CN104906045A - Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof - Google Patents
Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN104906045A CN104906045A CN201510359524.4A CN201510359524A CN104906045A CN 104906045 A CN104906045 A CN 104906045A CN 201510359524 A CN201510359524 A CN 201510359524A CN 104906045 A CN104906045 A CN 104906045A
- Authority
- CN
- China
- Prior art keywords
- weight heparin
- molecular weight
- low molecular
- slow releasing
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a low-molecular-weight heparin slow-release preparation, as well as a preparation method and application thereof; the low-molecular-weight heparin slow-release preparation contains nano-particles prepared from polypeptides and low-molecular-weight heparin; and polypeptides are crosslinked through chemical bonds. The low-molecular-weight heparin slow-release preparation is prepared in the invention by adopting any one technology or a combination of at least two technologies selected from a group including a self-assembly technology, a freezing and drying technology, a homogenizing technology, an emulsifying technology, an embedding technology or a spraying and drying technology. The release ratio of the low-molecular-weight heparin slow-release preparation disclosed by the invention is only about 20% within 500 h in vitro; therefore, the low-molecular-weight heparin slow-release preparation has the slow-release effect; the blood half-life period can be effectively prolonged; the medicinal peak valley fluctuation can be avoided; the risk in massive haemorrhage due to the low-molecular-weight heparin solution preparation is massively used can be reduced; the toxic and side effects of medicines are reduced; the compliance of patients is improved; the disease treatment efficiency is increased; the low-molecular-weight heparin slow-release preparation can be used for preventing and treating thrombus diseases; and the low-molecular-weight heparin slow-release preparation is simple in preparation method and wide in application prospect.
Description
Technical field
The invention belongs to medical art, relate to a kind of low molecular weight heparin slow releasing preparation and its preparation method and application.
Background technology
China has stepped into aged tendency of population society, and aging population quantity increases gradually.In old people colony, thrombus disease is one of the most common disease, and sickness rate is high, and patient group is wide, and thrombotic sequela has a strong impact on the ability to work of patient, even disables.
In antithrombotic class medicine, heparin class medicine is derived from the biotic component in life entity because of it, has the advantages such as therapeutic effect is good, biocompatibility, safety and receives much concern.In heparin class medicine, Low molecular heparin (LMWH) has the effects such as anticoagulation, antithrombotic, Adjust-blood lipid, be mainly used in the disease such as treatment and control dvt, pulmonary infarction, disseminated inravascular coagulation clinically, be widely used in Prevention of cardiovascular disease field.LMWH is the heparin fragment of the low-molecular-weight (Mw) obtained through the method that chemistry or enzymolysis gather by common unfraction heparin (UFH), or the heparin component of low Mw that classified method obtains, its Mw scope is generally 3000 ~ 8000Da, and average Mw is about 5000Da.
Compared with UFH, LMWH has subcutaneous injection good absorbing, long half time, and bioavailability is high, with the advantage such as blood plasma, platelet affinity be little.But the deficiency that when LMWH exists Half-life in vivo short and solution intravenous injection, massive hemorrhage side effect is comparatively serious, and need frequent drug administration, greatly reduce the compliance of the therapeutic efficiency of medicine, the safety of medication and patient, therefore study its slow releasing injection and there is important clinical meaning.
CN101024086 A discloses the complex and preparation and preparation method that Chitosan-phospholipid complex and low molecular weight heparin formed, the Nano/micron complex that this invention utilizes Chitosan-phospholipid complex and low molecular weight heparin to be formed as carrier to increase the picked-up of cell to Low molecular heparin, utilize the bioadhesive of Chitosan-phospholipid complex and of short durationly open intercellular close-connected characteristic and increase Low molecular heparin further through biomembranous amount, improved the stability of Low molecular heparin by the formation of complex, thus improve the bioavailability of medication.But chitosan only have dissolve under strongly acidic conditions, dissolubility is low under physiological condition, chitosan has higher toxicity and poor biocompatibility simultaneously, and these defects seriously limit its application in vivo.In addition, the complex that Chitosan-phospholipid complex and low molecular weight heparin are formed and preparation do not adopt crosslinked means to be solidified by nano-particle, granule, under the intervention of Physiological Medium, easily causes low molecular weight heparin rapid, high volume to discharge, and easily affects body internal stability and the therapeutic efficiency of preparation.
Therefore, in this area, expecting to obtain one can slow releasing LMWH, the Half-life in vivo of effective prolongation LMWH, reduces the risk of massive hemorrhage when LMWH uses, avoids blood drug level " peak valley " to fluctuate larger, make release steady, reduce the toxic and side effects of medicine, improve the compliance of patient, improve the low molecular weight heparin preparation of the therapeutic efficiency to disease.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of low molecular weight heparin slow releasing preparation and its preparation method and application.
