rate measuring obtained solubilized thrombin nano-particle is 75.81%, and mean diameter is 250nm.Observe under obtained solubilized thrombin nano-particle is carried out scanning electron microscope, electromicroscopic photograph as shown in Figure 3.
Embodiment 3
Example 1 gained amylose-polylysine nano-particle 40mg, inserts in three-neck flask, adds the acetic acid aqueous solution 1500ml of mass fraction 2%, stirs and makes it dissolve completely; Separately take thrombin (being purchased from sigma company of the U.S.) 6000u, add phosphate buffer (pH7.4) 150ml, stirring and dissolving, form thrombin buffer solution; At room temperature stir with the speed of 100 ~ 800 revs/min, thrombin buffer solution is added drop-wise in above-mentioned amylose-polylysine acetic acid aqueous solution, time for adding is 40min, is warming up to 40 DEG C, drips mass fraction 0.1% glutaraldehyde solution 40ml, with the speed continuous stirring 40min of 300 revs/min, stop stirring, be poured into in 500ml liquid paraffin, stir 35min, add the Tween 80 of 100ml, within 4 hours, carry out emulsifying with the speed high-speed stirred of 500 revs/min; Get the glutaraldehyde solution 150ml that emulsion adds mass fraction 25% again, stirring 40min makes it be uniformly dispersed, be placed in 40 DEG C of water-baths and carry out crosslinked self assembly 10h, product is cooled to room temperature, take out, filter, obtain product washing with acetone 3 times, each 300ml, obtains the translucent nanoparticle of gel; After more successively with the ethanol of the ethanol of the ethanol of volume fraction 70%, volume fraction 80%, volume fraction 90% and each 200ml washing dehydration of anhydrous alcohol, obtain solid nano microsphere; Obtained solid nano microsphere is placed in vacuum desiccator 30 DEG C of vacuum dryings, obtains solubilized thrombin nano-particle, the envelop rate measuring obtained solubilized thrombin nano-particle is 65.26%, and mean diameter is 220nm.
Embodiment 4
Example 1 gained amylose-polylysine nano-particle 30mg, inserts in three-neck flask, adds the acetic acid aqueous solution 1000ml of mass fraction 2%, stirs and makes it dissolve completely; Separately take thrombin (being purchased from sigma company of the U.S.) 5000u, add phosphate buffer (pH7.4) 150ml, stirring and dissolving, form thrombin buffer solution; At room temperature stir with the speed of 100 ~ 800 revs/min, thrombin buffer solution is added drop-wise in above-mentioned amylose-polylysine acetic acid aqueous solution, time for adding is 40min, is warming up to 50 DEG C, drips mass fraction 0.5% sodium alginate 30ml, with the speed continuous stirring 30min of 250 revs/min, stop stirring, be poured into in 500ml liquid paraffin, stir 25min, add the poloxamer of 150g, within 3 hours, carry out emulsifying with the speed high-speed stirred of 800 revs/min; Get the extra large bath acid sodium solution 250ml that emulsion adds mass fraction 15% again; stirring 50min makes it be uniformly dispersed; be placed in 50 DEG C of water-baths and carry out crosslinked self assembly 12h; product is cooled to room temperature; one takes out, and filters, obtains product washing with acetone 3 times; each 400ml, obtains the translucent nanoparticle of gel; After more successively with the ethanol of the ethanol of the ethanol of volume fraction 70%, volume fraction 80%, volume fraction 90% and each 250ml washing dehydration of anhydrous alcohol, obtain solid nano microsphere; Obtained solid nano microsphere is placed in vacuum desiccator 30 DEG C of vacuum dryings, obtains solubilized thrombin nano-particle, the envelop rate measuring obtained solubilized thrombin nano-particle is 55.12%, and mean diameter is 200nm.
Nano thrombin activity stability is analyzed:
(1) the solubilized thrombin nano-particle deionized water dissolving will prepared in embodiment 2-4 respectively, with the densitometer of thrombin, be mixed with the solubilized thrombin nano-particle aqueous solution of 50U/mL respectively, prepare the identical free thrombin aqueous solution of concentration of thrombin simultaneously, all preserve under the preservation condition of 110rpm/37 DEG C, the ability of platelet aggregation in remaining enzyme induction PRP is measured in different time points, (be the solubilized thrombin nano-particle of embodiment 1 and the activity change curve of free thrombin in figure as shown in Figure 4, the activity change curve chart of solubilized thrombin nano-particle of embodiment 3 and 4 and the activity change Similar Broken Line of embodiment 1), the nanoparticle activated change of solubilized thrombin is similar to drug release patterns, burst drug release is there is about when 5h, about when 72h, enzymatic activity reaches maximum, start slow decline afterwards, free enzyme is then the process of a decay of activity.
