CN103638527A - Hemopoietin drug-carried nanoparticles and application thereof - Google Patents
Hemopoietin drug-carried nanoparticles and application thereof Download PDFInfo
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Abstract
The invention relates to the field of medicine, and particularly relates to hemopoietin drug-carried nanoparticles and application thereof. Specifically, chitosan-stearic acid grafted micelle and polyethylene glycol (PEG) modified lipid nanoparticles are used as carriers, under electrostatic interaction, the carriers are combined with human erythropoietin (hEPO) which presents negative electricity in water solution, thus, drug-carried nanoparticles are formed. Application of the hemopoietin drug-carried nanoparticles in preparation of cerebral ischemic injury treatment medicine and a nasal delivery preparation of the hemopoietin are also disclosed. According to the invention, the defect of adopting ventricular delivery or using high-dose EPO (erythropoietin) to treat cerebral ischemia in the current experiments and clinical tests is overcome. Moreover, treatment effect also can be achieved.
Description
Technical field
The present invention relates to field of medicaments, refer in particular to erythropoietin drug-carrying nanometer particle and application thereof.The lipid nanoparticle of specifically using respectively CSO-SA micelle and Polyethylene Glycol (PEG) to modify is carrier, pass through electrostatic interaction, make carrier and human forcing erythrogenin erythropoietin (hEPO) combination that is negative electricity in aqueous solution, form drug-carrying nanometer particle, for the application of nasal mucosa medicine administration treatment cerebral ischemia.
Background technology
Cerebral ischemia is one of commonly encountered diseases that sickness rate, disability rate and mortality rate are the highest, serious harm human health.Because its pathophysiological mechanism is very complicated, relate to the polynary mechanism such as excititoxic, oxidative damage, inflammatory reaction, Ca2+ overload, osmotic effect, in recent years research shows that erythropoietin (EPO) draws result likely in preliminary experiment, causes researcher extensive concern and profound thinking.
Erythropoietin (EPO) is mainly produced by liver at first, a kind of glycoprotein that turns to gradually kidney to produce after birth, and its molecular weight is 34KDa.Think that at first EPO mainly regulates erythrocytic generation by the differentiation of apoptosis inhibit and the red owner's cell of stimulation with propagation.Study and show recently, EPO receptor (EPOR) is expressed in various cells and tissue widely, comprises neuron, glial cell and brain inner capillary tube endotheliocyte.EPO and EPOR have functional expression at the nervous system of nibbling muroid, primates and the mankind, and have significantly and change in the process of growing, and point out it in neurodevelopment, to play an important role.In addition, EPO and the EPOR expression when cerebral ischemia, anoxia and anemia increases, and pointing out it may be also a kind of endogenic neuroprotective.Cell and Integral animal experiment show, EPO has protective effect to the excitotoxic injury of the inductions such as cerebral ischemia, anoxia.The result in our early stage proves that EPO has the excitotoxic injury effect of neuroprotective unit on whole and cell equally.These results show, EPO may provide for the treatment of clinical cerebral ischemia a new thinking.
But, because EPO is a kind of polypeptides matter, be difficult to directly enter blood brain barrier.Although when cerebral ischemia, the permeability of blood brain barrier may increase, also there is the EPO of thinking to enter in brain by receptor-mediated active transport, a series of research shows, the EPO entering in brain only has 1% left and right.Therefore, in experiment and clinical trial, had to adopt the EPO of ventricles of the brain administration or high dose to treat cerebral ischemia.But ventricles of the brain dosage regimen often inevitably produces the side effect such as promoting erythrocyte generation in the unrealistic property of clinical practice and the EPO of high dose.In addition, also there is viewpoint to think that periphery gives the growth that high dose EPO may promote tumor simultaneously.Recently, the recombinant epo of high dose treatment cerebral ischemia enters clinical first phase test in the U.S., but due to side effect such as its peripheries, is faced with and stops the further danger of test.Therefore, for safety, effectively, non-invasively EPO is entered in brain, improve its medicinal potential, each research group of the whole world has been attempted many methods.But the interior usefulness of the brain that does not also effectively improve so far EPO solves the periphery side-effect problem of EPO simultaneously.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of erythropoietin drug-carrying nanometer particle that easily enters blood brain barrier.
Erythropoietin drug-carrying nanometer particle, comprises with CSO-SA micelle or polyethyleneglycol modified nanoparticle and the erythropoietin of being combined with nanoparticle by electrostatic interaction.
Further, with the micelle modified nanoparticle of CSO-SA, the mass ratio of described erythropoietin and carrier is 10-20%; The particle diameter of nanoparticle is 200-270nm, and envelop rate is between 43-87%.