For reaching this goal of the invention, the present invention by the following technical solutions:
On the one hand, the invention provides a kind of low molecular weight heparin preparation, described low molecular weight heparin slow releasing preparation comprises the nano-particle formed by polypeptide and low molecular weight heparin, is cross-linked between described polypeptide by chemical bond.
Low molecular weight heparin slow releasing preparation of the present invention comprises polypeptide and low molecular weight heparin, the two forms nano-particle by electrostatic interaction, based on the crosslinked low molecular weight heparin that makes between peptide molecule, there is slow release effect, its blood halflife of effective prolongation, avoids medicine " peak valley fluctuation ", effectively steadily release, reduce the risk of massive hemorrhage when its solution heavy dose uses, reduce the toxic and side effects of medicine, improve the compliance of patient, improve the therapeutic efficiency to disease.
The heparin preparations now reported all does not adopt crosslinked means, the nano-particle formed is only based on the interaction of electrostatic force, poor stability, easily causes the prominent of heparin to be released, effectively cannot ensure drug safety under the intervention of Physiological Medium (as phosphate buffer).The advantages such as the polypeptide that the present invention uses, by the Amino acid profile in organism, has biodegradable, good biocompatibility, and under physiological condition dissolubility is high, its safety is better.Simultaneously the present invention by chemical bond as polypeptide is cross-linked by the combineds effect such as covalent bond, ionic bond, hydrogen bond, the effect of slow releasing heparin is reached by the slow degraded of polypeptide, reduce the side effect of heparin, reduce the risk of massive hemorrhage when LMWH uses, decrease administration frequency, improve the compliance of patient, the body internal stability of preparation is more excellent, the therapeutic efficiency ensureing clinical drug safety and improve disease.
In low molecular weight heparin slow releasing preparation of the present invention, the mass ratio of described polypeptide and low molecular weight heparin is 1:100 ~ 100:1, such as 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:15, 1:10, 1:5, 1:3, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, preferred 1:20 ~ 1:100, preferred 1:20 ~ 1:50 further, under selected preferred proportion, the particle diameter of granule is more even, more easily control at below 100nm, and current potential is more controlled, the favorable reproducibility of technique.
In low molecular weight heparin slow releasing preparation of the present invention, the molecular weight of described polypeptide is 500 ~ 50000Da, such as 500Da, 1000Da, 1500Da, 2000Da, 2500Da, 3000Da, 4000Da, 6000Da, 8000Da, 10000Da, 15000Da, 20000Da, 25000Da, 30000Da, 35000Da, 40000Da, 45000Da or 50000Da, preferably 1000 ~ 30000Da.
Preferably, described polypeptide is the polypeptide formed by any one in arginine, histidine, lysine, cysteine, glycine, phenylalanine, aspartic acid, glutamic acid, serine, alanine or leucine or at least two kinds.
Preferably, described chemical bond is any one or at least two kinds in disulfide bond, ionic bond or hydrogen bond.
In low molecular weight heparin slow releasing preparation of the present invention, the molecular weight < 8000Da of described low molecular weight heparin, such as 7500Da, 7000Da, 6500Da, 6000Da, 5500Da, 5000Da, 4500Da, 4000Da, 3500Da or 3000Da, preferably 3000 ~ 7000Da.
Preferably, described low molecular weight heparin is the combination of any one or at least two kinds in heparin sodium, dalteparin sodium, nadroparin calcium, fondaparinux sodium or Enoxaparin Sodium.
In low molecular weight heparin slow releasing preparation of the present invention, the mean diameter of described nano-particle is 50 ~ 200nm, such as 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm or 200nm, preferably 60 ~ 150nm.
In low molecular weight heparin slow releasing preparation of the present invention, the surface potential of described nano-particle is-20 ~ 20mV, such as-20mV ,-18mV ,-15mV ,-13mV ,-10mV ,-8mV ,-5mV, 0mV, 5mV, 8mV, 10mV, 13mV, 15mV, 18mV, 20mV, preferably-20 ~-5mV.
In low molecular weight heparin slow releasing preparation of the present invention, described low molecular weight heparin slow releasing preparation is any one in lyophilized injectable powder, sterile powder injection or nanometer solution agent.
On the other hand, the invention provides the preparation method of low molecular weight heparin slow releasing preparation as described in relation to the first aspect, described preparation method adopts the combination of any one or at least two kinds in self-assembling technique, Freeze Drying Technique, homogeneous technology, emulsifying technology, embedding techniques or spray drying technology, the combination of preferred self-assembling technique, homogeneous technology and Freeze Drying Technique.
