CN102475686A - Low molecular heparin iron nanoparticles and preparation method thereof - Google Patents
Low molecular heparin iron nanoparticles and preparation method thereof Download PDFInfo
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- CN102475686A CN102475686A CN201010563472XA CN201010563472A CN102475686A CN 102475686 A CN102475686 A CN 102475686A CN 201010563472X A CN201010563472X A CN 201010563472XA CN 201010563472 A CN201010563472 A CN 201010563472A CN 102475686 A CN102475686 A CN 102475686A
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Abstract
The present invention relates to a low molecular heparin iron nanoparticle biological product. The invention further relates to a preparation method for the low molecular heparin iron nanoparticles. According to the present invention, the preparation method for the low molecular heparin iron nanoparticles with excellent slow release function is provided; with the method, the low molecular heparin iron nanoparticles capable of slow release of LMWH in vivo can be obtained, and the heparin administration time and the times of the heparin administration can be effectively reduced with the drug.
Description
Technical field
The present invention relates to a kind of Low molecular heparin iron nano-particle biological product.
The invention still further relates to the method for preparing of this Low molecular heparin iron nano-particle.
Technical background
Heparin is one type of glycosaminoglycans, the mixture of the polysaccharide chain that the repetition disaccharide unit that is coupled together with 1 → 4 glycosidic bond by alduronic acid and glucamine is formed.Heparin is prevented and treated thrombosis through combining antithrombin (AT-III) Trombin inhibiting efficiently thereby delay blood coagulation, impels thromboembolism.
The molecular weight of heparin about 3000～15000, average 16000.Low molecular heparin (LMWH) molecular weight is lower than 6000～10000, and LMWH has the active effect of selectivity anticoagulin X a, and thrombin and other thrombins are not had obvious influence.And its anticoagulation of unfractionated heparin mainly is to combine with the AT-III; Quicken of the deactivation of AT-III, make thrombin factor, X a deactivation thrombin, and the activity of inhibitive factor IX a, XI a, XII a and VIII a; Factor also there is direct repression, but a little less than the effect.LMWH and unfractionated heparin relatively, its half-life is longer, antithrombotic effect is good, and the anticoagulant bleeding tendency a little less than, the trend that replaces unfractionated heparin is arranged.In recent years clinical commonly used having: dalteparin sodium (Fragmin), Enoxaparin Sodium (Ke Sai), low molecular heparin calcium (fraxiparine, nadroparin calcium).
Clinical heparin commonly used and LMWH need the every day of administration repeatedly, give to be in hospital, non-inpatient's medication makes troubles, if single administration, drug effect can continue a couple of days, and that will bring huge economy and social effect.
It is big that nano-particle has specific surface area, has the good slow release function; Nanometer particle size is less than below the 200nm, and cells in vivo obviously descends to the phagocytosis of nano-particle, when simultaneously the nano-particle Zeta potential is negative value, is unfavorable for that also phagocyte to the engulfing of nano-particle, helps nano-particle and in body, exists for a long time.The Low molecular heparin nano-particle will help medicine slow release in vivo, can effectively reduce administration number of times and prolong administration time.
LMWH is the electronegative straight chain biomacromolecule chemical compound that is rich in sulfate radical; Precision architecture is not clear; It is generally acknowledged that it is to connect into " tetrose " as construction unit by α-L-iduronic acid-2-sulfuric ester, the amino Fructus Vitis viniferae 6-of N-sulfo group-α-D-sulfuric ester, β-D-glucuronic acid and N-sulfo group-alpha-D aminoglucose-6-sulfuric ester by glycosidic bond, aggregates into polysaccharide by " tetrose " again.On a LMWH molecule, just contain groups such as a large amount of carboxyls, hydroxyl, amino, sulfonic group.
Iron ion is the essential trace element of body, and body is had no side effect.An iron ion and six hydrones form coordination compound, in the hydrated iron ion, add alkali (OH
-) can in the hydrion (H that comes out with ionization
+), coordinate hydrone is just corresponding to be decomposed, thereby on iron ion, produce more hydroxyl solution is deepened.This experiment slowly adds FeCl with NaOH solution
3In the solution, Fe in molar ratio
3+: OH
-After=1: 2 mixing, form dihydroxy ferrum (III) ion ([Fe (OH)
2]
+) solution (being abbreviated as DHOF).Its hydrolysis equation is: [Fe (H
2O)
6]
3+2OH
-=[Fe (OH)
2(H
2O)
4]
++ 2H
2O.
