Hole-hole composite micro-nano structure polysaccharide microsphere is in the purposes for preparing bleeding-stopping dressing
Technical field
The present invention relates to a kind of hole-hole composite micro-nano structure polysaccharide microspheres in the purposes for preparing bleeding-stopping dressing, belongs to height
Molecular material preparation technical field.
Background technique
Natural macromolecule amylose have resourceful, green non-pollution, good biocompatibility, biodegradable advantage,
Possess the macromolecule polysaccharide microballoon of porous structure in hemostasis, load medicine, tissue engineering bracket, protein purification, adsorption of metal ions point
There is very big application from equal fields.
A kind of preparation of core-shell structure composite porous microspheres of patent report that number of patent application is 201610011690.X
Method is shell material using sodium alginate, chitosan, cellulose, gelatin etc., porous micro- with chitosan porous microsphere, cellulose
Ball, porous polylactic acid microball, Porous Gelatin Microspheres By Using etc. are that kernel is obtained good dispersion, do not reunited by double emulsion method, microballoon,
But its surface crust is fine and close, does not have hole-pore structure.The patent that number of patent application is 201611024927.4 is with emulsion-crosslinking method
The calcium alginate of preparation is core balls, has successively coated chitosan, sodium alginate and shell in outer layer using LBL self-assembly method and has gathered
Sugar obtains ten micron-sized multilayer core shell mould drug bearing microspheres, and wherein kernel has loaded endothelial growth factors, and outer shell has loaded ten thousand
Ancient mycin has both and promotes angiogenesis and antibacterial functions, and still, this microspherulite diameter is small, the micropore that surface is not evenly distributed.
Cmc soln is ejected by the patent that number of patent application is 201710935220.7 with electrostatic spraying method
In dilute sulfuric acid coagulating bath, regeneration molding is washed out and dries, obtains the porous hemostatic microsphere of carboxymethyl cellulose, party's legal system
Fast, the environmentally protective, uniform particle diameter for rate, prepared microballoon has pore structure abundant, but its surface does not have micron order
Cheat shape structure.Gelatin solution is passed through emulsification-dehydration-washing-drying-sieve by the patent that number of patent application is 201710318416.1
Filtration journey obtains microballoon crude product, then obtains gelatine microsphere, prepared gelatine microsphere small toxicity, degradation again with genipin cross-linked
Effect is good.In short, using microballoon made from above method, there are structures, and single, microscopic appearance is difficult to, particle diameter distribution is excessive
The problem of.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of hole-hole composite micro-nano structure polysaccharide microsphere
In the purposes for preparing bleeding-stopping dressing.
Technical solution of the present invention is summarized as follows:
Hole-hole composite micro-nano structure polysaccharide microsphere is in the purposes for preparing bleeding-stopping dressing.
The hole-hole composite micro-nano structure polysaccharide microsphere is made of following methods:
(1) natural polysaccharides are molten into ultrapure water, it is made into the poly sugar aqueous solution that mass concentration is 2%~10%;
(2) emulsifier is added in oily phase, makes the mass fraction 0.4%-5% of emulsifier, at 40-80 DEG C, stirred molten
Solution, obtains the oily phase containing emulsifier;The poly sugar aqueous solution is added drop-wise in the oily phase containing emulsifier at room temperature, stirring cream
Change, obtains homogeneous latex emulsion;The oil containing emulsifier is mutually and the mass ratio of poly sugar aqueous solution is (10-3): 1;
(3) polyvalent metal crosslinking agent is dissolved in ultrapure water and is made into the cross-linking agent aqueous solution that mass concentration is 20%~30%,
Cross-linking agent aqueous solution is added drop-wise in the homogeneous latex emulsion that the step (2) under stirring obtains, the cross-linking agent aqueous solution and uniformly cream
The volume ratio (2-5) of liquid: 10, continue to be stirred to react 2~12h after dripping off;
(4) step (3) are added to 3~10 times of liquid volume of the n-hexane or petroleum ether that are equivalent to step (3) acquisition
In the liquid of acquisition, upper oil phase layer is poured out after solution layering, retains water phase, cleans water phase with n-hexane or petroleum ether;It uses again
Ethyl alcohol cleaning, freeze-drying obtain hole-hole composite micro-nano structure polysaccharide microsphere.
