CN105943497A - Self-assembly nanometer hemostatic as well as preparation method and applications thereof - Google Patents
Self-assembly nanometer hemostatic as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a self-assembly nanometer hemostatic as well as a preparation method and applications of the self-assembly nanometer hemostatic. The nanometer hemostatic is composed of a hydrophilic cationic polymer and a water repellent compound containing carboxyl groups, wherein the mass ratio of the hydrophilic cationic polymer to the water repellent compound containing carboxyl groups is between (1:0.1) to (1:5), and the grain diameter of the nanometer hemostatic is 20nm-800nm. The preparation method comprises the following steps: dissolving the hydrophilic cationic polymer into a strong polar solvent, dissolving the water repellent compound containing carboxyl groups into an organic solvent miscible with water, slowly adding the organic solution containing the carboxyl compound into the polymer solution under the effect of ultrasonic, placing the mixed solution into a dialysis bag, and carrying out dialysis in deionized water under the magnetic stirring to remove the organic solvent. The nanometer hemostatic can be dropwise added or applied to the local part of the tissue for stopping bleeding, and also can be injected to prevent bleeding. The nanometer hemostatic is easy to synthesize and low in cost, the self-assembly method is simple and feasible, the organic solvent can be easily removed, the stability is good, and no immunogenicity exists.
Description
Technical field
The present invention relates to nanometer hemostatic material field, specifically a kind of self-assembled nanometer hemorrhage, this hemorrhage
Preparation method and preventing and treating body hemorrhage in application.
Background technology
In wound and clinical operation, bleeding profusely of body is one of murderous principal element.Blood
Liquid coagulation is the first step of post-trauma wound healing.Damaging not under serious conditions, such as some thin vessels
Destruction can stimulate body to produce physiological haemostasis.But if the most serious body injury, it is necessary for adopting
Take series of measures to reach to stop blooding fast and effectively.Clinical operation and wound generally should in processing at present
Hemostasis mainly include chemical hemostasis method, thermal hemostasis method and mechanical hemostasis method.
Chemical method mainly changes the blood coagulation activity of blood, such as vasoconstrictor (thrombosis by chemical reagent
Element A2) vasoconstriction can be caused, reduce blood flowing, thus promote blood coagulation.Heat setting hemostasis includes
Calcination hemostasis and laser hemostasis.Mechanical hemostasis method is mainly by pressing, ligaturing and use binder to stop blooding.
The most employing chemistry and mechanical means combine hemostasis, conventional Fibrinogen, thrombin and calcium ion shape
Fibroblast cells glue is as platelet substitutes.While it is true, the method is the most much limited, such as
Some reagent is solid, it is impossible to arrives wound and plays the effect of hemostasis；Some liquid hemostatic agent are (such as cyanogen
Acrylates) need competence exertion anastalsis in a dry environment；And partial hemostasis material easily produces
Raw immunogenicity or graft versus host disease, thus body is caused secondary injury, or during survival itself
Between short, need face with now joining.
In order to overcome the problems referred to above, the most progressively develop some novel hemostatic techniques, diligent including making
Energy property liposome, block copolymer, synthesis platelet and nano material etc..Wherein, nanometer hemostatic agent because of
Its good haemostatic effect and the further flexibility of use, have bigger clinical value, enjoy and grind
Study carefully the concern of personnel.The nanofiber of such as some self-assembling polypeptides, can form similar extracellular matrix
Barrier, resolve into natural aminoacid simultaneously, thus promote the recovery of wound, improve hemostasis efficiency.
And there are some researches show that carbon nanoparticle and Clottocytes nanoparticle can activate platelet, reduce body
Bleeding time.The block copolymer (PLGA-PLL) of poly-(lactic acid-ethanol) and polylysine, passes through
The far-end bonding RGD small peptide of the Polyethylene Glycol (PEG) introduced wherein, is self-assembly of by it and receives
The grain of rice, such nanoparticle can activate platelet thus promote blood coagulation, therefore be referred to as " artificial platelets ".
RGD and platelet receptor glycoprotein II b-III a and integrin element αvβ3In conjunction with, activate platelet thus promote
Enter platelet aggregation.
Although the above-mentioned research about nanometer hemostatic agent makes some progress, but is stopped by nanoparticle
Blood nevertheless suffers from much limiting, such as short at body circulation time, itself there is nanometer toxicity, body made
Become irreversible damage, make the risk increase etc. of thrombosis relevant disease.Additionally the hemorrhage of research at present is still
There is the problems such as poor storage stability, synthesis technique is complicated, preparation cost is high.
Summary of the invention
It is an object of the invention to provide the composition of a kind of self-assembled nanometer hemorrhage, preparation method, the most also test
Demonstrate,prove this nanometer hemostatic agent effect in different Hemorrhage Models.
