Shitosan hemostatic material that covalent cross-linking is formed and preparation method thereof
Technical field
Shitosan hemostatic material formed the present invention relates to covalent cross-linking and preparation method thereof.
Background technology
The bleeding of human body can occur in various situations, such as, and surgical operation, arteriopuncture(Arterial intervention diagnose and
Interventional therapy), size wound in war wound and daily life.Substantial amounts of the losing blood of human body can cause serious consequence, so that prestige
Side of body life.It is the second reason of civilian deaths in daily life that wound non-controlling is lost blood, and is first cause of the death on battlefield.Cause
This, is always domestic and international focus of attention for the research and development of hemostatic material.
So far, in the treatment of surgery operative hemorrhage, the hemostatic material for clinically using is mainly degradable day
The derived material of right polymer, such as gelfoam, oxycellulose, microfibrillar collagen powder, Fibrin Glue and collagen sponge.
But because their a variety of deficiencies cannot meet widespread demand clinically(Hu Guoying, Gu Hanqing, styptic basis with face
Bed application study new development;Dialysis and the .6 of artificial organs [J] 2010:21 (2):29-35).
Shitosan, also known as poly-GlucoseAmine (1-4) -2- amino-beta-D glucose, is a kind of to be widely present in nature
Chitin is by the linear cationic polysaccharide that obtains after partially deacetylated.Due to itself being considered to have good biofacies
Capacitive, degradability, antibacterial activity and promote Wound healing the advantages of and be widely studied.In the hair such as nineteen eighty-three Malette
After the table result of the hemostatic function of shitosan, scientists begin attempt to develop hemostatic material using shitosan.2002 9
Month, U.S. FDA have approved the HemCon hemostasia products produced by HemCon Medical Technologies companies of the U.S., and
And this product was used in Afghan War in 2003 by US military.So far, U.S. FDA has had been approved by HemCon
The shitosan class hemostasia products listing such as hemostasis bandage, CELOX, Clo-Sur P.A.D.
Meanwhile, also there is domestic and international patent to disclose the result of study to shitosan class styptic, such as, United States Patent (USP)
US4532134 relate to solution concentration and molten after chitosan material deacetylation, molecular weight, dissolving solvent for use, dissolving
The optimization of the conditions such as liquid pH, is prepared for chitose haemostatic, and demonstrates chitose haemostatic by zoopery not only having
Good haemostatic effect, while also having, the effects such as fibroplasia, promotion organization regeneration can be suppressed.It is using shitosan directly
The patent that raw material carries out styptic sponge preparation includes, Chinese invention patent ZL03153820.7,200810198164.4,
201010291789.2 with Chinese invention patent application 02109638.4,200680030822.8,200110115108.5,
201110382475.8、201210545888.8、201210033793.8.Wherein, Chinese invention patent 201010291789.2
New shitosan extracting method is disclosed, shitosan styptic sponge is made by oneself in the new technology extracted, obtains porous after freezing
The hemostatic material for easily absorbing, and proved by zoopery, this hemostatic material has superior anthemorrhagic performance.There is research to send out
It is existing, the use of shitosan is directly that chitosan sponge mechanical strength prepared by raw material is poor, therefore occur in that some correlations successively
The patent of improved method, such as, and Chinese invention patent 200810198164.4 and Chinese invention patent application
200710115108.5th, 02109638.4,20101048730.8 disclose and carried out using other natural polysaccharide materials and shitosan
Mix, prepare the correlation technique of styptic sponge.But, the complicated and resulting styptic humid stability of preparing craft
It is poor(Meet blood dissolving or become gel-like), such as current most study with shitosan and gelatin as main component is prepared
Into porous styptic sponge, easily bleed profusely wound dissolving, it is impossible to play hemoglutination(The such as Wu Bin, Sun Yun are a kind of
For styptic sponge preparation method [P] the Chinese invention patent applications 201210545888.8,2012-12-14 of auxiliary material).
In order to solve the above problems, the technology for being crosslinked to shitosan using crosslinking agent and being prepared styptic is sent out
Exhibition, such as patent CN9110678.4 is disclosed during glycerine added into chitin solution, then carries out crosslinking curing with formaldehyde, is freezed
The technology of formaldehyde section encapsulation is washed away after shaping.It is said that using the styptic sponge soft texture obtained by the method, having with human body
It is good histocompatbility, nontoxic, it is non-stimulated to the surface of a wound;Patent CN03112762.2 also provides the polysaccharide such as one kind shitosan
For raw material carries out the method that crosslinking prepares sponge using glutaraldehyde or formaldehyde.But, the involved crosslinking for using in these patents
Agent has toxicity in itself.In addition, Chinese patent application 201110382475.8,201210033793.8 discloses addition and coagulates
The technology of preparing of the bioactie agents such as hemase, bacteriostatic agent, plasticizer and foaming agent in shitosan styptic sponge.