Autonomous packing technique can the size of Effective Regulation granule, has that grain diameter is controlled, process stabilizing, is easy to the advantage of producing; Further effectively can be controlled the particle size distribution of preparation by homogeneous technology, get rid of bulky grain to the impact of preparation security; By Freeze Drying Technique, effectively reduce the degradation rate (as hydrolysis and oxidation etc.) of medicine and adjuvant, improve the long-time stability of preparation.By the control of above Multiple techniques, expect the slow release nanometer formulation obtaining uniform particle sizes, good stability, determined curative effect.
As optimal technical scheme, the preparation method of low molecular weight heparin slow releasing preparation of the present invention comprises the following steps:
(1) add in glucose solution by polypeptide, form polypeptide solution, then add Traut ' s reagent, stirring reaction obtains sulfhydrylation polypeptide;
(2) in sulfhydrylation polypeptide, add glucose solution, then add low molecular weight heparin solution, mixing, homogenizing, aging, described low molecular weight heparin slow releasing preparation is made in lyophilization.
In the preparation method of low molecular weight heparin slow releasing preparation of the present invention, step (1) and step (2) described glucose solution are the glucose solution of 5%.
Preferably, relative to 100mg polypeptide, the addition of step (1) described glucose solution is 5 ~ 100mL, such as 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 14mL, 16mL, 18mL, 20mL, 25mL, 28mL, 30mL, 35mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL or 100mL.
Preferably, the concentration of step (1) described polypeptide solution is 1mg/mL ~ 20mg/mL, such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL or 20mg/mL, be preferably 5mg/mL ~ 15mg/mL.
Preferably, the concentration of step (1) described Traut ' s reagent is 0.1 ~ 50mg/mL, such as 0.1mg/mL, 0.5mg/mL, 0.8mg/mL, 1mg/mL, 3mg/mL, 5mg/mL, 8mg/mL, 10mg/mL, 13mg/mL, 15mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 28mg/mL, 30mg/mL, 32mg/mL, 35mg/mL, 38mg/mL, 40mg/mL, 43mg/mL, 45mg/mL, 48mg/mL or 50mg/mL, be preferably 0.1 ~ 30mg/mL.
Preferably, relative to 1mL polypeptide solution, the addition of step (1) described Traut ' s reagent is 0 ~ 10mL, such as 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL or 10mL.When containing sulfydryl in polypeptide, then can not add Traut ' s reagent, namely not carry out sulfhydrylation step, and rely on the sulfydryl that polypeptide itself contains to complete being cross-linked between polypeptide.When not carrying out sulfhydrylation containing needing to add Traut ' s reagent during sulfydryl in polypeptide, to realize being cross-linked between polypeptide.
Preferably, relative to 1mL polypeptide solution, the addition of step (2) described glucose solution is 2mL ~ 100mL, such as 2mL, 5mL, 10mL, 15mL, 20mL, 25mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL or 100mL, be preferably 10mL ~ 40mL.
Preferably, the described low molecular weight heparin solution of step (2) is the low molecular weight heparin solution formed in glucose solution low molecular weight heparin being dissolved in 5%.
Preferably, the described low molecular weight heparin solution concentration of step (2) is 1mg/mL ~ 30mg/mL, such as 1mg/mL, 3mg/mL, 5mg/mL, 8mg/mL, 10mg/mL, 12mg/mL, 14mg/mL, 16mg/mL, 20mg/mL, 22mg/mL, 24mg/mL, 26mg/mL, 28mg/mL or 30mg/mL, preferred 5mg/mL ~ 20mg/mL.
Preferably, the mass ratio of step (1) described polypeptide and step (2) described low molecular weight heparin is 1:100 ~ 100:1, such as 1:100,1:90,1:80,1:70,1:60,1:50,1:40,1:30,1:20,1:15,1:10,1:5,1:3,1:1,2:1,5:1,8:1,10:1,15:1,18:1,20:1,25:1,30:1,35:1,40:1,45:1,50:1,60:1,70:1,80:1,90:1 or 100:1, preferred 1:20 ~ 1:100, further preferred 1:20 ~ 1:50.
In the preparation process in accordance with the present invention, use the object of glucose solution to be the balance keeping osmotic pressure in preparation process, avoid the release causing low molecular weight heparin due to the change of osmotic pressure.
On the other hand, the low molecular weight heparin slow releasing preparation that the invention provides as described in relation to the first aspect is preparing the application in antithrombotic reagent.
Slow releasing preparation made by the present invention effectively can extend the blood halflife of low molecular weight heparin, avoid medicine " peak valley fluctuation ", reduce the risk of massive hemorrhage when its solution heavy dose uses, effectively steadily release, there is curative effect high, the advantage that good stability, side effect are little, is easy to suitability for industrialized production; Its slow release nanometer formulation prepared can be used for prevention and the treatment of thrombus disease, has good therapeutic effect.