The present invention is by the multiple non-covalent bond between hydrophilic cationic polymer and carboxylic hydrophobic compound
The self assembly that power (including electrostatic interaction, hydrogen bond action and hydrophobic interaction) mediates is to build novel nano hemostasis
Agent, by promoting the contraction of thin vessels, promotes platelet aggregation and the startup of blood coagulation system, with erythrocyte
Middle iron ion combines and forms the grumeleuse minimizing body bleeding time.And owing to this nanoparticle surface is with positive charge,
It can with blood in the matter interaction such as leukocyte, erythrocyte and platelet of negative charge, combination,
Form blood coagulation bolt, final its hemostatic effect of performance, drastically increase hemostasis efficiency.Stop blooding relative to other
Agent, the nanometer hemostatic agent that the present invention is prepared by self assembly has many advantages, thus has broader application
Prospect.
In consideration of it, the technical solution used in the present invention is as follows: a kind of self-assembled nanometer hemorrhage is by hydrophilic sun
Ionomer and carboxylic hydrophobic compound composition, wherein said hydrophilic cationic polymer with containing carboxylic
The mass ratio of the hydrophobic compound of base is between 1:0.1 to 1:5, and the particle diameter of described nanometer hemostatic agent is at 20nm
Between 800nm.Wherein hydrophilic cationic polymer (includes that branched polyethylene is sub-selected from polymine
Amine and L-PEI), PLL hydrobromate and poly-(L-arginine) hydrochlorate, point
Son amount is between 500Da to 50000Da；Carboxylic hydrophobic compound selected from cholic acid (Cholic acid),
Ursodesoxycholic acid (Ursodeoxycholic acid), chenodeoxycholic acid (Chenodeoxycholic acid), pig
Deoxycholic acid (Hyodeoxycholic acid), lithocholic acid (Lithocholic acid), deoxycholic acid
(Deodeoxycholic acid), dehydrocholic acid (Dehydrocholic acid), ibuprofen (Ibuprofen),
Ketoprofen (Ketoprofen), fenoprofen (Fenoprofen), flurbiprofen (Flurbiprofen), sand difficult to understand
The general Qin (Oxaprozin), naproxen (Naproxen), indomethacin (Indomethacin), sulindac
(Sulindac), etodolac (Etodolac), mefenamic acid (Mefenamic acid), meclofenamic acid
(Meclofenamic acid), flufenamic acid (Flufenamic acid), tolfenamic acid (Tolfenamic acid),
Lumiracoxib (Lumiracoxib), Licofelone (Licofelone), diflunisal (Diflunisal), Ah
A department woods (Aspirin), gemfibrozil (Gemfibrozil), ciprofloxacin (Ciprofloxacin), promise fluorine
Sha Xing (Norfloxacin) and ofloxacin (Ofloxacin).
The present invention also provides for the preparation method of a kind of self-assembled nanometer hemorrhage, comprises the following steps: first will
Hydrophilic cationic polymer is dissolved in a certain amount of intensive polar solvent, obtains polymer solution, will contain carboxyl
Hydrophobic compound be dissolved in water-miscible organic solvent, then organic by dissolved with carboxylic hydrophobic compound
Solution is slowly added in polymer solution under ultrasonication, is placed in bag filter by above-mentioned mixed liquor afterwards,
Under magnetic agitation, organic solvent is removed in dialysis in deionized water, i.e. can get self-assembled nanometer hemorrhage.
Wherein, above-mentioned intensive polar solvent selected from deionized water, dimethyl sulfoxide, DMF,
DMAC N,N' dimethyl acetamide.Water-miscible organic solvent is selected from acetone, acetonitrile, ethanol, dimethyl sulfoxide, N, N-
Dimethylformamide, DMAC N,N' dimethyl acetamide, acetic acid.
The concentration of above polymer solution between 1mg/mL to 100mg/mL, carboxylic hydrophobic compound
The concentration of the organic solution of thing is between 1mg/mL to 100mg/mL.
Invention further provides the application of self-assembled nanometer hemorrhage, including by outside described nanometer hemostatic agent
For tissue local (including spraying, smearing or be externally applied to tissue local), or nanometer hemostatic agent is used for vein
Injection, reaches hemostasis or prevents hemorrhage purpose.
The present invention has the advantage that
1) hydrophilic cationic polymer used in the present invention all has commercial prod, and its synthesis is simple, valency
Lattice relative moderate, therefore it is easily achieved the industrialization of corresponding preparations.
2) self-assembly method that the present invention uses is simple, and the organic solvent used easily removes, it is ensured that
The feasibility of final nanometer hemostatic agent application and safety.
3) size of the nanometer hemostatic agent prepared by the present invention can be regulated and controled by preparation technology parameter.
4) the nanometer hemostatic agent prepared by the present invention is liquid, flexible and convenient to use.
5) stability of the nanometer hemostatic agent prepared by the present invention preferably, non-immunogenicity, it is ensured that internal peace
Quan Xing.
6) the nanometer hemostatic agent prepared by the present invention both can use with partial smearing, it is also possible to intravenous injection uses.
