CN103386114A - Application of artificial platelet PLAG-PEG-RCD to preparing systemic nanometer styptic for veins - Google Patents
Application of artificial platelet PLAG-PEG-RCD to preparing systemic nanometer styptic for veins Download PDFInfo
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Abstract
The invention discloses a vein nanometer styptic PLAG-PEG-RCD for preventing and treating systemic haemorrhage comprising traumatic hemorrhages, organ internal hemorrhages, deep hemorrhages and operation hemorrhages, and a preparation technology. Experiments prove that the artificial platelet PLAG-PEG-RCD with RGD tripeptide loaded by PLGA-PEG nanometer particles has high hemostatic effect, is applicable to systemic nanometer styptic material for veins, and provides more medicament selection for hemostasis treatment on complex traumas, organ internal hemorrhages and surgical operations, and has a wide application prospect.
Description
Technical field
The present invention relates to a kind of artificial platelet of amphipathic macromolecular material load polypeptide, be specifically related to a kind of artificial platelet PLGA-PEG-RGD and preparation method thereof of novel PLGA-PEG nano-particle load RGD tripeptides with take it as the active fraction preparation vein with the application in general nanometer hemostatic medicine.
Background technology
The wound mortality rate that causes of losing blood greatly is all very high with wartime at ordinary times.Lose blood and can be divided into internal hemorrhage and external haemorrhage, trauma patient at ordinary times has the therapeutic time of " gold one hour ", and for the wounded on battlefield, only has " platinum 5 minutes ".Traditional wound hemostasis material (first-aid kit, four-tailed bandage, tourniquet and binder etc.) has solved the hemostasis problem of serious external haemorrhage substantially, and the internal hemorrhage major part is to cause because of Viscera rupture, because using the external hemostatic material, make wound rear " very first time " hemostasis possibility hardly, the incidence rate of hemorrhagic shock and the wounded's mortality rate are high.Therefore, how in the very first time, fast and effeciently give treatment to and lose blood greatly and the internal hemorrhage patient is the major issue for the treatment of of war wound.
Recombinant human blood coagulation factor VII (rFVII) is the representative of whole body with hemorrhage, but the shortcomings such as rFVII has, and molecular weight is large, production cost is high, expensive, expression is low, easy inactivation, difficult preservation, greatly limited its application (Nunez TC in treatment of war wound, Cotton BA.Transfusion therapy in hemorrhagic shock.Curr Opin Crit Care2009,15:536-541).In addition, platelet product, because of defects such as the donor source is limited, immunogenicity, difficult storage, easy inactivations, has also limited its application in emergency aid and treatment.
In recent years, researcher builds artificial hematoblastic research with macromolecular material load polypeptide becomes a new direction of nanometer hemostatic material.Lavik group has reported a kind of synthetic platelet (Bertram JP of the macromolecular material load styptic activity factor, Williams CA, Robinson R, et al.Intravenous Hemostat:Nanotechnology to Halt Bleeding.Sci Transl Med2009,1:1-8).This artificial platelet has been used the hydrophobic inner core of PLGA-PLL as macromolecule carrier, and PEG, as hydrophilic crown skin, is grafted to the GRGDS pentapeptide on PEG.By intravenously administrable, the nanometer hemostatic granule of load polypeptide can targeting be combined with the platelet of activation, promotes platelet aggregation and further excites clotting mechanism, the purpose of quick-acting haemostatic powder.(Shoffstall AJ in the femoral artery of mice is subjected to the dirty internal hemorrhage model of wound model regulating liver-QI, Atkins KT, Groynom RE, et al.Intravenous hemostatic nanoparticles increase survival following blunt trauma injury.Biomacromolecules2012,13:3850-3857) all shown good haemostatic effect.In addition, this artificial platelet stable existence at room temperature.Combine with clot in thrombosed process; In 24 hours, artificial platelet is absorbed fully; In 7 days after use, do not find obvious side effect.But there is the shortcoming of synthetic method relative complex in this artificial platelet.
Summary of the invention
Under above-mentioned background, the present invention utilizes high molecular polymer PLGA (FDA ratifies pharmaceutic adjuvant), PEG and the design of RGD tripeptides to synthesize a kind of novel vein general nanometer hemostatic medicine, therefore, the object of the invention is to provide the artificial platelet PLGA-PEG-RGD of a kind of novel PLGA-PEG block copolymer load RGD.