Relative to prior art, the present invention has following beneficial effect:
The present invention utilizes polypeptide and low molecular weight heparin to form nano-particle, low molecular weight heparin is made to have slow release effect, the release rate of low molecular weight heparin slow releasing preparation of the present invention in 500 hours is only about 20%, can effectively extend its blood halflife, avoid medicine " peak valley fluctuation ", effectively steadily release, reduce the risk of massive hemorrhage when low molecular weight heparin solution heavy dose uses, reduce the toxic and side effects of medicine, improve the compliance of patient, improve the therapeutic efficiency to disease, can be used for prevention and the treatment of thrombus disease.The preparation method of low molecular weight heparin slow releasing preparation of the present invention is simple, is easy to operation, the adding of organic solvent-free in preparation process, belongs to energy-conserving and environment-protective green and produces, have broad application prospects.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of low molecular weight heparin slow releasing preparation prepared by the embodiment of the present invention 1;
Fig. 2 A is the grain size distribution of low molecular weight heparin slow releasing preparation prepared by the embodiment of the present invention 2;
The surface electrical bitmap of the low molecular weight heparin slow releasing preparation of Fig. 2 B embodiment of the present invention 2 preparation;
Fig. 3 A is the grain size distribution of the low molecular weight heparin slow releasing preparation prepared under different concentration of heparin;
Fig. 3 B is the potential change curve chart of the low molecular weight heparin slow releasing preparation prepared under different concentration of heparin;
Fig. 3 C is the grain size distribution of the low molecular weight heparin slow releasing preparation that the polypeptide preparation of different sulfhydrylation degree obtains;
Fig. 3 D is the potential change curve chart of the low molecular weight heparin slow releasing preparation that the polypeptide preparation of different sulfhydrylation degree obtains;
Fig. 4 is the tablets in vitro curve chart of low molecular weight heparin slow releasing preparation of the present invention;
Fig. 5 is the safety evaluatio result figure of low molecular weight heparin slow releasing preparation of the present invention at cellular level;
Fig. 6 be low molecular weight heparin slow releasing preparation of the present invention animal body in anticoagulation therapeutic evaluation result figure.
Detailed description of the invention
Technical scheme of the present invention is further illustrated below by detailed description of the invention.Those skilled in the art should understand, described embodiment is only help to understand the present invention, should not be considered as concrete restriction of the present invention.
embodiment 1
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg polylysine (PLL, M
w=13000Da) in centrifuge tube, add 5% glucose solution of 5mL, ultrasonic mixing; The PLL solution measuring 5mL 15mg/mL, in centrifuge tube, then adds 6mg/mL Traut ' s reagent 5mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, PLL (PLL-SH) solution of obtained sulfhydrylation; To in PLL (PLL-SH) solution of sulfhydrylation, add the mixing of 60mL 5% glucose solution, 100mL 20mg/mL heparin sodium (LMWH) solution is added fast in this mixed solution, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, the formulation soln after redissolution is used for follow-up sign.
Carry out scanning electron microscope (SEM) sign to the nano-particle that the present embodiment prepares, as shown in Figure 1, mean diameter is about 120nm to result, the smooth rounding of particle surface.
embodiment 2
The mass ratio being only to adjust PLL and low molecular weight heparin in the difference of the present embodiment and embodiment 1 is 5:6, all the other preparation methoies and condition are all identical with embodiment 1, the low molecular weight heparin slow releasing preparation nano-particle prepared are measured particle diameter and the current potential of nano-particle by dynamic light scattering technique (DLS).
As shown in Figure 2, wherein Fig. 2 A is the grain size distribution of low molecular weight heparin slow releasing preparation to result, and as seen from the figure, mean diameter is about 105nm; Fig. 2 B is the surface electrical bitmap of low molecular weight heparin slow releasing preparation; As shown in the figure, surface potential is about-16mV.
embodiment 3
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg polyhistidyl (M
w=1000Da) in centrifuge tube, add 5% glucose solution of 5mL, ultrasonic mixing; The PLL solution measuring 5mL 20mg/mL, in centrifuge tube, then adds 0.1mg/mL Traut ' s reagent 10mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, the polyhistidyl solution of obtained sulfhydrylation; To in the polyhistidyl solution of sulfhydrylation, add the mixing of 50mL 5% glucose solution, 5mL 30mg/mL dalteparin sodium (LMWH) solution is added fast in this mixed solution, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 110nm, and surface potential is-20mV, the smooth rounding of particle surface.