Accompanying drawing explanation
Fig. 1 is that molecular weight is the cladodification polymine of 25000Da and ursodesoxycholic acid is prepared by self assembly
The transmission electron microscope picture of the mass ratio obtained nanoparticle between 1:1 to 1:4.
Fig. 2 is blank group (0.9% normal saline) and treatment group (nanometer hemostatic agent) promotion platelet
The phase contrast microscope figure of coagulation, wherein scale is 10 μm.
Fig. 3 is blank group (0.9% normal saline) and treatment group (nanometer hemostatic agent) promotion platelet
The common focused view of coagulation, wherein scale is 5 μm.
Fig. 4 be rat femoral intravenous injection A (0.9% normal saline), B (Cy5 labelling ursodesoxycholic acid/
Polyethylene imine nanometer grain, the one section of femoral artery not processed) and C (Cy5 labelling ursodesoxycholic acid/poly-second
Alkene imine nanometer grain, one section of femoral artery puncturing of local) after solution, living imaging observes blood coagulation situation.
Fig. 5 be molecular weight be that the cladodification polymine of 25000Da and ursodesoxycholic acid are with 1:1's to 1:4
Nanometer hemostatic agent prepared by mass ratio is to mouse tail, rat femoral, liver and intravenous injection nanometer only
The impact in the bleeding time of femoral artery after blood agent.
Fig. 6 is that mouse tail, rat stock are moved by the nanometer hemostatic agent of variable concentrations (1,2 and 4mg/mL)
The impact in the bleeding time of femoral artery after arteries and veins, liver and intravenous injection nanometer hemostatic agent.
For different polymine, (molecular weight is cladodification polymine PEI1800 of 1800Da and divides Fig. 7
Son amount is the L-PEI PEI10000 of 10000Da) and the nanometer hemostatic agent prepared of ursodesoxycholic acid
To the bleeding time of femoral artery after mouse tail, rat femoral, liver and intravenous injection nanometer hemostatic agent
Impact.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the summary of the invention of the present invention is described in further detail.Should be understood that
Embodiments of the invention are served only for illustrating the present invention without limiting the present invention, without departing from the technology of the present invention thought
In the case of, according to ordinary skill knowledge and customary means, the various replacements made and change, all
Should be included within the scope of the invention.
The present invention is elaborated by the embodiment below in conjunction with indefiniteness.In self assembling process, permissible
Control the particle diameter of nanometer hemostatic agent between 20nm to 800nm.
Embodiment 1
The cladodification polymine that 5mg molecular weight is 25000Da is dissolved in 5mL deionized water, polymerization
The concentration of thing solution is 1mg/mL；25mg ursodesoxycholic acid is dissolved in 1mL dimethyl sulfoxide, wherein
The concentration of ursodesoxycholic acid is 25mg/mL；Under ul-trasonic irradiation, by molten for the acetone of 1mL ursodesoxycholic acid
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 200～500nm.
Embodiment 2
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL dimethyl sulfoxide,
The concentration of polymer solution is 10mg/mL；Being dissolved in 1mL acetonitrile by 1mg cholic acid, wherein cholic acid is dense
Degree is 1mg/mL；Under ul-trasonic irradiation, the acetonitrile solution of 1mL cholic acid is slowly added in polymer solution；
Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da, under magnetic agitation in deionized water
Dialysis, temperature 20 DEG C, change deionized water every 30min；Collect dialysis solution after 24h i.e. can be contained
The aqueous solution of self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent is between 200-400nm.
Embodiment 3
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL N, N-dimethyl formyl
In amine, the concentration of polymer solution is 10mg/mL；2mg chenodeoxycholic acid is dissolved in 1mL ethanol,
Wherein the concentration of chenodeoxycholic acid is 2mg/mL；Under ul-trasonic irradiation, by the ethanol of 1mL chenodeoxycholic acid
Solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the dialysis that molecular cut off is 10000Da
In Dai, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 150-300nm.
Embodiment 4
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL N, N-dimethylacetamide
In amine, the concentration of polymer solution is 10mg/mL；5mg Hyodeoxycholic Acid is dissolved in 1mL dimethyl sub-
In sulfone, wherein the concentration of Hyodeoxycholic Acid is 5mg/mL；Under ul-trasonic irradiation, by 1mL Hyodeoxycholic Acid
Dimethyl sulphoxide solution be slowly added in polymer solution；Above-mentioned mixed liquor is placed in molecular cut off is
In the bag filter of 8000Da, dialysing in deionized water under magnetic agitation, temperature 20 DEG C, every 30min
Change deionized water；Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage,
The particle diameter of nanometer hemostatic agent is between 100-300nm.
