CN108619569A - Three-layer artificial blood vessel electrospinning membrane loaded with micro nucleic acid and preparation method and application thereof - Google Patents
Three-layer artificial blood vessel electrospinning membrane loaded with micro nucleic acid and preparation method and application thereof Download PDFInfo
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Abstract
the invention relates to a three-layer artificial blood vessel electrospun fibrous membrane loaded with micro nucleic acid, a preparation method and application thereof, wherein the artificial blood vessel is composed of three layers of electrospun membranes, the inner layer is a polyethylene glycol-b-poly (L-lactide-co-caprolactone) electrospun fibrous membrane, the interior of the three layers of artificial blood vessel electrospun fibrous membrane is loaded with particles formed by trimethyl chitosan-graft-polyethylene glycol-REDV composite miRNA-126 and PE L C L-REDV, the middle layer is a poly (glycolide-co-lactide) electrospun fibrous membrane, the interior of the three layers of artificial blood vessel electrospun fibrous membrane is loaded with particles formed by trimethyl chitosan-graft-polyethylene glycol-VAPG composite miRNA-145, and the outer layer is a poly-caprolactone electrospun fibrous membrane.
Description
Technical field
The present invention relates to a kind of three layers of artificial blood vessel's electrospun fiber membrane and its preparation method and application of the small nucleic acid of load,
Belong to organizational project and biomedical materials field.
Background technology
Artificial blood vessel's substitute is the treatment method of clinical common solution blood vessel blockage, but since restenosis easily occurs,
Internal diameter less than 6mm small-caliber artificial blood vessel be still need solve problem (Pashneh-Tala S, MacNeil S,
Claeyssens F.The tissue-engineered vascular graft-past,present,and
future.Tissue Engineering Part B-Reviews,2016,22(1): 68-100).Utilize electrospinning process
Superfine fibre film can be prepared, extracellular matrix can be simulated.Aliphatic poly ester material is poly- (L- lactide-co-caprolactones)
(PLCL), poly- (glycolide-co- lactides) (PLGA) and poly-epsilon-caprolactone (PCL) all have good biocompatibility and power
Performance is learned, can be used for preparing small-caliber artificial blood vessel electrospinning film (Awad NK, Niu H, Ali U, Morsi YS, Lin T.
Electrospun fibrous scaffolds for small-diameter blood vessels:A
review.Membranes,2018,8(1):15)。
Arteries has three-decker, separately includes three kinds of different vascular cells.Internal layer is endothelial cell, thicker
Middle level mainly contains smooth muscle cell, outermost layer contain fibroblast (Wise S G, Byrom M J, Waterhouse A,
Bannon P G,Ng M KC, Weiss A S.A multilayered synthetic human elastin/
polycaprolactone hybrid vascular graft with tailored mechanical
properties.Acta Biomaterialia,2011,7(1):295-303).In order to simulate native blood vessels structure, construction with
The artificial blood vessel that native blood vessels performance matches, can be used bilayer or multilayer electrospun fiber membrane (Wu T, Zhang J, Wang Y,
Li D,Sun B,Hany E,Meng Y, Mo X.Fabrication and preliminary study of a
biomimetic tri-layer tubular graft based on fibers and fiber yarns for
vascular tissue engineering.Materials Science&Engineering C,2018,82:121-129)。
But common artificial blood vessel bracket, at the initial stage of implanting, since endodermis is not yet formed, blood directly connects with biomaterial
After touch, it is easy to cause adherency, activation and the accumulation etc. of blood platelet；The hyper-proliferative of smooth muscle cell can cause blood vessel narrow again simultaneously
It is narrow, it is unfavorable for revascularization.
Small nucleic acid (miRNAs) is the 18-25 nucleic acid molecule that be about of non-coding, and wherein miRNA-126 etc. can promote
Migration of vascular endothelial cells and proliferation (Urbich C, Kuehbacher A, Dimmeler S.Role of microRNAs in
vascular diseases, inflammation,and angiogenesis.Cardiovascular Research,
2008,79(4):581-588), the controllable vascular smooth muscle cells phenotypes of miRNA-145 inhibit its hyper-proliferative (Gadde
S,Rayner KJ.Nanomedicine meets microRNA:Current advances in RNA-based
nanotherapies for atherosclerosis.Arteriosclerosis Thrombosis and Vascular
Biology,2016, 36(9):e73-e79).But miRNAs is easy inactivation in vivo, need to be protected by the method that carrier contains
miRNAs.Have in document and not same drug is loaded into respectively in core/shell structure of electrospinning fibre using coaxial electrically spun method, to adjust
Control rate of release (Jiang YN, Mo HY, the Yu DG.Electrospun drug-loaded core-sheath of drug
PVP/zein nanofibers for biphasic drug release. International Journal of
Pharmaceutics,2012,438(1-2):232-239), but these method and processes are complex.