The natural polysaccharides are degree of substitution: >=80%, carboxymethyl chitosan that viscosity is 10-80mPa.s, viscosity are
1500-4500mPa.s carboxymethyl cellulose, weight average molecular weight are the Sensor Chip CM 5 of 50000-100000, weight average molecular weight
It is at least one for 100000-500000 Sodium Hyaluronate and viscosity 100-200mpa.s sodium alginate.
Preferably, step (2) oil is mutually at least one of atoleine, vegetable oil, n-hexane, the vegetable oil
For peanut oil, soybean oil or rapeseed oil.
Emulsifier be preferably in span 85, sorbester p17, sorbester p18, Tween-80, Tween-60 and Triton X-100 at least
It is a kind of.
Polyvalent metal crosslinking agent is preferably CaCl2、ZnCl2、CuCl2Or FeCl3At least one of.
The rate of the stirring and emulsifying of the step (2) is preferably 500~800rpm, and mixing time is 1~4h.
The stirring rate of step (3) is preferably 300~500rpm.
Step (4) is preferred are as follows: is added with 3~10 times of liquid volume of the n-hexane or petroleum ether that are equivalent to step (3) acquisition
Enter in the liquid obtained to step (3), pour out upper oil phase layer after solution layering, retain water phase, then with being equivalent to water phase volume 3
~10 times of n-hexane or petroleum ether cleans water phase 1-3 times;It is cleaned 2-4 times with the ethyl alcohol for being equivalent to 3~10 times of water phase volume again,
Freeze-drying, obtains hole-hole composite micro-nano structure polysaccharide microsphere.Hole-hole composite micro-nano structure polysaccharide of above method preparation is micro-
Ball.
Advantages of the present invention:
(1) hole of the invention-hole composite micro-nano structure polysaccharide microsphere, surface have micron-sized pit, cheat inner surface
There is nanoscale pore again, there is unique surface " hole-hole " composite micro-nano rice structure.
(2) hole of the invention-hole composite micro-nano structure polysaccharide microsphere particle diameter distribution is uniform, and porosity is high, and water absorption rate is high,
Strong adsorption.
(3) preparation process is simple, does not need to heat, low energy consumption.Prepared hole-hole composite micro-nano structure polysaccharide microsphere
In the application for preparing bleeding-stopping dressing.
(4) hole-hole obtained according to GB/T16886.5-2003 vitro cytotoxicity judgment criteria, various embodiments of the present invention
Composite micro-nano structure polysaccharide microsphere leaching liquor has 0 grade of cytotoxicity in 10~40 μ g/mL, shows good bio-compatible
Property.
(5) through detecting, hole of the invention-hole composite micro-nano structure polysaccharide microsphere can efficiently accomplish in 20s~50s
Hemostasis.
It is can absorb porous styptic powder high comprehensive performance according to what this technology was prepared, convenient for operation and is saved, and
Preparation process is simple, is suitble to industrialized production.
(6) through detecting, hole of the invention-hole composite micro-nano structure polysaccharide microsphere has good physicochemical property.Hemolysis rate
Respectively less than 5%, meet national standard.
(7) the result shows that hole-hole composite micro-nano structure polysaccharide microsphere has moment hemostasis for the hepar damnification surface of a wound
Function (bleeding stopping period is less than 5s), and after 72h, hemostatic material is degradable, without any residual.Femoral artery is measured greatly
Blood, hole-hole composite micro-nano structure polysaccharide microsphere can complete hemostasis in 20s, and surgical wound surface is back to normal after 1 week, and nothing is appointed
Blood microsphere sample remains far more than.
Detailed description of the invention
Fig. 1 is the SEM figure that hole obtained by embodiment 1-hole composite micro-nano structure polysaccharide microsphere amplifies 300 times.
Fig. 2 is the SEM figure that hole obtained by embodiment 2-hole composite micro-nano structure polysaccharide microsphere amplifies 1200 times.
Fig. 3 is the SEM figure that hole obtained by embodiment 3-hole composite micro-nano structure polysaccharide microsphere amplifies 10000 times.
Fig. 4 is the SEM figure that hole-hole composite micro-nano structure polysaccharide microsphere cross section obtained by embodiment 1 amplifies 1000 times.
Fig. 5 is the SEM figure that hole-hole composite micro-nano structure polysaccharide microsphere cross section obtained by embodiment 2 amplifies 5000 times.
Fig. 6 is the SEM figure that hole obtained by embodiment 3-hole composite micro-nano structure polysaccharide microsphere surface amplifies 10000 times.
Fig. 7 is the cytotoxicity statistical result of embodiment 1, microballoon prepared by embodiment 2 and embodiment 3.