The artificial platelet PLGA-PEG-RGD of PLGA-PEG block copolymer load RGD provided by the present invention, connect RGD by high molecular polymer poly lactic-co-glycolic acid (PLGA) connection Polyethylene Glycol (PEG) again and obtain.
Its molecular structural formula is suc as formula shown in I,
Wherein, x represents the number of lactic acid in PLGA, x=233-400; Y represents the number of hydroxyacetic acid in PLGA, y=233-400; N represents Polyethylene Glycol (CH
2-CH
2-O) the number of fragment, n=20-134.
Described PLGA model is preferably Resomer503H (50: 50lactic to glycolic acid ratio, Mw=25kDa); The PEG model is preferably the different Polyethylene Glycol (NH of functional group
2-PEG-COOH, Mw=3400Da).
Described artificial platelet PLGA-PEG-RGD is nano-particle.
The present invention also provides a kind of method for preparing above-mentioned artificial platelet PLGA-PEG-RGD, can comprise the following steps:
Step 1: PLGA-NHS's is synthetic
According to following chemical equation, with the PLGA (Resomer503H) of end carboxyl and the synthetic Acibenzolar PLGA-NHS of NHS;
In formula, the restriction of x, y as hereinbefore;
Step 2: PLGA-PEG block copolymer PLGA-PEG-COOH's is synthetic
Polyethylene Glycol (NH with Acibenzolar PLGA-NHS and different modified with functional group
2-PEG-COOH) by amido link, connect the block copolymer PLGA-PEG-COOH of the PLGA-PEG that obtains holding carboxyl;
In formula, the restriction of n as hereinbefore;
Step 3: PLGA-PEG-NHS's is synthetic
According to following chemical equation, the end carboxyl of PLGA-PEG block copolymer PLGA-PEG-COOH is obtained corresponding Acibenzolar PLGA-PEG-NHS with the NHS reaction under the EDC effect;
Step 4: PLGA-PEG-RGD's is synthetic
According to following chemical equation, with Acibenzolar PLGA-PEG-NHS and RGD tripeptides (arginyl-glycyl-aspartic acid, end amino Arg-Gly-Asp) docks and obtains PLGA-PEG-RGD under alkali condition;
Step 5: the preparation of nano-particle and collection
The preparation method of Nano microsphere is: with the PLGA-PEG-RGD of step 4 preparation be dissolved in can be miscible with water solvent, during the macromolecular solution that will obtain under ultrasonication slowly is added dropwise to pure water solution, at room temperature stir and evaporate into the formation suspension, drip again polyacrylic acid solution (1g/100ml), there is flocculent deposit to separate out, standing, centrifugal, the nano-particle solid is used distilled water wash three times again, be the aqueous solution of 5-25mg/mL (being preferably 10mg/mL) with this solid preparation solubility, use the liquid nitrogen quick freezing, lyophilization obtains the powder nanometer granule.
A present invention also purpose is to provide a kind of vein general nanometer hemostatic medicine, and its active component is described artificial platelet PLGA-PEG-RGD.This medicine is intravenous fluid, with normal saline or PBS, forms after with the PLGA-PEG-RGD nanoparticulate dispersed.
Disperse to comprise following process: with normal saline (0.9%NaCl solution) or PBS (formula: 1.75mM KH
2PO
4, Na
2HPO
4, 10.06mM NaCl, 2.68mM KCl, pH7.2-7.4) and suspended nano microsphere to its concentration is 5-25mg/mL, and ultra-sonic dispersion (200-300 watt, 60-120min), obtains the dispersion liquid of Nano microsphere.
In dispersion liquid, Nano microsphere concentration is preferably 20mg/mL, and ultra-sonic dispersion is preferably ultrasonic 60min under 250 watts.
Described artificial platelet PLGA-PEG-RGD loses blood greatly and the vein of internal hemorrhage also belongs to the present invention with the application in hemorrhage for general in preparation.
Comprise described artificial platelet PLGA-PEG-RGD or described vein and also belong to content of the present invention with the first-aid kit of general nanometer hemostatic medicine.