Embodiment 5
The cladodification polymine that 10mg molecular weight is 1800Da is dissolved in 1mL DMAC N,N' dimethyl acetamide
In, the concentration of polymer solution is 10mg/mL；10mg lithocholic acid is dissolved in 1mL N, N-dimethyl methyl
In amide, wherein the concentration of lithocholic acid is 10mg/mL；Under ul-trasonic irradiation, by the N of 1mL lithocholic acid, N-
Dimethyl formamide solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in molecular cut off is
In the bag filter of 3500Da, dialysing in deionized water under magnetic agitation, temperature 20 DEG C, every 30min
Change deionized water；Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage,
The particle diameter of nanometer hemostatic agent is between 100-200nm.
Embodiment 6
The cladodification polymine that 10mg molecular weight is 800Da is dissolved in 1mL dimethyl sulfoxide, poly-
The concentration of polymer solution is 10mg/mL；20mg deoxycholic acid is dissolved in 1mL dimethyl sulfoxide, its
The concentration of middle deoxycholic acid is 20mg/mL；Under ul-trasonic irradiation, the dimethyl of 1mL deoxycholic acid is sub-
Sulfolane solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the dialysis that molecular cut off is 3500Da
In Dai, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 200-300nm.
Embodiment 7
The cladodification polymine that 10mg molecular weight is 500Da is dissolved in 1mL dimethyl sulfoxide, polymerization
The concentration of thing solution is 10mg/mL；30mg dehydrocholic acid is dissolved in 1mL dimethyl sulfoxide, wherein goes
The concentration of hydrogen cholic acid is 30mg/mL；Under ul-trasonic irradiation, by molten for the dimethyl sulfoxide of 1mL dehydrocholic acid
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 150-250nm.
Embodiment 8
The L-PEI that 10mg molecular weight is 2500Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；20mg ibuprofen is dissolved in 1mL acetone, wherein ibuprofen
Concentration is 20mg/mL；Under ul-trasonic irradiation, the acetone soln of 1mL ibuprofen is slowly added to polymer
In solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da, is going under magnetic agitation
Ionized water is dialysed, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
Obtaining the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent is between 200-300nm.
Embodiment 9
The L-PEI that 10mg molecular weight is 5000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；20mg ketoprofen is dissolved in 1mL acetonitrile, wherein ketoprofen
Concentration is 20mg/mL；Under ul-trasonic irradiation, the acetonitrile solution of 1mL ketoprofen is slowly added to polymer
In solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da, is going under magnetic agitation
Ionized water is dialysed, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
Obtaining the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent is between 100-200nm..
Embodiment 10
The L-PEI that 20mg molecular weight is 5000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 20mg/mL；60mg fenoprofen is dissolved in 1mL dimethyl sulfoxide, Qi Zhongfei
The concentration of promise ibuprofen is 60mg/mL；Under ul-trasonic irradiation, by molten for the dimethyl sulfoxide of 1mL fenoprofen
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 200-350nm.
Embodiment 11
The L-PEI that 20mg molecular weight is 20000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 20mg/mL；100mg flurbiprofen is dissolved in 1mL dimethyl sulfoxide, wherein
The concentration of flurbiprofen is 100mg/mL；Under ul-trasonic irradiation, by the dimethyl sulfoxide of 1mL flurbiprofen
Solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 300-450nm.
Embodiment 12
The L-PEI that 20mg molecular weight is 10000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 20mg/mL；80mg oxaprozin is dissolved in 1mL dimethyl sulfoxide, Qi Zhongao
The concentration of Sha Puqin is 80mg/mL；Under ul-trasonic irradiation, by molten for the dimethyl sulfoxide of 1mL oxaprozin
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 300-480nm.
Embodiment 13
The cladodification polymine that 50mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 50mg/mL；50mg naproxen is dissolved in 1mL dimethyl sulfoxide, wherein Nabumetone
Raw concentration is 50mg/mL；Under ul-trasonic irradiation, by slow for the dimethyl sulphoxide solution of 1mL naproxen
Add in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da, magnetic force
Dialyse in deionized water under stirring, temperature 20 DEG C, change deionized water every 30min；Collect after 24h
Dialysis solution i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, and the particle diameter of nanometer hemostatic agent is at 100-200
Between nm.
Embodiment 14
The cladodification polymine that 80mg molecular weight is 1800Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 80mg/mL；40mg indomethacin is dissolved in 1mL N,N-dimethylacetamide,
Wherein the concentration of indomethacin is 40mg/mL；Under ul-trasonic irradiation, by the N of 1mL indomethacin, N-bis-
Methyl vinyl amine aqueous solution is slowly added in polymer solution；It is 3500 that above-mentioned mixed liquor is placed in molecular cut off
In the bag filter of Da, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change every 30min
Deionized water；Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, nanometer
The particle diameter of hemorrhage is between 150-250nm.