In sum, from the point of view of shitosan hemostatic material technology of preparing disclosed at present, cannot also for various reasons
Fully meet the demand of clinic.
The content of the invention
Shitosan hemostatic material formed it is an object of the invention to provide covalent cross-linking and preparation method thereof.
The technical solution used in the present invention is:
The shitosan hemostatic material that covalent cross-linking is formed, it is to carry out chemistry by covalent bond to shitosan using crosslinking agent
Crosslinking is obtained;Described crosslinking agent is polyethyleneglycol diglycidylether.
The preparation method of the shitosan hemostatic material that covalent cross-linking is formed, step is as follows:
1)Shitosan is made the aqueous solution;
2)By in crosslinking agent addition chitosan aqueous solution, it is thoroughly mixed, is subsequently poured into mould, freeze-drying is
Can;
Described crosslinking agent is polyethyleneglycol diglycidylether.
The chemical formula of described polyethyleneglycol diglycidylether is:
。
n=1-20。
Polyethyleneglycol diglycidylether is 0.001 with the molar ratio of monosaccharide units in chitosan molecule:1~1:1.
Chitosan solution is that the shitosan being configured to the aqueous solution, the aqueous solution containing organic solvent or aqueous acid is water-soluble
Liquid, wherein, the acid for using is organic acid and inorganic acid.
Chitosan solution is to be dissolved in aqueous acetic acid being made by shitosan.
In described chitosan solution, the concentration of shitosan is 1% -3%(w/v).
In described acetum, the concentration of acetic acid is 1% -3%(v/v).
The material of the mould is the aluminium sheet material of polytetrafluoroethylene (PTFE) plated film.
The beneficial effects of the invention are as follows:
The present invention uses polyethyleneglycol diglycidylether, and shitosan is crosslinked in acid condition.Wherein, poly- second
In acid condition, there is ring-opening reaction in the ethylene oxide group in Hexanediol diglycidyl ether, lead to the hydroxyl on shitosan
Cross the product for generating ehter bond and forming crosslinking.The crosslinking agent that the inventive method is used is comparatively safe, and preparation is simple, raw
Produce cost relatively low.
Brief description of the drawings
Fig. 1 is addition crosslinking agent GDGE(0.05eq)Prepared chitosan sponge, is immersed in shake in 2% aqueous acetic acid
Swing the form after 30min.
Fig. 2 is addition crosslinking agent GDGE(0.05eq)(It is right)With not plus crosslinking agent(It is left)Prepared chitosan sponge, leaching
Bubble stands the form after 10min in aqueous.
Fig. 3 is the blood coagulation rate of different crosslinking agents 2% chitosan sponge of covalent cross-linking under different mol ratio example(Treatment
1min)With the comparison diagram of the existing hemostasia products blood coagulation rate of in the market.
Fig. 4 is the blood coagulation rate of different crosslinking agents 2% chitosan sponge of covalent cross-linking under different mol ratio example(Treatment
3min)With the comparison diagram of the existing hemostasia products blood coagulation rate of in the market.
Fig. 5 is the blood coagulation rate of different crosslinking agents 3% chitosan sponge of covalent cross-linking under different mol ratio example(Treatment
1min)With the comparison diagram of the existing hemostasia products blood coagulation rate of in the market.
Fig. 6 is the blood coagulation rate of different crosslinking agents 3% chitosan sponge of covalent cross-linking under different mol ratio example(Treatment
3min)With the comparison diagram of the existing hemostasia products blood coagulation rate of in the market.
Specific embodiment
The shitosan hemostatic material that covalent cross-linking is formed, it is to carry out chemistry by covalent bond to shitosan using crosslinking agent
Crosslinking is obtained;Described crosslinking agent is polyethyleneglycol diglycidylether.
The preparation method of the shitosan hemostatic material that covalent cross-linking is formed, step is as follows:
1)Shitosan is made the aqueous solution;
2)By in crosslinking agent addition chitosan aqueous solution, it is thoroughly mixed, is subsequently poured into mould, freeze-drying is
Can;
Described crosslinking agent is polyethyleneglycol diglycidylether.
The chemical formula of described polyethyleneglycol diglycidylether is:
。
Preferably, n=1-20, it is further preferred that being n=1 or 10 or 20.