embodiment 4
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg polylysine (PLL, M
w=30000Da) in centrifuge tube, add 5% glucose solution of 100mL, ultrasonic mixing; The PLL solution measuring 5mL 1mg/mL, in centrifuge tube, then adds 1mg/mL Traut ' s reagent 5mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, PLL (PLL-SH) solution of obtained sulfhydrylation; The mixing of 200mL 5% glucose solution is added in PLL-SH solution, in this mixed solution, add 200mL 1mg/mL fondaparin (LMWH) solution fast, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 130nm, and surface potential is-15mV, the smooth rounding of particle surface.
embodiment 5
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg poly arginine (M
w=500Da) in centrifuge tube, add 5% glucose solution of 50mL, ultrasonic mixing; The PLL solution measuring 5mL 2mg/mL, in centrifuge tube, then adds 50mg/mL Traut ' s reagent 1mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, the poly arginine solution of obtained sulfhydrylation; The mixing of 500mL 5% glucose solution is added in the poly arginine solution of sulfhydrylation, 400mL 5mg/mL nadroparin calcium (LMWH) solution is added fast in this mixed solution, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 60nm, and surface potential is-8mV, the smooth rounding of particle surface.
embodiment 6
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg polylysine (PLL, M
w=50000Da) in centrifuge tube, add 5% glucose solution of 20mL, ultrasonic mixing; The PLL solution measuring 6mL 5mg/mL, in centrifuge tube, then adds 30mg/mL Traut ' s reagent 6mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, PLL (PLL-SH) solution of obtained sulfhydrylation; The mixing of 250mL 5% glucose solution is added in PLL-SH solution, in this mixed solution, add 500mL 10mg/mL nadroparin calcium (LMWH) solution fast, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 50nm, and surface potential is-20mV, the smooth rounding of particle surface.
embodiment 7
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg polyglycine-poly arginine (M
w=13000Da) in centrifuge tube, add 5% glucose solution of 40mL, ultrasonic mixing; The PLL solution measuring 5mL 2.5mg/mL, in centrifuge tube, then adds 30mg/mL Traut ' s reagent 10mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, the polyglycine-poly arginine solution of obtained sulfhydrylation; In the polyglycine-poly arginine solution of sulfhydrylation, add the mixing of 50mL 5% glucose solution, 1mL 1mg/mL Enoxaparin Sodium (LMWH) solution is added fast in this mixed solution, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 150nm, and surface potential is 10mV, the smooth rounding of particle surface.
embodiment 8
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 100mg and gather cysteine (MW=13000Da) in centrifuge tube, add 5% glucose solution of 10mL, ultrasonic mixing; Measure the PLL solution of 5mL 10mg/mL in beaker, add the mixing of 10mL 5% glucose solution, 1mL 5mg/mL Enoxaparin Sodium (LMWH) solution is added fast in this mixed solution, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 130nm, and surface potential is-5mV, the smooth rounding of particle surface.
embodiment 9
In the present embodiment, prepare low molecular weight heparin slow releasing preparation by the following method, said method comprising the steps of:
Take 1.2mg polylysine (PLL, M
w=13000Da) in centrifuge tube, add 5% glucose solution of 1mL, ultrasonic mixing; The PLL solution measuring 5mL 1.2mg/mL, in centrifuge tube, then adds 0.1mg/mL Traut ' s reagent 50mL, adds magneton, is placed in magnetic stirring apparatus stirs 2h to carry out sulfhydrylation, PLL (PLL-SH) solution of obtained sulfhydrylation; The mixing of 10mL 5% glucose solution is added in PLL-SH solution, in this mixed solution, add 120mL 1mg/mL Enoxaparin Sodium (LMWH) solution fast, homogenizing after mixing, aging 24h is to treat nanoparticles stable, lyophilizing, prepares low molecular weight heparin slow releasing preparation.
Characterize through scanning electron microscope and dynamic light scattering, the mean diameter of low molecular weight heparin slow releasing preparation prepared by the present embodiment is 200nm, and surface potential is 20mV, the smooth rounding of particle surface.
embodiment 10
In the present embodiment, the stable preparation process of low molecular weight heparin slow releasing preparation nano-particle is investigated, method is as follows: prepare according to the method described in embodiment the PLL-SH solution that sulfhydrylation degree is 10%, and the PLL-SH solution of 10% sulfhydrylation and LMWH solution being kept the mass ratio of 6:5 to prepare LMWH final concentration is 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, the nano-particle of 0.8mg/mL, 0.9mg/mL, 1.0mg/mL, nanoparticles stable is treated, lyophilizing after aging 24h.Similarly, adjustment sulfhydrylation degree is 5% ~ 30%, prepares nanometer formulation respectively.Measured particle diameter and the current potential of all samples by dynamic light scattering technique after redissolution.