It is artificial that the present invention using aliphatic polyester as raw material, by lotion electrospinning method prepares a kind of load three layers of miRNAs
Blood vessel electrospinning fibre membrane material.The artificial blood vessel loads miRNA-126 in internal layer tunica fibrosa, accelerates its rate of release, and promote
Vascular endothelial cell adherency, proliferation.MiRNA-145 is loaded in mean fiber film, it is smooth by long-term slow release modulating vascular
Myocytic phenotype inhibits its hyper-proliferative.The present invention has good application prospect in small-caliber artificial blood vessel Material Field, should
Item work has not been reported at present.
Invention content
Present invention aims at prepare a kind of three layers of artificial blood vessel of load miRNAs.Technical scheme is as follows：
A kind of three layers of artificial blood vessel of load microRNAs, it is characterised in that the artificial blood vessel is made of three layers of tunica fibrosa,
Internal layer is poly- (L- lactide-co- 6-caprolactones) electrospun fiber membranes of polyethylene glycol-b-, and internal load has trimethyl chitosan chloride-
The particle and PELCL-REDV that the compound miRNA-126 of grafting-polyethylene glycol-REDV (TMC-g-PEG-REDV) are formed, middle level is
Poly- (glycolide-co- lactides) electrospun fiber membrane, internal load have trimethyl chitosan chloride-grafting-polyethylene glycol-VAPG
The particle of the formation of (TMC-g-PEG- VAPG) compound miRNA-145, outer layer are poly-epsilon-caprolactone electrospun fiber membrane.
It is described that Chinese patent CN 105153430A (Yuan Xiao are referred to the protection of miRNAs and the preparation method of carrier
Swallow, Zhou Fang, Zhao Yunhui appoint a kind of trimethyl chitosan chloride-grafting-polyethylene glycol-REDV copolymers of beautiful rosy clouds and preparation method
.CN 105153430A,2015).The preparation method of the PELCL-REDV refers to Chinese patent CN 106729976A (Yuan Xiao
Swallow, Zhou Fang, Ren Lixia, Zhao Yunhui, Cui Ce, a kind of PELCL/ polycaprolactones-REDV electrospun fiber membranes of Wen Meiling and preparation side
Method CN 106729976A, 2017).
Poly- (L- lactide-co- 6-caprolactones) tunica fibrosas of polyethylene glycol-b-, it is characterised in that inside the tunica fibrosa
It is thick containing the compound miRNA-126 compounds of PELCL-REDV and trimethyl chitosan chloride-grafting-polyethylene glycol-REDV
Degree is 100~300 μm.
Poly- (glycolide-co- lactides) tunica fibrosa, it is characterised in that it is poly- that tri-methylated shell is loaded inside the fiber
MiRNA-145 compounds compound sugar-grafting-polyethylene glycol-VAPG, thickness are 100~300 μm.
The poly-epsilon-caprolactone tunica fibrosa, it is characterised in that the tunica fibrosa is made of the fiber of 1~2 μm of diameter.
Poly- (L- lactide-co- 6-caprolactones) number-average molecular weights of inner fiber film polyethylene glycol-b- be (5~9) ×
104, molar ratio LA:CL (lactides:Caprolactone)=3:1.
The number-average molecular weight of the mean fiber film poly- (glycolide-co- lactides) is (4~8) × 104, molar ratio
LA:GA (lactides:Glycolide)=3:1.
The number-average molecular weight of the outer layer fiber film poly-epsilon-caprolactone is (5~10) × 104。
The preparation method of three layers of artificial blood vessel of the present invention, i.e., by from inside to outside to each layer of progress electrostatic spinning, shape
At the artificial blood vessel bracket with different three-deckers, it is characterized in that including following procedure：
(1) by polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones) and PELCL-REDV according to mass ratio be (1~
5):1 is dissolved in chloroform and n,N-Dimethylformamide (DMF) mixed solution, chloroform:DMF volume ratios are (4~8):1, it is configured to
A concentration of 100~200mg/mL solution is as oil phase, with carrier TMC-g-PEG-REDV by P in N and miRNAs in chitosan
Ratio (N/P) is than being (12~18):1 load miRNA-126 prepares stable internal layer electrospinning lotion as water phase, stirring.