As can be seen from above-described embodiment, solubilized thrombin nano-particle provided by the invention, maintains the biological activity of thrombin, and activity stabilized, have slow-release function, drug treating time is long.
(2) temperature stability experiment:
Respectively the solubilized thrombin nano-particle prepared in embodiment 2-4 and common thrombin are placed in suitable clean culture dish to seal, place at 25 DEG C, 0 DEG C and-20 DEG C respectively, respectively at 0 day, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days and sampling in 60 days, detect thrombin activity with reference to thrombin activity assay method in Chinese Pharmacopoeia.As shown in Fig. 5, Fig. 6 and Fig. 7, under 25 DEG C, 0 DEG C and-20 DEG C of conditions, the activity of two groups of materials all presents and extends downward trend gradually in time; Wherein under 25 DEG C of conditions, common thrombin activity declines the most remarkable, and 60 days time, activity preservation rate drops to 40%, and nano thrombin activity activity preservation rate 60 days time drops to 60%; Under 0 DEG C and-20 DEG C of conditions, nano thrombin activity decrease all declines slowly than common thrombin activity, and two groups are compared P<0.05, and the activity preservation rate of P nano thrombin group in 60 days is higher than common thrombin group.
(3) ph stability experiment:
Respectively the solubilized thrombin nano-particle prepared in embodiment 2-4 and common thrombin are placed in suitable clean culture dish, evenly drip with the phosphate buffer of different PH and infiltrate and seal, the material pH value under wet condition is made to be in 6.5-6.8 respectively, 7.0-7.4, and in 7.6-8.0 scope, in sampling in the 3rd and the 5th day, detect with reference to above-mentioned thrombin activity assay method.As during preservation material desiccation, then measure pH value after first infiltrating with deionized water, afterwards according to result again with phosphate buffer adjust ph to former set point.Shown in result table 1 and table 2: the activity preservation rate of two groups of materials all has reduction; Within the scope of the pH value 7.0-7.4 close to neutrality, two groups of materials, 5 days activity preservation rate are respectively the common thrombin group of 74.8%() and 84.9%(nano thrombin group); At same time point, under weak acid (pH value 6.5-6.8) and weak base (pH value 7.6-8.0) environment, the activity preservation rate of two groups of materials is all lower than neutral environment (pH value 7.0-7.4), larger on the impact of material activity under weak acid environment, 5 days activity preservation rate, two groups of materials are all lower than 70%; Nano thrombin group material activity retention rate under the same terms is higher than common thrombin group.
(4) light durability experiment:
Respectively the solubilized thrombin nano-particle prepared in embodiment 2-4 and common thrombin are placed in suitable clean culture dish, seal in suitable clean culture dish, be placed in YB-II type clarity detecting apparatus, be place 10 days under the condition of 4500lx ± 500lx in illumination, in sampling in the 5th and the 10th day, detect with reference to above-mentioned thrombin activity assay method.Result is as shown in table 3: the activity preservation rate of two groups of materials under illumination condition all has reduction, and the activity preservation rate of 5 days common thrombins is respectively 72.2%, and the activity preservation rate of nano thrombin is respectively 80.6%; The activity preservation rate of 10 days common thrombins is respectively 67.5%, and the activity preservation rate of nano thrombin is respectively 74.9%; Nano thrombin group material activity retention rate under the same terms is higher than common thrombin group.
The common thrombin of table 1 is to ph stability
Table 2 thrombin nano-particle is to ph stability
Table 3 thrombin nano-particle is to the stability of illumination
Nano thrombin haemostatic effect is analyzed:
(1) test in laboratory method:
Get 1.5 × 15cm test tube, add the Fibrinogen normal saline solution 5ml of mass concentration 3%, the nano-particle material got about containing 5U thrombin is placed in test tube, observes and forms coagulative situation.There is coagulum to be formed in 5min, think and effectively stop blooding.As shown in Figure 8, nano thrombin is placed in fibrinogen solution, visible significantly fibrin coagulum deposition during 5min.Fig. 9 be nano thrombin variable concentrations gradient to the haemostatic effect of blood, show in figure, along with nano thrombin concentration raises gradually, haemostatic effect strengthens gradually.