To sum up, the invention discloses a kind of hemorrhagely for prevention and treatment general, comprise that wound is lost blood, the vein of internal organs internal hemorrhage, Hemorrhage in Deep and operative hemorrhage is with nanometer hemostatic medicine PLGA-PEG-RGD and preparation technology thereof.The present invention has the following advantages:
1) be applicable to the hemostasis that general is lost blood, especially to the treatment that on internal organs internal hemorrhage and battlefield, perforating wound is hemorrhage, provide new Therapeutic Method;
2) intravenous administration is suitable for the trauma care of complex condition, such as improving individual soldier's self and mutual medical aid ability on battlefield, and the wounded's self-saving ability in the major accidents such as raising traffic accident; Has simultaneously the prophylactic treatment dual-use function;
3) the RGD nano-particle stable performance in the present invention, can be made into lyophilized powder and use, and is beneficial to preservation, is adapted at using under field condition,, such as equipping our troops, improves complicated war trauma care ability;
4) cheap: as to have the haemostatic effect that is better than rFVII, but overcome the defects such as biological product (such as the rFVII factor) are expensive, expression is low, easy inactivation, difficult preservation;
5) do not have immunogenicity, can not propagate infectious disease;
6) preparation method is simple, and is easy to operate.The present invention compares with the artificial platelet of PLGA-PLL-PEG grafting GRGDS pentapeptide well known in the prior art the progress that two aspects are arranged: the synthesis step of a. macromolecule carrier is simplified, use the PLGA-PEG block copolymer to replace PLGA-PLL-PEG---the synthetic of PLGA-PEG block copolymer comprises: the PLGA of end carboxyl obtains corresponding Acibenzolar with the NHS activation, this Acibenzolar obtains the PLGA-PEG block copolymer with the amino reaction of Polyethylene Glycol under alkali condition, only need two-step reaction; And the synthetic of PLGA-PLL-PEG comprises: the PLGA of end carboxyl is connected and has obtained PLGA-PLL by amido link with the polylysine of benzyloxycarbonyl group protection; slough protecting group with hydrogen bromide/acetum again and obtain holding amino PLGA-PLL; PEG first activates with carbonyl dimidazoles; further with the PLGA-PLL of end amino, be connected and obtained PLGA-PLL-PEG, need altogether four-step reaction.B. use the RGD tripeptides to replace the GRGDS pentapeptide, saved synthetic cost.
In sum, the artificial platelet PLGA-PEG-RGD of PLGA-PEG nano-particle load RGD tripeptides of the present invention has efficient haemostatic effect, can be used as vein general nanometer hemostatic material,, for complexity war wound, dirty phase internal hemorrhage and operating hemostasis treatment provide more medicament selection, have a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Description of drawings
Fig. 1 is the size that dynamic light scattering (DLS) detects the PLGA-PEG-RGD nano-particle
Fig. 2 is the result of scanning electron microscopic observation PLGA-PEG-RGD nano-particle
Fig. 3 is PBS, rgd peptide, the PLGA-PEG-RGD nano-particle impact on clotting time
Fig. 4 is the TEG figure as a result of representational a certain person-portion
Fig. 5 is the tangent plane of bleeding wounds after the Liver Damage in Rats intravenously administrable
Fig. 6 is the variation of blood pressure after the Liver Damage in Rats intravenously administrable
The specific embodiment
RGD is the Arg-Gly-Asp aminoacid sequence, is present in the most of attachment proteinses of human body, is the main recognition site of the special receptor protein interaction of attachment proteins and cell surface.The formation of thrombosis starts from hematoblastic gathering.Hematoblastic gathering needs the special integrin GP II b-III a of platelet and the interaction that contains the solubility blood protein (as Fibrinogen) of RGD sequence, and the latter gathers together platelet aggregation as adaptin.Participate in Fibrinogen (being thrombin F I), fibronectin (FN) and the Feng-von willebrand's factor (von Willebrand Factor, vWF) etc. of coagulation process, all contain conservative RGD aminoacid sequence district., because fibrinogenic RGD sequence is combined with being positioned at activated blood platelet GP II b/IIIa, be platelet aggregation and thrombotic co-channel.Therefore, the RGD sequence is platelet aggregation and the pass key sequence that sticks.