Polyethyleneglycol diglycidylether is 0.001 with the molar ratio of monosaccharide units in chitosan molecule:1~1:1;It is excellent
Choosing, be 0.05:1 or 0.10:1 or 0.20:1.The mole of monosaccharide units is determined as common knowledge in chitosan molecule.
Chitosan solution is that the shitosan being configured to the aqueous solution, the aqueous solution containing organic solvent or aqueous acid is water-soluble
Liquid, wherein, the acid for using is organic acid and inorganic acid;Preferably, it is aqueous acid;Still more preferably, it is water-soluble acetic acid
Liquid.
Chitosan solution is to be dissolved in aqueous acetic acid being made by shitosan.
In described chitosan solution, the concentration of shitosan is 1% -3%(w/v);Preferably, it is 2% or 3%.
In described acetum, the concentration of acetic acid is 1% -3%(v/v);Preferably, it is 2%.
The material of the mould is the aluminium sheet material of polytetrafluoroethylene (PTFE) plated film.
With reference to specific embodiment, the present invention is described further:
Embodiment 1:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.34g, the mole of monosaccharide units is
21.2mmol)It is dissolved in 2% acetum(V/v, 17ml), obtain 2% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h,
It is completely dissolved it.Add polyethyleneglycol diglycidylether(n = 1)(18.4mg , 0.05eq), after mechanical agitation is mixed
12h is stood in the mould for pouring into a diameter of 6cm, freeze-drying obtains the spongy product that thickness is 5mm.
Embodiment 2:
Identical with the step of embodiment 1, its difference is to add polyethyleneglycol diglycidylether(n = 1)Amount be
36.8mg(0.10eq).
Embodiment 3:
Identical with the step of embodiment 1, its difference is to add polyethyleneglycol diglycidylether(n = 1)Amount be
73.6mg(0.20eq).
Embodiment 4:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.51g)It is dissolved in 2% acetum(V/v,
17ml), obtain 3% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h, it is completely dissolved it.Polyethylene glycol two is added to contract
Water glycerin ether(n = 1)(27.6mg , 0.05eq), 12h is stood in the mould that a diameter of 6cm is poured into after mechanical agitation mixing,
Freeze-drying, obtains the spongy product that thickness is 5mm.
Embodiment 5:
Identical with the step of embodiment 4, its difference is to add polyethyleneglycol diglycidylether(n = 1)Amount be
55.1mg(0.10eq).
Embodiment 6:
Identical with the step of embodiment 4, its difference is to add polyethyleneglycol diglycidylether(n = 1)Amount be
110.2mg (0.20eq).
Embodiment 7:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.34g)It is dissolved in 2% acetum(V/v,
17ml), obtain 2% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h, it is completely dissolved it.Polyethylene glycol two is added to contract
Water glycerin ether(n = 10)(52.7mg , 0.05eq), 12h is stood in the mould that a diameter of 6cm is poured into after mechanical agitation mixing,
Freeze-drying, obtains the spongy product that thickness is 5mm.
Embodiment 8:
Identical with the step of embodiment 7, its difference is to add polyethyleneglycol diglycidylether(n = 10)Amount
It is 105.4mg(0.10eq).
Embodiment 9:
Identical with the step of embodiment 7, its difference is to add polyethyleneglycol diglycidylether(n = 10)Amount
It is 210.8mg(0.20eq).
Embodiment 10:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.51g)It is dissolved in 2% acetum(V/v,
17ml), obtain 3% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h, it is completely dissolved it.Polyethylene glycol two is added to contract
Water glycerin ether(n = 10)(79.1mg , 0.05eq), 12h is stood in the mould that a diameter of 6cm is poured into after mechanical agitation mixing,
Freeze-drying, obtains the spongy product that thickness is 5mm.
Embodiment 11:
Identical with the step of embodiment 10, its difference is to add polyethyleneglycol diglycidylether(n = 10)Amount
It is 158.2mg(0.10eq).
Embodiment 12:
Identical with the step of embodiment 10, its difference is to add polyethyleneglycol diglycidylether(n = 10)Amount
It is 316.4mg(0.20eq).
Embodiment 13:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.34g)It is dissolved in 2% acetum(, v/v,
17ml), obtain 2% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h, it is completely dissolved it.Polyethylene glycol two is added to contract
Water glycerin ether(n = 20)(105.4mg , 0.05eq), stood in the mould that a diameter of 6cm is poured into after mechanical agitation mixing
12h, freeze-drying obtains the spongy product that thickness is 5mm.