(2) poly- (glycolide-co- lactides) is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform:
DMF volume ratios are (4~8):1, a concentration of 150~250mg/mL solution is configured to as oil phase, with carrier TMC-g-PEG-
VAPG is (12~18) by N/P ratios:1 load miRNA-145 prepares stable middle level electrospinning lotion as water phase, stirring.
(3) poly-epsilon-caprolactone is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform:DMF volume ratios are
(4~8):1, it is configured to a concentration of 200~300mg/mL solution.
(4) electrospinning will be carried out by the lotion obtained by step (1), electrospinning condition is：Injection rate is 0.2~0.7mL/h, electricity
The voltage of spinning is 10~15kV, and it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the electrospinning fibre of 100~300 μ m thicks
Film.Later, electrospinning will be carried out by the lotion obtained by step (2), electrospinning condition is：Injection rate is 0.2~0.8mL/h, electrospinning
Voltage be 10~15kV, receive distance be 10~15cm, 1~3h of electrospinning obtains the tunica fibrosa of 100~300 μ m thicks.Finally,
Electrospinning will be carried out by the solution obtained by step (3), electrospinning condition is：Injection rate is 0.2~0.8mL/h, and the voltage of electrospinning is
10~15kV, it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the tunica fibrosa of 100~300 μ m thicks.
The preparation method of above-mentioned carrier TMC-g-PEG-REDV and TMC-g-PEG-VAPG is according to Chinese patent CN
Prepared by 105153430A, REDV is replaced into VAPG.
The preparation method of above-mentioned PELCL-REDV is prepared according to Chinese patent CN106729976A, by poly-epsilon-caprolactone
It is replaced into PELCL.
Trilaminate material oil phase solvent is chloroform and n,N-Dimethylformamide mixed solvent, and internal layer is with polyethylene glycol-b-
As oil phase, water phase is the compound miRNA- of TMC-g-PEG-REDV by poly- (L- lactide-co- 6-caprolactones) and PELCL-REDV
126, it is mixed to form lotion and prepares electrospun fiber membrane.Middle level is using poly- (glycolide-co- lactides) as oil phase, TMC-g-PEG-
The aqueous solution of the compound miRNA-145 of VAPG carries out lotion electrospinning as water phase.Outer layer with poly-epsilon-caprolactone be dissolved in oil phase solvent into
Row electrospinning.Internal layer electrospun fiber membrane hydrophily is improved, and REDV is conducive to endothelial cell adherency, and by loading miRNA-
126 promote endothelial cell proliferation.MiRNA-145 rates of release are relatively low in mean fiber film, reach long period regulation smooth muscle cell
The effect of phenotype.Outer layer fiber film promotes growth of the cell to material internal by larger hole, and improves intravascular stent machine
Tool performance.
Three layers of artificial blood vessel of the load microRNAs of the present invention are used for vascular graft field.
It is an advantage of the current invention that three layers of artificial blood vessel can efficiently control the rate of release of two kinds of miRNAs.Internal layer
Tunica fibrosa diameter is smaller, in electro-spinning process, promotes endothelial cell to adhere in film surface using PELCL-REDV, is loaded with miRNA-
126 compound promotes particle quick release close to electrospinning fibre silk table face, meanwhile, it can make under the action of wall shear stress
There is defect in Electrospun surface, to accelerate miRNA-126 diffusion, release and Human Umbilical Vein Endothelial Cells effect.In addition, compound
Introducing improve the hydrophily of electrospun fiber membrane, so that it is more conducive to cell adherence.Mean fiber film lotion electrospinning makes to be loaded with
The particle of miRNA-145 is scattered in electricity and puts in filament, since the effect of inside and outside two layers of electrospinning film physical separation makes Particle diffusion
It is more difficult, therefore delayed the release process of miRNA-145.The thick fiber diameters of outer layer possess the hole of bigger, are conducive to thin
Born of the same parents promote the formation of multi-layer cellular to the growth inside tunica fibrosa, and can provide and be similar to naturally for intravascular stent
The mechanical performance of blood vessel.The quick release of miRNA-126 is conducive to quick endothelialization, reduces the risk of thrombosis.
MiRNA-145's delays to discharge, and can effectively regulate and control the phenotype of smooth muscle cell, inhibit its hyper-proliferative, reduce blood vessel weight
Mould the risk of middle and later periods restenosis.The present invention is applied to small-caliber artificial blood vessel material and vasotransplantation bio-medical material is led
Domain.
Description of the drawings
Fig. 1：The SEM photograph of three layers of artificial blood vessel of load microRNAs prepared by embodiment 1.