Embodiment 15
The cladodification polymine that 100mg molecular weight is 50000Da is dissolved in 1mL deionized water, poly-
The concentration of polymer solution is 100mg/mL；100mg sulindac is dissolved in 1mL dimethyl sulfoxide, wherein
The concentration of sulindac is 100mg/mL；Under ul-trasonic irradiation, by the dimethyl sulphoxide solution of 1mL sulindac
It is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da,
Dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；After 24h
Collecting dialysis solution and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 100-200nm.
Embodiment 16
The cladodification polymine that 10mg molecular weight is 1800Da is dissolved in 1mL dimethyl sulfoxide, poly-
The concentration of polymer solution is 10mg/mL；30mg etodolac is dissolved in 1mL acetone, wherein degree of support
The concentration of acid is 30mg/mL；Under ul-trasonic irradiation, the acetone soln of 1mL etodolac is slowly added to
In polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da, magnetic agitation
Under dialyse in deionized water, temperature 20 DEG C, every 30min change deionized water；Dialysis is collected after 24h
Liquid i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, and the particle diameter of nanometer hemostatic agent is at 150-250nm
Between.
Embodiment 17
The cladodification polymine that 10mg molecular weight is 1800Da is dissolved in 1mL dimethyl sulfoxide, poly-
The concentration of polymer solution is 10mg/mL；25mg mefenamic acid is dissolved in 1mL acetonitrile, wherein mefenamic acid
Concentration is 25mg/mL；Under ul-trasonic irradiation, the acetonitrile solution of 1mL mefenamic acid is slowly added to polymer
In solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da, is going under magnetic agitation
Ionized water is dialysed, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
Obtaining the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent is between 150-250nm.
Embodiment 18
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL dimethyl sulfoxide, poly-
The concentration of polymer solution is 10mg/mL；25mg meclofenamic acid is dissolved in 1mL dimethyl sulfoxide, its
The concentration of middle meclofenamic acid is 25mg/mL；Under ul-trasonic irradiation, by the diformazan of 1mL meclofenamic acid
Base sulfoxide solution is slowly added in polymer solution；It is 3500Da that above-mentioned mixed liquor is placed in molecular cut off
Bag filter in, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, every 30min change go from
Sub-water；Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, nanometer hemostatic
The particle diameter of agent is between 140-200nm.
Embodiment 19
The PLL hydrobromate that 10mg molecular weight is 2000Da is dissolved in 1mL deionized water
In, the concentration of polymer solution is 10mg/mL；20mg flufenamic acid is dissolved in 1mL dimethyl sulfoxide,
Wherein the concentration of flufenamic acid is 20mg/mL；Under ul-trasonic irradiation, by the dimethyl of 1mL flufenamic acid
Sulfoxide solution is slowly added in polymer solution；It is the saturating of 3500Da that above-mentioned mixed liquor is placed in molecular cut off
In analysis bag, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 170-230nm.
Embodiment 20
The PLL hydrobromate that 10mg molecular weight is 5000Da is dissolved in 1mL deionized water
In, the concentration of polymer solution is 10mg/mL；10mg tolfenamic acid is dissolved in 1mL dimethyl sulfoxide,
Wherein the concentration of tolfenamic acid is 10mg/mL；Under ul-trasonic irradiation, by the dimethyl sulfoxide of 1mL tolfenamic acid
Solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 200-400nm.
Embodiment 21
The PLL hydrobromate that 10mg molecular weight is 15000Da is dissolved in 1mL deionized water
In, the concentration of polymer solution is 10mg/mL；15mg lumiracoxib is dissolved in 1mL dimethyl sulfoxide,
Wherein the concentration of lumiracoxib is 15mg/mL；Under ul-trasonic irradiation, by the dimethyl of 1mL lumiracoxib
Sulfoxide solution is slowly added in polymer solution；It is the saturating of 7000Da that above-mentioned mixed liquor is placed in molecular cut off
In analysis bag, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 180-230nm.
Embodiment 22
Poly-(L-arginine) hydrochlorate that 10mg molecular weight is 15000Da is dissolved in 1mL deionized water,
The concentration of polymer solution is 10mg/mL；15mg Licofelone is dissolved in 1mL dimethyl sulfoxide, its
The concentration of middle Licofelone is 15mg/mL；Under ul-trasonic irradiation, the dimethyl of 1mL Licofelone is sub-
Sulfolane solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the dialysis that molecular cut off is 7000Da
In Dai, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 230-300nm.
Embodiment 23
Poly-(L-arginine) hydrochlorate that 20mg molecular weight is 15000Da is dissolved in 1mL deionized water,
The concentration of polymer solution is 20mg/mL；20mg diflunisal is dissolved in 1mL dimethyl sulfoxide, its
The concentration of middle diflunisal is 20mg/mL；Under ul-trasonic irradiation, the dimethyl of 1mL diflunisal is sub-
Sulfolane solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the dialysis that molecular cut off is 7000Da
In Dai, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 300-400nm.
