CN104856960A - Nanometer particles with iron chelators encapsulated in amphiphilic polymers and preparation method and application thereof - Google Patents
Nanometer particles with iron chelators encapsulated in amphiphilic polymers and preparation method and application thereof Download PDFInfo
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 26
- 239000002245 particle Substances 0.000 title abstract description 23
- 229920000642 polymer Polymers 0.000 title abstract description 8
- 239000002738 chelating agent Substances 0.000 title abstract 10
- UBQYURCVBFRUQT-UHFFFAOYSA-N N-benzoyl-Ferrioxamine B Chemical compound CC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCN UBQYURCVBFRUQT-UHFFFAOYSA-N 0.000 claims abstract description 110
- 229960000958 deferoxamine Drugs 0.000 claims abstract description 109
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000003814 drug Substances 0.000 claims abstract description 14
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 238000004945 emulsification Methods 0.000 claims abstract description 7
- 239000002105 nanoparticle Substances 0.000 claims description 116
- 150000005846 sugar alcohols Polymers 0.000 claims description 69
- 229940075525 iron chelating agent Drugs 0.000 claims description 58
- 239000000797 iron chelating agent Substances 0.000 claims description 58
- 239000000839 emulsion Substances 0.000 claims description 30
- 239000004094 surface-active agent Substances 0.000 claims description 27
- 239000007864 aqueous solution Substances 0.000 claims description 26
- 239000003960 organic solvent Substances 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- 229920000382 poly(ethylene glycol) methyl ether-block-poly(L-lactide-co-glycolide) Polymers 0.000 claims description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
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- 238000003756 stirring Methods 0.000 claims description 9
- 229920001400 block copolymer Polymers 0.000 claims description 7
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 7
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 6
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 229920000375 Poly(ethylene glycol)-block-poly(ε−caprolactone) methyl ether Polymers 0.000 claims description 5
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
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- 238000001262 western blot Methods 0.000 description 3
- 208000035126 Facies Diseases 0.000 description 2
- TZXKOCQBRNJULO-UHFFFAOYSA-N Ferriprox Chemical compound CC1=C(O)C(=O)C=CN1C TZXKOCQBRNJULO-UHFFFAOYSA-N 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 239000013522 chelant Substances 0.000 description 2
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- 229960001489 deferasirox Drugs 0.000 description 2
- FMSOAWSKCWYLBB-VBGLAJCLSA-N deferasirox Chemical compound C1=CC(C(=O)O)=CC=C1N(N\C(N\1)=C\2C(C=CC=C/2)=O)C/1=C\1C(=O)C=CC=C/1 FMSOAWSKCWYLBB-VBGLAJCLSA-N 0.000 description 2
- 229960003266 deferiprone Drugs 0.000 description 2
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229920001427 mPEG Polymers 0.000 description 2
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- 229920000573 polyethylene Polymers 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 238000008575 Iron Assay Methods 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
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- 108010087230 Sincalide Proteins 0.000 description 1
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- 208000005980 beta thalassemia Diseases 0.000 description 1
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- 150000003278 haem Chemical class 0.000 description 1
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- 235000014655 lactic acid Nutrition 0.000 description 1
- 229910052746 lanthanum Inorganic materials 0.000 description 1
- FZLIPJUXYLNCLC-UHFFFAOYSA-N lanthanum atom Chemical compound [La] FZLIPJUXYLNCLC-UHFFFAOYSA-N 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a kind of nanometer particles with iron chelators encapsulated in amphiphilic polymers and a preparation method and application thereof. The nanometer particles are formed in the mode that water-soluble chelators are encapsulated in amphiphilic polymers. According to the method, the amphiphilic polymers serve as carriers, and the nanometer particles with iron chelators encapsulated in amphiphilic polymers are obtained through a multiple emulsion method. The obtained nanometer particles are stable, the half-life period of the iron chelators in blood circulation in vivo is prolonged, the iron chelators are transported to target organs in a targeting manner, nanometer drugs containing the nanometer particles with iron chelators encapsulated in amphiphilic polymers can be used for treating iron-overloaded diseases, the iron chelators release slowly so that deferoxamine can metabolize in a human body, more iron chelators can enrich in viscera, the curative effect of the drugs is improved, the toxic and side effect on cells is reduced, and the iron chelators can remove iron in cells more efficiently.
Description
Technical field
The invention belongs to pharmaceutical technology sectors, relate to a kind of amphipathic nature polyalcohol bag nanoparticle carrying iron chelating agent and its preparation method and application.
Background technology
There is accurate ferrum steady-state system in human body, normal human takes in the ferrum of 1-2mg every day by duodenum, and these ferrum, in conjunction with entering ferrikinetics in body after transferrins, mainly participate in the synthesis of haemachrome.And the macrophage of liver can engulf the erythrocyte of aging or necrosis, thus discharge heme iron again, thus recycle or store.When causing that in body, ferrum is had more than needed by heredity or other reason in human body, owing to lacking the mechanism excreted by ferrum, too much ferrum can be enriched in the organ such as liver, spleen, may cause the disease such as myocardial dysfunction, liver cirrhosis under long-term behavior, What is more entail dangers to life.Ferrum overload disease is mainly divided into heritability ferrum to transship (congenital hemochromatosis) or Secondary cases ferrum overload (transfusion ferrum overload etc.) according to the cause of disease.
Mainly use Deferioxamine (deferoxamine, DFO), Deferiprone (deferiprone) and Deferasirox (Deferasirox) three kinds of iron chelating agents clinically now, wherein deferoxamine is most widely used general.Deferoxamine in conjunction with the ferrum in blood, can be discharged after chelated iron from urine and feces.Main employing injected s. c is treated, and because the half-life is shorter, treatment time is generally 8-12 hour every day, on every Fridays sky.In view of above treatment predicament, deferoxamine specificity how is made to enter in cells of organs, urgently to be resolved hurrily.
Along with the development of nanotechnology, due to dimensional effect and macrophage is isocellular engulfs, nano-particle energy passive target, in internal organs, has become the common recognition of this area research person.Therefore, nano-particle can be adopted to wrap up deferoxamine to improve the utilization ratio of deferoxamine, ferrum overload disease is treated.
The people such as Schlicher, E.J. (Int.J.Pharm.Vol.153,1997,235-245) describe the microparticle compositions of desferrioxamine (deferoxamine).Described microgranule is prepared by PLGA (Poly(D,L-lactide-co-glycolide), but the particle diameter of described microgranule is micron order, and particle diameter is large, and poor stability in vivo, circulating half-life in vivo is short, without organ targeting, more difficult by cellular uptake in vivo.
US 2005/175684 A1 discloses the target liposomes compositions of iron chelating agent.Described compositions sustained release can be provided and long time active.By combining described chelating agen, one or more form the lipid of vesicles and the agent of at least one targeting, are prepared by liposomal encapsulated mode with described iron chelating agent.The shortcoming of these liposomees is particle easily in vivo because the reason such as osmotic pressure or mechanical external force is broken, and causing prominent releasing, and shape is variable, and it is low that bag carries rate, especially carry rate for the bag of hydrophilic drugs extremely low, and its preparation process is comparatively complicated.
Therefore, in the art, expect that developing a kind of nanoparticle carrying iron chelating agent that can effectively wrap effectively treats ferrum overload disease.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of amphipathic nature polyalcohol bag nanoparticle carrying iron chelating agent and its preparation method and application.The advantages such as the targeting that the present invention has in conjunction with amphipathic nature polyalcohol nanoparticle, stability and plasma metabolism speed are slow, realize transporting the targeting of hydrophilic iron chelating agent and delaying the clearance rate of iron chelating agent.
For reaching this goal of the invention, the present invention by the following technical solutions:
On the one hand, the invention provides the nanoparticle that a kind of amphipathic nature polyalcohol bag carries iron chelating agent, described nanoparticle take amphipathic nature polyalcohol as the nanoparticle that carrier bag carries the formation of water-soluble iron chelating agen.
The present invention is using amphipathic nature polyalcohol as carrier, because amphipathic nature polyalcohol has hydrophobic fragment and hydrophilic segment, hydrophilic core hydrophilic iron chelating agent being wrapped in the stable nanoparticles that amphipathic nature polyalcohol is formed can be realized in self assembling process, the nano-particle formed can stable existence in body circulation, extension body circulating half-life, the iron chelating agent simultaneously making bag be loaded in its core more effectively can enter organ, there is the effect of targeting and slow release, improve curative effect of medication, reduce toxic and side effects.
Carry in the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, described amphipathic nature polyalcohol is for selecting good biocompatibility, safety, the material that can use people is ratified by FDA, described amphipathic nature polyalcohol can for but be not limited to the combination of any one or at least two kinds in PEG-PLA (polyethylene glycol-polylactic acid block copolymer), PEG-PCL (PEG-PCL block copolymer) or mPEG-PLGA (mono methoxy polyethylene glycol-PLGA), be preferably mPEG-PLGA.
Preferably, described iron chelating agent is deferoxamine (DFO).
On the other hand, the invention provides the preparation method that amphipathic nature polyalcohol bag of the present invention carries the nanoparticle of iron chelating agent, described method take amphipathic nature polyalcohol as carrier, carries water-soluble iron chelating agen form nanoparticle by multi-emulsion method bag.
The preparation method that amphipathic nature polyalcohol bag of the present invention carries the nanoparticle of iron chelating agent comprises the following steps:
(1) amphipathic nature polyalcohol is dissolved in organic solvent, adds the aqueous solution of iron chelating agent wherein, ultrasonic, form the first emulsion;
(2) the first emulsion prepared by step (1) is added in the aqueous solution of surfactant, ultrasonic, form the second emulsion;
(3) the second emulsion that step (2) is formed is added in the aqueous solution of surfactant, stir;
(4) remove organic solvent, obtain nanoparticle suspension, be separated, obtain the nanoparticle that described amphipathic nature polyalcohol bag carries iron chelating agent.
The present invention adopts multi-emulsion method to prepare the nanoparticle that amphipathic nature polyalcohol bag carries iron chelating agent, described multi-emulsion method refers to the method utilizing twice emulsifying, first time emulsifying amphipathic nature polyalcohol forms hydrophilic core and hydrophobic shell, deferoxamine is wrapped in the hydrophilic kernel of amphipathic nature polyalcohol formation, second time emulsifying makes nanoparticle top layer form hydrophilic layer, and makes nanoparticle structure more stable.Therefore, the nanoparticle utilizing method of the present invention to obtain is the hydrophilic core of the Bao Zaitie intercalating agent that innermost layer is formed for amphipathic nature polyalcohol hydrophilic segment, its skin is the hydrophobic layer that the hydrophobic fragment of amphipathic nature polyalcohol is formed, make hydrophobic layer form hydrophilic layer outward by second time emulsifying, improve its body circulation in stability.And the stable appearance of the nanoparticle formed is high, can bear external force mechanical strength, prominent the releasing of the medicine (deferoxamine) not easily causing bag to carry.
Utilize the method for the invention can control within 10-300nm by nanometer particle size, this granule size is adapted at enrichment in internal organs, easily by identifications such as macrophages, thus is transported in internal organs by nanoparticle.If granularity is too large, easy in pulmonary's enrichment, cause pulmonary's disease of immune system, granularity is too little easily directly to be excreted by kidney.
Carry in the preparation method of the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, the mass ratio of step (1) described amphipathic nature polyalcohol and iron chelating agent is 4:1-150:1, such as 4:1,5:1,10:1,15:1,20:1,25:1,30:1,40:1,50:1,60:1,70:1,80:1,90:1,100:1,110:1,120:1,130:1,140:1 or 150:1, be preferably 10:1-40:1.
Preferably, step (1) described organic solvent is the combination of any one or at least two kinds in dichloromethane, dimethyl sulfoxide, oxolane or acetone.
Preferably, the volume ratio of the aqueous solution of step (1) described organic solvent and iron chelating agent is 4:1-10:1, such as 4:1,5:1,6:1,7:1,8:1,9:1 or 10:1.The volume of organic facies is made to be greater than aqueous phase volume, so that make amphipathic nature polyalcohol form the nano-particle with hydrophilic core and hydrophobic shell, to be loaded onto in hydrophilic core by hydrophilic iron chelating agent bag in first time emulsion process.
Preferably, step (1) is described ultrasonicly to be utilized ultrasonic cell disruption instrument to realize, power is 190W-380W, such as 190W, 200W, 210W, 220W, 230W, 240W, 250W, 270W, 290W, 300W, 320W, 340W, 350W, 360W, 370W or 380W, ultrasonic time is 5-10min, such as 5min, 6min, 7min, 8min, 9min or 10min.
Carry in the preparation method of the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, step (2) and step (3) described surfactant are the combination of any one or at least two kinds in polyvinyl alcohol, polyoxyethylene polyoxypropylene ether block copolymer, Polysorbate, dodecyl sodium sulfate or hexadecyltrimethylammonium chloride.
Preferably, the concentration of step (2) described aqueous surfactant solution is 1%-5%, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.
Preferably, the aqueous solution of step (2) described surfactant and the volume ratio of step (1) described organic solvent are 2:1-5:1, such as 2:1,2.5:1,3:1,3.5:1,4:1,4.5:1 or 5:1.In second time emulsion process, strengthen aqueous phase volume, make to carry out emulsifying in water environment thus form hydrophilic layer in the nanoparticle surface formed, so as nanoparticle can in water environment stable existence.
Carry in the preparation method of the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, step (2) is described ultrasonicly to be utilized ultrasonic cell disruption instrument to realize, power is 190W-285W, such as 190W, 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W or 285W, ultrasonic time is 5-10min, such as 5min, 6min, 7min, 8min, 9min or 10min.
First emulsion adds after in the aqueous solution of surfactant by step of the present invention (2), mixed liquor can be stirred, and then carries out ultrasonic again, and the object stirred is that the first emulsion is scattered in aqueous surfactant solution better.
Of the present invention twice ultrasonic in, the ultrasonic power of step (1) is made to be a bit larger tham or to equal the ultrasonic power of step (2), this is to obtain relatively little particle diameter after step (1) is ultrasonic, and time again ultrasonic in step (2), ensure that nanoparticle is too not little, therefore slightly little power is adopted, because if the nano particle diameter adopting too large power may make to produce is too little, easily directly to be excreted by kidney, thus reduce its targeting for particular organization, be unfavorable for treatment use.
Preferably, the concentration of step (3) described aqueous surfactant solution is 0.2%-1%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.
When surfactant is polyvinyl alcohol, polyoxyethylene polyoxypropylene ether block copolymer, Polysorbate, in dodecyl sodium sulfate or hexadecyltrimethylammonium chloride during the combination of at least two kinds, step (2) or step (3) described concentration refer to often kind of composition concentration in the solution in combination and are 1%-5% or 0.2%-1% such as when surfactant is the combination of dodecyl sodium sulfate and hexadecyltrimethylammonium chloride, the aqueous surfactant solution of 1% concentration, the concentration referring to dodecyl sodium sulfate and hexadecyltrimethylammonium chloride in aqueous surfactant solution is 1%.
The aqueous surfactant solution object that step (2) adopts concentration higher makes the first emulsion obtain more stable nanometer system, and step (3) adopts the aqueous surfactant solution of low concentration to be that the second emulsion because being formed by the first emulsion and surfactant is relatively stable, in order to obtain stable system further, need again to add surfactant to stir, but do not need too large concentration, the aqueous surfactant solution of 0.2%-1% concentration is just enough to reach this object.
Carry in the preparation method of the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, step (3) described mixing time is 10-40min, such as 10min, 15min, 20min, 25min, 30min, 35min or 40min.
Preferably, the volume ratio of step (3) described aqueous surfactant solution and step (1) described organic solvent is 5:1-15:1, such as 5:1,6:1,7:1,8:1,9:1,10:1,11:1,12:1,13:1,14:1 or 15:1.In step (3), the second emulsion being put into aqueous surfactant solution makes this system aqueous phase volume be greater than the volume of organic facies, be in order to obtain further can in water environment the nanoparticle of stable existence.
Carry in the preparation method of the nanoparticle of iron chelating agent at amphipathic nature polyalcohol bag of the present invention, the described removing organic solvent of step (4) adopts existing techniques in realizing, such as, remove organic solvent with Rotary Evaporators; The described separation of step (4) adopts existing techniques in realizing, such as, utilize the method for centrifugalize.
Raw materials used in the present invention, such as Amphipathilic block polymer and ferrum intercalating agent, surfactant and organic solvent etc. all can business buy.
On the other hand, the invention provides a kind of iron chelating agent Nano medication, described iron chelating agent Nano medication comprises the nanoparticle that amphipathic nature polyalcohol bag of the present invention carries iron chelating agent.
On the other hand, the invention provides the application of nanoparticle in the medicine for the preparation for the treatment of ferrum overload disease that described amphipathic nature polyalcohol bag carries iron chelating agent.
Iron chelating agent targeting can be transported in target organ by the nanoparticle that amphipathic nature polyalcohol bag of the present invention carries iron chelating agent, utilize the iron chelating agent treatment ferrum overload disease of slow releasing, especially Secondary cases ferrum overload disease, as transfusion ferrum overload and β type thalassemia, the nano-particle carrying iron chelating agent can the macrophage of the direct deposited iron element such as targeting major hepatic, spleen, makes treatment with a definite target in view.
Relative to prior art, the present invention has following beneficial effect:
The present invention has adopted the method for twice emulsifying to prepare and has wrapped the amphipathic nature polyalcohol nanoparticle being loaded with iron chelating agent, obtain the nanoparticle of 10-300nm, granule size is adapted at enrichment in internal organs, it is higher that stable in properties and deferoxamine bag carry rate, iron chelating agent is wrapped in amphipathic nature polyalcohol carrier by this nanoparticle, improve the half-life of iron chelating agent in vivo in blood circulation, iron chelating agent targeting is enable to be transported in target organ, the iron chelating agent of slow releasing can be utilized to carry out the treatment of ferrum overload disease, slow down the metabolic rate of deferoxamine in human body, improve the curative effect of medicine, when DFO concentration is 20-80 μM, the survival rate of NPs-DFO group cell is all more than 70%, relative to free DFO, significantly reduce the toxicity to cell, when DFO concentration is 80 μMs, NPs-DFO makes total iron content in mouse monokaryon macrophage be reduced to 10 μ g/10
10about individual cell, relative to free DFO (280 μ g/10
10individual cell), significantly improve DFO and efficiency is dispelled to ferrum in cell, improve treatment feasibility, alleviate doctors and patients' burden.And preparation method of the present invention is simple, cost is low, and the material toxicity adopted is little, and safety is high, can laboratory small-scale production, also can industrialization large-scale production.
Accompanying drawing explanation
Fig. 1 detects the uv scan figure that bag prepared by embodiment 1 obtains when carrying DFO in the nanoparticle of DFO;
Fig. 2 is that bag prepared by embodiment 1 carries the grain size distribution of the nanoparticle of DFO;
Fig. 3 is that bag prepared by embodiment 1 carries nanoparticle transmission electron microscope (TEM) figure of DFO;
Fig. 4 is that the particle diameter that the bag nanoparticle system that carries DFO prepared by embodiment 1 is placed 0-3 days is at various ph values schemed over time;
Fig. 5 is that nanoparticle is to the envelop rate figure of DFO under the different quality ratio of amphipathic nature polyalcohol with DFO;
Fig. 6 is under pH 4.4 with pH 7.4 condition, the release profiles of DFO in nanoparticle prepared by embodiment 1;
Fig. 7 is under different DFO concentration, and the nanoparticle (NPs-DFO) that free DFO and bag carry DFO affects cell survival rate, and wherein * represents P<0.05, n=5;
Fig. 8 is the result figure with total iron content in cell after nanoparticle (NPs-DFO) cultured cell of free DFO and bag year DFO in embodiment 6;
Fig. 9 detects iron level in cell with western blotting in embodiment 7 to be correlated with the result figure of ferritin and the change of TfR 1 expression.
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, preparation bag carries the amphipathic nature polyalcohol nanoparticle (NPs-DFO) of deferoxamine by the following method, and described method specifically comprises the following steps:
(1) by 20mg amphipathic nature polyalcohol mPEG-PLGA (purchased from Jinan Dai Gang biological engineering company limited, in mPEG-PLGA molecule, the molecular weight of mPEG fragment is 5000, in PLGA, the fragment mol ratio of lactic acid and hydroxyacetic acid is 75:25, the mass ratio of mPEG and PLGA is 1:4) be dissolved in 2mL dichloromethane, (mass ratio of amphipathic nature polyalcohol and DFO is 20:1 to add the aqueous solution of 0.5mL 2mg/mL deferoxamine (DFO) wherein, organic solvent and deferoxamine aqueous solution volume ratio are 4:1), utilize ultrasonic cell disruption instrument under 250W power ultrasonic 5 minutes, form the first emulsion,
(2) the first emulsion prepared by step (1) is dissolved in dichloromethane, slowly add in polyvinyl alcohol (PVA) aqueous solution of 4mL 2%, utilize ultrasonic cell disruption instrument ultrasonic 5min under 200W power, form the second emulsion;
(3) the second emulsion that step (2) is formed slowly is added in the aqueous solution of the PVA of 0.6% of 10mL, stir 10min;
(4) remove organic solvent dichloromethane with Rotary Evaporators, obtain nanoparticle suspension, 13000rpm centrifugalize 10 minutes, obtain wrapping the amphipathic nature polyalcohol nanoparticle carrying iron chelating agent DFO.
comparative example 1
This comparative example does not add deferoxamine as different from Example 1 in preparation process, and in addition other conditions are identical with embodiment 1, is prepared into unloaded mPEG-PLGA nanoparticle (NPs-B).
Whether successfully wrap a year DFO to the nanoparticle of embodiment 1 preparation to detect, method is dissolve with dichloromethane the nanoparticle prepared, the DFO then wrapped up can discharge from nanoparticle, then add standard ferrum and carry out uv scan, if wrapped up DFO in nanoparticle, then can there is the absworption peak of chelate with ferrum chelating in DFO at 430nm place.Result is shown in Figure 1, can be drawn by Fig. 1, and amphipathic nature polyalcohol nanoparticle prepared by embodiment 1 successfully wraps and carried iron chelating agent DFO.
Utilize nano particle size and Zeta potential analyser (Zetasizer Nano ZS, Malvern Instr Ltd. of Britain) characterize the particle diameter of nanoparticle (NPs-DFO) and surface charge prepared by embodiment 1, and utilize 200K lanthanum hexaboride transmission electron microscope (Tecnai G2 20S-TWIN, FEI Co. of the U.S.) characterize particle diameter and the form of NPs-DFO, Fig. 2 is that dynamic light scattering characterizes the particle size results obtained, Fig. 3 is the transmission electron microscope results of NPs-DFO, what nanoparticle prepared by embodiment 1 and comparative example 1 had been shown in table 1 characterizes through dynamic light scattering the mean diameter and surface charge that obtain, the particle size distribution of NPs-DFO is between 60nm-300nm as seen from Figure 2, Fig. 3 can find out, NPs-DFO prepared by embodiment 1 is the double-deck nanoparticle of nucleocapsid, little particle diameter is tens nanometers, large particle diameter is at about 160nm, this conforms to the result of dynamic light scattering.The mean diameter giving NPs-DFO in table 1 is 158.15 ± 1.62nm, and relative to NPs-B, particle diameter slightly reduces, but change is little, illustrates that the bag of DFO carries the increase that can not cause particle diameter; The Zeta potential of NPs-DFO is-17.64 ± 0.74mV, therefore, shows that this stability of solution is better.
Table 1 (end value is meansigma methods ± SD, n=3)
In order to characterize the stability of NPs-DFO nanoparticle prepared by embodiment 1 further, by NPs-DFO nanoparticle dispersion in PBS buffer, the 0th day is designated as when preparing the dispersion of nanoparticle, the nanoparticle of preparation is divided into three parts respectively, be adjusted to pH value and be respectively 4.4, 5.3 and 7.4, measure the mean diameter of nanoparticle now, at this system is placed in 37 DEG C, stir, rotating speed is 110rpm, every day same timing nanoparticle mean diameter, result as shown in Figure 4, can find out that mean diameter does not change within the time of 3 days, therefore prove that this nanoparticle system is more stable.
The impact of mass ratio on DFO envelop rate of different amphipathic nature polyalcohol and DFO is investigated, method is as follows: under the mass ratio of different amphipathic nature polyalcohol and DFO, prepare the amphipathic nature polyalcohol nanoparticle (NPs-DFO) wrapping and carry deferoxamine, described preparation method and selected material are all identical with embodiment 1, difference is only the mass ratio of amphipathic nature polyalcohol and DFO to be set as 4:1, 10:1, 20:1, 40:1, 100:1 and 150:1, preparation bag carries the mPEG-PLGA nanoparticle (NPs-DFO) of deferoxamine respectively, its envelop rate is investigated, result as shown in Figure 5.As seen from Figure 5, along with the increase of the mass ratio of amphipathic nature polyalcohol and DFO, the envelop rate of DFO raises, after mass ratio is greater than 40:1, though envelop rate has certain rising, but elevation amplitude is very little, and now concentrations of nanoparticles increases, the stability of system is declined to some extent, therefore the consideration of comprehensive envelop rate and stability aspect, the mass ratio of preferred amphiphilic polymer and DFO is 10:1-40:1.
In the NPs-DFO nanoparticle prepare embodiment 1, the release conditions of DFO pH 4.4 and 7.4 time is investigated, and method is as follows:
First the aqueous solution of variable concentrations DFO is prepared, add equivalent standard ferrum wherein and carry out uv scan, measure the absorbance of chelate at 430nm of ferrum and DFO, make the standard curve of DFO concentration and absorbance, bag prepared by embodiment 1 is carried deferoxamine nanoparticle NPs-DFO (30mg) to be scattered in 5mL pH value and to be respectively in the PBS buffer of 4.4 and 7.4, be placed in 4 DEG C of stirrings, rotating speed is 110rpm, in different time points, supernatant is got after 12000rpm is centrifugal, add the absorbance that standard ferrum detects 430nm, the amount of the DFO that deferoxamine nanoparticle NPs-DFO discharges is drawn according to standard curve, per cents according to the DFO of cumulative release goes out drug release patterns, as shown in Figure 6.
As shown in Figure 6, under pH 7.4 and pH 4.4 condition, in first 20 hours, major part DFO can discharge and under pH 7.4 condition rate of release slower, relative to free DFO medicine itself in blood 20-30min namely fallen by metabolism and discharged by kidney, nanoparticle of the present invention can be consumed completely at DFO, and DFO is loaded into internal organs.
embodiment 2
In the present embodiment, preparation bag carries the amphipathic nature polyalcohol nanoparticle (NPs-DFO) of deferoxamine by the following method, and described method specifically comprises the following steps:
(1) 10mg amphipathic nature polyalcohol PEG-PLA is dissolved in 1mL dimethyl sulfoxide, (mass ratio of amphipathic nature polyalcohol and DFO is 10:1 to add the aqueous solution of 0.2mL 5mg/mL deferoxamine (DFO) wherein, organic solvent and deferoxamine aqueous solution volume ratio are 5:1), utilize ultrasonic cell disruption instrument under 200W power ultrasonic 10 minutes, form the first emulsion;
(2) the first emulsion prepared by step (1) is dissolved in dimethyl sulfoxide, slowly add in the Tween-20 of 3mL 5% and the aqueous solution of dodecyl sodium sulfate (concentration of Tween-20 and dodecyl sodium sulfate is 10%), utilize ultrasonic cell disruption instrument ultrasonic 10min under 200W power, form the second emulsion;
(3) the second emulsion that step (2) is formed slowly is added in the Tween-20 of 1% of 10mL and the aqueous solution of dodecyl sodium sulfate (concentration of Tween-20 and dodecyl sodium sulfate is 1%), stir 30min;
(4) with the method removing organic solvent dimethyl sulfoxide utilizing dialysis in water, obtain nanoparticle suspension, 13000rpm centrifugalize 10 minutes, obtain the nanoparticle that amphipathic nature polyalcohol bag carries iron chelating agent.
Through characterizing the particle size distribution of preparation-obtained nanoparticle between 40nm-300nm, mean diameter is at 151.25 ± 1.21nm, and Zeta potential is-17.31 ± 0.50mV, and nanoparticle is more stable.
embodiment 3
In the present embodiment, preparation bag carries the amphipathic nature polyalcohol nanoparticle (NPs-DFO) of deferoxamine by the following method, and described method specifically comprises the following steps:
(1) 20mg amphipathic nature polyalcohol PEG-PCL is dissolved in 2mL oxolane, add the aqueous solution (mass ratio of amphipathic nature polyalcohol and DFO is 20:1) of 0.2mL 5mg/mL deferoxamine (DFO) wherein, utilize ultrasonic cell disruption instrument under 380W power ultrasonic 10 minutes, form the first emulsion;
(2) the first emulsion prepared by step (1) is dissolved in oxolane, slowly add in the aqueous solution of the hexadecyltrimethylammonium chloride of 10mL 1%, utilize ultrasonic cell disruption instrument ultrasonic 10min under 200W power, form the second emulsion;
(3) the second emulsion that step (2) is formed slowly is added in the aqueous solution of the hexadecyltrimethylammonium chloride of 0.2% of 20mL, stir 40min;
(4) remove organic solvent tetrahydrofuran with Rotary Evaporators, obtain nanoparticle suspension, 13000rpm centrifugalize 10 minutes, obtain the nanoparticle that amphipathic nature polyalcohol bag carries iron chelating agent.
Through characterizing the particle size distribution of preparation-obtained nanoparticle between 10nm-300nm, mean diameter is at 162.10 ± 2.13nm, and Zeta potential is-17.25 ± 0.72mV, and nanoparticle is more stable.
embodiment 4
In the present embodiment, preparation bag carries the amphipathic nature polyalcohol nanoparticle (NPs-DFO) of deferoxamine by the following method, and described method specifically comprises the following steps:
(1) mixture of 20mg amphipathic nature polyalcohol PEG-PCL and PEG-PLA is dissolved in the mixture of 1mL acetone and oxolane, add the aqueous solution (mass ratio of amphipathic nature polyalcohol and DFO is 40:1) of 0.2mL 2.5mg/mL deferoxamine (DFO) wherein, utilize ultrasonic cell disruption instrument under 300W power ultrasonic 8 minutes, form the first emulsion;
(2) the first emulsion prepared by step (1) is dissolved in the mixture of acetone and oxolane, slowly add in the polyoxyethylene polyoxypropylene ether block copolymer of 4mL 3% and the aqueous solution of Tween-20 and dodecyl sodium sulfate (three's concentration is 3%), utilize ultrasonic cell disruption instrument ultrasonic 8min under 285W power, form the second emulsion;
(3) the second emulsion that step (2) is formed slowly is added in the polyoxyethylene polyoxypropylene ether block copolymer of 0.2% of 15mL and the aqueous solution of Tween-20 and dodecyl sodium sulfate (three's concentration is 0.2%), stir 40min;
(4) with Rotary Evaporators removing organic solvent-acetone and oxolane, obtain nanoparticle suspension, 13000rpm centrifugalize 10 minutes, obtain the nanoparticle that amphipathic nature polyalcohol bag carries iron chelating agent.
Through characterizing the particle size distribution of preparation-obtained nanoparticle between 10nm-280nm, mean diameter is at 142.54 ± 1.71nm, and Zeta potential is-17.68 ± 0.35mV, and nanoparticle is more stable.
embodiment 5
In the present embodiment, investigate the impact of NPs-DFO nanoparticle on cell survival rate prepared by embodiment 1, method is as follows:
The mouse monokaryon macrophage furnishing 1 × 10 of exponential phase will be in
3individual/hole is inoculated in 96 well culture plates, directly add the free deferoxamine DFO of variable concentrations respectively, the mPEG-PLGA nanoparticle (NPs-B of zero load prepared by comparative example 1, namely containing the NPs-DFO of 0 μM of DFO) and the bag prepared of embodiment 1 carry deferoxamine nanoparticle NPs-DFO (in NPs-DFO, the concentration of DFO and the concentration of free DFO are consistent), often often kind of parallel 5 holes of concentration in group.Every hole adds 100 μ L 10% Ox blood serum DMEM culture fluid, and after cultivating 24h at 37 DEG C respectively, utilize CCK-8 method to detect nanoparticle to the apoptosis-promoting effect effect of cell, testing result as shown in Figure 7.
As can be seen from Figure 7, relative to direct dosing, the nano particle cell toxicity being loaded with deferoxamine is more weak, minimum to the damage of cell.Also find that different pharmaceutical proportional concentration is also different to cytotoxicity, when deferoxamine concentration is higher, toxicity is larger simultaneously.When DFO concentration is 20-80 μM, the survival rate of NPs-DFO group cell, all more than 70%, relative to free DFO, significantly reduces the toxicity to cell.
embodiment 6
In the present embodiment, investigate the efficiency of dispelling of NPs-DFO nanoparticle to iron content in cell prepared by embodiment 1, method is as follows:
The mouse monokaryon macrophage furnishing 1 × 10 of exponential phase will be in
5individual/ware is inoculated in six orifice plates, directly add the deferoxamine of variable concentrations (0,20,40 and 80 μM) respectively, the mPEG-PLGA nanoparticle (NPs-B of zero load prepared by comparative example 1, namely containing the NPs-DFO of 0 μM of DFO) and the bag prepared of embodiment 1 carry deferoxamine nanoparticle NPs-DFO (in NPs-DFO, the concentration of DFO and the concentration of free DFO are consistent), often organize often kind of parallel 3 holes of concentration.Give 10% Ox blood serum DMEM culture fluid, after cultivating 48h at 37 DEG C respectively, measure iron content in each group of cell by ferrum detection kit (Quanti Chrom Iron Assay Kit DIFE-250), testing result as shown in Figure 8.
As seen from Figure 8, after the NPs-DFO process cell 48h utilizing the present invention to prepare, in cell, iron content is starkly lower than the iron content utilized after DFO process cell 48h in cell, such as when DFO concentration is 80 μMs, NPs-DFO makes total iron content in mouse monokaryon macrophage be reduced to 10 μ g/10
10about individual cell, relative to free DFO (280 μ g/10
10individual cell), significantly improve DFO and efficiency is dispelled to ferrum in cell.
embodiment 7
In the present embodiment, on embodiment 1 prepare NPs-DFO nanoparticle on iron level in cell be correlated with ferritin and TfR 1 expression change impact investigate, method is as follows:
Mouse monokaryon macrophage (Raw264.7) furnishing 4 × 10 of exponential phase will be in
5individual/ware is inoculated in 10cm culture dish, directly adds the deferoxamine of variable concentrations (20,40 and 80 μMs), the mPEG-PLGA nanoparticle (NPs-B) of zero load and the bag of embodiment 1 preparation respectively and carries deferoxamine nanoparticle NPs-DFO (in NPs-DFO, the concentration of DFO and the concentration of free DFO are consistent).Murine hepatocarcinoma cell (HepG 2) furnishing 4 × 10 of exponential phase will be in
5individual/ware is inoculated in 10cm culture dish, directly adds bag prepared by 60mM ferric citrate amine (FAc), the deferoxamine of variable concentrations (20,40 and 80 μMs), unloaded mPEG-PLGA nanoparticle (NPs-B) and embodiment 1 respectively and carries deferoxamine nanoparticle NPs-DFO (in NPs-DFO, the concentration of DFO and the concentration of free DFO are consistent).Give 10% Ox blood serum DMEM culture fluid, after cultivating 48h respectively, with Western blotting (western blot) method detect iron level in each group of cell be correlated with ferritin and TfR 1 (TfR1) expression change, testing result is as shown in Figure 9.
As seen from Figure 9, in Raw264.7 and HepG2 two kinds of liver derived cell systems, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) for internal reference, DFO group and NPs-DFO group are compared to the normal cell of matched group, ferritin FTL (light chain ferritin) expression all reduces, and TfR1 expression all raises.
Applicant states, the present invention illustrates amphipathic nature polyalcohol bag nanoparticle carrying iron chelating agent 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, namely do not 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. amphipathic nature polyalcohol bag carries a nanoparticle for iron chelating agent, it is characterized in that, described nanoparticle take amphipathic nature polyalcohol as the nanoparticle that carrier bag carries the formation of water-soluble iron chelating agen.
2. amphipathic nature polyalcohol bag according to claim 1 carries the nanoparticle of iron chelating agent, it is characterized in that, described amphipathic nature polyalcohol is the combination of any one or at least two kinds in PEG-PLA, PEG-PCL or mPEG-PLGA, is preferably mPEG-PLGA;
Preferably, described iron chelating agent is deferoxamine.
3. amphipathic nature polyalcohol bag according to claim 1 and 2 carries the preparation method of the nanoparticle of iron chelating agent, it is characterized in that, described method take amphipathic nature polyalcohol as carrier, carries water-soluble iron chelating agen form nanoparticle by multi-emulsion method bag.
4. preparation method according to claim 3, is characterized in that, said method comprising the steps of:
(1) amphipathic nature polyalcohol is dissolved in organic solvent, adds the aqueous solution of iron chelating agent wherein, ultrasonic, form the first emulsion;
(2) the first emulsion prepared by step (1) is added in the aqueous solution of surfactant, ultrasonic, form the second emulsion;
(3) the second emulsion that step (2) is formed is added in the aqueous solution of surfactant, stir;
(4) remove organic solvent, obtain nanoparticle suspension, be separated, obtain the nanoparticle that described amphipathic nature polyalcohol bag carries iron chelating agent.
5. preparation method according to claim 4, is characterized in that, the mass ratio of step (1) described amphipathic nature polyalcohol and iron chelating agent is 4:1-150:1, is preferably 10:1-40:1;
Preferably, step (1) described organic solvent is the combination of any one or at least two kinds in dichloromethane, dimethyl sulfoxide, oxolane or acetone;
Preferably, the volume ratio of the aqueous solution of step (1) described organic solvent and iron chelating agent is 4:1-10:1;
Preferably, step (1) is described ultrasonicly to be utilized ultrasonic cell disruption instrument to realize, and power is 190W-380W, and ultrasonic time is 5-10min.
6. the preparation method according to claim 4 or 5, it is characterized in that, step (2) and step (3) described surfactant are the combination of any one or at least two kinds in polyvinyl alcohol, polyoxyethylene polyoxypropylene ether block copolymer, Polysorbate, dodecyl sodium sulfate or hexadecyltrimethylammonium chloride;
Preferably, the concentration of step (2) described aqueous surfactant solution is 1%-5%;
Preferably, the aqueous solution of step (2) described surfactant and the volume ratio of step (1) described organic solvent are 2:1-5:1.
7. the preparation method according to any one of claim 4-6, is characterized in that, step (2) is described ultrasonicly to be utilized ultrasonic cell disruption instrument to realize, and power is 190W-285W, and ultrasonic time is 5-10min.
8. the preparation method according to any one of claim 4-7, is characterized in that, the concentration of step (3) described aqueous surfactant solution is 0.2%-1%;
Preferably, the volume ratio of step (3) described aqueous surfactant solution and step (1) described organic solvent is 5:1-15:1;
Preferably, step (3) described mixing time is 10-40min.
9. an iron chelating agent Nano medication, is characterized in that, described iron chelating agent Nano medication comprises the nanoparticle that amphipathic nature polyalcohol bag according to claim 1 and 2 carries iron chelating agent.
10. amphipathic nature polyalcohol bag according to claim 1 and 2 carries the application of nanoparticle in the medicine for the preparation for the treatment of ferrum overload disease of iron chelating agent.
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