Result as shown in Figure 3, wherein, Fig. 3 A is the grain size distribution of the low molecular weight heparin slow releasing preparation prepared under different concentration of heparin, as seen from the figure, under the concentration of heparin of 0.5-1.0mg/mL, the change of size of low molecular weight heparin slow releasing preparation is not obvious, and Particle dispersity (PDI) all remains on comparatively fractional value, proof particle dispersion is good, and granule is more stable; Fig. 3 B is the potential change curve chart of the low molecular weight heparin slow releasing preparation prepared under different concentration of heparin, and as seen from the figure, under the concentration of heparin of 0.5-1.0mg/mL, the current potential of low molecular weight heparin slow releasing preparation changes without significance; Fig. 3 C is the grain size distribution of the low molecular weight heparin slow releasing preparation that the polypeptide preparation of different sulfhydrylation degree obtains, as seen from the figure, when polypeptide sulfhydrylation degree is 5% ~ 30%, the change of size of low molecular weight heparin slow releasing preparation is not obvious, Particle dispersity (PDI) all remains on comparatively fractional value, good dispersion, granule is more stable; Fig. 3 D is the potential change curve chart of the low molecular weight heparin slow releasing preparation that the polypeptide preparation of different sulfhydrylation degree obtains, and as seen from the figure, when polypeptide sulfhydrylation degree is 5% ~ 30%, the current potential of low molecular weight heparin slow releasing preparation changes without significance.Therefore, along with the change of sulfhydrylation degree and LMWH concentration, there are no significant the change of the particle diameter of nano-particle and current potential, presents good technology stability.
embodiment 11
In the present embodiment, measure the tablets in vitro curve of the nano-particle of low molecular weight heparin slow releasing preparation nano-particle, method is as follows:
Respectively nano-particle (slow release nanometer formulation) the 500 μ L that the PLL-SH (sulfhydrylation ratio 10%) and LMWH that add 4mg/mL free heparin sodium aqua 500 μ L (heparin sodium aqua agent) and 4mg/mL are formed is added bag filter, sealing; The 50mL centrifuge tube filling 20mL PBS is put into respectively by often organizing 3 samples, magneton is added in each centrifuge tube, be placed in the upper stirring of temperature control stirring device (37 DEG C), sample respectively at different time points, and add the PBS fluid infusion of equivalent after sampling; Utilize toluidine blue spectrophotography to carry out the mensuration of LMWH release to sample, and draw In-vitro release curves.
The tablets in vitro curve that the present embodiment measures as shown in Figure 4, as seen from the figure, low molecular weight heparin slow releasing preparation slow releasing heparin sodium in 500 hours of preparation prepared by the present invention, in 500 hours, the release rate of low molecular weight heparin slow releasing preparation of the present invention is only about 20%, has good slow release effect.
embodiment 12
In the present embodiment, evaluate the safety of low molecular weight heparin slow releasing preparation, method is as follows:
Human umbilical vein endothelial HUVEC cell is inoculated in 96 orifice plates with the density in 5000 every holes, cultivate in incubator after 24 hours, discard culture medium, PBS cleans, add the culture medium that 180 μ L are fresh, then add the Lipo-Hepin that 20 μ L have embodiment 1 preparation of Concentraton gradient (final concentration 20 μ g/mL, 200 μ g/mL, 400 μ g/mL) respectively, each concentration arranges 6 multiple holes, after cultivating 48h, mtt assay measures the survival rate of cell.
As shown in Figure 5, result shows that the safety of heparin sodium slow releasing preparation is good to the present embodiment measurement result, with heparin sodium aqua agent in safety without significant difference.
embodiment 13
In the present embodiment, evaluate anticoagulation curative effect in the body of low molecular weight heparin slow releasing preparation nano-particle, method is as follows:
SD rat divides into groups the previous day by experiment, and the low molecular weight heparin slow releasing preparation (slow release nanometer formulation group) being divided into free LMWH solution group (free LMWH group) and embodiment 1 to prepare, often organizes 6,12h fasting before experiment.Free LMWH group and slow release nanometer formulation group tail vein injections appropriate amount of drug respectively, gets blood, 3000rpm, 15min centrifugal treating blood sample according to the interval of setting, and get supernatant taking-up and put into centrifuge tube ,-20 DEG C of preservations after subpackage, multigelation avoided by sample.The Anti-Xa factor activity of enzyme-linked immunosorbent assay (ELISA) test kit to all samples is adopted to detect.
In the body of this enforcement, anticoagulation therapeutic evaluation result as shown in Figure 6, as seen from the figure, the present invention prepare low molecular weight heparin slow releasing preparation group (slow release nanometer formulation group) Anti-Xa factor activity be significantly higher than free heparin sodium aqua agent, this show low molecular weight heparin slow releasing preparation of the present invention in animal body antithrombotic curative effect be better than heparin sodium aqua agent.