Embodiment 24
Poly-(L-arginine) hydrochlorate that 10mg molecular weight is 15000Da is dissolved in 1mL deionized water,
The concentration of polymer solution is 10mg/mL；50mg aspirin is dissolved in 1mL dimethyl sulfoxide, its
The concentration of middle aspirin is 50mg/mL；Under ul-trasonic irradiation, the dimethyl of 1mL aspirin is sub-
Sulfolane solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the dialysis that molecular cut off is 7000Da
In Dai, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；
Collect dialysis solution after 24h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the grain of nanometer hemostatic agent
Footpath is between 200-300nm.
Embodiment 25
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；20mg gemfibrozil is dissolved in 1mL dimethyl sulfoxide, Qi Zhongji
The concentration of non-Betsy is 20mg/mL；Under ul-trasonic irradiation, by molten for the dimethyl sulfoxide of 1mL gemfibrozil
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 180-220nm.
Embodiment 26
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；10mg ciprofloxacin is dissolved in 1mL acetic acid, wherein ciprofloxacin
Concentration be 10mg/mL；Under ul-trasonic irradiation, it is slowly added to gather by the acetum of 1mL ciprofloxacin
In polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da, under magnetic agitation
Dialyse in deionized water, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
I.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent 180-250nm it
Between.
Embodiment 27
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；15mg norfloxacin is dissolved in 1mL acetic acid, wherein norfloxacin
Concentration be 15mg/mL；Under ul-trasonic irradiation, it is slowly added to gather by the acetum of 1mL norfloxacin
In polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da, under magnetic agitation
Dialyse in deionized water, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
I.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent 200-260nm it
Between.
Embodiment 28
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；20mg ofloxacin is dissolved in 1mL acetic acid, wherein ofloxacin
Concentration be 20mg/mL；Under ul-trasonic irradiation, it is slowly added to gather by the acetum of 1mL ofloxacin
In polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da, under magnetic agitation
Dialyse in deionized water, temperature 20 DEG C, change deionized water every 30min；Dialysis solution is collected after 24h
I.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent 180-160nm it
Between.
Embodiment 29
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL dimethyl sulfoxide, poly-
The concentration of polymer solution is 10mg/mL；15mg indomethacin is dissolved in 1mL dimethyl sulfoxide, wherein
The concentration of indomethacin is 15mg/mL；Under ul-trasonic irradiation, by the dimethyl sulfoxide of 1mL indomethacin
Solution is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 3500Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 20-60nm.
Embodiment 30
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；20mg dehydrocholic acid is dissolved in 1mL dimethyl sulfoxide, wherein goes
The concentration of hydrogen cholic acid is 20mg/mL；Under ul-trasonic irradiation, by molten for the dimethyl sulfoxide of 1mL dehydrocholic acid
Liquid is slowly added in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da
In, dialyse in deionized water under magnetic agitation, temperature 20 DEG C, change deionized water every 30min；24
Collecting dialysis solution after h and i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, the particle diameter of nanometer hemostatic agent exists
Between 500-600nm.
Embodiment 31
The cladodification polymine that 10mg molecular weight is 25000Da is dissolved in 1mL deionized water, polymerization
The concentration of thing solution is 10mg/mL；30mg lithocholic acid is dissolved in 1mL dimethyl sulfoxide, wherein stone gallbladder
The concentration of acid is 30mg/mL；Under ul-trasonic irradiation, by slow for the dimethyl sulphoxide solution of 1mL lithocholic acid
Add in polymer solution；Above-mentioned mixed liquor is placed in the bag filter that molecular cut off is 7000Da, magnetic force
Dialyse in deionized water under stirring, temperature 20 DEG C, change deionized water every 30min；Collect after 24h
Dialysis solution i.e. can obtain the aqueous solution containing self-assembled nanometer hemorrhage, and the particle diameter of nanometer hemostatic agent is at 700-800
Between nm.
In the nanometer hemostatic agent being prepared as described above, the transmission electron microscope picture of nanoparticle is as shown in Figure 1.
Fig. 2 is that blank group (0.9% normal saline) and treatment group (nanometer hemostatic agent) promote that platelet is coagulated
The phase contrast microscope figure of collection, wherein scale is 10 μm.Sprague-Dawley male rat (230-250g)
Taking blood after anesthesia, under room temperature, 1000rpm is centrifuged 10min, takes supernatant and can obtain platelet rich plasma (PRP),
Take 100 μ L PRP to put in 96 orifice plates, add 10 μ L ursodesoxycholic acid (UDCA) and molecular weight is 25000
Cladodification polymine self assembly (mass ratio is 2:1) the nanoparticle solution of Da is as stimulating (figure before blood coagulation
2 experimental grouies) or add normal saline as blank (Fig. 2 blank group).Sample adds peer
2% long-pending glutaraldehyde fixes 1h, observes hematoblastic poly-after incubated at room temperature 10min under phase contrast microscope
Collection state (Fig. 2).