Based on above analysis, the present invention proposes a kind of artificial platelet of novel PLGA-PEG nano-particle load RGD tripeptides, called after PLGA-PEG-RGD.
Artificial platelet PLGA-PEG-RGD provided by the present invention, its molecular structural formula be suc as formula shown in I, is to connect Polyethylene Glycol (PEG) by high molecular polymer poly lactic-co-glycolic acid (PLGA), then connects that the RGD amino acid residue sequence obtains.Its structural formula is suc as formula shown in I:
Wherein, x represents the number of lactic acid in PLGA, x=233-400; Y represents the number of hydroxyacetic acid in PLGA, y=233-400; N represents Polyethylene Glycol (CH
2-CH
2-O) the number of fragment, n=20-134.
The model of described high molecular polymer poly lactic-co-glycolic acid (PLGA) is preferably Resomer503H (50: 50lactic to glycolic acid ratio, Mn=25kDa); The model of Polyethylene Glycol (PEG) is preferably the different Polyethylene Glycol (NH of functional group
2-PEG-COOH, Mw=3400Da); RGD is (arginyl-glycyl-aspartic acid, Arg-Gly-Asp) tripeptides.
The present invention also provides a kind of method for preparing above-mentioned artificial platelet PLGA-PEG-RGD.
The raw material that uses in described preparation method all can be bought from the market, and those skilled in the art can preparation in accordance with the present invention prepare described any one compound.
The method of the artificial platelet PLGA-PEG-RGD of preparation provided by the present invention can comprise the following steps:
Step 1: PLGA-NHS's is synthetic
According to following chemical equation, with the PLGA (Resomer503H) of end carboxyl and the synthetic Acibenzolar PLGA-NHS of NHS (N-maloyl imines);
X, y implication are identical with aforementioned restriction.
Step 2: PLGA-PEG block copolymer PLGA-PEG-COOH's is synthetic
Polyethylene Glycol (NH with Acibenzolar PLGA-NHS and different modified with functional group
2-PEG-COOH) by amido link, connect the block copolymer PLGA-PEG-COOH of the PLGA-PEG that obtains holding carboxyl;
The implication of n is identical with aforementioned restriction.
Step 3: PLGA-PEG-NHS's is synthetic
According to following chemical equation, the end carboxyl of PLGA-PEG block copolymer PLGA-PEG-COOH is obtained corresponding Acibenzolar PLGA-PEG-NHS with the NHS reaction under EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) effect;
Step 4: PLGA-PEG-RGD's is synthetic
According to following chemical equation, with Acibenzolar PLGA-PEG-NHS and RGD tripeptides (arginyl-glycyl-aspartic acid, end amino Arg-Gly-Asp) docks and obtains PLGA-PEG-RGD under alkali condition;
Step 5: preparation and the collection of Nano microsphere (nano-particle)
The preparation method of Nano microsphere is: with the PLGA-PEG-RGD of step 4 preparation be dissolved in can be miscible with water solvent, in under ultrasonication, this macromolecular solution slowly being added dropwise to pure water solution, at room temperature stir volatilization and cause the formation of nano-particle, re-use hydrogen bond flocking settling method and obtain cotton-shaped nanoparticle aggregate thing, use and filter or centrifugal method collection, obtain the PLGA-PEG-RGD nano-particle.
Experimental results show that, artificial platelet PLGA-PEG-RGD nano-particle has efficient haemostatic effect, can it be active fraction preparation vein general nanometer hemostatic medicine, this hemorrhage can be used for prevention and the treatment general is hemorrhage, comprises that wound is lost blood, internal organs internal hemorrhage, Hemorrhage in Deep and operative hemorrhage etc.
vein provided by the present invention general nanometer hemostatic medicine, need just can to carry out intravenous injection with normal saline or PBS after with the PLGA-PEG-RGD nanoparticulate dispersed, hemorrhage for prevention and treatment general, the preparation of general nanometer hemostatic medicine of described vein, comprise the process of nanoparticulate dispersed: with normal saline (0.9%NaCl solution) or 0.1M PBS (formula: 1.75mM KH2PO4, Na2HPO4, 10.06mM NaCl, 2.68mM KCl, pH7.2-7.4) suspension PLGA-PEG-RGD nano-particle (5-25mg/mL, be preferably 20mg/mL), and ultra-sonic dispersion (200-300 watt, 60-120min, be preferably 250 watts, 60min), obtain nanoparticulate dispersion.