(2) zoopery assessment haemostatic effect:
1. the hemostasis experiment of rats'liver angiorrbagia model:
Healthy male SD rat, body weight 300--350g, the room temperature 23-25 DEG C cage that places an order is raised, ad lib, water inlet, 12 h light/dark.Rat and standard rat chow provide by Zhongshan University's Experimental Animal Center.Rat is with 10% chloral hydrate (0.3ml/100g) intraperitoneal injection anesthesia, and dorsal position is fixed, and sterilization, gets abdominal part median incision and be about 5cm, successively cut off skin, muscle and peritoneum, enter abdomen.Expose liver.The square border of getting 1 × 1cm is drawn with sharp knife, deeply about 0.5cm in its center after fully being exposed by liver lobus dexter.With tissue shear, hepatic tissue in border is removed, and strike off wound surface with tissue clamps, make liver wound surface occur obvious oozing of blood.By preprepared double-layer folding hemostatic material (3 × 3cm, 5U/cm
2) be placed in wound surface, fully cover and attach hepatic tissue, with dry cotton ball suitably compressing also timing above outer material side wound surface, the complete dwell time of record oozing of blood.Matched group adopts gelfoam and common dry yarn ball is capable operates equally.Often kind of material parallel measures 10 times, assessment haemostatic effect.As shown in Figure 10, nano thrombin has good anastalsis to liver oozing of blood model.
2. rat iliac artery Hemorrhage Model hemostasis experiment:
With preceding method anesthesia, enter abdomen, rat intestinal tube is pushed to abdominal cavity, right side, with saline cotton swab blunt separation ventral aorta front posterior peritoneum and periarterial fatty tissue, appear ventral aorta.Common iliac artery on the right side of the downward blunt separation in ventral aorta lower end, free about 1cm.Silk thread labelling is far away, near-end prepare ligation.Mention free section, will be prepared in advance nano thrombin.With the mid point of the 5ml syringe needle puncture right common iliac artery section of dissociating, point of puncture is covered with preset subject material immediately after the ejection of arterial blood pulsation, ensure that material is well attached at common iliac artery outer wall, remove subject material after 5min and record without active hemorrhage.As shown in figure 11, nano thrombin has good anastalsis to angiorrhea model.
Solubilized thrombin nano-particle provided by the invention, maintains the biological activity of thrombin; At room temperature stable preservation; Activity stabilized; There is slow-release function, be suitable for intravenous injection; Drug treating time is long, and bioavailability is high; Enter in body and be not easy to be degraded, reduce dosage; Amylose-polylysine material non-toxic, bio-compatibility are good, make solubilized thrombin nano-particle economic security provided by the invention; By preparing targeted drug preparation to nanoparticle surface modified, nano thrombin provided by the invention has good drug development potentiality, surgical field can be widely used in, tissue as general surgery, orthopaedics, cardiothoracic surgery, neurosurgery, department of obstetrics and gynecology etc. stops blooding, tissue closure and tissue adhesion, has broad application prospects.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a preparation method for solubilized thrombin nano-particle, is characterized in that comprising following operating procedure:
(1) amylose-polylysine nano-particle is synthesized by click chemistry;
(2) amylose-polylysine nano-particle is joined in acetic acid aqueous solution, is stirred to and dissolves completely, form amylose-polylysine acetic acid aqueous solution;
(3) thrombin is added in the buffer of pH6.0 ~ 8.0, stirring and dissolving, forms thrombin buffer solution;
(4) thrombin buffer solution is joined in amylose-polylysine acetic acid aqueous solution, adding cross-linking agent to mass concentration is 0.01 ~ 0.10%, be warming up to 30 ~ 50 DEG C, stir 20 ~ 60 minutes with the speed of 100 ~ 800 revs/min, be poured into in liquid paraffin, stir, then to add dispersant to mass concentration be 1 ~ 10%, within 2 ~ 8 hours, carry out emulsifying with the speed high-speed stirred of 300 ~ 1500 revs/min, obtain emulsion;
(5) in step (4) gained emulsion, adding cross-linking agent to cross-linking agent mass concentration is 0.5 ~ 5.0%, stirs 20 ~ 60 minutes, makes it be uniformly dispersed, be placed in 30 ~ 50 DEG C of water-baths and carry out crosslinked 8 ~ 12 hours, product is cooled to room temperature, takes out, filter;
(6) filtrate washing with acetone, makes acetone surface without floating oil mark, obtains the translucent nanoparticle of gel; Again with washing with alcohol dehydration, obtain solid nano microsphere;
(7) by solid nano microsphere vacuum drying at 10 ~ 40 DEG C of temperature, solubilized thrombin nano-particle is obtained.