Embodiment 14:
Identical with the step of embodiment 13, its difference is to add polyethyleneglycol diglycidylether(n = 20)Amount
It is 210.8mg(0.10eq).
Embodiment 15:
Identical with the step of embodiment 13, its difference is to add polyethyleneglycol diglycidylether(n = 20)Amount
It is 421.6mg(0.20eq).
Embodiment 16:
Weigh shitosan(Viscosity 100mPas, deacetylation 90%)(0.51g)It is dissolved in 2% acetum(V/v,
17ml), obtain 3% chitosan solution(w/v), in 40 DEG C of shaking table vibration 24h, it is completely dissolved it.Polyethylene glycol two is added to contract
Water glycerin ether(n = 20)(158.2mg , 0.05eq), stood in the mould that a diameter of 6cm is poured into after mechanical agitation mixing
12h, freeze-drying obtains the spongy product that thickness is 5mm.
Embodiment 17:
Identical with the step of embodiment 16, its difference is to add polyethyleneglycol diglycidylether(n = 20)Amount
It is 316.4mg(0.10eq).
Embodiment 18:
Identical with the step of embodiment 16, its difference is to add polyethyleneglycol diglycidylether(n = 20)Amount
It is 632.8mg(0.20eq).
Solubility experiment
In order to prove that the chitosan sponge for preparing is by being cross-linked to form, to not adding crosslinking agent and adding what crosslinking agent was obtained
Chitosan sponge product carries out dissolubility test.
1、2%(V/V)Aqueous acetic acid
Choose the sponge product for adding minimum crosslinking agent, i.e. embodiment 1 and do not add the sponge that crosslinking agent is formed each
15mg, is respectively placed in 10ml centrifuge tubes, each to add 2% aqueous acetic acid 5ml.It is put into oscillator(Rotating speed is 200-
300rpm)Concussion 30min, as a result shows, does not add the sponge of crosslinking agent to be dissolved completely in 2% aqueous acetic acid, and embodiment 1
It is only swelling and undissolved.Swelling situation is as shown in Figure 1.
2nd, the aqueous solution
Choose the sponge product for adding minimum crosslinking agent, i.e. embodiment 1 and do not add the sponge each about 1 of crosslinking agent formation
× 1cm, is placed in batch cultur ware, each to add aqueous solution 5ml.10min is stood, is as a result shown, the sponge of crosslinking agent is not added
In gel, embodiment 1 is changed into swelling spongy.As shown in Figure 2.
Absorbency is tested
With reference to (pair dogface, Wu Zhigu, modern wound dressings theory and practice [M], Beijing:Chemical Industry Press,
2007:651) experimental technique carries out the test of absorbency to the styptic sponge product in specific embodiment in.Due to phosphoric acid
Buffer solution will be tested distilled water used and be changed to phosphate buffer in testing than water closer to human body fluid in bibliography
pH 7.4。
1st, styptic sponge product prepared in Example 1-18 respectively, it is ensured that product is weighed in the dry state, note
It is Wdry(g);
2nd, phosphate buffer pH 7.4, soaking at room temperature 2h will be added in product;
3rd, the phosphate buffer suction pipe and filter paper that will be absorbed by the sponge after 2h are blotted, and weighing is designated as Wwet(g).
4th, the absorbency of each product is calculated using formula 1.
Absorbency=(Wwet - Wdry)/ Wdry (formula 1)
The absorbency of the chitosan sponge of the covalent cross-linking of table 1.+
+Absorbency:Per the liquid quality (g) that 1g chitosan sponges product absorbs
* 2%CS products:2% chitosan solution resulting product; GDGE:Polyethyleneglycol diglycidylether(n = 1);
PEG500:Polyethyleneglycol diglycidylether(n = 10); PEG1000:Polyethyleneglycol diglycidylether(n = 20)
* bracket inner digitals are the molar ratio of crosslinking agent and shitosan monosaccharide units.
From table 1 it follows that the imbibition quality of styptic sponge product of the present invention is 21 ~ 39 times of sponge in itself.It is different
Concentration articles slightly have difference, and 2% chitosan sponge product is slightly higher compared with 3% chitosan sponge product.
Clotting assay
With reference to (Pei-Leun Kang, Shwu Jen Chang, Ioannis Manousakas, Chen Wei
Lee, Chun-Hsu Yao,Feng-Huei Lin, Shyh Ming Kuo Development and assessment of
hemostasis chitosan dressings, Carbohydrate Polymers[J]2011, 85:565-570) in
Experimental technique carries out clotting assay to the styptic sponge product in specific embodiment.