Positively charged DHOF can combine with sulfonate radical, the carboxylate radical in the heparin molecule, forms difficult dissociated iron salt through ionic bond; Iron ion can provide a plurality of unoccupied orbitals; Can form chelate through coordinate bond with a plurality of parts of lone pair electrons that provide; So DHOF can also with heparin molecule in provide the group (carboxyl, sulfonic group, hydroxyl, amino) of lone pair electrons to combine through electrostatic force, coordinate bond, form the LMWH/DHOF chelate.
Summary of the invention
The present invention is to provide Low molecular heparin nano controlled-release granule; Through being cross-linking agent with dihydroxy ferrum (be called for short DHOF); Utilize Low molecular heparin (being called for short LMWH) to contain groups such as great amount of hydroxy group, carboxyl, amido and sulfate radical; Through Electrostatic Absorption, coordinate bond, ionic bond active force, form nano-particle under certain conditions with good sustained release performance.
Low molecular heparin nano controlled-release granule provided by the invention has following characteristics: nano-particle is the rectangle nanocrystal, mean diameter in 100 nanometers, Zeta potential about-19mV～-35mV between, envelop rate is 25%～93%.
The preparation of bibliographical information unfractionated heparin iron nano-particle is arranged at present, but the material that uses with nano-particle that we make, the manufacture method of nano-particle, the form of nano-particle and particle diameter, the Zeta potential of nano-particle all has very big difference.
The present invention finds that also Zeta potential changes with the DHOF that adds different amounts with envelop rate.
The present invention is to provide the particulate manufacture method of Low molecular heparin nano controlled-release, step is following:
The configuration of A.DHOF solution:
Use deionized water dissolving FeCl
36H
2O; Under the condition of magnetic agitation, press Fe
3+: OH
-Mol ratio 1: 2 slowly splashes into FeCl with NaOH solution
3In the solution, add the concentration of an amount of water adjustment DHOF again, the DHOF solution of preparation pH value 3.0 mass concentrations between 1～20g/L;
The preparation of B.LMWH solution:
Low molecular sodium heparin is dissolved in the deionized water, and mass concentration and adds sodium chloride in the Low molecular heparin sodium solution between 1～20g/L, and the concentration that makes sodium chloride is between 1～5g/L;
C. the making of Low molecular heparin iron nano-particle:
Under the condition of ultrasonic concussion, DHOF and LMWH, join DHOF solution in the LMWH solution in certain mass ratio 0.2～2 in certain mass, and under ultrasonic concussion condition, put 10-30 minute;
Add the lyophilized powder adjuvant in D, the Low molecular heparin ferrum nanometer solution, be prepared into lyophilized powder and preserve.
In above-mentioned preparation process, DHOF and LMWH form nano crystal particles under the effect of the active force of ionic bond, coordinate bond and Electrostatic Absorption etc.
When in the said method DHOF being mixed with LMWH, adopting sonic oscillation is one of key factor that forms qualified nanometer particle size.If adopt common alr mode, then do not reach the particle diameter of requirement.
The present invention is to provide the method for preparing of Low molecular heparin nano-particle with good slow-release function; Can obtain slowly to discharge in vivo the Low molecular heparin iron nano-particle of LMWH through the method, this medicine can reduce the administration time and the administration number of times of heparin effectively.
Description of drawings
Fig. 1 Low molecular heparin iron nano-particle particle diameter detects figure
Fig. 2 Low molecular heparin iron nano-particle zeta current potential detects figure
Fig. 3 Low molecular heparin iron nano-particle scanning electron microscope picture
Fig. 4 Low molecular heparin iron nano-particle and low molecular sodium heparin fourier infrared abosrption spectrogram
Specific embodiment
The preparation of Low molecular heparin iron nano-particle and detection
Method: (1) preparation dihydroxy (III) iron ion (DHOF) solution:
DHOF solution quality concentration is at 1～20g/L, and is wherein proper between 5～10g/L.With deionized water with FeCl
36H
2O is dissolved into the twice of desired concn; Press molal quantity Fe: OH=1 again: the amount of 2 weighing NaOH, with the deionized water dissolving of same volume.Under the condition of magnetic agitation, NaOH solution is slowly splashed into FeCl
3In the solution, be made into pH value 3.0 dihydroxy (III) ferric ion solutions.
(2) preparation of Low molecular heparin (being abbreviated as LMWH) solution:
The mass concentration of LMWH is at 1～20g/L, and is wherein proper between 5～10g/L.And in LMWH solution, adding sodium chloride, the concentration of sodium chloride is at 1～5g/L.
(3) preparation of Low molecular heparin iron nano-particle:
Under the ultrasonic concussion condition of room temperature, be respectively 0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,2.0 ratios in the mass ratio of DHOF and LMWH, DHOF solution rapid and uniform is joined in the LMWH solution ultrasonic concussion 10～30 minutes.