2. preparation method according to claim 1, is characterized in that: the described amylose of step (1)-polylysine nano-particle molecular weight is between 200-300kDa; Described amylose-polylysine nano-particle obtains according to following preparation process:
(1) by amidation process synthesis first generation dendroid polylysine: by 1.73g L-2,6-bis-tertbutyloxycarbonyl lysine and 5.5mmol propargylamine are dissolved in 20mL dry DMF altogether, at logical stirred under nitrogen 10min, are then cooled to 0 DEG C, add 2.18g BTA-N again, N, N', N'-tetramethylurea hexafluorophosphate and 0.74g1-hydroxy benzo triazole, continue at room temperature to stir 16h, after reaction terminates, add 200mL ethyl acetate, use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification, and then rotary evaporation can obtain the first generation dendroid polylysine of mass fraction 90%;
(2) synthesize second filial generation dendroid polylysine: at room temperature, take 0.96g first generation dendroid polylysine and be dissolved in 15mL trifluoroacetic acid-dichloromethane mixed solution, after reaction 2h, vacuum removes solvent; Add 30mL dry DMF, add 4mL triethylamine and 1.75gL-2 subsequently, 6-bis-tertbutyloxycarbonyl lysine, then add 2.48g BTA-N, N, N', N'-tetramethylurea hexafluorophosphate and 0.86g1-hydroxy benzo triazole, at room temperature stir 24h, after reaction terminates, add 250mL ethyl acetate, use saturated NaHCO
3the NaHSO of solution, 0.1mol/L
4, saturated NaHCO
3solution and salt rinse successively, dry by rotary evaporation afterwards, and the raw product obtained is passed through column chromatography purification, and then rotary evaporation can obtain the second filial generation dendroid polylysine of mass fraction 63%;
(3) third generation dendroid polylysine is synthesized: take 1.12g second filial generation dendroid polylysine, add in 20mL trifluoroacetic acid-dichloromethane mixed solution, L-2 is removed after at room temperature leaving standstill 2h, 6-bis-tertbutyloxycarbonyl, the solid of acquisition is added 40mL dry DMF, adds 6mL triethylamine and 2.08gL-2,6-bis-tertbutyloxycarbonyl lysine subsequently, at logical stirred under nitrogen 10min, be then cooled to 0 DEG C; After reaction terminates, dry by rotary evaporation, the raw product obtained is passed through column chromatography purification, and then rotary evaporation can obtain the third generation dendroid polylysine of mass fraction 60%;
(4) take 0.515g third generation dendroid polylysine, add in 15mL trifluoroacetic acid-dichloromethane mixed solution, after at room temperature leaving standstill 2h, remove residual L-2,6-bis-tertbutyloxycarbonyl; After being evaporated by solvent, add 0.05g amylose and 10mL dimethyl sulfoxide dissolves; Under logical nitrogen, add 18mgCuSO
45H
2o and 65mg sodium ascorbate, and mixed solution is heated to 40 DEG C of constant temperature 48h, after reaction terminates, dialyse product in distilled water 3d, can obtain the amylose-polylysine nano-particle of mass fraction 65% after lyophilizing.
3. preparation method according to claim 2, is characterized in that: in the described trifluoroacetic acid-dichloromethane mixed solution of step (2) ~ (4), the volume ratio of trifluoroacetic acid and dichloromethane is 1:1.
4. preparation method according to claim 1, is characterized in that: the mass concentration of step (2) described acetic acid aqueous solution is 0.5 ~ 2.5%; In described amylose-polylysine acetic acid aqueous solution, the mass concentration of amylose-polylysine is 0.5 ~ 8%.
5. preparation method according to claim 1, is characterized in that: step (3) described thrombin is human thrombin, pig thrombiase, thrombin of beef or sheep thrombin; In thrombin in step (3) described thrombin buffer solution and step (2) described amylose-polylysine acetic acid aqueous solution, the mass ratio of amylose-polylysine is 4:0.5 ~ 2.
6. preparation method according to claim 1, is characterized in that: step (4) is described thrombin buffer solution is joined in amylose-polylysine acetic acid aqueous solution while to stir with the speed of 100 ~ 800 revs/min.
7. preparation method according to claim 1, is characterized in that: step (4) and (5) described cross-linking agent are Polyethylene Glycol, sodium alginate or glutaraldehyde; Step (4) described dispersant is sorbester p17, Tween 80 or poloxamer.
8. preparation method according to claim 1, is characterized in that: the number of times of step (6) described washing with acetone is 2 ~ 5 times; Step (6) described washing with alcohol to be ethanol, the percent by volume being 70% with percent by volume be 80% ethanol, percent by volume be 90% ethanol and dehydrated alcohol dewater successively.
9. a solubilized thrombin nano-particle prepared by the method according to any one of claim 1 ~ 8, is characterized in that: the particle diameter of this solubilized thrombin nano-particle is 200 ~ 350nm, and its envelop rate is 50 ~ 75%.
10. solubilized thrombin nano-particle according to claim 9 is preparing the application in haemostatic medicament or hemostasis spray.
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| CN102614128A (en) * | 2012-03-15 | 2012-08-01 | 江苏晔铭生物医药科技有限公司 | Thrombin inhibitor SSR182289A magnetic nanometer microsphere and preparation method thereof |
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| CN102614128A (en) * | 2012-03-15 | 2012-08-01 | 江苏晔铭生物医药科技有限公司 | Thrombin inhibitor SSR182289A magnetic nanometer microsphere and preparation method thereof |
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