Fig. 3 is blank group (0.9% normal saline) and treatment group (nanometer hemostatic agent) promotion platelet
The common focused view of coagulation, wherein scale is 5 μm.Precision measures the cladodification polyethyleneimine of 1mL 20mg/mL
The DMSO solution of amine PEI (MW=25000), adds 1mL 2.36mg/mL Cy5NHS ester's
DMSO solution, reacts 12h in 40 DEG C of lucifuges, obtains PEI-Cy5 solution.Then, PEI-Cy5 is drawn
The DMSO solution of the 20mg/mL UDCA of solution 1mL with 1mL mixes, and is placed in molecular weight 3500
Bag filter in, pure water dialysis 24h, obtain the nanoparticle of Cy5 labelling.The PRP taking 500 μ L adds 3
The THB (tyrode) of mL, with 50 μ L CD61/FITC labellings, after hatching 40min altogether under room temperature, 1600g
Centrifugal 5min, dilutes with 1.5mL PPP and i.e. obtains reconstruction platelet.Take 150 μ L and rebuild platelet addition 15
PEI/UDCA (10:20) nanoparticle solution or 15 μ L THB that μ L Cy5 modifies add as after blank
15 μ L formaldehyde are fixed；Copolymerization Jiao observes hematoblastic coherent condition (Fig. 3).
Fig. 4 be rat femoral intravenous injection A (0.9% normal saline), B (Cy5 labelling ursodesoxycholic acid/
Polyethylene imine nanometer grain, the one section of femoral artery not processed) and C (Cy5 labelling ursodesoxycholic acid/poly-second
Alkene imine nanometer grain, one section of femoral artery puncturing of local) after solution, living imaging observes blood coagulation situation.
After Sprague-Dawley male rat (230-250g) anesthesia, tail vein injection 0.5mL Cy5-PEI-UDCA
Nanoparticle or normal saline, expose rat femoral after 5min, moves with the needle pierces stock of 0.45mm
Arteries and veins, after stopping blooding, cuts femoral artery and observes fluorescence distribution.
Fig. 5 be molecular weight be that the cladodification polymine of 25000Da and ursodesoxycholic acid are with 1:1's to 1:4
Nanometer hemostatic agent prepared by mass ratio is to mouse tail, rat femoral, liver and intravenous injection nanometer only
The impact in the bleeding time of femoral artery after blood agent.
Fig. 6 is that mouse tail, rat stock are moved by the nanometer hemostatic agent of variable concentrations (1,2 and 4mg/mL)
The impact in the bleeding time of femoral artery after arteries and veins, liver and intravenous injection nanometer hemostatic agent.
For different polymine, (molecular weight is cladodification polymine PEI1800 of 1800Da and divides Fig. 7
Son amount is the L-PEI PEI10000 of 10000Da) and the nanometer hemostatic agent prepared of ursodesoxycholic acid
To the bleeding time of femoral artery after mouse tail, rat femoral, liver and intravenous injection nanometer hemostatic agent
Impact.
Foundation and the evaluation methodology of different Hemorrhage Models described in Fig. 5-7 are as follows:
1) mouse tail Hemorrhage Model and evaluation
20～25g C57BL mouses, after anesthesia, cut transversely cutting at anesthetized mice tail point 0.5cm by profit,
Suck with filter paper after blood flows out first automatically, Mouse Tail-tip is respectively placed in normal saline,
PEI25000, PEI1800, PEI10000 aqueous solution and different proportion (10:10,10:20,10:30 and 10:40),
Variable concentrations (1,2 and 4mg/mL) and the polymine (bPEI1800 and bPEI10000) of different molecular weight
Nanoparticle suspension, after mouse tail and nanoparticle suspension are fully contacted, take out and starts timing, every 15
Second filter paper sucks blood, until stopping bleeding completely, records the bleeding time.
2) rat femoral Hemorrhage Model and evaluation
Take 250～300g Sprague-Dawley rats, after anesthesia, make hind leg launch to expose femoribus internus,
Cutting skin, cut overlying muscle, expose femoral artery and sciatic nerve, femoral artery injury produces high pressure and makes
Mouse is bled profusely, after femoral artery injury respectively at arterial injury add normal saline, PEI25000,
PEI1800 and PEI10000 aqueous solution, different proportion (10:10,10:20,10:30 and 10:40), different
Concentration (1,2 and 4mg/mL) and the polymine (bPEI1800, bPEI10000) of different molecular weight
Nanoparticle suspension 300 μ L, records the bleeding time.
3) liver Hemorrhage Model and evaluation
Take 250～300g Sprague-Dawley rats, after anesthesia, expose after scalpel hara kiri
Liver, and laterally cut in the liver middle period from beak tail direction, make liver form the wound of an about 4mm,
Normal saline, PEI25000, PEI1800 and PEI10000 aqueous solution it is separately added at hepar damnification, different
Ratio (10:10,10:20,10:30 and 10:40), variable concentrations (1,2 and 4mg/mL) from different points
Polymine (bPEI1800 and bPEI10000) the nanoparticle suspension 300 μ L of son amount, records hemorrhage
Time.