Can be hemorrhage for prevention and treatment general by the PLGA-PEG-RGD nanoparticulate dispersion that intravenous mode will obtain, injection volume is generally 30-50mg/kg.
Described PLGA-PEG-RGD nano-particle or the vein that also can be used as that packages nanoparticulate dispersion are provided in first-aid kit with general nanometer hemostatic medicine, are used for first aid or equip our troops.
In following embodiment, method therefor is conventional method if no special instructions, concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
Described percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/V) percent concentration if no special instructions.
Be described in embodiment the approach that obtains of various biomaterials only be to provide approach that a kind of experiment obtains to reach concrete disclosed purpose, should not become the restriction to biological material source of the present invention.In fact, the source of biomaterial used is widely, any keep on the right side of the law and the moral ethics biomaterial that can obtain can be replaced and use according to the prompting in embodiment.
Embodiment implements under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, and embodiment will help to understand the present invention, but protection scope of the present invention is not limited to following embodiment.
The artificial platelet PLGA-PEG-RGD of embodiment 1, preparation LGA-PEG load RGD tripeptides
The present embodiment is described the process of the artificial platelet PLGA-PEG-RGD of preparation LGA-PEG load RGD tripeptides in detail:
Step 1: PLGA-NHS's is synthetic
according to following chemical equation, with the PLGA (Resomer503H) of end carboxyl and the synthetic Acibenzolar PLGA-NHS of NHS, concrete synthetic method is: under nitrogen protection, with PLGA (Resomer503H, x=272, y=272) 3g is dissolved in the anhydrous methylene chloride of 10mL, add again EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) 76 μ L (4.3equiv) and NHS (N-maloyl imines) 46mg (4equiv), stirring at room 6 hours, reactant liquor is concentrated into 2-3mL, add the ether sedimentation, standing, filter, obtain the thick product of white, thick product dichloromethane-ether sedimentation 3 times, vacuum drying, obtain 2.6 gram white solids,
Step 2: PLGA-PEG block copolymer PLGA-PEG-COOH's is synthetic
Polyethylene Glycol (NH with Acibenzolar PLGA-NHS and different modified with functional group
2-PEG-COOH) by amido link, connecting the block copolymer PLGA-PEG-COOH of the PLGA-PEG that obtains holding carboxyl, concrete synthetic method is: under nitrogen protection, PLGA-NHS1.5g is dissolved in the anhydrous chloroform of 6mL, then adds NH
2-PEG-COOH (n=72, molecular weight 3400) 200mg (1.3equiv) and N, N '-diisopropylethylamine 30 μ L (4equiv), stirring at room 48 hours, be concentrated into 2-3mL with reactant liquor, add and freeze the methanol sedimentation, standing, filter and obtain the thick product of white, with chloroform-methanol sedimentation 3 times of thick product, vacuum drying, obtain 1 gram white solid;
Step 3: PLGA-PEG-NHS's is synthetic
according to following chemical equation, the end carboxyl of PLGA-PEG block copolymer PLGA-PEG-COOH is obtained corresponding Acibenzolar PLGA-PEG-NHS with the NHS reaction under the EDC effect, concrete synthetic method is: under nitrogen protection, PLGA-PEG-COOH1g is dissolved in the anhydrous methylene chloride of 10mL, add again EDC26 μ L (5equiv) and NHS12mg (4equiv), stirring at room 6 hours, reactant liquor is concentrated into 2-3mL, add the ether sedimentation, standing, filtration obtains the thick product of white, with the dichloromethane-ether sedimentation 3 times of thick product, vacuum drying, obtain 0.7 gram white solid,
Step 4: PLGA-PEG-RGD's is synthetic
according to following chemical equation, with Acibenzolar PLGA-PEG-NHS and RGD tripeptides (arginyl-glycyl-aspartic acid, Arg-Gly-Asp) end amino docks and obtains PLGA-PEG-RGD under alkali condition, concrete synthetic method is: under nitrogen protection, PLGA-PEG-NHS0.5g is dissolved in the dry DMF (N of 6mL, dinethylformamide), add again RGD20mg (4equiv) and N, N '-diisopropylethylamine 22 μ L (9equiv), stirring at room 48 hours, with the reactant liquor bag filter of packing into, with the distilled water 12h that dialyses, liquid freezing in bag filter is dry, obtain white solid 0.38g,
Step 5: preparation and the collection of nano-particle (or Nano microsphere)
50mg PLGA-PEG-RGD is dissolved in the 5mL acetonitrile, then slowly be added drop-wise in the distilled water of 50mL, the ultra-sonic dispersion while dripping (power is 250 watts), after dripping, continue to stir 3h, then drip polyacrylic acid solution (1g/100ml), there is flocculent deposit to separate out (nanoparticle aggregate thing), standing, centrifugal, solid is used three nanoparticle aggregate things of distilled water wash again; Be the aqueous solution of 5-25mg/mL (being preferably 10mg/mL) with this nanoparticle aggregate thing preparation solubility, use the liquid nitrogen quick freezing, lyophilization obtains the powdery nano-particle.
Detect through nuclear-magnetism and elementary analysis, conclusive evidence is the high molecular polymer of PLGA-PEG block copolymer load RGD tripeptides with the white solid that said method preparation process four obtains, it connects Polyethylene Glycol (PEG) by high molecular polymer poly lactic-co-glycolic acid (PLGA), connect again RGD aminoacid (arginyl-glycyl-aspartic acid, Arg-Gly-Asp).Its molecular structural formula is:
In addition, according to aforesaid operations, the value of x and y in feed change PLGA, the value of n in PEG, can obtain PLGA-PEG-RGD white solid and artificial platelet PLGA-PEG-RGD nano-particle equally.Empirical tests, represent the x=233-400 of lactic acid number in PLGA, represents the y=233-400 of hydroxyacetic acid number in PLGA, represents the n=20-134 of (CH2-CH2-O) fragment number of Polyethylene Glycol.Therefore, the universal architecture of the present invention one artificial platelet PLGA-PEG-RGD of class is suc as formula shown in I:
The sign of embodiment 2, PLGA-PEG-RGD nano-particle
Embodiment 1 is obtained nano-particle is suspended in PBS buffer solution (concentration is 5-25mg/mL, is preferably 20mg/mL), by ultra-sonic dispersion (power is 250 watts), again obtain Nano microsphere solution for detection of.
By the effective diameter of this PLGA-PEG-RGD nano-particle of Dynamic Light Scattering Determination, by the pattern of scanning electron microscopic observation nano-particle.While using acetonitrile, under the ultrasonication of sonicator, what obtain is nano level granule.(abscissa represents the diameter of the PLGA-PEG-RGD that obtains nano-particle for the test result of dynamic light scattering (DLS) such as Fig. 1, vertical coordinate represents the quantity that detects) shown in, effective grain size is 91.3nm, confirms that the size of PLGA-PEG-RGD nano-particle is nano level; The stereoscan photograph of PLGA-PEG-RGD nano-particle as shown in Figure 2, due to the scope of nano-particle generally at 1-100nm, thereby confirm that further the size of PLGA-PEG-RGD nano-particle is nano level.
The external coagulation function of embodiment 3, PLGA-PEG-RGD Nano microsphere granule detects
Thrombelastogram (thrombelastography, TEG) be blood coagulation analyzer for whole blood sample, as a kind of detection method of measuring coagulation function, observe the dynamic change of Blood Coagulation Process, comprise thrombin, platelet, fibrin and Fibrinolytic whole process.Can analyze whether balance of blood coagulation system by the blood coagulation situation of measuring each stage.R value in TEG is the blood coagulation response time, refers to that blood sample comes into operation can detect to first the blood clot that obtains and form the required time, i.e. fibrin time of starting to form.Extract healthy volunteer's venous blood (the 3.8% fresh anticoagulation of sodium citrate) 10mL, do TEG thrombelastogram (U.S. Haemoscope company).Establish respectively the PBS matched group, rgd peptide group (0.1,0.2,2.0mg/mL), PLGA-PEG-RGD group (nanoparticulate dispersion, two groups of minute 2.5mg/mL, 5mg/mL).
The testing result of TEG as shown in Figure 3, the TEG result of representational a certain person-portion as shown in Figure 4, in Fig. 4, A) PBS matched group; B) PLGA-PEG-RGD group.Thrombelastogram result (TEG) statistical table of PBS matched group, rgd peptide and PLGA-PEG-RGD nano-particle is as shown in table 1, can find out, the clotting time of PLGA-PEG-RGD was 4.6 minutes, less than the standard of 5-10 minute; It is 2.2 minutes that clot forms speed, between 1-3 minute standard; The α angle is 60.3 degree, between the 53-72 degree; Maximum clot thickness is 54mm, between 50-70mm; LY30 and EPL fail to detect; Compare with RGD (2mg/mL) with the PBS matched group, the PLGA-PEG-RGD that the present invention synthesizes (5mg/mL) group can significantly shorten clotting time, promotes blood coagulation, proves that the PLGA-PEG-RGD that the present invention synthesizes has the effect that promotes blood coagulation.
Thrombelastogram result (TEG) statistical table of table 1PBS matched group, rgd peptide group, PLGA-PEG-RGD nano-particle group
The preparation of embodiment 4, nanometer hemostatic medicine, the detection of the body intravascular coagulation ability of PLGA-PEG-RGD nano-particle
Vein of the present invention is with general nanometer hemostatic medicine, needs just can carry out intravenous injection with normal saline or PBS after with the PLGA-PEG-RGD nanoparticulate dispersed, and described vein with the preparation method of general nanometer hemostatic medicine, can comprise the following steps:
1) prepare the PLGA-PEG-RGD nano-particle by the method in embodiment 1;
2) dispersion of Nano microsphere: with normal saline (0.9%NaCl solution) or PBS (formula: 1.75mM KH
2PO
4, Na
2HPO
4, 10.06mM NaCl, 2.68mM KCl, pH7.2-7.4) and suspended nano granule (5-25mg/mL is preferably 20mg/mL), and ultra-sonic dispersion (200-300 watt, 60-120min are preferably 250 watts, 60min), obtain nanoparticulate dispersion.
Detect the body intravascular coagulation ability of PLGA-PEG-RGD Nano microsphere granule with following method: SD rat (male, 200g left and right), pentobarbital sodium anesthesia (50mg/kg).Cut open the belly, cut liver middle period 50% left and right, make liver internal hemorrhage model.Minute PBS matched group, RGD test group (500 μ l/ only, 0.2mg/mL), the PLGA-PEG-RGD experimental group (500 μ l/ only, 5mg/mL), the tail intravenously administrable.
After the Liver Damage in Rats intravenously administrable, the tangent plane of bleeding wounds is as shown in Fig. 5 (arrow is the clot forming position), can find out, the liver middle period cut surface of PBS matched group is smooth, form there are no grumeleuse, and in the incision of RGD and PLGA-PEG-RGD experimental group, all have grumeleuse to form, hemorrhage to reduce, and the blood clot that PLGA-PEG-RGD nano-particle experimental group forms is more larger, has better played Blood clotting.And with matched group, compare, the blood pressure of administration group 1-2min after losing blood is elevated to normal level (as shown in Figure 6) very soon.Can be found out by the above results, the PLGA-PEG-RGD nano-particle of PLGA-PEG load RGD tripeptides of the present invention has efficient haemostatic effect, can be as vein general nanometer hemostatic medicine.
Claims (10)
1. the artificial platelet PLGA-PEG-RGD of a PLGA-PEG block copolymer load RGD, connect RGD by high molecular polymer poly lactic-co-glycolic acid (PLGA) connection Polyethylene Glycol (PEG) again and obtain.
2. artificial platelet PLGA-PEG-RGD according to claim 1 is characterized in that: its molecular structural formula is suc as formula shown in I,
Wherein, x represents the number of lactic acid in PLGA, x=233-400; Y represents the number of hydroxyacetic acid in PLGA, y=233-400; N represents Polyethylene Glycol (CH
2-CH
2-O) the number of fragment, n=20-134.
3. artificial platelet PLGA-PEG-RGD according to claim 1 and 2, it is characterized in that: described PLGA model is preferably Resomer503H (50: 50 lactic to glycolic acid ratio, Mw=25kDa); The PEG model is preferably the different Polyethylene Glycol (NH of functional group
2-PEG-COOH, Mw=3400Da).