(4) the Low molecular heparin iron nano-particle detects:
Getting capable respectively nanometer particle size of 2ml nanometer solution and zeta current potential detects.
Nanometer solution after 45 minutes, with 500 times of supernatant dilutions, with the content of remaining LMWH in the toluidine blue colorimetry detection supernatant, is calculated the envelop rate of nano-particle through the 14000rpm high speed centrifugation.
Nova NanoSEM230 scanning electron microscope behind the precipitate natural drying after centrifugal is detected.
The result: scanning electron microscope shows: the rectangular nanostructured of Low molecular heparin ferrum granule.The nano-particle mean diameter is lower than 100nm, and the zeta current potential is a negative value.Along with the increase that adds the DHOF amount, the zeta current potential increases gradually, and envelop rate increases; When mass ratio DHOF/LMWH is lower than 1.2, forms the nano-particle mean diameter and be lower than 100nm; When mass ratio DHOF/LMWH sodium greater than 1.2 the time, micron particles (its result sees table 1) appears.Granule footpath and zeta current potential are seen accompanying drawing 1, accompanying drawing 2 respectively, and the scanning electron microscope testing result is seen accompanying drawing 3.
The not commensurability DHOF of table 1 to Low molecular heparin iron nano-particle particle diameter, zeta current potential with and the comparison of envelop rate
Conclusion: LMWH and DHOF can form the heparin iron nano-particle under certain conditions, and it makes heparin iron nano-particle optimal proportion between 0.8～1.2.
The infrared absorption spectroscopy of Low molecular heparin iron nano-particle
Method: the mass ratio by DHOF and LMWH is 1.0; Under the condition of ultrasonic concussion; The Low molecular heparin nanoparticles solution (manufacture method is with embodiment 1) of preparation; With the preparation the Low molecular heparin nanoparticles solution at-54 ℃ of vacuum dryings; With Low molecular heparin iron nano-particle powder 10mg and LMWH powder respectively with potassium bromide powder 90mg mix homogeneously tabletting after under the nitrogen purging of 99.999% purity, use the fourier infrared appearance to measure Low molecular heparin iron nano-particle and LMWH infrared absorption spectroscopy.
Result: the infrared absorption spectroscopy broadly similar of Low molecular heparin iron nano-particle and LMWH (the fourier infrared abosrption spectrogram is seen accompanying drawing 4).Because [Fe (OH)
2]
+Interact with LMWH, the O-H on the Low molecular heparin iron nano-particle, N-H, various bond energys such as O-S occur all that cloud density descends, the mechanical constant of key weakens, O-H wherein, and the N-H wave number is 3444cm respectively
-, 1619cm
-To 3405cm
-, 1629cm
-Slight moving, and 1732cm appears
-, 1549cm
-, 690cm
-, 423cm
-Absworption peak, wherein 1732cm everywhere
-, 1549cm
-Two place's absworption peaks are carbonyl absorption peaks, 690cm
-, 423cm
-Two places are O-Fe absworption peaks.Prompting: O-H, N-H and [Fe (OH) in the LMWH molecule
2]
+Between have coordinate bond to form, make O-H, N-H wave number had slight moves; In the LMWH molecule carboxylic acid can with [Fe (OH)
2]
+Chemical reaction takes place, and forms carboxyl ferrum or carbonyl iron; There is the O-Fe key in the Low molecular heparin iron nano-particle.
Conclusion: DHOF forms Low molecular heparin ferrum with after LMWH mixes; Low molecular heparin iron molecule structure is similar with LMWH.
The external slow release of Low molecular heparin iron nano-particle detects
Method: the Low molecular heparin nanoparticles solution of preparation (manufacture method is with embodiment 1), nanoparticles solution through 10000rpm high speed centrifugation 20 minutes, is abandoned supernatant; Deionized water washing sediment 3 times takes by weighing 10mg after the drying, with ultra-sonic dispersion is even again after the concussion evenly of 10mlPBS liquid; Using the dialysis molecular weight is 12000 bag filter splendid attires, it is inserted in the 190ml PBS liquid 80rpm concussion; Respectively at 1d, 2d, 1w, 2w, the 4w 100ul that from PBS, takes a sample, detect the concentration of its LMWH.
The result: the Low molecular heparin nano-particle is bigger first day burst size, accounts for 5.1% of total amount, is thereafter even release, and to 4 weeks, burst size accounts for 43.8% of total amount.The result sees table 2.
Table 2. different time Low molecular heparin iron nano-particle burst size
Conclusion: the Low molecular heparin nanoparticle structure is stable, slow in PBS liquid, evenly release.