(A) cell survival rate of mtt assay measurement is utilized;
(B) method for using fluorescent staining, cell after dyeing 24 hours show the survival shape of cell number and cell
State.
Fig. 8 is liver hemostasis experiment (A) and SD rat femoral hemostasis experiment of 3 thus obtained microsphere of embodiment for SD rat
(B)。
Fig. 9 is the whole blood clotting index test result of hemostatic microsphere obtained by embodiment 1-3, wherein wound does not do any place
Reason is as a control group.
Specific embodiment
Particle size, appearance structure and specific surface area of porous microsphere etc. have a significant impact to its performance, and the present invention uses
Natural polysaccharides are main raw material(s), by the methods of the molecular weight, solution viscosity, preparation process condition for regulating and controlling component, are overcome
Problem of the existing technology, has obtained a kind of hole-hole composite micro-nano structure polysaccharide microsphere.
Below by way of specific embodiment, the present invention is further illustrated.With reference to embodiments and attached drawing is further
Illustrate summary of the invention and embodiment of the invention, it is only for reference and illustrate use, the limit to the scope of the present invention is not constituted
System.
Embodiment 1
Hole-hole composite micro-nano structure polysaccharide microsphere preparation method, includes the following steps:
(1) ratio for being 1:2:2 by mass ratio, by degree of substitution: >=80%, carboxymethyl chitosan that viscosity is 10mPa.s,
Weight average molecular weight is that 100000 Sodium Hyaluronates and viscosity 100mpa.s sodium alginate are molten into ultrapure water, and being made into mass concentration is
2% poly sugar aqueous solution;
(2) by mass ratio be 1:1 span 85 and Tween-80 mix emulsifier be added atoleine in, make emulsifier
Mass fraction be 0.4%, at 40 DEG C, stirring and dissolving obtains the oily phase containing emulsifier;Poly sugar aqueous solution is added dropwise at room temperature
Into the oily phase containing emulsifier, 500rpm stirring and emulsifying, mixing time 4h obtains homogeneous latex emulsion;Containing emulsifier oil mutually with
The mass ratio of poly sugar aqueous solution is 10:1;
(3) by CaCl2It is dissolved in ultrapure water and is made into the cross-linking agent aqueous solution that mass concentration is 20%, by cross-linking agent aqueous solution,
It is added drop-wise in the homogeneous latex emulsion that the step (2) under 300rpm stirring obtains, the body of the cross-linking agent aqueous solution and homogeneous latex emulsion
Product is continuing to be stirred to react 2h in 300rpm than 2:10 after dripping off;
(4) liquid of step (3) acquisition is added to 10 times of liquid volume of petroleum ether for being equivalent to step (3) acquisition
In, upper oil phase layer is poured out after solution layering, retains water phase, then clean water phase 1 with the petroleum ether for being equivalent to 10 times of water phase volume
It is secondary;It is cleaned 2 times with the ethyl alcohol for being equivalent to 10 times of water phase volume again, is lyophilized, obtains hole-hole composite micro-nano structure polysaccharide microsphere.
Gained hole-hole composite micro-nano structure polysaccharide microsphere average grain diameter is 10.01 μm, and microsphere surface has average diameter
For 0.98 μm of circular pit, it is the hole of 50.05nm that pit inner surface, which has average pore size, has the average pore size to be inside microballoon
0.502 μm of hole, is shown in Fig. 1 and Fig. 4.
It is demonstrated experimentally that with degree of substitution: >=80%, viscosity is that the carboxymethyl chitosan of 80mPa.s substitutes taking for the present embodiment
Dai Du: >=80%, viscosity is the carboxymethyl chitosan of 10mPa.s;And the present embodiment is substituted with viscosity 200mpa.s sodium alginate
Viscosity 100mpa.s sodium alginate, other same the present embodiment, obtain hole-hole composite micro-nano structure polysaccharide microsphere, character
It is similar to hole-hole composite micro-nano structure polysaccharide microsphere that the present embodiment obtains.
It is demonstrated experimentally that substituting the Tween-80 in the present embodiment with Tween-60, other same the present embodiment can the property prepared
Shape is similar to hole-hole composite micro-nano structure polysaccharide microsphere that the present embodiment obtains.