4. according to claim 1 and 2 or 3 described artificial platelet PLGA-PEG-RGD, it is characterized in that: described artificial platelet PLGA-PEG-RGD is nano-particle.
5. method for preparing claim 1 or 2 or 3 or 4 described artificial platelet PLGA-PEG-RGD comprises the following steps:
Step 1: PLGA-NHS's is synthetic
According to following chemical equation, with the PLGA (Resomer503H) of end carboxyl and the synthetic Acibenzolar PLGA-NHS of NHS;
In formula, the restriction of x, y is identical with claim 1;
Step 2: PLGA-PEG block copolymer PLGA-PEG-COOH's is synthetic
Polyethylene Glycol (NH with Acibenzolar PLGA-NHS and different modified with functional group
2-PEG-COOH) by amido link, connect the block copolymer PLGA-PEG-COOH of the PLGA-PEG that obtains holding carboxyl;
In formula, the restriction of n is identical with claim 1;
Step 3: PLGA-PEG-NHS's is synthetic
According to following chemical equation, the end carboxyl of PLGA-PEG block copolymer PLGA-PEG-COOH is obtained corresponding Acibenzolar PLGA-PEG-NHS with the NHS reaction under the EDC effect;
Step 4: PLGA-PEG-RGD's is synthetic
According to following chemical equation, with Acibenzolar PLGA-PEG-NHS and RGD tripeptides (arginyl-glycyl-aspartic acid, end amino Arg-Gly-Asp) docks and obtains PLGA-PEG-RGD under alkali condition;
Step 5: the preparation of nano-particle and collection
The preparation method of Nano microsphere is: with the PLGA-PEG-RGD of step 4 preparation be dissolved in can be miscible with water solvent, during the macromolecular solution that will obtain under ultrasonication slowly is added dropwise to pure water solution, at room temperature stir and evaporate into the formation suspension, drip again polyacrylic acid solution (1g/100ml), there is flocculent deposit to separate out, standing, centrifugal, the nano-particle solid is used distilled water wash three times again, be the aqueous solution of 5-25mg/mL (being preferably 10mg/mL) with this solid preparation solubility, use the liquid nitrogen quick freezing, lyophilization obtains the powder nanometer granule.
6. a vein is with general nanometer hemostatic medicine, and its active component is claim 1 or 2 or 3 or 4 described artificial platelet PLGA-PEG-RGD, is intravenous fluid, with normal saline or PBS, forms after with the PLGA-PEG-RGD nanoparticulate dispersed.
7. vein according to claim 6 is with general nanometer hemostatic medicine, and it is characterized in that: the dispersion of Nano microsphere comprises following process: with normal saline (0.9%NaCl solution) or PBS (formula: 1.75mM KH
2PO
4, Na
2HPO
4, 10.06mM NaCl, 2.68mM KCl, pH7.2-7.4) and suspended nano microsphere to its concentration is 5-25mg/mL, and ultra-sonic dispersion (200-300 watt, 60-120min), obtains the dispersion liquid of Nano microsphere.
8. vein according to claim 7 is with general nanometer hemostatic medicine, and it is characterized in that: in dispersion liquid, Nano microsphere concentration is preferably 20mg/mL, and ultra-sonic dispersion is preferably ultrasonic 60min under 250 watts.
Claim 1 or 2 or 3 or 4 described artificial platelet PLGA-PEG-RGD preparation lose blood greatly for general and the vein of internal hemorrhage with the application in hemorrhage.
10. comprise the first-aid kit of the arbitrary described vein of the arbitrary described artificial platelet PLGA-PEG-RGD of claim 1 to 4 or claim 6 to 8 with general nanometer hemostatic medicine.
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| WO2024040363A1 (en) * | 2022-08-20 | 2024-02-29 | 中国人民解放军空军军医大学 | Staphylococcus aureus vaccine and preparation method therefor, use of plga-peg copolymer in vaccine preparation |
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| CN115944714B (en) * | 2022-10-20 | 2023-12-15 | 苏州大学 | Nanometer material capable of simultaneously resisting interleukin 1 and tumor necrosis factor inflammation, and preparation method and application thereof |
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