Applicant states, the present invention illustrates low molecular weight heparin slow releasing preparation of the present invention and its preparation method and application by above-described embodiment, but the present invention is not limited to above-described embodiment, does not namely mean that the present invention must rely on above-described embodiment and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (10)
1. a low molecular weight heparin slow releasing preparation, is characterized in that, described low molecular weight heparin slow releasing preparation comprises the nano-particle formed by polypeptide and low molecular weight heparin, is cross-linked between described polypeptide by chemical bond.
2. low molecular weight heparin slow releasing preparation according to claim 1, is characterized in that, the mass ratio of described polypeptide and low molecular weight heparin is 1:100 ~ 100:1, preferred 1:20 ~ 1:100, further preferred 1:20 ~ 1:50.
3. low molecular weight heparin slow releasing preparation according to claim 1 and 2, is characterized in that, the molecular weight of described polypeptide is 500 ~ 50000Da, preferably 1000 ~ 30000Da;
Preferably, described polypeptide is the polypeptide formed by any one in arginine, histidine, lysine, cysteine, glycine, phenylalanine, aspartic acid, glutamic acid, serine, alanine or leucine or at least two kinds;
Preferably, described chemical bond is any one or at least two kinds in disulfide bond, ionic bond or hydrogen bond.
4. the low molecular weight heparin slow releasing preparation according to any one of claim 1-3, is characterized in that, the molecular weight < 8000Da of described low molecular weight heparin, preferably 3000 ~ 7000Da;
Preferably, described low molecular weight heparin is the combination of any one or at least two kinds in heparin sodium, dalteparin sodium, nadroparin calcium, fondaparinux sodium or Enoxaparin Sodium.
5. the low molecular weight heparin slow releasing preparation according to any one of claim 1-4, is characterized in that, the mean diameter of described nano-particle is 50 ~ 200nm, preferably 60 ~ 150nm;
Preferably, the surface potential of described nano-particle is-20 ~ 20mV, preferably-20 ~-5mV.
6. the low molecular weight heparin slow releasing preparation according to any one of claim 1-5, is characterized in that, described low molecular weight heparin slow releasing preparation is any one in lyophilized injectable powder, sterile powder injection or nanometer solution agent.
7. the preparation method of the low molecular weight heparin slow releasing preparation according to any one of claim 1-6, it is characterized in that, described preparation method adopts the combination of any one or at least two kinds in self-assembling technique, homogeneous technology, Freeze Drying Technique, emulsifying technology, embedding techniques or spray drying technology, the combination of preferred self-assembling technique, homogeneous technology and Freeze Drying Technique.
8. preparation method according to claim 7, is characterized in that, said method comprising the steps of:
(1) add in glucose solution by polypeptide, form polypeptide solution, then add Traut ' s reagent, stirring reaction obtains sulfhydrylation polypeptide;
(2) in sulfhydrylation polypeptide, add glucose solution, then add low molecular weight heparin solution, mixing, homogenizing, aging, described low molecular weight heparin slow releasing preparation is made in lyophilization.
9. preparation method according to claim 8, is characterized in that, step (1) and step (2) described glucose solution are the glucose solution of 5%;
Preferably, relative to 100mg polypeptide, the addition of step (1) described glucose solution is 5 ~ 100mL;
Preferably, the concentration of step (1) described polypeptide solution is 1mg/mL ~ 20mg/mL, is preferably 5mg/mL ~ 15mg/mL;
Preferably, the concentration of step (1) described Traut ' s reagent is 0.1 ~ 50mg/mL, is preferably 0.1 ~ 30mg/mL;
Preferably, relative to 1mL polypeptide solution, the addition of step (1) described Traut ' s reagent is 0 ~ 10mL;
Preferably, relative to 1mL polypeptide solution, the addition of step (2) described glucose solution is 2mL ~ 100mL, preferred 10mL ~ 40mL;
Preferably, the described low molecular weight heparin solution of step (2) is the low molecular weight heparin solution formed in glucose solution low molecular weight heparin being dissolved in 5%;
Preferably, the described low molecular weight heparin solution concentration of step (2) is 1mg/mL ~ 30mg/mL, preferred 5mg/mL ~ 20mg/mL;
Preferably, the mass ratio of step (1) described polypeptide and step (2) described low molecular weight heparin is 1:100 ~ 100:1, preferred 1:20 ~ 1:100, further preferred 1:20 ~ 1:50.