Embodiment 2
Hole-hole composite micro-nano structure polysaccharide microsphere preparation method, includes the following steps:
(1) be 4500mPa.s carboxymethyl cellulose by the viscosity that mass ratio is 1:1:1, weight average molecular weight is 100000
Sensor Chip CM 5, weight average molecular weight be 500000 Sodium Hyaluronates it is molten into ultrapure water, be made into mass concentration be 10% it is more
Water solution;
(2) emulsifier span 80 is added in soybean oil, makes the mass fraction 5% of emulsifier, at 80 DEG C, stirred molten
Solution, obtains the oily phase containing emulsifier;Then poly sugar aqueous solution is added drop-wise in the oily phase containing emulsifier at room temperature, 800rpm is stirred
Emulsification is mixed, mixing time 1h obtains homogeneous latex emulsion;Oil containing emulsifier is mutually 3:1 with the mass ratio of poly sugar aqueous solution;
(3) by ZnCl2It is dissolved in ultrapure water and is made into the cross-linking agent aqueous solution that mass concentration is 30%, by cross-linking agent aqueous solution,
In the homogeneous latex emulsion that the step (2) being added drop-wise under 500rpm stirring obtains, the volume of the cross-linking agent aqueous solution and homogeneous latex emulsion
Than 5:10, continuing to be stirred to react 12h in 500rpm after dripping off;
(4) it is added in the liquid of step (3) acquisition with 3 times of liquid volume of n-hexane for being equivalent to step (3) acquisition,
Upper oil phase layer is poured out after solution layering, retains water phase, then cleaned water phase 3 times with the n-hexane for being equivalent to 3 times of water phase volume;Again
It is cleaned 4 times with the ethyl alcohol for being equivalent to 3 times of water phase volume, is lyophilized, obtains hole-hole composite micro-nano structure polysaccharide microsphere.
Gained hole-hole composite micro-nano structure polysaccharide microsphere average grain diameter is 198 μm, and microsphere surface is with average diameter
5.23 μm of circular pit, it is the hole of 200.04nm that pit inner surface has average pore size again, has average pore size inside microballoon
For 2.1 μm of hole, Fig. 2 and Fig. 5 are seen.
It is demonstrated experimentally that be the viscosity that 1500mPa.s carboxymethyl cellulose substitutes the present embodiment with viscosity being 4500mPa.s carboxylic
Methylcellulose;It is 100000 with the weight average molecular weight that the Sensor Chip CM 5 that weight average molecular weight is 50000 substitutes the present embodiment
Sensor Chip CM 5, other same the present embodiment obtain hole-hole composite micro-nano structure polysaccharide microsphere, character and this implementation
Hole-hole composite micro-nano structure polysaccharide microsphere that example obtains is similar.
It is demonstrated experimentally that the other vegetable oil of soybean oil in the present embodiment, if peanut oil or rapeseed oil substitute, acquisition is obtained
Hole-hole composite micro-nano structure polysaccharide microsphere is obtained, the hole that character is obtained with the present embodiment-hole composite micro-nano structure polysaccharide is micro-
Ball is similar.
Embodiment 3
Hole-hole composite micro-nano structure polysaccharide microsphere preparation method, includes the following steps:
(1) by the weight average molecular weight that mass ratio is 2:1:1 be 80000 Sensor Chip CM 5, weight average molecular weight be
400000 Sodium Hyaluronates, viscosity 150mpa.s sodium alginate are molten into ultrapure water, are made into the poly syrup that mass concentration is 6%
Solution;
(2) by mass ratio be 4:1 sorbester p18 and Triton X-100 mix emulsifier be added mass ratio be 5:1 liquid
In body paraffin and n-hexane, make the mass fraction 1% of emulsifier, at 50 DEG C, stirring and dissolving obtains the oily phase containing emulsifier;So
Poly sugar aqueous solution is added drop-wise in the oily phase containing emulsifier at room temperature afterwards, 600rpm stirring and emulsifying, mixing time 2h is obtained
To homogeneous latex emulsion;Oil containing emulsifier is mutually and the mass ratio of poly sugar aqueous solution is 5:1;
(3) by CuCl2It is dissolved in ultrapure water and is made into the cross-linking agent aqueous solution that mass concentration is 25%, by cross-linking agent aqueous solution,
In the homogeneous latex emulsion that the step (2) being added drop-wise under 400rpm stirring obtains, the volume of the cross-linking agent aqueous solution and homogeneous latex emulsion
Than 3:10, continuing to be stirred to react 6h in 400rpm after dripping off;
(4) it is added in the liquid of step (3) acquisition with 5 times of liquid volume of petroleum ether for being equivalent to step (3) acquisition,
Upper oil phase layer is poured out after solution layering, retains water phase, then cleaned water phase 2 times with the petroleum ether for being equivalent to 5 times of water phase volume;Again
It is cleaned 3 times with the ethyl alcohol for being equivalent to 5 times of water phase volume, is lyophilized, obtains hole-hole composite micro-nano structure polysaccharide microsphere.
Gained hole-hole composite micro-nano structure polysaccharide microsphere average grain diameter is 100.04 μm, and microsphere surface has average straight
The circular pit that diameter is 2.88 μm, it is the hole of 99.99nm that pit inner surface has average pore size again, has average hole inside microballoon
The hole that diameter is 1.1 μm, is shown in Fig. 3 and Fig. 6.
Experimental example 1
Hole-hole composite micro-nano structure polysaccharide microsphere in the purposes for preparing bleeding-stopping dressing, with following experiments have shown that.
Cytotoxicity experiment
Experimental cell uses the eugonic L929 cell line cell (commercially available) of 48h~72h.
Culture medium is the RAPI1640 that 10% (V/V) fetal calf serum is added.
Negative control group is the RAPI1640 that 10% (V/V) fetal calf serum is added;
Experimental group (hole-hole composite micro-nano structure the poly for taking 0.5g embodiment 1, embodiment 2 and embodiment 3 to prepare respectively
The RAPI1640 of 10% (V/V) fetal calf serum is added in sugared microballoon and 10mL, for 24 hours in 37 DEG C of extractions, obtains leaching liquor);
Positive controls (phenol solution that mass fraction is 5%);
Test carries out on 96 orifice plates.Cell (L929 cell line cell) density of initial incubation liquid is about 50,000/mL, often
200 μ L cell culture fluids are added in hole, make number of cells about 10000 in every hole.Every hole in triplicate, in 37 DEG C of 5% (V/V) two
Culture for 24 hours, then discards former culture medium in carbonoxide/air constant incubator.
Add 200 μ L in negative control group;
Experimental group adds 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L leaching liquors respectively, add negative controls make it is molten in every hole
Liquid product is 200 μ L;
Positive controls add respectively 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L mass fractions be 5% phenol solution, then plus
Entering negative controls makes 200 μ L of liquor capacity in every hole.
Then, which is put into after being further cultured for 48h in 37 DEG C of constant incubators and takes out culture plate, discarded in culture plate
20 μ L MTT solution are added in solution, every hole, continue to cultivate 4h, then suck stoste, 150 μ L dimethyl sulfoxides are added, concussion is very
Clock measures absorbance value (ABS) at 570nm wavelength in immune microplate reader.
Versus cell activity (%)=(ABS570 experimental groups/ABS570 negative controls) × 100%
According to GB/T16886.5-2003 vitro cytotoxicity judgment criteria, described in embodiment 1, embodiment 2 and embodiment 3
Microballoon leaching liquor 10~40 μ g/mL have 0 grade of cytotoxicity, show good biocompatibility.As a result such as Fig. 7 institute
Show.The data obtained shows that the cytotoxicity of 3 thus obtained microsphere of embodiment 1- embodiment is 0 grade in Fig. 7 (A), without apparent cell
Toxicity.Fig. 7 (B) is 3 cell fluorescence coloration result of embodiment 1- embodiment, and diagram cell growth state is good, the growth of cell
State is not affected by the influence of microballoon leaching liquor.
Experimental example 2
The test of body surface anthemorrhagic performance
To hole-hole composite micro-nano structure polysaccharide microsphere that embodiment 1, embodiment 2 and embodiment 3 obtain, carry out external
Hemostatic function detection, specific detection method are as follows:
It is fixed by SD rat anesthesia, 4 circular wounds (diameter 2cm, depth are symmetrically manufactured at left and right sides of back
0.5cm), after bleeding, in bleeding part, the hole 0.05g-hole composite micro-nano structure polysaccharide microsphere is spilt in painting.
Through detecting, hole of the invention-hole composite micro-nano structure polysaccharide microsphere can efficiently accomplish only in 20s~50s
Blood (table 3).
Table 1SD is embodiment 1-3 rat body surface bleeding stopping period statistical result
Number |
Negative control |
Embodiment 1 |
Embodiment 2 |
Embodiment 3 |
Bleeding stopping period (s) |
170±20 |
20±3 |
25±5 |
22±2 |
Note: negative control be state's pan create (chitosan styptic powder) (trade name:Chitosan styptic powder, the general love in Hubei
Pharmaceutcal corporation, Ltd)
Experimental example 3
Hemolysis rate: the hole embodiment 1-3-hole composite micro-nano structure poly is measured according to GB/T 16175-1996 prescriptive procedure
The hemolysis rate of sugared microballoon.Through detecting, hole of the invention-hole composite micro-nano structure polysaccharide microsphere has good physicochemical property.It is micro-
The hemolysis rate of ball is respectively less than 5% (table 2), meets national standard.
Table 2 is hole-hole composite micro-nano structure polysaccharide microsphere hemolysis rate statistical result of embodiment 1-3
Note: positive controls are distilled water;Negative control group is physiological saline;Experimental group is hole-hole composite micro-nano structure
The physiological saline leaching liquor of polysaccharide microsphere;X is absorbance under 545nm wavelength;S is absorbance measurement deviation.
Experimental example 4
Using the hepar damnification bleeding of SD rat as model:
To hole-hole composite micro-nano structure polysaccharide microsphere that embodiment 3 obtains, internal hemostatic function detection is carried out, specifically
Detection method are as follows: using the hepar damnification bleeding of SD rat as model, by chloraldurate aqueous solution intraperitoneal injection of anesthesia, with operation
Knife manufactures 1cm × 1cm wound in liver surface.Quantity for spray is hole-hole composite micro-nano structure polysaccharide microsphere of 20 μ g in the surface of a wound.
Blood feelings are observed, record time and amount of bleeding, such as Fig. 8 A, wherein a is formed for the surface of a wound, starts to bleed;Hole-hole is compound micro- in b spraying
Micro-nano structure polysaccharide microsphere;C removes surface microballoon with physiological saline, and surface of a wound hemostasis is completed.It is postoperative, suture abdomen.After 1 week, open
Line observes recovery situation.The result shows that hole-hole composite micro-nano structure polysaccharide microsphere prepared by embodiment 3, for hepar damnification
The surface of a wound has moment hemostatic function (bleeding stopping period is less than 5s), and after 72h, remaining microballoon is degradable.
Experimental example 5
Hole-hole composite micro-nano structure polysaccharide microsphere hemostatic function test (femoral artery Hemorrhage Model):
Hole-hole the composite micro-nano structure largely obtained at Hemorrhage Model, testing example 3 with the femoral artery injury of SD rat
The anthemorrhagic performance of polysaccharide microsphere.
After SD rat anesthesia, femoral artery is exposed in dissection, manufactures big bleeding using scalpel shearing artery.0.2g sample is spilt
In wound, until hemostatic closure, observing bleeding and hemostasis, (Fig. 8 B, a are formed for the surface of a wound, are started to spray blood;Hole-in b spraying
Hole composite micro-nano structure polysaccharide microsphere;C removes surface microballoon with physiological saline).It is postoperative, suture abdomen.After 1 week, sight of bursting at the seams
Examine recovery situation.The experimental results showed that bleed profusely for femoral artery, hole-hole composite micro-nano structure poly that embodiment 3 obtains
Sugared microballoon can complete hemostasis in 20s, and surgical wound surface restores normal after 1 week, no any microsphere sample residual.
Experimental example 6.
The measurement of whole blood clotting index:
To hole prepared by embodiment 1-3-hole composite micro-nano structure polysaccharide microsphere, the measurement of whole blood clotting index is carried out
Specific experiment step: 20mg sample is placed on vinyl disc, and 5min is placed at 37 DEG C, and 200 μ L anticoagulations slowly drip
Sample surfaces are added to, 20 μ l 0.2M calcium chloride waters are then added, continue 37 DEG C of culture 5min.25ml distilled water is added, shakes
Bed 30rpm shake culture 10min, separates supernatant, and the absorbance that supernatant is measured at 540nm is denoted as A value, anticoagulation go from
Absorbance in sub- water, which is used as, refers to B value.
Blood clotting index calculates:Acquired results are as shown in Figure 9.
Whole blood clotting index is the result shows that compared with the control group, the clotting index of embodiment 1-3 significantly reduces.This shows reality
Porous microsphere prepared by a 1-3 is applied, has and promotees coagulation function well.
Above embodiments are the present invention preferably embodiment relatively, and the present invention is not limited to the above embodiments, is not taking off
Under the premise of spirit and scope of the invention, the present invention also has various change, improvement and equivalent replacement, is included in the present invention
Protection scope within.