Fig. 2：Three layers of electrospinning film accumulation to miRNA-126 and miRNA-145 respectively in the last fortnight prepared by embodiment 1
Release profiles.
Specific implementation mode
Below by case study on implementation, technical scheme of the present invention will be further described, and following case study on implementation is to this hair
Bright further explanation is not intended to limit the scope of application of the present invention.
Load microRNAs three layers of artificial blood vessel, be made of three layers of tunica fibrosa, internal layer be load have miRNA-126 and
Poly- (L- lactide-co- 6-caprolactones) tunica fibrosas of polyethylene glycol-b- of PELCL-REDV, by the fibre of a diameter of 500~800nm
Dimension is constituted, and thickness is 100~300 μm.Middle level is poly- (glycolide-co- lactides) tunica fibrosa that load has miRNA-145,
It is made of the fiber of a diameter of 500~800nm, thickness is 100~300 μm.Outer layer is poly-epsilon-caprolactone tunica fibrosa, by diameter
It is constituted for 1~2 μm of fiber, thickness is 100~300 μm.
Above-mentioned poly- (L- lactide-co- 6-caprolactones) number-average molecular weights of polyethylene glycol-b- are (5~9) × 104。
The number-average molecular weight of above-mentioned poly- (glycolide-co- lactides) is (4~8) × 104。
The number-average molecular weight of above-mentioned poly-epsilon-caprolactone is (5~10) × 104。
The preparation method of three layers of artificial blood vessel of above-mentioned load microRNAs, it is characterised in that including following procedure：
(1) by polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones) and PELCL-REDV according to mass ratio be (1~
5):1 is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform:DMF volume ratios are (4~8):1, it is configured to concentration
It is 100~200mg/mL solution as oil phase, with carrier TMC-g-PEG-REDV by N/P ratios for (12~18):1 load
MiRNA-126 prepares stable lotion as water phase, stirring.
(2) poly- (glycolide-co- lactides) is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform:
DMF volume ratios are (4~8):1, a concentration of 150~250mg/mL solution is configured to as oil phase, with carrier TMC-g-PEG-
VAPG is (12~18) by N/P ratios:1 load miRNA-145 prepares stable lotion as water phase, stirring.
(3) poly-epsilon-caprolactone is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform：DMF volume ratios are
(4~8):1, it is configured to a concentration of 200~300mg/mL solution.
(4) electrospinning will be carried out by the lotion obtained by step (1), electrospinning condition is：Injection rate is 0.2~0.7mL/h, electricity
The voltage of spinning is 10~15kV, and it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the electrospinning fibre of 100~300 μ m thicks
Film.Later, electrospinning will be carried out by the lotion obtained by step (2), electrospinning condition is：Injection rate is 0.2~0.8mL/h, electrospinning
Voltage be 10~15kV, receive distance be 10~15cm, 1~3h of electrospinning obtains the tunica fibrosa of 100~300 μ m thicks.Finally,
Electrospinning will be carried out by the solution obtained by step (3), electrospinning condition is：Injection rate is 0.2~0.8mL/h, and the voltage of electrospinning is
10~15kV, it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the tunica fibrosa of 100~300 μ m thicks.
The preparation method of carrier TMC-g-PEG-REDV and TMC-g-PEG-VAPG are according to Chinese patent CN
Prepared by 105153430A：
(1) matter of the adjacent two thiopyridines-polyethylene glycol of raw material-succinimide acetic acid vinegar and trimethyl chitosan chloride (TMC)
Amount is than being 1:5~1, a concentration of 10mg/ml of trimethyl chitosan chloride is dissolved in deionized water, it is stirred to react 3 at room temperature~
10h.Product uses deionized water dialysis, freeze-drying to obtain trimethyl chitosan chloride-g- polyethylene glycol-two thiopyridines of neighbour.
(2) two thiopyridines copolymer of trimethyl chitosan chloride-g- polyethylene glycol-neighbour and the mass ratio of REDV small peptides be 10~
20:1, REDV small peptide is dissolved in deionized water, a concentration of 0.25~0.5mg/ml solution being configured to, be stirred at room temperature reaction 2~
6h.The small peptide for not participating in reaction is removed by dialysing, and trimethyl chitosan chloride-g- polyethylene glycol-REDV is made.Above-mentioned carrier
The preparation method of TMC-g-PEG-VAPG is similar with the above-mentioned preparation method of carrier TMC-g-PEG-REDV.