Embodiment 4
The lyophilizing of Low molecular heparin nano-particle is preserved
Method: the Low molecular heparin nanoparticles solution of preparation (manufacture method is with embodiment 1), get each 5ml of nanoparticles solution of 5 groups of prepared fresh.Wherein directly go nanometer particle size and the detection of zeta current potential for one group; Evenly add 50mg, 100mg, 200mg, 300mg Dextran-10 in the remaining set nanometer solution respectively; Ultrasonic concussion 10 minutes; Freezing back uses the 5ml deionized water with the dried granules powder dissolution respectively-54 ℃ of drying under vacuum overnight.Respectively getting capable respectively nanometer particle size of 2ml and zeta current potential detects.
The result: after lyophilizing was dissolved again, the zeta current potential was respectively organized difference little (similar with fresh group).Nanometer particle size is found: dissolve after adding 50mg, the lyophilizing of 100mg Dextran-10 group, micron particles is more again; Other group nanometer particle sizes are similar with the prepared fresh nanometer particle size, and the result sees table 3.
Table 3. adds the not commensurability lyophilizing group of Dextran-10 and fresh group of particle diameter and the comparison of zeta current potential
Conclusion: Dextran-10 can effectively reduce the reunion of Low molecular heparin iron nano-particle in freezing dry process, uses Dextran-10 concentration greater than 20g/L, and lyophilization is little to nano particle diameter and the influence of zeta current potential.
Claims (2)
1. Low molecular heparin nano controlled-release granule is characterized in that nano-particle is the rectangle nanocrystal, mean diameter in 100 nanometers, Zeta potential about-19mV～-35mV between, envelop rate is 25%～93%.
2. the particulate manufacture method of Low molecular heparin nano controlled-release is characterized in that comprising the steps:
1) configuration of DHOF solution:
Use deionized water dissolving FeCl
36H
2O; Under stirring condition, press Fe
3+: OH
-Mol ratio 1: 2 slowly splashes into FeCl with NaOH solution
3In the solution, add the concentration of an amount of water adjustment DHOF again, the DHOF solution of preparation pH value 3.0 concentration between 1～20g/L;
2) preparation of LMWH solution:
Low molecular sodium heparin is dissolved in the deionized water, and mass concentration and adds sodium chloride in the Low molecular heparin sodium solution between 1～20g/L, and the concentration that makes sodium chloride is between 1～5g/L.
3) making of Low molecular heparin iron nano-particle:
Under the condition of ultrasonic concussion, DHOF and LMWH join DHOF solution rapid and uniform in the LMWH solution in certain mass ratio 0.2～2, and under ultrasonic concussion condition, put 10～30 minutes;
4) add a certain amount of lyophilized powder adjuvant in the Low molecular heparin ferrum nanometer solution and be prepared into the lyophilized powder preservation.
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|---|---|---|---|
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|---|---|---|---|
| CN201010563472.XA CN102475686B (en) | 2010-11-29 | 2010-11-29 | Low molecular heparin iron nanoparticles and preparation method thereof |
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| CN102475686B CN102475686B (en) | 2015-05-06 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104906045A (en) * | 2015-06-25 | 2015-09-16 | 中国科学院过程工程研究所 | Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof |
| CN108186675A (en) * | 2018-02-08 | 2018-06-22 | 山东大学 | Heparin iron complexes are to the treatment use of chronic inflam matory anemia |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024086A (en) * | 2007-03-09 | 2007-08-29 | 沈阳药科大学 | Composition formed from chitosan and its derivatives and low-molecular heparin and pneparation and preparing method |
-
2010
- 2010-11-29 CN CN201010563472.XA patent/CN102475686B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024086A (en) * | 2007-03-09 | 2007-08-29 | 沈阳药科大学 | Composition formed from chitosan and its derivatives and low-molecular heparin and pneparation and preparing method |
Non-Patent Citations (1)
| Title |
|---|
| LU YU ET AL: "Novel Hollow Microcapsules Based on Iron-Heparin Complex Multilayers", 《LANGMUIR》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104906045A (en) * | 2015-06-25 | 2015-09-16 | 中国科学院过程工程研究所 | Low-molecular-weight heparin slow-release preparation as well as preparation method and application thereof |
| CN104906045B (en) * | 2015-06-25 | 2018-04-13 | 中国科学院过程工程研究所 | A kind of low molecular weight heparin sustained release preparation and its preparation method and application |
| CN108186675A (en) * | 2018-02-08 | 2018-06-22 | 山东大学 | Heparin iron complexes are to the treatment use of chronic inflam matory anemia |
| CN108186675B (en) * | 2018-02-08 | 2020-10-02 | 山东大学 | Application of heparin-iron complex in treatment of chronic inflammatory anemia |
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| Publication number | Publication date |
|---|---|
| CN102475686B (en) | 2015-05-06 |
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