4) femoral artery hemorrhage after the agent of intravenous injection nanometer hemostatic
Take 250～300g Sprague-Dawley rats, after anesthesia, tail vein injection 0.5mL normal saline,
PEI25000, PEI1800 and PEI10000 aqueous solution, different proportion (10:10,10:20,10:30 and 10:40),
Variable concentrations (1,2 and 4mg/mL) and the polymine (bPEI1800 and bPEI10000) of different molecular weight
Nanoparticle suspension.After 5min, rat femoral is exposed, with the needle pierces femoral artery of 0.45mm,
The record bleeding time.
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Claims (9)
1. a self-assembled nanometer hemorrhage, is characterized in that: described nanometer hemostatic agent is gathered by hydrophilic cationic
Compound and carboxylic hydrophobic compound composition, wherein said hydrophilic cationic polymer is dredged with carboxylic
The mass ratio of hydrate is between 1:0.1 to 1:5, and the particle diameter of described nanometer hemostatic agent is at 20nm extremely
Between 800nm.
The most according to claim 1, a kind of self-assembled nanometer hemorrhage, is characterized in that: described hydrophilic sun
Ionomer is selected from cladodification or L-PEI, PLL hydrobromate and poly-(L-essence ammonia
Acid) hydrochlorate, the molecular weight of described hydrophilic cationic polymer is between 500Da to 50000Da.
The most according to claim 1, a kind of self-assembled nanometer hemorrhage, is characterized in that: described carboxylic
Hydrophobic compound is selected from cholic acid, ursodesoxycholic acid, chenodeoxycholic acid, Hyodeoxycholic Acid, lithocholic acid, deoxidation
Cholic acid, dehydrocholic acid, ibuprofen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, naproxen,
Indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, sieve
U.S. former times cloth, Licofelone, diflunisal, aspirin, gemfibrozil, ciprofloxacin, norfloxacin and
Ofloxacin.
4. the preparation method of self-assembled nanometer hemorrhage described in any one of Claim 1-3, is characterized in that,
Comprise the following steps: first hydrophilic cationic polymer is dissolved in a certain amount of intensive polar solvent, obtains
Polymer solution, is dissolved in carboxylic hydrophobic compound in water-miscible organic solvent, then will be dissolved with containing carboxylic
The organic solution of the hydrophobic compound of base is slowly added in polymer solution under ultrasonication, afterwards by above-mentioned
Mixed liquor is placed in bag filter, and under magnetic agitation, organic solvent is removed in dialysis in deionized water, the most available
Self-assembled nanometer hemorrhage.
The most according to claim 4, the preparation method of self-assembled nanometer hemorrhage, is characterized in that: described by force
Polar solvent is selected from deionized water, dimethyl sulfoxide, N,N-dimethylformamide, DMAC N,N' dimethyl acetamide.
The most according to claim 4, the preparation method of self-assembled nanometer hemorrhage, is characterized in that: described water
Solubleness organic solvent is selected from acetone, acetonitrile, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, N, N-
Dimethyl acetylamide, acetic acid.
The most according to claim 4, the preparation method of self-assembled nanometer hemorrhage, is characterized in that: described poly-
The concentration of polymer solution between 1mg/mL to 100mg/mL, having of described carboxylic hydrophobic compound
The concentration of machine solution is between 1mg/mL to 100mg/mL.
8. the application of self-assembled nanometer hemorrhage described in any one of Claim 1-3, is characterized in that: by institute
State nanometer hemostatic agent outer for tissue local, or nanometer hemostatic agent is used for intravenous injection.
The most according to claim 8, the application of self-assembled nanometer hemorrhage, is characterized in that: described external bag
Include sprinkling, smear or external application.
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| CN112245406A (en) * | 2020-11-09 | 2021-01-22 | 重庆理工大学 | A kind of multifunctional hemostatic nanoparticle with low thrombosis risk, preparation method and application thereof |
| CN113943753A (en) * | 2021-10-11 | 2022-01-18 | 中国人民解放军陆军军医大学 | High-efficiency low-toxicity self-assembled cationic nanoparticles and preparation method and application thereof |
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| WO2001054802A1 (en) * | 2000-01-27 | 2001-08-02 | Hospal Industrie | Non-thrombogenic semipermeable membrane and method for making same |
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| 周兴: "基于酵母微囊构建新型口服巨噬细胞靶向递送系统的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112245406A (en) * | 2020-11-09 | 2021-01-22 | 重庆理工大学 | A kind of multifunctional hemostatic nanoparticle with low thrombosis risk, preparation method and application thereof |
| CN113943753A (en) * | 2021-10-11 | 2022-01-18 | 中国人民解放军陆军军医大学 | High-efficiency low-toxicity self-assembled cationic nanoparticles and preparation method and application thereof |
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