1st, styptic sponge product prepared in Example 1-18, is cut to 1cm × 1cm sizes respectively, is put into burning
In cup;
2nd, in the slow new fresh poultry blood addition styptic sponge product by 0.25ml(Chicken blood:Anticoagulant heparin agent=5: 1);
3rd, at room temperature(25℃)Place the time set in advance(1min、3min)Afterwards, by 20ml purified waters along walls of beaker
Slowly pour into beaker, try not to have influence on solidification of the blood on sponge in toppling process;
4th, the erythrocyte not absorbed by styptic sponge product after placing ten minutes can be dissolved in the solution, using ultraviolet
Spectrophotometer detects absorption value at wavelength 540nm, and is dissolved in 20ml purified waters as negative control with 0.25ml chicken bloods;
5th, the blood coagulation rate of styptic sponge product is calculated using formula 2.
Blood coagulation rate=(1-product absorbance/control absorbance) × 100% (formula 2)
The blood coagulation rate of 2% chitosan sponge of the covalent cross-linking of table 2.(Treatment 1min)
* control group is the existing absorbable gelatin sponge product in market.
**GDGE:Polyethyleneglycol diglycidylether(n = 1); PEG500:Polyethyleneglycol diglycidylether(n =
10); PEG1000:Polyethyleneglycol diglycidylether(n = 20)
***Crosslinking agent equivalent:The molar ratio of crosslinking agent and shitosan monosaccharide units
+Blood coagulation rate is six average values of measurement, and bracket inner digital is SD(That is standard deviation)
The blood coagulation rate of 2% chitosan sponge of the covalent cross-linking of table 3.(Treatment 3min)
* control group is the existing absorbable gelatin sponge product in market.
**GDGE:Polyethyleneglycol diglycidylether(n = 1); PEG500:Polyethyleneglycol diglycidylether(n =
10); PEG1000:Polyethyleneglycol diglycidylether(n = 20)
***Crosslinking agent equivalent:The molar ratio of crosslinking agent and shitosan monosaccharide units
+Blood coagulation rate is six average values of measurement, and bracket inner digital is SD
The blood coagulation rate of 3% chitosan sponge of the covalent cross-linking of table 4.(Treatment 1min)
* control group is the existing absorbable gelatin sponge product in market.
**GDGE:Polyethyleneglycol diglycidylether(n = 1); PEG500:Polyethyleneglycol diglycidylether(n =
10); PEG1000:Polyethyleneglycol diglycidylether(n = 20)
***Crosslinking agent equivalent:The molar ratio of crosslinking agent and shitosan monosaccharide units
+Blood coagulation rate is six average values of measurement, and bracket inner digital is SD
The blood coagulation rate of 3% chitosan sponge of the covalent cross-linking of table 5.(Treatment 3min)
* control group is the existing absorbable gelatin sponge product in market.
**GDGE:Polyethyleneglycol diglycidylether(n = 1); PEG500:Polyethyleneglycol diglycidylether(n =
10); PEG1000:Polyethyleneglycol diglycidylether(n = 20)
***Crosslinking agent equivalent:The molar ratio of crosslinking agent and shitosan monosaccharide units
+Blood coagulation rate is six average values of measurement, and bracket inner digital is SD
As can be seen that shitosan styptic sponge product blood coagulation rate of the present invention exists from table 2-5 and Fig. 3, Fig. 4, Fig. 5, Fig. 6
Between 93% -99%, with good coagulant property, blood coagulation rate also has obvious advantage compared with the hemostasia products of in the market.
Blood coagulation rate illustrates that product of the invention has quick coagulating effectiveness without significant difference during 1min and 3min.
Above test result indicate that, styptic sponge product anthemorrhagic performance of the present invention is excellent.The hemostatic mechanism of people is mainly and leans on
Clotting factor is activated after platelet aggregation and forms clot.Being currently known the blood coagulation of clotting mechanism at least needs 90s just to start(Luo Xiao
Waterside chitose haemostatic [P] Chinese invention patent application, 200710028001.7,2007-5-14).The hemostasis of chitosan sponge
Effect increases the red blood cell and hematoblastic concentration on its surface it is believed that quickly absorb serum by the capillarity of sponge, makes
It is gathered in sponge surface and closes wound, stops blood before blood clot is formed, and reduces the loss of blood.Meanwhile, shitosan
The positive charge of sponge surface can activate the clotting mechanism of erythrocyte participation, so as to accelerate the such as test tube of hepari blood containing anticoagulant
The blood coagulation of liquid.