Further, with polyethyleneglycol modified lipid nanoparticle, the mass ratio of described erythropoietin and carrier is 1%; The particle diameter of described nanoparticle is 171.3 ± 0.71nm, and the surface potential of described nanoparticle is 7.8 ± 0.28mV.
In addition, the present invention also provides the preparation method of erythropoietin drug-carrying nanometer particle, concrete grammar is: first, select chitosan-stearic acid self aggregation in water to form " core-shell " structure micelle, then, by electrostatic interaction, " core-shell " structure micelle of positively charged is combined with the erythropoietin that is negative electricity in aqueous solution, form erythropoietin drug-carrying nanometer particle; Or first take glyceryl monostearate, stearylamine, PEG (2000) is raw material, adopts solvent diffusion method to prepare SLN, then, by electrostatic interaction, in aqueous solution, be the erythropoietin of negative electricity and the SLN of positively charged is combined, form erythropoietin drug-carrying nanometer particle.
The present invention also provides erythropoietin drug-carrying nanometer particle for the preparation of the application in treatment cerebral ischemia damnification medicament.
The present invention provides again the nasal cavity administrated preparation of erythropoietin, comprises the nanoparticle that is combined with erythropoietin, and described preparation is targeted to brain by nasal cavity by erythropoietin.
Further, described nanoparticle be erythropoietin by electrostatic interaction with CSO-SA micelle or polyethyleneglycol modified nanoparticle, be combined the erythropoietin drug-carrying nanometer particle forming.
Further, described dosage form is nanoparticle solution.
Further, erythropoietin valid density is 1000iu/ml.
Further, each nasal-cavity administration dosage is 10-20iu/kg.
Recently; the proposition of brain targeting drug delivery system concept; adopt particulate delivery system; as liposome, nanoparticle, microemulsion can award nerve growth factor by nasal cavity can be through nervi olfactory-olfactory bulb-tractus olfactorius path; final arrival in cerebral deep and cerebrospinal fluid; thereby play the effect of nutrition and protection CNS, it will solve the peripherally administered contradiction of CNS disease effectively.In addition, to have bioavailability high for nasal-cavity administration.Meanwhile, be also applicable to due to a variety of causes cannot oral administration patient.Compare with drug administration by injection, nasal-cavity administration is easy to use, medication voluntarily, and the light or nothing to body injury, patient compliance is good, and fibre-less toxicity and irritating pharmaceutical preparation are suitable for long term administration, and can reduce the propagation of infectious disease.According to above imagination, we are by the structure of EPO nasal drug delivery system, EPO intranasal administration, through Nasal Epithelial Cells, absorb, by olfactory pathway, enter maincenter, to reach the object of inside and outside week drug treatment cerebral ischemia, avoided insecurity that EPO may be brought and be clinically difficult to the problems such as application, and greatly having improved economic benefit.This will be to make the more feasible and economy of its clinical practice, thereby set up new method and the means for the treatment of cerebral ischemia.In addition, the mentality of designing of EPO nasal drug delivery system not only has potential treatment prospect to cerebral ischemia, also will in the treatment research of the various diseases such as maincenter, provide new thinking for polypeptide drug.
For improving the brain availability of fat-soluble medicine, the present invention adopts particulate delivery system, as nasal-cavity administration again after liposome, nanoparticle, microemulsion.This patent is carrier with CSO-SA micelle and polyethyleneglycol modified lipid nanoparticle respectively, pass through electrostatic interaction, make carrier and human forcing erythrogenin erythropoietin (hEPO) combination that is negative electricity in aqueous solution, form drug-carrying nanometer particle, make nasal mucosa medicine administration dosage form, be used for the treatment of cerebral ischemia.
The present invention adopts chitosan-stearic acid polymer as the cerebral ischemia of EPO carrier nasal mucosa medicine administration brain targeted therapy: selecting chitosan-stearic acid (CSO-SA) is raw material, its self aggregation in water forms " core-shell " structure micelle, has good permeates cell membranes ability and lower cytotoxicity.By electrostatic interaction, make the CSO-SA micelle and human forcing erythrogenin erythropoietin (hEPO) combination that is negative electricity in aqueous solution of positively charged, form drug-carrying nanometer particle.With Fluorescein isothiocyanate FITC labelling CSO-SA, follow the trail of in brain and distribute.Particle diameter and potential change before and after particle size and surface potential analysis-e/or determining FITC labelling CSO-SA medicine carrying.Select SD rat as animal pattern, after nasal mucosa medicine administration, homogenate method is measured the carrier concn of administration 0.5h and 1h tissues following MCAO in rats.In addition, select SD evaluating focal brain ischemia in rats, after nasal mucosa medicine administration, observe cerebral infarction volume and function of nervous system's scoring.