10. the low molecular weight heparin slow releasing preparation according to any one of claim 1-6 is preparing the application in antithrombotic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510359524.4A CN104906045B (en) | 2015-06-25 | 2015-06-25 | A kind of low molecular weight heparin sustained release preparation and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510359524.4A CN104906045B (en) | 2015-06-25 | 2015-06-25 | A kind of low molecular weight heparin sustained release preparation and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104906045A true CN104906045A (en) | 2015-09-16 |
| CN104906045B CN104906045B (en) | 2018-04-13 |
Family
ID=54075778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510359524.4A Active CN104906045B (en) | 2015-06-25 | 2015-06-25 | A kind of low molecular weight heparin sustained release preparation and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104906045B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102475686A (en) * | 2010-11-29 | 2012-05-30 | 吴忠仕 | Low molecular heparin iron nanoparticles and preparation method thereof |
| CN102652834A (en) * | 2012-01-13 | 2012-09-05 | 诸敏 | Preparation method and composite of low-molecular heparin nanopolymer |
| CN104288776A (en) * | 2014-06-30 | 2015-01-21 | 昆明医科大学 | Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof |
-
2015
- 2015-06-25 CN CN201510359524.4A patent/CN104906045B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102475686A (en) * | 2010-11-29 | 2012-05-30 | 吴忠仕 | Low molecular heparin iron nanoparticles and preparation method thereof |
| CN102652834A (en) * | 2012-01-13 | 2012-09-05 | 诸敏 | Preparation method and composite of low-molecular heparin nanopolymer |
| CN104288776A (en) * | 2014-06-30 | 2015-01-21 | 昆明医科大学 | Self-assembled polypeptide-apoptin gene composite nanoparticle, and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHAO DENG等: "Functional polypeptide and hybrid materials Precision synthesis via α-amino acid N-carboxyanhydride polymerization and merging biomedical applications", 《PROGRESS IN POLYMER SCIENCE》 * |
| QI TAN等: "Controlled release of chitosan/heparin nanoparticle delivered VEGF enhances regeneration of decellularized tissue-engineered scaffolds", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| 邵明香等: "自组装多肽在新型药物制剂研发领域中的应用", 《中国生物工程杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104906045B (en) | 2018-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Peng et al. | Injectable and biodegradable thermosensitive hydrogels loaded with PHBHHx nanoparticles for the sustained and controlled release of insulin | |
| Hu et al. | Effect of bases with different solubility on the release behavior of risperidone loaded PLGA microspheres | |
| Zhao et al. | Synthesis of surfactant-free hydroxypropyl methylcellulose nanogels for controlled release of insulin | |
| Avadi et al. | Ex vivo evaluation of insulin nanoparticles using chitosan and arabic gum | |
| WO2017186073A1 (en) | Preparation method of sustained release microparticulates, sustained release microparticulates thereby and use thereof | |
| US8017156B2 (en) | Long-acting colloidal insulin formulation and its preparation | |
| AU2019219792B2 (en) | Bendamustine anionic-cationic cyclopolysaccharide compositions | |
| WO2018233095A1 (en) | Biological self-assembled nanocrystal injection having lymphatic targeting function and preparation method | |
| CN102370622A (en) | Medicament carrying nano particles and preparation method and application thereof | |
| Zheng et al. | Novel iron–polysaccharide multilayered microcapsules for controlled insulin release | |
| CN102727899A (en) | Protein-medicament-carrying PLGA composite microspheres and preparation method thereof | |
| WO2017186074A1 (en) | Method for preparing sustained release microparticles, prepared sustained release microparticles and application thereof | |
| KR101307729B1 (en) | Injectable composition comprising microparticles with reduced initial drug release and method for preparing thereof | |
| Mao et al. | A novel self-assembly nanocrystal as lymph node-targeting delivery system: higher activity of lymph node targeting and longer efficacy against lymphatic metastasis | |
| EP3616726B1 (en) | Protein particle wrapped with medicine insoluble in water and preparation method therefor | |
| Zhang et al. | Controlled Interfacial Polymer Self‐Assembly Coordinates Ultrahigh Drug Loading and Zero‐Order Release in Particles Prepared under Continuous Flow | |
| CN104906045A (en) | Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof | |
| Fu et al. | Therapeutic effects of paclitaxel loaded polyethylene glycol-polylactic acid-glycolic acid copolymer nanoparticles on pancreatic cancer in rats | |
| CN104546741A (en) | Preparation method and application of 2-methoxyestradiol albumin nano freeze-dried agent | |
| KR20090116494A (en) | Poly(lactide-co-glycolide) microsphere, a process for the preparatrion thereof, and a protein or peptide drug formulation comprising the same | |
| WO2022051247A1 (en) | Methods for treating or preventing inflammatory events | |
| WO2020229536A1 (en) | Dry microparticles | |
| Suzuki et al. | Complex of branched cyclodextrin and lidocaine prolonged the duration of peripheral nerve block | |
| CN104840432A (en) | Taxane long-circulating nanoparticles and preparation method thereof | |
| CN1806801A (en) | Pharmaceutical composition of sodium houttuyfonate for treating respiratory infection and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |