CN109602953A - A kind of New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm and preparation method thereof - Google Patents
A kind of New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
The present invention relates to nano biological medical material technical field, a kind of New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm and preparation method thereof is specifically disclosed.Nanofiber diaphragm obtained by the method for the invention has the property that 1. pairs of tissue nontoxicitys, will not generate harmful substance after degradation in vivo, has good biocompatibility；2. growth factor can be caused to inactivate to avoid factors such as organic solvent, strong acid and strong base, high temperature, guarantees that the growth factor loaded on diaphragm keeps good activity, play optimal therapeutic effect；3. avoiding the problem that wound caused by ventricles of the brain direct injection growth factor and being unable to control concentration, have the function of good promotion blood vessel and nerve regneration, it is insufficient to can be applied to the indirect vascular bypass post-surgical vascular regeneration of neurosurgery, the therapeutic effect for improving related ischemic cerebrovascular disease, reduces the occurrence risk again of cerebral arterial thrombosis.
Description
Technical field
The present invention relates to nano biological medical material technical fields, specifically, be a kind of New-type long-acting sustained release VEGF and
BFGF degradable biological nanometer diaphragm and preparation method thereof.
Background technique
In recent years, porous electrostatic spinning film because its diameter it is close with body cell epimatrix, and have large specific surface area,
Diameter and the features such as aperture is adjustable, tridimensional network, shows in optics, Electrical Appliances, pharmacy as the matrix of composite material
Huge application potential out.Nano material is prepared using electrostatic spinning technique, in high voltage electric field, by degradable polymer solution
Or melt drawn (such as Nanowire of pla-pcl, poly lactose, poly lactide-glycolide acid, collagen, fibroin albumen
Tie up film), drop is spouting from spinning nozzle tip, jet stream is formed, and under the interaction of electrostatic repulsion and surface tension,
Jet stream refinement produces tens nanometers of continuity superfine fibre films not waited to micron.The very thin film of the nanofiber is a kind of non-
Immunogenicity nanometer materials degradable can absorb, and have excellent cell adhesion and biocompatibility, but it is in biology
Application in medicine, Neuscience, especially ischemic brain damage is very rare, therefore is worth innovation and explores.
Vascular endothelial growth factor (vascularendothelial growthfactor, VEGF) is a kind of endothelial cell
Specific mitogen and secreting type glycoprotein polyprotein precursor are the key factors for promoting angiogenesis, can adjust blood vessel endothelium in real time
The division and proliferation of cell participate in a variety of pathological processes, such as tissue damage reparation, brain trauma, cerebral apoplexy.Alkalinity is at fibre
Tieing up Porcine HGF (basic fibroblast growth factor, bFGF) is a kind of with various biological effect
Neurotrophic factor has and promotes angiogenesis, embryonic development, cell differentiation and proliferation, is conducive to neuronal survival, nerve again
Raw, Synaptic formation effect.There are close reciprocation, the two in angiogenesis and process of neurogenesis by VEGF, bFGF
Nerve is played a crucial role with revascularization and reparation.However, at present using VEGF and (or) bFGF as target spot
In the treatment of ischemic brain damage, VEGF, bFGF factor or direct injection for mostly using part or intracerebroventricular to synthesize in vitro are high
The stem cell of VEGF expression, bFGF.These treatment methods do not simply fail to the concentration for effectively controlling intracerebral VEGF, bFGF, and
It may cause local occupation time process or puncture bleeding.The treatment of stem cell is even more the risk in face of intracerebral tumor formation, limits its clinic
Conversion.VEGF, bFGF are adsorbed in the polyelectrolyte with opposite charges by the present invention, by layer-by-layer, then
Polyelectrolyte with opposite charges can be successively adsorbed on the fiber of nano fibrous membrane.The nano material of preparation, is covered on
Ischemic region brain tissue surface can effectively control the release and intracerebral concentration of VEGF, bFGF by this method, promote ischemic region
Nerve with blood vessel network metaplasia at, be expected to collaboration promote neural blood vessel regeneration, improve ischemic brain damage.
In current existing report, a kind of Chinese patent " preparation side of biodegradable diaphragm for promoting regeneration of periodontal tissue
Method " (China Patent No.: CN201010105573.2, Authorization Notice No.: CN101791431A) is disclosed through electrostatic spinning side
The biodegradable material film (materials such as polylactic acid) with nanostructure is made in method, and ERK/P38 cell pathway is had suppression
It makes of and the growth factor of periodontal bone tissue regeneration is promoted to be loaded on macromolecule material film respectively, although also being controlled
Therapeutic effect, but inhibitor also plays certain inhibiting effect to the growth of surrounding normal cell, in addition its controlled release period and control
It is also not good enough to release concentration.Chinese patent CN201611087329.1 discloses a kind of applied to the indirect vascular bypass art of neurosurgery
Biodegradable polymer material is dissolved in polarity, in volatile organic solvent, passed through by Biodegradable nano fiber diaphragm
The diaphragm being made of the superfine fibre that diameter is nanoscale is made in electrospinning process；It will be promoted by layer-by-layer
Into brain tissue revascularization and/or the growth factor of nerve regneration is promoted to be loaded on diaphragm, through dry obtained product.The present invention
With the main distinction technical characteristic of the above-mentioned prior art are as follows: (1) blood-brain barrier be completely maintain cerebral function normally basic,
General drug or macromolecular are difficult through blood-brain barrier, and direct intracerebral injection drug or albumen have very big bleeding risk, and dense
Degree and timeliness are difficult to control and monitor, and for the application based on brain there are the particularity of blood-brain barrier, innovative preparation can be in brain
The degradable nano diaphragm of the sustained release growth factor in spinal fluid；(2) two kinds of growth factors are adsorbed simultaneously, and however, it is not limited to this, can
It is applied to a variety of with identical or opposite charges growth factor absorption；(3) theory based on neural blood vessel unit, from facing
Bed problem is set out, and selecting the sub- factor of life for having dual regulating and controlling effect to nerve and blood vessel, (VEGF mainly promotes angiogenesis, right
Nerve also has certain protective role；BFGF mainly promotes nerve growth, also there is certain facilitation to angiogenesis),
By the number of plies screening to nanofiber diaphragm, slow-release bio effect (cell growth and the detection of nanometer diaphragm Electronic Speculum diameter) is selected
The optimal number of plies, slow-release time are also extended, and the effective concentration for controlling growth factor and sustained release timeliness promote ischemic region
Blood vessel network metaplasia is at revascularization is not during being expected to overcome the outer reconstructing blood vessel of the indirect encephalic for the treatment of ischemic cerebrovascular disease
The defect of foot, it is basic to improve patient's prognosis；(4) compared with the CN201611087329.1 patent applied before this seminar, this
The nanofiber diaphragm that invention prepares supported V EGF and bFGF can obviously reduce nerve cell apoptosis, have more superior nerve
Protective effect.
In conclusion VEGF and bFGF are the growth factors with clinical value, this research from clinical practice,
Novelty application a kind of nonimmunogenic nanoscale genophore (nano material degradable can absorb, to tissue without
Toxicity will not generate harmful substance after degradation in vivo, have excellent cell adhesion and biocompatibility) sustained release VEGF
And bFGF, it is different from traditional cell transplantation or intracerebroventricular, immune response, cell loss or injection wound etc. is avoided and asks
Topic is a kind of new treatment concept of ischemic neuronal damage and function reparation, has innovative clinical conversion meaning.
Summary of the invention
The first purpose of this invention be aiming at the shortcomings in the prior art, provide a kind of New-type long-acting sustained release VEGF and
BFGF degradable biological nanometer diaphragm.
Second object of the present invention is aiming at the shortcomings in the prior art, to provide degradable biological nanometer film as described above
The purposes of piece.
To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:
A kind of New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm, is prepared by method as described below:
By biodegradable polymer material, it is dissolved in polarity, in volatile organic solvent, by electrospinning process, is made to have and receive
The Biodegradable nano fiber diaphragm of metrical scale structure；Growth factor is loaded to Biodegradable nano tunica fibrosa on piece,
It is freeze-dried, to get required product after sterilization packaging；Wherein: the applied high direct voltage range of electrospinning process are as follows: 15-
20KV, flow 0.1-1ml/h, time 3-5h, distance of the spinning head apart from collection device are as follows: 20-100cm；The life
Biodegradable polymer material is polycaprolactone, in polylactic acid, poly lactide-glycolide acid, fibroin albumen, collagen
One or several；The growth factor is vascular endothelial growth factor VEGF and basic fibroblast growth factor bFGF, raw
The method of long factor loads is as follows: step 1, the Biodegradable nano fiber diaphragm after drying being immersed positively charged poly- electrolysis
In matter solution, it is washed with deionized；The Biodegradable nano fiber diaphragm after washing is immersed in containing growth factor again
It is adsorbed, is washed with deionized in solution；Step 2: Biodegradable nano fiber diaphragm successively being immersed electronegative
In polyelectrolyte solution, growth factor solution and positively charged polyelectrolyte solution, it is washed with deionized water after impregnating every time de-；
Above-mentioned steps 1 and step 2 are a circulation, are recycled 10 times, the nanofiber diaphragm of 10 layers of VEGF and bFGF of preparation load, both
Obtain final products.
In above-mentioned New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm, as a preferred embodiment, institute
State polarity, volatile organic solvent are as follows: chloroform, methylene chloride, N,N-dimethylformamide, methanol, ethyl alcohol, hexafluoro isopropyl
The mixture of one or more of alcohol, trifluoroacetic acid, formic acid, acetic acid or acetone solvent.
In above-mentioned New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm, as a preferred embodiment, institute
Stating positively charged polyelectrolyte is one or more of polyethyleneimine, polyallylamine hydrochloride, collagen, chitosan；
The electronegative polyelectrolyte is one of kayexalate, sodium alginate, hyaluronic acid, chondroitin sulfate or several
Kind.
In above-mentioned New-type long-acting sustained release VEGF and bFGF degradable biological nanometer diaphragm, as a preferred embodiment, by
Method as described below is prepared: polylactic acid being dissolved in chloroform/alcohol mixed solution, it is 10%wt's that concentration, which is made,
Solution；This mixture solution is added in the electrostatic spinning syringe controlled by syringe pump, the electricity of high pressure generator is set
Pressure is 20KV, and collecting distance is 15cm, the diaphragm of the superfine fibre composition of nanoscale is obtained, then by this diaphragm deionization
It is stand-by after being dried in vacuo after water washing；Step 1: the diaphragm after drying being immersed in polyethyleneimine 0.5-2mg/ml solution, leaching
Then bubble 30-60 minutes impregnates minute with deionized water；The mixing that 0.1-10 μ g/ml concentration is made in VEGF and bFGF again is raw
Long factor solutions are placed in diaphragm to impregnate in this growth factor solution and adsorb, are then washed with deionized；Step 2: by diaphragm
It successively immerses in hyaluronic acid 0.5-2mg/ml solution, growth factor solution and polyethylenimine solution, is used after impregnating every time
Deionized water elution；Above-mentioned steps 1 and step 2 are a circulation, are recycled 10 times, and preparation loads receiving for 10 layers of VEGF and bFGF
Rice fiber diaphragm.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:
Any long-acting slow-release VEGF and bFGF degradable biological nanometer diaphragm as above are used for neuro-surgery in preparation
Application in the Biodegradable nano fiber diaphragm of indirect Coronary Artery Bypass, for controlling for ischemic neuronal damage and function reparation
It treats.
In the present invention, for the polymer solution of electrostatic spinning, be added polymer, polymeric blends not
Together and its difference of molecular weight, the viscosity of solution system are also different.Specifically electrostatic field gravitation Cheng Siwei can be passed through
Preferably.
In the present invention, using layer-by-layer, refer to positively charged and electronegative polyelectrolyte self assembly layer by layer
Method, this method are realized based on the weak interaction force (electrostatic attraction, Fan Dehua power, hydrogen bond etc.) between macromolecular to biology
The ordered fabrication of macromolecular and polyelectrolyte can also be each in addition to the bioactivity and space structure that can keep large biological molecule
It plants and is realized on the material surface with complex space configuration.
Nanofiber diaphragm obtained by the method for the invention has the property that 1. is obtained by the method for the invention
Nanofiber diaphragm will not generate harmful substance after degradation in vivo, have good bio-compatible to tissue nontoxicity
Property；2. blood vessel and (or) nerve growth factor this method are loaded in nano fibrous membrane on piece by layer-by-layer, it can
Cause growth factor to inactivate to avoid factors such as organic solvent, strong acid and strong base, high temperature, it is ensured that the growth loaded on diaphragm because
Son keeps good activity, plays optimal therapeutic effect；It is discharged 3. the nanofiber diaphragm can control at cerebral ischemia position
Growth factor, it is possible thereby to revascularization and (or) nerve regneration in induction regulating controlling ischemic tissue of brain.Present invention can apply to
The indirect vascular bypass post-surgical vascular regeneration of neurosurgery is insufficient, improves the therapeutic effect of related ischemic cerebrovascular disease, reduces
The occurrence risk again of cerebral arterial thrombosis.
The present invention has potential clinical conversion meaning, can be applied to the treatment of ischemic neuronal damage Yu function reparation,
It after the indirect Coronary Artery Bypass of neurosurgery, is pasted between brain surface and temporalis, slow release growth factor plays biological effect.
The Biodegradable nano fiber diaphragm is non-toxic, will not generate harmful substance after degradation in vivo, has good biofacies
Capacitive.It is growth factor-loaded in nano fibrous membrane on piece by layer-by-layer, it can be strong to avoid organic solvent, strong acid
The factors such as alkali, high temperature cause growth factor to inactivate, and the polyelectrolyte of assembling can guarantee that the growth factor loaded on diaphragm is kept
Good activity.The nanofiber diaphragm can cerebral vessels region of obstruction control for a long time release VEGF, bFGF etc. growth because
Son, it is possible thereby to the revascularization at induction regulating controlling brain tissue angiemphraxis position and nerve regneration.
The present invention preferably provides 10 layers of supported V EGF and bFGF nanofiber diaphragm, and diaphragm under this structure is not only
It can guarantee the control-release function of the growth factor of dual regulating and controlling effect, can be in 30 days or more controlled release growth factors, and the material
Material shows optimal biological slow-released effect (cell growth and the detection of nanometer diaphragm Electronic Speculum diameter), and slow-release time significantly extends,
Can be effectively controlled growth factor concentration and sustained release timeliness, promote ischemic region blood vessel network metaplasia at.Application of the invention is expected to
Overcome the insufficient defect of revascularization during the outer reconstructing blood vessel of indirect encephalic for the treatment of ischemic cerebrovascular disease, basic improve suffers from
Person's prognosis.
Detailed description of the invention
Fig. 1: the ideograph of nanofiber diaphragm is prepared.(in figure: 1. syringes；2. high pressure connects injection needle；3. polymerization
Object solution；4. metal receiver board；5. speed adjuster；6. polycationic compounds；7. poly- VEGF compound；8. poly- bFGF compound；
9. layer assembly process；10. the nanofiber diaphragm of supported V EGF+bFGF).
Fig. 2: supported V EGF and bFGF nanofiber diaphragm pictorial diagram.
Fig. 3: supported V EGF and bFGF nanofiber diaphragm electron microscope.
Fig. 4: the different layers of release profiles of supported V EGF and bFGF nano fibrous membrane.
Fig. 5: the different numbers of plies adsorb VEGF and bFGF nanofiber diaphragm.(in figure: compared with 0 layer of control, as the number of plies increases
Add, raw sub- factor amount absorption gradually increases, and fiber filament average diameter gradually increases；15 layers of diameter and 0 layer, 5 layers, 10 layers of diameter
It compares, difference is statistically significant, P < 0.05, and for 20 layers of diameter compared with 0 layer, 5 layers, 10 layers of diameter, difference is statistically significant,
P<0.05)。
Fig. 6: sustained release VEGF and bFGF nano fibrous membrane can promote the growth of nerve cell.
Fig. 7: sustained release VEGF and bFGF nano fibrous membrane can promote vascular endothelial cell into pipe.
Fig. 8: the more superior anti-apoptotic effect of sustained release VEGF and bFGF nano fibrous membrane.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Embodiment 1
Polylactic acid is dissolved in chloroform/alcohol mixed solution, the solution that concentration is 10% (wt) is made.This is mixed
Polymer solution is added in the electrostatic spinning syringe controlled by syringe pump, is set the voltage of high pressure generator as 20KV, is received
Integrate distance as 15cm, the diaphragm that forms of superfine fibre by diameter for nanoscale can be obtained, then by this diaphragm spend from
Sub- water is stand-by after being dried in vacuo after repeatedly washing.Step 1: it is molten that the diaphragm after drying being immersed into polyethyleneimine (0.5-2mg/ml)
In liquid, impregnates 30-60 minutes, then impregnated 10 minutes with deionized water.It is dense that 0.1-10 μ g/ml is made in VEGF and bFGF again
The mixed growth factor solutions of degree are placed in diaphragm to impregnate 10-20 minutes in this growth factor solution and be adsorbed, then spent
Ion water washing.Step 2: and then successively immerse hyaluronic acid (0.5-2mg/ml) solution, growth factor solution and polyethyleneimine
It in amine aqueous solution, impregnates 10-20 minutes, is washed with deionized water after impregnating every time de- every time.Above-mentioned steps 1 and step 2 are one
Circulation, the nanofiber diaphragm of one layer of VEGF and bFGF of each circulation preparation load；5,10,15,20 are successively recycled respectively
It is secondary, the nanofiber diaphragm of 5,10,15,20 layers of VEGF and bFGF of load is prepared respectively.It is dry with stream of nitrogen gas after the completion.To obtain the final product
To the Biodegradable nano fiber diaphragm for being applied to the indirect Coronary Artery Bypass of neuro-surgery.As shown in Fig. 1, Nanowire of the present invention
Tie up the ideograph of diaphragm preparation.Fig. 2 is supported V EGF and bFGF the nanofiber diaphragm pictorial diagram of preparation.Fig. 3 is the negative of preparation
Carry VEGF and bFGF nanofiber diaphragm electron microscope.
Embodiment 2
Polylactic acid is dissolved in chloroform/alcohol mixed solution, the solution that concentration is 8% (wt) is made.This is mixed
Polymer solution is added in the electrostatic spinning syringe controlled by syringe pump, is set the voltage of high pressure generator as 15KV, is received
Integrate distance as 15cm, the diaphragm that forms of superfine fibre by diameter for nanoscale can be obtained, then by this diaphragm spend from
Sub- water is stand-by after being dried in vacuo after repeatedly washing.Step 1: it is molten that the diaphragm after drying being immersed into polyethyleneimine (0.5-2mg/ml)
In liquid, impregnates 30-60 minutes, then impregnated 10 minutes with deionized water.It is dense that 0.1-10 μ g/ml is made in VEGF and bFGF again
The mixed growth factor solutions of degree are placed in diaphragm to impregnate 10-20 minutes in this growth factor solution and be adsorbed, then spent
Ion water washing.Step 2: and then successively immerse hyaluronic acid (0.5-2mg/ml) solution, growth factor solution and polyethyleneimine
It in amine aqueous solution, impregnates 10-20 minutes, is washed with deionized water after impregnating every time de- every time.Above-mentioned steps 1 and step 2 are one
Circulation, the nanofiber diaphragm of one layer of VEGF and bFGF of each circulation preparation load；5,10,15,20 are successively recycled respectively
It is secondary, the nanofiber diaphragm of 5,10,15,20 layers of VEGF and bFGF of load is prepared respectively.It is dry with stream of nitrogen gas after the completion.To obtain the final product
To the Biodegradable nano fiber diaphragm for being applied to the indirect Coronary Artery Bypass of neuro-surgery.
Embodiment 3
Polylactic acid is dissolved in chloroform/alcohol mixed solution, the solution that concentration is 9% (wt) is made.This is mixed
Polymer solution is added in the electrostatic spinning syringe controlled by syringe pump, is set the voltage of high pressure generator as 20KV, is received
Integrate distance as 15cm, the diaphragm that forms of superfine fibre by diameter for nanoscale can be obtained, then by this diaphragm spend from
Sub- water is stand-by after being dried in vacuo after repeatedly washing.Step 1: it is molten that the diaphragm after drying being immersed into polyethyleneimine (0.5-2mg/ml)
In liquid, impregnates 30-60 minutes, then impregnated 10 minutes with deionized water.It is dense that 0.1-10 μ g/ml is made in VEGF and bFGF again
The mixed growth factor solutions of degree are placed in diaphragm to impregnate 10-20 minutes in this growth factor solution and be adsorbed, then spent
Ion water washing.Step 2: and then successively immerse hyaluronic acid (0.5-2mg/ml) solution, growth factor solution and polyethyleneimine
It in amine aqueous solution, impregnates 10-20 minutes, is washed with deionized water after impregnating every time de- every time.Above-mentioned steps 1 and step 2 are one
Circulation, the nanofiber diaphragm of one layer of VEGF and bFGF of each circulation preparation load；5,10,15,20 are successively recycled respectively
It is secondary, the nanofiber diaphragm of 5,10,15,20 layers of VEGF and bFGF of load is prepared respectively.It is dry with stream of nitrogen gas after the completion.To obtain the final product
To the Biodegradable nano fiber diaphragm for being applied to the indirect Coronary Artery Bypass of neuro-surgery.
Effect example
One, ELISA kit detects VEGF and bFGF sustained release amount
1. experimental method
(1) supported V EGF and bFGF the nanofiber diaphragm that Example 1 prepares 5,10,15,20 numbers of plies respectively is placed in carefully
In born of the same parents' culture solution.
(2) equal cell culture solution is taken daily, using Rat VEGF ELISA kit (ab100786, abcam, USA)
VEGF and bFGF release concentration is detected with Rat bFGF ELISA kit (ELR-bFGF, RayBio, GA).
(3) ELISA detection method illustrates to carry out according to kit, briefly as follows: by known VEGF and bFGF antigen diluent
To 1~10 μ g/ml, the every hole of 6 orifice plates adds 0.1ml, is placed in 4 DEG C of refrigerator overnights；Second day adds a certain amount of 0.1ml measuring samples
In the reacting hole that (cell culture fluid of storage nanometer diaphragm) Yu Shangshu has been coated with, it is placed in 37 DEG C of incubators and is incubated for 1 hour；Instead
Enzyme mark secondary antibody (anti-rat) 0.1ml, the 37 DEG C of incubation 30-60min of diluted fresh are added in Ying Kongzhong, while doing blank control
(antibody is not added)；Substrate solution 0.1ml is separately added into every hole, 37 DEG C are incubated for 10~30 minutes, colour developing；It is added in each reacting hole
2M sulfuric acid 0.05ml terminates reaction；On ELISA detector, 450nm detects each hole average optical density value.
(4) it continuously collects and detects 40 days, finally count, draw.
2. result
As shown in figure 4, the specific time (day) is set to: 1,2,3,4,6,8,10,12,16,20,24,28,32.In figure
As the result is shown: with the increase of number of days, each group sustained release amount increases；Sustained release amount is most in 10 days of 5 layers of group, is sustained at the 10th day
Amount is up to 88.3%, and the 12nd day sustained release amount is 93.2%, and the 12nd day to the 32nd day sustained release amount basically reaches plateau, increases without significant
Add；The 10th day sustained release amount of 10 layers of group is 81%, and the 10th day sustained release amount of 15 layers of group is 80.5%；The 10th day sustained release amount of 20 layers of group be
80%, without significant difference between each group；10 layers of group are 97.4% in 24 days sustained release amounts；The 24th day sustained release amount of 15 layers of group be
96.8%, the 24th day sustained release amount of 20 layers of group is 97.2%, without significant difference between each group.10 layers of sustained release amount in group 16 days, than 15,
20 layers of group sustained release amount are more, no significant difference (P > 0.05), from result figure it can be seen that 10 layers of elution profiles, between 5
Between layer and 15 layers, the nanofiber diaphragm that 10,15 layer of initial option.
Two, Electronic Speculum detects filament diameter
1. experimental method
(1) difference Example 1 prepares supported V EGF and bFGF the nanofiber diaphragm of 5,10,15,20 numbers of plies.
(2) electron microscopic picture is shot under (10nm) under identical multiple.
(3) Image J image measurement software (NIH, Bethesda, USA) is used, is surveyed under identical multiple, the different numbers of plies are received
The average diameter of rice fiber diaphragm.
(4) it is analyzed using 20.0 statistical software of SPSS.
2. result
As shown in figure 5, as the number of plies increases, raw sub- factor amount absorption gradually increases, and fiber filament is flat compared with 0 layer of control
Equal diameter gradually increases；15 layers of diameter are compared with 0 layer, 5 layers, 10 layers of diameter, and difference is statistically significant, P < 0.05；20 layers straight
Diameter is compared with 0 layer, 5 layers, 10 layers of diameter, and difference is statistically significant, P < 0.05；In electron microscope as can be seen that 15,20 layers because
Diameter is too big, and slit is too small between each fiber filament, and filament diameter reflects the gap between fiber filament indirectly, between 15 layers, 20 layers
Gap is unfavorable for the adherency growth of cell, therefore complex chart 4 and Fig. 5 result select 10 layers.
Three, culture of primary neurons supports experiment
1. experimental method (the nanofiber diaphragm of 10 layers of VEGF and bFGF of the preparation load of Example 1 is tested)
(1) under aseptic condition, Rat hippocampus is taken.
(2) it is pre-chilled in buffer, separation and removal pia mater, eye scissors shred hippocampal tissue repeatedly.
(3) it is put into culture dish, 37 DEG C of digestion 30min of trypsase is added.
(4) 3 washings, after centrifugation, remove supernatant.
(5) culture solution 1ml is suspended, and blows and beats repeatedly, cell count, culture dish or culture orifice plate culture cell.
(6) culture for 24 hours to cell it is adherent after, change full nutrient solution culture 3d, control group, OGD group (oxygen sugar is deprived), OGD+
VEGF+bFGF group neuron-specific marker NSE identification of cell.
(7) fluorescent fiber mirror is taken pictures.
2. result
As shown in fig. 6, compared with the control group, OGD model group NSE positive cell number is obvious under equivalent microscope multiple
It reduces, difference has statistical significance (P < 0.05)；Compared with OGD model group, OGD+VEGF+bFGF group positive cell number is obvious
Increase, difference has statistical significance (P < 0.05), the results showed that sustained release VEGF and bFGF nano fibrous membrane can promote neural thin
The growth of born of the same parents.
Four, vascular endothelial cell official jargon forming experiment
1. experimental method (the nanofiber diaphragm of 10 layers of VEGF and bFGF of the preparation load of Example 1 is tested)
(1) matrigel is embedded with trash ice, is placed in 4 DEG C of refrigerators, thawed overnight.
(2) by liquid transfer gun head, 48 orifice plates etc. are placed in 4 DEG C of pre-coolings.
(3) into 48 orifice plates of pre-cooling, 150 μ l matrigels are added in every hole, and shaking orifice plate paves glue.37 DEG C of incubations
30min allows matrigel to polymerize.
(4) control group will after mixing after centrifugation is resuspended by the vascular endothelial cell of logarithmic growth phase, trypsin digestion cell
Glue hole is added in cell suspension；OGD model group cell is trained with serum free medium Nature enemy 12h, OGD+VEGF+bFGF group cell
It supports and contains VEGF+bFGF nano fibrous membrane.
(5) the pretreated endothelial cell of each group is shaken up uniformly to be layered on gelatin.
(6) 48 orifice plates are placed in cell incubator, 37 DEG C of culture 18h.
(7) after cultivating, orifice plate is placed in light microscopic observation, is taken pictures.
2. result
As shown in fig. 7, compared with the control group, the every um2 blood vessel number of OGD model group obviously subtracts under equivalent microscope multiple
Few, difference has statistical significance (P < 0.05)；Compared with OGD model group, the every um of OGD+VEGF+bFGF group2Blood vessel number is obvious
Increase, difference has statistical significance (P < 0.05), the results showed that sustained release VEGF and bFGF nano fibrous membrane can promote intravascular
Chrotoplast is at pipe.
Five, the expression of the apoptosis-related protein of immunoblotting analysis experiment detection nerve cell
1. experimental method (compared with the nanofiber diaphragm prepared in Chinese patent CN201611087329.1 project, this
Influence of the secondary supported V EGF+bFGF nanofiber diaphragm to nerve cell apoptosis)
(1) experiment points 4 groups: control group, OGD group, OGD+ supported V EGF/NGF diaphragm group (Chinese patent
Nano fibrous membrane prepared by CN201611087329.1 embodiment 1), (present invention is real for the diaphragm group of OGD+ supported V EGF and bFGF
Apply the nano fibrous membrane of the preparation of example 1).
(2) albumen of traditional extraction uses BCA kit (green skies biotech company) protein quantification.
(3) in the lauryl sodium sulfate polyacrylamide gels of the albumen loading 10-12% of equivalent.Under 60mV voltage,
After electrophoresis, Kynoar (polyvinylidene difluoride, PVDF) film is gone to room in 5% skim milk
Temperature closing 1h.
(4) apoptosis-related protein --- Cysteine-aspartic acid protease-3 (cysteine asparate is added
Protease, Caspase-3) antibody, diluted concentration 1:500.
(5) pvdf membrane is incubated at room temperature 1h in IgG two corresponding anti-solution is added.
(6) chemical luminescence reagent kit method is developed, and is averaged using Image J (NIH, Bethesda, MD, USA) software
Photodensitometry.
2. result
As shown in figure 8, compared with normal control, OGD recombinant apoptosis proteins Caspase-3 expression is significant to be obviously increased (P <
0.05)；Compared with OGD group, OGD+VEGF/NGF recombinant apoptosis proteins Caspase-3 expression is significant to significantly reduce (P < 0.05)；With
OGD+VEGF/NGF group is compared, and OGD+VEGF+bFGF recombinant apoptosis proteins Caspase-3 expression is significant to be significantly reduced, and difference has
Statistical significance (P < 0.05).The result shows that compared with the nanofiber diaphragm of supported V EGF/NGF, supported V EGF's and bFGF
Nanofiber diaphragm can obviously reduce nerve cell apoptosis, have more superior neuroprotection.
In summary experimental result preferably provides 10 layers of supported V EGF and bFGF nanofiber diaphragm, in the structure
Under diaphragm can not only guarantee dual regulating and controlling effect growth factor control-release function, can the growth of 30 days or more controlled releases because
Son, and show optimal biological slow-released effect (cell growth and the detection of nanometer diaphragm Electronic Speculum diameter), slow-release time is significantly prolonged
It is long, the concentration and sustained release timeliness of growth factor can be effectively controlled, promote ischemic region blood vessel network metaplasia at.The material is expected to overcome
Treat the insufficient defect of revascularization during the outer reconstructing blood vessel of indirect encephalic of ischemic cerebrovascular disease, basic to improve patient pre-
Afterwards.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (5)
1. a kind of New-type long-acting is sustained VEGF and bFGF degradable biological nanometer diaphragm, which is characterized in that by method as described below
It is prepared: by biodegradable polymer material, being dissolved in polarity, in volatile organic solvent, pass through electrospinning process, system
There must be the Biodegradable nano fiber diaphragm of nano-scale structures；Growth factor is loaded to Biodegradable nano fiber
It is freeze-dried on diaphragm, to get required product after sterilization packaging；Wherein: the applied high direct voltage range of electrospinning process
Are as follows: 15-20KV, flow 0.1-1ml/h, time 3-5h, distance of the spinning head apart from collection device are as follows: 20-100cm；Institute
The biodegradable polymer material stated is polycaprolactone, polylactic acid, poly lactide-glycolide acid, fibroin albumen, collagen
One or several in albumen；The growth factor is vascular endothelial growth factor VEGF and basic fibroblast growth factor
The method of bFGF, growth factor load are as follows: step 1, the Biodegradable nano fiber diaphragm after drying being immersed positively charged
Polyelectrolyte solution in, be washed with deionized；The Biodegradable nano fiber diaphragm after washing is immersed in containing life again
It is adsorbed, is washed with deionized in the solution of the long factor；Step 2: Biodegradable nano fiber diaphragm is successively immersed
In electronegative polyelectrolyte solution, growth factor solution and positively charged polyelectrolyte solution, every time impregnate after spend from
Sub- water elution；Above-mentioned steps 1 and step 2 are a circulation, are recycled 10 times, the nanofiber of 10 layers of VEGF and bFGF of preparation load
Diaphragm both obtains final products.
2. New-type long-acting is sustained VEGF and bFGF degradable biological nanometer diaphragm according to claim 1, which is characterized in that institute
State polarity, volatile organic solvent are as follows: chloroform, methylene chloride, N,N-dimethylformamide, methanol, ethyl alcohol, hexafluoro isopropyl
The mixture of one or more of alcohol, trifluoroacetic acid, formic acid, acetic acid or acetone solvent.
3. New-type long-acting is sustained VEGF and bFGF degradable biological nanometer diaphragm according to claim 1, which is characterized in that institute
Stating positively charged polyelectrolyte is one or more of polyethyleneimine, polyallylamine hydrochloride, collagen, chitosan；
The electronegative polyelectrolyte is one of kayexalate, sodium alginate, hyaluronic acid, chondroitin sulfate or several
Kind.
4. New-type long-acting is sustained VEGF and bFGF degradable biological nanometer diaphragm according to claim 1, which is characterized in that by
Method as described below is prepared: polylactic acid being dissolved in chloroform/alcohol mixed solution, it is 10%wt's that concentration, which is made,
Solution；This mixture solution is added in the electrostatic spinning syringe controlled by syringe pump, the electricity of high pressure generator is set
Pressure is 20KV, and collecting distance is 15cm, the diaphragm of the superfine fibre composition of nanoscale is obtained, then by this diaphragm deionization
It is stand-by after being dried in vacuo after water washing；Step 1: the diaphragm after drying being immersed in polyethyleneimine 0.5-2mg/ml solution, leaching
Then bubble 30-60 minutes impregnates minute with deionized water；The mixing that 0.1-10 μ g/ml concentration is made in VEGF and bFGF again is raw
Long factor solutions are placed in diaphragm to impregnate in this growth factor solution and adsorb, are then washed with deionized；Step 2: by diaphragm
It successively immerses in hyaluronic acid 0.5-2mg/ml solution, growth factor solution and polyethylenimine solution, is used after impregnating every time
Deionized water elution；Above-mentioned steps 1 and step 2 are a circulation, are recycled 10 times, the nanometer of 10 layers of VEGF and bFGF of preparation load
Fiber diaphragm.
5. any long-acting slow-release VEGF of claim 1-4 and bFGF degradable biological nanometer diaphragm are used for cranial nerve in preparation
Application in the Biodegradable nano fiber diaphragm of the indirect Coronary Artery Bypass of surgery.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111388766A (en) * | 2020-05-09 | 2020-07-10 | 刘成刚 | Biodegradable nano-film for vascular surgery and preparation method thereof |
| CN113846490A (en) * | 2021-10-13 | 2021-12-28 | 北京科技大学 | Preservative film with intelligent microbial self-inhibition effect and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1729949A (en) * | 2005-07-15 | 2006-02-08 | 清华大学 | Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold |
| CN106390193A (en) * | 2016-12-01 | 2017-02-15 | 上海市同济医院 | Production method of biodegradable nanometer fiber diaphragm applied to neurosurgery indirect vascular bypass |
| CN108339151A (en) * | 2018-02-08 | 2018-07-31 | 上海市同济医院 | A kind of nano layered double hydroxides-multiple-factor combined system for promoting nerve regneration to repair spinal cord injury |
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2019
- 2019-01-31 CN CN201910098438.0A patent/CN109602953A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1729949A (en) * | 2005-07-15 | 2006-02-08 | 清华大学 | Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold |
| CN106390193A (en) * | 2016-12-01 | 2017-02-15 | 上海市同济医院 | Production method of biodegradable nanometer fiber diaphragm applied to neurosurgery indirect vascular bypass |
| CN108339151A (en) * | 2018-02-08 | 2018-07-31 | 上海市同济医院 | A kind of nano layered double hydroxides-multiple-factor combined system for promoting nerve regneration to repair spinal cord injury |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111388766A (en) * | 2020-05-09 | 2020-07-10 | 刘成刚 | Biodegradable nano-film for vascular surgery and preparation method thereof |
| CN111388766B (en) * | 2020-05-09 | 2020-11-10 | 中卫科创(北京)医学研究院有限公司 | Biodegradable nano-film for vascular surgery and preparation method thereof |
| CN113846490A (en) * | 2021-10-13 | 2021-12-28 | 北京科技大学 | Preservative film with intelligent microbial self-inhibition effect and preparation method thereof |
| CN113846490B (en) * | 2021-10-13 | 2022-10-21 | 北京科技大学 | Preservative film with intelligent microbial self-inhibition effect and preparation method thereof |
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