The present invention adopts PEG to modify lipid nanoparticle as the cerebral ischemia of EPO carrier nasal mucosa medicine administration brain targeted therapy: with isosulfocyanic acid fluorescence labelling stearylamine (ODA-FITC), take glyceryl monostearate (MS), stearylamine (ODA-FITC), PEG (2000) is raw material, adopts solvent diffusion method to prepare SLN.By electrostatic interaction, the human forcing erythrogenin erythropoietin (hEPO) that is negative electricity in aqueous solution is combined with the SLN of positively charged, forms drug-carrying nanometer particle.The particle diameter of particle size and surface potential analysis-e/or determining FITC labelling SLN.By ELISA test kit method, investigate SLN and EPO hydrolysising condition.Select SD rat as model, by FITC labelling PEG-SLN nasal mucosa medicine administration, with acetonitrile extraction ODA-FITC, follow the trail of interior distribution of brain of SLN.In addition, select SD evaluating focal brain ischemia in rats, after nasal mucosa medicine administration, observe cerebral infarction volume and function of nervous system's scoring.
The present invention has overcome and in current experiment and clinical trial, has adopted the EPO of ventricles of the brain administration or high dose to treat the defect of cerebral ischemia.And can reach therapeutic effect.
Specific implementation method
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that following examples are only not used in and limit the scope of the invention for the present invention is described.
In the following example, method therefor if no special instructions, is conventional method.Needed material or reagent in following examples, be if no special instructions market and buy.The approach that obtains of the various biomaterials that are described in embodiment be only the approach that obtains of a kind of experiment to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.
Described percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/V) percent concentration unless otherwise noted.
The reagent such as chitosan, stearic acid, Macrogol 2000 are purchased from Shanghai Chemical Reagent Co., Ltd., Sinopharm Group, analytical pure.
EPO stock solution is provided by Sansheng pharmacy Co., Ltd., Shenyang city, and concentration is 30000iu/ml.
Embodiment 1: chitosan-stearic acid polymer is as the nasal mucosa medicine administration brain targeted therapy cerebral ischemia of the nasal cavity administrated preparation of EPO carrier
(1) experimental technique
The preparation of 1.1EPO drug-carrying nanometer particle solution
Get EPO stock solution, by distilled water diluting concentration, approximately to 1000IU/ml (being equivalent to 0.0082mg/ml), under magnetic agitation, add the 0.082mg/ml CSO-SA solution of different volumes, make EPO/ carrier mass ratio be respectively 10%, 15%, 20%.Stirring at room 5min, obtains the CSO-SA/EPO drug-carrying nanometer particle solution of different drug loading, is denoted as respectively CSO-SA/10%,, CSO-SA/15%, CSO-SA/20%.
The mensuration of 1.2 envelop rates
Get the some volumes of CSO-SA/EPO drug-carrying nanometer particle solution, as for ultra-filtration centrifuge tube (Microcon YM-10, MWCO10,000, Millipore Co., USA).The centrifugal 5min of 10,000rpm, 5000 times of sample diluting liquid dilutions for ultrafiltrate.Computational envelope rate and drug loading as follows.Envelop rate=(the drug/lipid body Chinese medicine total amount of sealing in liposome) * 100%.By ELISA test kit method, measure EPO content.
1.3CSO-SA/EPO vitro release
As 1.1 preparation CSO-SA/EPO drug-carrying nanometer particle solution, making EPO/CSO-SA is 20%(w/w).Get above-mentioned solution 100 μ l in filter tube, add the PBS400 μ l of pH7.4,37 ℃ of vibrations, timing sampling.Ultrafiltration centrifugal condition is 10000rpm, 10min.And add fresh PBS.Content with ELISA kit measurement EPO.
1.4 labeled vector
The CSO-SA carrier solution 2ml of preparation 10mg/ml, 20:1(carrier/FITC in mass ratio) add the FITC solution 1ml of 4mg/ml, stirring reaction spends the night.Dialysis 12h, every 1h changes water one time.Be settled to 4ml, obtain the carrier solution of 5mg/ml, be denoted as FITC-CSO-SA.The 1.5 tissue extraction method response rate
The cerebral tissue of getting 0.25g left and right, makes the final concentration after sample treatment be respectively 0.01 μ g/ml, 0.048 μ g/ml, and 0.16 μ g/ml, and be supplemented to g*5ml, homogenate with PBS solution.Centrifugal (15000rpm, 15min), gets supernatant 0.8ml, adds acetonitrile 1.2ml, and 37 ° of C are hatched 30min, centrifugal (15000rpm, 15min).Get supernatant 1.5ml, dry up, add water 0.3ml, mix, centrifugal (15000rpm, 15min), gets supernatant and measures fluorescent value A.With PBS solution preparation 0.01 μ g/ml, 0.048 μ g/ml, the carrier solution of 0.16 μ g/ml is as standard solution, and recording fluorescence is B.
A is the fluorescence intensity of sample solution, A
0blank fluorescence intensity for sample solution
B is the fluorescence intensity of standard solution, B
0blank fluorescence intensity for standard solution
1.6 nasal mucosa medicine administration
Select SD male rat (250g-300g), fasting 24h before administration, by 3ml/kg with after 10% chloral hydrate anesthesia rat anesthesia, cut cervical region, separated trachea, ligation esophagus, do tracheal intubation for breathing, and seal nose palates to stop medicine from nasal cavity inflow entrance with 502 binding agents.Administration 0.5h, after 1h, eye socket is got blood, and broken end is got brain (n=2).Administration concentration is 4mg/ml, and administration volume is 25 μ l/ nostrils, μ l～30, by the dosed administration of 1mg/kg.
1.7 tissues and blood sample are processed
Tissue treatment methods and 1.5 similar adds appropriate carrier with blank cerebral tissue simultaneously, prepares the standard curve of carrier.
By blood sample centrifugal (3000rpm, 10min), get supernatant 0.4ml, add acetonitrile 0.6ml, vortex mixes, and centrifuging and taking supernatant, dries up, and adds water 0.3ml, and vortex mixes, centrifugal, gets supernatant and measures fluorescent value.Use preprepared blank plasma, the final concentration after preparation makes to process with sample same method is respectively 0,0.5 μ g/ml, 1 μ g/ml, and 2 μ g/ml, 3 μ g/ml, the plasma solutions 0.4ml of 5 μ g/ml, operates equally with sample.
1.8 evaluating focal brain ischemia in rats (MCAO)
10% chloral hydrate solution 400mg/kg, intraperitoneal injection of anesthesia animal.Rat dorsal position is fixed, cervical region medisection skin, and each layer tissue of blunt separation, exposes right carotid (CCA).Be separated to after internal carotid artery, external carotid artery bifurcated one section, carefully separation avoids damaging vagus nerve and trachea, puts line standby.
In neck, common carotid artery place closes with bulldog clamp folder, external carotid artery proximal part and distal end ligation, cut off centre.External carotid artery free-end is pulled to internal carotid artery in alignment, nylon wire is inserted by external carotid artery, insert rear with No. 0 silk thread ligation, hemorrhage to prevent, open internal carotid artery place bulldog clamp, nylon wire is inserted to internal carotid artery, continue to be inserted to intracranial, the about 18.5mm of insertion depth, to micro-sense resistance, makes nylon wire head end by MCA section start, arrives thinner anterior cerebral artery, the blood flow of now realizing right side middle cerebral artery blocks, ligation internal carotid artery with fixing nylon wire and prevent hemorrhage, layer-by-layer suture, nylon wire stump 1cm is longer than outside skin.Recombinant human epo (rhEPO) or normal saline give after 1h at the rear 10min of MCAO operation with after pouring into again.
The mensuration of 1.9 infarct sizes
After operation, 24h broken end is got brain, removes olfactory bulb and brain stem, is cut into continuously the crown brain sheet that 2mm is thick, in 2% TTC (TTC) solution, hatches 30min, and the white portion not dyeed by TTC is ischemic infarction district.Stained brain sheet is with CCD digital camera (WV-CP-230, Panasonic) input computer after shooting, software (Analy Power1.0) with this laboratory designed, designed calculates the infarct size of each brain sheet, then is multiplied by thickness, calculates the approximation of brain sheet volume and infarct volume.With infarction of brain volume, divided by homonymy cerebral hemisphere volume, then be multiplied by 100% and obtain infarction percentage rate.In addition, measure respectively brain left and right sides volume, using ischemia side/non-ischemia side ratio as the index of evaluating cerebral edema.
1.10 function of nervous system's scoring
24h after ischemia operation, the symptom score method of take records nervous symptoms: sick of impassivity sign is 0 minute; It is 1 minute that the offside forelimb of performing the operation while carrying tail can not stretch; Except the export-oriented operation of 1 grade of sign drops to 2 minutes to collateral resistance; Except 1,2 grade of sign, when movable, oriented operation is 3 minutes to sideway swivel sign.Behavioristics and morphological observation all adopt single blind method, and observer does not know experiment grouping.2, climb that plate test arm is postoperative 24,48h, to climb plate test quantitative assay balance coordination function.Mice is placed on the lucite flat board that a 23.5cm * 17.5cm is surrounded by copper mesh (aperture is 0.2mm), first keep dull and stereotyped at horizontal level, then gradually flat board is holded up, until mice can not climb and fall from flat board, the angle (holding angle) while recording this between dull and stereotyped axis and level.Each mice is repeated 3 said process, averages.
1.11 statistical analysis
With SPSS statistics software processes, result represents with S ± SD.Between group, relatively with SPSS, carry out nonparametric Mann-Whitney U check.
(2) experimental result
The particle diameter of 2.1 blank carriers and drug-carrying nanometer particle
Change of size before and after table 1 carrier medicine carrying
After EPO and CSO-SA are compound, medicine carrying micelle particle diameter reduces.This is that the compressed change of micelle structure is compacted, and micelle particle diameter is reduced due to after electronegative EPO is combined with CSO-SA on schedule of band.Along with the raising of drug loading, the particle diameter of micelle does not have significant change.
The variation of diameter of carrier before and after table 2FITC labelling
It can be seen from the table, before and after FITC labelling, the particle diameter of carrier does not have significant change, shows that FITC labelling does not cause larger change to support.
From experimental result, the Z of carrier is 330nm, and number is all in 20nm left and right.Generally, after nanoparticle nasal mucosa medicine administration, must cause the cell " interior digestion " of different endocytosis approach.The result of digestion depends on particle diameter and the surface physicochemical property of nanoparticle, to the change of nanoparticle particle diameter and finishing, is also for improving the method for olfactory epithelium cellular uptake.After the nanoparticle nasal mucosa medicine administration of the general >20nm of bibliographical information, in theory can transcellular transport.
The molecule mechanism of cell endocytic is a lot, as giant cell drink, clathrin mediation endocytosis, caveolin mediation endocytosis, engulf etc.Laser Scanning Confocal Microscope finds that the nanoparticle of particle diameter <200nm take the endocytosis of clathrin mediation as main, and particle diameter be take the endocytosis of caveolin mediation as main at the nanoparticle of 200-1000nm.Particle diameter is faster than the nanoparticle endocytosis of 200nm at the nanoparticle of 50-100nm in addition, although mechanism unclear.
In addition, affect the surface potential that also has nanoparticle of particle endocytic pathway.The nanoparticle of surface band positive potential can more easily have the accumulation of high concentration at brain, this may be electronegative with cell membrane, and positive and negative charge is attracted each other relevant.This tests FITC-CSO-SA carrier surface current potential used is+60mV left and right to be conducive to particle penetration cell membrane, by cellular uptake.
2.2EPO envelop rate, drug loading and release in vitro
The envelop rate of carrier to EPO under the different dosages of table 3
| Sample | CSO-SA/10% | CSO-SA/15% | CSO-SA/20% |
| Entrapment?Efficiency/% | 43.15 | 63.29 | 86.69 |
Along with dropping into the increase of EPO, CSO-SA also increases the envelop rate of EPO.Ultra-filtration centrifuge tube is 14.5% to the rejection of EPO.CSO-SA/EPO, at the PBS of pH7.4, does not almost have EPO to discharge after 120h.Illustrate that EPO is combined with CSO-SA closely, under neutrallty condition, be difficult for being hydrolyzed.
2.3 carriers organize the response rate
The absolute recovery of table 4 carrier when high, medium and low concentration
| Carrier concn/μ g/ml | Absolute recovery/% |
| 0.01 | 54.62 |
| 0.048 | 50.27 |
| 0.16 | 53.49 |
The absolute recovery of carrier extracting method in tissue, at 50.27%-54.62%, meets biological tissue extracted and requires (50%-80%).
The cerebral tissue of 2.4 carrier nasal mucosa medicine administrations distributes
The distribution (1mg/kg) of table 5 carrier nasal mucosa medicine administration in rat brain
| Tissue distribution %/g or %/ml | 0.5h | 1h |
| Brain | 0.022±0.005 | 0.033±0.017 |
| Cerebellum | 0.039±0.018 | 0.033±0.027 |
| Blood plasma | 0.55 | 1.68 |
From experimental result, after carrier nasal mucosa medicine administration, at cerebral tissue, distribute less.Possible reason is nasal mucosa medicine administration agent quantitative limitation, and again due to the existence of blood brain barrier, the carrier concn that makes to arrive cerebral tissue is very low, and the susceptiveness of detection method is had higher requirement.This tests spectrofluorophotometer method used can not finely meet this point, makes testing result on the low side.
2.5EPO nanoparticle nasal-cavity administration improves focal cerebral ischemia in rats damage
Table 6EPO nanoparticle nasal-cavity administration improves focal cerebral ischemia in rats damage
| Medicine | Dosage | Cerebral infarction volume (%) | Function of nervous system's scoring |
| Control | 0 | 28.34±2.64 | 2.54±0.24 |
| rhEPO（in） | 1iu | 26.13±2.31 | 2.23±0.21 |
| ? | 5iu | 20.46±1.85 | 1.87±0.16 |
| ? | 10iu | 14.53±1.28* | 1.75±0.16* |
| ? | 20iu | 12.83±1.26* | 1.52±0.17* |
| rhEPO（iv） | 5000iu | 12.54±1.19* | 1.57±0.18* |
From experimental result, EPO nanoparticle nasal-cavity administration significantly improves cerebral infarction volume and the function of nervous system's scoring after focal cerebral ischemia in rats, wherein at 20iu, has reached maximum improvement effect, suitable with the effect of vein 5000iu.
Embodiment 2:PEG modifies lipid nanoparticle as the cerebral ischemia of EPO carrier nasal mucosa medicine administration brain targeted therapy
(1) experimental technique
Synthesizing of 2.1 stearylamines-fluorescein isothiocyanate (fitc) grafting (ODA-FITC)
Precision takes ODA20mg and is dissolved in 5ml dehydrated alcohol, under lucifuge and magnetic agitation (400rpm), adds 28mg fluorescein isothiocyanate (fitc) FITC, after reaction 24h, adds enough water, makes product be cotton-shaped and separates out.Adopt 0.45 μ m filtering with microporous membrane, with suitable quantity of water washing 2 times, be dried, obtain the FITC label (ODA-FITC) of stearylamine, 4 ℃ keep in Dark Place.
2.2 preparation PEG-SLN
Precision takes 27.6mg monoglyceride, 2.4mg PEG, and 6mg ODA-FITC, is dissolved in 3ml dehydrated alcohol, is heated to 67 ℃.Under electromagnetic agitation, organic facies pin is noted below the synthermal water liquid level of 30ml, stirred 5min.-65 ℃ of freezing 3h, are adjusted to pH1.2 with the HCl of 2M, and centrifugal (20000rpm, 15min), with 0.1%Tween80 ultrasonic (400w, 30 times) redispersion, is adjusted to pH6.32 with 0.1mol/L NaOH.Particle diameter and the current potential of particle size and surface potential analysis-e/or determining FITC labelling SLN.
2.3PEG-SLN and EPO hydrolysising condition are investigated
The above SLN solution of getting 5ml1mg/ml, adds 1% (EPO/SLN, w/w) EPO, stirs 30min, makes EPO drug-carrying nanometer particle solution.Particle diameter and the current potential of PEG-SLN after particle size and surface potential analysis-e/or determining medicine carrying.
Get two parts of identical SLN/EPO solution, with HCl and NaOH, be adjusted to pH1.2 and pH13.4 respectively, room temperature sample lucifuge hydrolysis 4-5h.The centrifugal 20min of 20000rpm, gets 10000 times of supernatant dilutions, with ELISA kit measurement EPO.
2.4SLN nasal mucosa medicine administration
Select SD male rat (250g-300g), fasting 24h before administration, by cutting cervical region after the chloral hydrate anesthesia rat anesthesia of 3ml/kg10%, separated trachea.Ligation esophagus, does tracheal intubation for breathing, and seals nose palates to stop medicine from nasal cavity inflow entrance with 502 binding agents.After administration a period of time (0.5h or 1h), eye socket is got blood, and broken end is got brain.Administration concentration is 5mg/ml, and by the dosed administration of 1mg/kg, administration volume is 35 μ l/ nostrils.Administration 1h posterior orbit is got blood, and broken end is got brain (n=2).
2.5 tissues and blood sample are processed
Get cerebral tissue 0.25g, add g*5ml PBS solution, homogenate.Get 0.9ml homogenate, add 4.5ml acetonitrile, hatch 30min for 37 ℃.Centrifugal (8000rpm, 10min), gets supernatant 4.5ml, dries up, and with the mixed solution (v/v, 1:1) of 0.3ml water and acetonitrile, redissolves.Vortex mixes, and centrifugal (15000rpm, 15min) gets supernatant and measure fluorescence.The standard curve of preparing SLN with blank cerebral tissue, the same operation.Whole blood is centrifugal, gets 0.45ml blood plasma, adds the acetonitrile of same volume, centrifugal, gets supernatant and measures fluorescence.With the SLN solution of blank plasma preparation variable concentrations, the same operation.
All samples and mark curve fluorescent spectrophotometer assay, testing conditions excitation wavelength 495nm, emission wavelength 520nm, slit is respectively 5nm, 5nm.
1.6 evaluating focal brain ischemia in rats (MCAO)
10% chloral hydrate solution 400mg/kg, intraperitoneal injection of anesthesia animal.Rat dorsal position is fixed, cervical region medisection skin, and each layer tissue of blunt separation, exposes right carotid (CCA).Be separated to after internal carotid artery, external carotid artery bifurcated one section, carefully separation avoids damaging vagus nerve and trachea, puts line standby.
In neck, common carotid artery place closes with bulldog clamp folder, external carotid artery proximal part and distal end ligation, cut off centre.External carotid artery free-end is pulled to internal carotid artery in alignment, nylon wire is inserted by external carotid artery, insert rear with No. 0 silk thread ligation, hemorrhage to prevent, open internal carotid artery place bulldog clamp, nylon wire is inserted to internal carotid artery, continue to be inserted to intracranial, the about 18.5mm of insertion depth, to micro-sense resistance, makes nylon wire head end by MCA section start, arrives thinner anterior cerebral artery, the blood flow of now realizing right side middle cerebral artery blocks, ligation internal carotid artery with fixing nylon wire and prevent hemorrhage, layer-by-layer suture, nylon wire stump 1cm is longer than outside skin.Recombinant human epo (rhEPO) or normal saline give after 1h at the rear 10min of MCAO operation with after pouring into again.
The mensuration of 1.7 infarct sizes
After operation, 24h broken end is got brain, removes olfactory bulb and brain stem, is cut into continuously the crown brain sheet that 2mm is thick, in 2% TTC (TTC) solution, hatches 30min, and the white portion not dyeed by TTC is ischemic infarction district.Stained brain sheet is with CCD digital camera (WV-CP-230, Panasonic) input computer after shooting, software (Analy Power1.0) with this laboratory designed, designed calculates the infarct size of each brain sheet, then is multiplied by thickness, calculates the approximation of brain sheet volume and infarct volume.With infarction of brain volume, divided by homonymy cerebral hemisphere volume, then be multiplied by 100% and obtain infarction percentage rate.In addition, measure respectively brain left and right sides volume, using ischemia side/non-ischemia side ratio as the index of evaluating cerebral edema.
The scoring of 1.8 function of nervous system
24h after ischemia operation, the symptom score method of take records nervous symptoms: sick of impassivity sign is 0 minute; It is 1 minute that the offside forelimb of performing the operation while carrying tail can not stretch; Except the export-oriented operation of 1 grade of sign drops to 2 minutes to collateral resistance; Except 1,2 grade of sign, when movable, oriented operation is 3 minutes to sideway swivel sign.Behavioristics and morphological observation all adopt single blind method, and observer does not know experiment grouping.2, climb that plate test arm is postoperative 24,48h, to climb plate test quantitative assay balance coordination function.Mice is placed on the lucite flat board that a 23.5cm * 17.5cm is surrounded by copper mesh (aperture is 0.2mm), first keep dull and stereotyped at horizontal level, then gradually flat board is holded up, until mice can not climb and fall from flat board, the angle (holding angle) while recording this between dull and stereotyped axis and level.Each mice is repeated 3 said process, averages.
1.9 statistical analysis
With SPSS statistics software processes, result represents with S ± SD.Between group, relatively with SPSS, carry out nonparametric Mann-Whitney U check.
(2) result and discussion
2.1PEG-SLN particle diameter and current potential
Particle diameter and potential change before and after table 11mg/ml PEG-SLN medicine carrying
It can be seen from the table, the number of PEG-SLN is all in 60nm left and right.After medicine carrying, particle diameter is without significant change, but current potential drops to 7.8mV by 13mV, shows the nanoparticle part positive charge and electronegative EPO neutralization of positively charged, makes the decline of nanoparticle surface potential.
2.2PEG-SLN and EPO hydrolysising condition are investigated
Hydrolysising experiment shows, by drug-carrying nanometer particle solution surface after basic hydrolysis, occurs one deck milky suspended solid, and ELISA test kit also shows that the EPO solution that basic hydrolysis obtains is still less impure, more approaches theoretical EPO level.So determine that the condition of hydrolysis year EPO nanoparticle is for using basic hydrolysis.
Table 25mg/ml PEG-SLN particle diameter
| PEG-SLN concentration | Zave(nm) | N(nm)area | PI |
| 5mg/ml | 223.85 | 113.5594.4% | 0.297 |
From particle diameter, PEG-SLN is when 5mg/ml, and number is all in 110nm left and right, and Z is all in 220nm left and right.De Lorenzo report sees by ultramicroscope, and the average diameter of the nervi olfactory aixs cylinder of 2 months large rabbits has 200nm, and majority is all less than 100nm.So in theory, the particle diameter of transcellular transport is all feasible below 200nm.Now, polydispersity is also less, and particle is uniformly dispersed.Illustrate that with the SLN administration of above concentration be feasible.
2.3PEG-SLN nasal mucosa medicine administration
In brain after table 3PEG-SLN nasal mucosa medicine administration 1h, distribute (1mg/kg)
| Testing result | Mus 1 | Mus 2 | average |
| Fluorescence intensity | 587.4 | 368.02 | 477.7 |
| Distribution %/g in cerebral tissue | 1.056 | 0.508 | 0.782±0.388 |
From experimental result, finally calculate after administration 1h at the 0.782%/g ± 0.388%/g that is distributed as of unit mass brain, lower in intracerebellar level, approach blankly, number is all unlisted.Existence due to blood brain barrier, make the concentration of a lot of medicines arrival brains very low, thereby can not bring into play drug effect, the bioavailability of general small-molecule drug nasal-cavity administration is very low (be less than dosage 0.12%) conventionally all, as sulfa drugs, dopamine, morphine.If rat cerebral tissue presses 1g and calculates, the PEG-SLN for preparing gained has 0.78% distribution at cerebral tissue at 1h after Nasal Mucosa Absorption, illustrate and have good brain targeting energy.
The protective effect of 2.4SLN-EPO nasal-cavity administration to focal cerebral ischemia in rats
The protective effect of table 4SLN-EPO nasal-cavity administration to focal cerebral ischemia in rats
| Medicine | Dosage | Cerebral infarction volume (%) | Function of nervous system's scoring |
| Control | 0 | 28.78±2.75 | 2.61±0.26 |
| SLN-EPO（in） | 1iu | 26.13±2.31 | 2.36±0.23 |
| ? | 5iu | 20.46±1.92 | 1.79±0.19 |
| ? | 10iu | 15.57±1.36 * | 1.68±0.17 * |
| ? | 20iu | 13.74±1.37 * | 1.61±0.14 * |
| EPO（iv） | 5000iu | 13.68±1.37 * | 1.62±0.18 * |
From experimental result, SLN-EPO nasal-cavity administration significantly improves cerebral infarction volume and the function of nervous system's scoring after focal cerebral ischemia in rats, wherein at 20iu, has reached maximum improvement effect, suitable with the effect of vein 5000iu.
Claims (10)
1. erythropoietin drug-carrying nanometer particle, comprises with CSO-SA micelle or polyethyleneglycol modified nanoparticle and the erythropoietin of being combined with nanoparticle by electrostatic interaction.
2. erythropoietin drug-carrying nanometer particle according to claim 1, is characterized in that: with the micelle modified nanoparticle of CSO-SA, the mass ratio of described erythropoietin and carrier is 10-20%; The particle diameter of nanoparticle is 200-270nm, and envelop rate is between 43-87%.
3. erythropoietin drug-carrying nanometer particle according to claim 1, is characterized in that: with polyethyleneglycol modified lipid nanoparticle, the mass ratio of described erythropoietin and carrier is 1%; The particle diameter of described nanoparticle is 171.3 ± 0.71nm, and the surface potential of described nanoparticle is 7.8 ± 0.28mV.
4. the preparation method of erythropoietin drug-carrying nanometer particle according to claim 1, it is characterized in that: concrete grammar is: first, select chitosan-stearic acid self aggregation in water to form " core-shell " structure micelle, then, pass through electrostatic interaction, " core-shell " structure micelle of positively charged is combined with the erythropoietin that is negative electricity in aqueous solution, forms erythropoietin drug-carrying nanometer particle; Or first take glyceryl monostearate, stearylamine, PEG (2000) is raw material, adopts solvent diffusion method to prepare SLN, then, by electrostatic interaction, in aqueous solution, be the erythropoietin of negative electricity and the SLN of positively charged is combined, form erythropoietin drug-carrying nanometer particle.
5. erythropoietin drug-carrying nanometer particle is for the preparation of the application in treatment cerebral ischemia damnification medicament.
6. the nasal cavity administrated preparation of erythropoietin, is characterized in that: comprise the nanoparticle that is combined with erythropoietin, described preparation is targeted to brain by nasal cavity by erythropoietin.
7. the nasal cavity administrated preparation of erythropoietin according to claim 6, is characterized in that: described nanoparticle be erythropoietin by electrostatic interaction with CSO-SA micelle or polyethyleneglycol modified nanoparticle, be combined the erythropoietin drug-carrying nanometer particle forming.
8. according to the nasal cavity administrated preparation of the erythropoietin described in claim 6 or 7, it is characterized in that: described dosage form is nanoparticle solution.
9. the nasal cavity administrated preparation of erythropoietin according to claim 6, is characterized in that: erythropoietin valid density is 1000iu/ml.
10. the nasal cavity administrated preparation of erythropoietin according to claim 9, is characterized in that: each nasal-cavity administration dosage is 10-20iu/kg.
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