The preparation method of above-mentioned PELCL-REDV is prepared according to Chinese patent CN106729976A：
(1) use N, N'- carbonyl dimidazoles (CDI) activated hydroxyl groups group that it is anhydrous that PELCL is made into 0.3~0.5mg/mL
Dichloromethane solution, according to one-ended hydroxy PELCL：CDI mole be 1：1 is addition CDI, N2In be stirred to react at room temperature for 24 hours,
Then excessive propene acid hydroxyl ethyl ester and mass fraction is added as 2%~4% catalyst 4- lutidines, stirring is anti-at room temperature
48~72h, the excessive methanol extraction of product, vacuum drying is answered to obtain white PELCL double bonds modified product.
(2) PELCL double bond modified products are made into the dichloromethane solution of 200~300mg/mL, according to double bond and REDV
Sulfydryl molar ratio is 1 in small peptide：1 is added REDV small peptides, and it is 0.2~1% photochemical catalyst 2,2- dimethoxies that mass fraction, which is added,
Base -2- phenyl acetophenones are stirred at room temperature uniformly, UV 10~15min of illumination of 365nm, product ethanol precipitation.
Embodiment 1：
Polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones)And PELCL-REDV
It is 1 according to mass ratio:1 is dissolved in chloroform and n,N-Dimethylformamide mixed solution, and solvent volume ratio is 4:1, it is configured to dense
It spends for 150mg/mL solution as oil phase, with carrier TMC-g-PEG-REDV by N/P ratios for 12:1 load miRNA-126 conducts
Water phase, stirring, prepares stable lotion.By poly- (glycolide-co- lactides)It is dissolved in chloroform and N, N- dimethyl
In formamide mixed solution, solvent volume ratio is 4:1, a concentration of 200mg/mL solution is configured to as oil phase, with carrier TMC-
G-PEG-VAPG is 12 by N/P ratios:1 load miRNA-145 prepares stable lotion as water phase, stirring.By poly-epsilon-caprolactoneIt is dissolved in chloroform and n,N-Dimethylformamide mixed solution, solvent volume ratio is 4:1, it is configured to a concentration of
200mg/mL solution.
Acquired solution is subjected to electrospinning, poly- (L- lactide-co- 6-caprolactones) the lotion electricity of internal layer polyethylene glycol-b- successively
It spins, condition is：Injection rate is 0.5mL/h, and the voltage of electrospinning is 13kV, and it is 13cm to receive distance, and electrospinning 2h obtains 300 μ m-thicks
The electrospun fiber membrane of degree is made of the fiber of a diameter of 500~800nm.Middle strata (glycolide-co- lactides) lotion later
Electrospinning is carried out, electrospinning condition is：Injection rate is 0.4mL/h, and the voltage of electrospinning is 12kV, and it is 13cm, electrospinning 2h to receive distance
The tunica fibrosa of 150 μm of thickness is obtained, is made of the fiber of a diameter of 500~800nm.Electrospinning, electricity finally are carried out to outer layer solution
Spinning condition is：Injection rate is 0.4mL/h, and the voltage of electrospinning is 13kV, and it is 13cm to receive distance, and electrospinning 2h obtains 250 μ m-thicks
The tunica fibrosa of degree, 1~2 μm of diameter.
The cross section SEM photograph of the artificial blood vessel, as shown in Figure 1.Three layers of electrospinning film of the artificial blood vessel discharge the tired of initial stage
Product release profiles, as shown in Figure 2.
Embodiment 2：
Polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones)And PELCL-REDV
It is 2 according to mass ratio:1 is dissolved in chloroform and n,N-Dimethylformamide mixed solution, and solvent volume ratio is 6:1, it is configured to dense
It spends for 100mg/mL solution as oil phase, with carrier TMC-g-PEG-REDV by N/P ratios for 15:1 load miRNA-126 conducts
Water phase, stirring, prepares stable lotion.By poly- (glycolide-co- lactides)It is dissolved in chloroform and N, N- diformazans
In base formamide mixed solution, solvent volume ratio is 6:1, a concentration of 150mg/mL solution is configured to as oil phase, with carrier
TMC-g-PEG-VAPG is 15 by N/P ratios:1 load miRNA-145 prepares stable lotion as water phase, stirring.By poly- ε-oneself
LactoneIt is dissolved in chloroform and n,N-Dimethylformamide mixed solution, solvent volume ratio is 6:1, it is configured to dense
Degree is 200mg/mL solution.
Acquired solution is subjected to electrospinning, poly- (L- lactide-co- 6-caprolactones) the lotion electricity of internal layer polyethylene glycol-b- successively
It spins, condition is：Injection rate is 0.2mL/h, and the voltage of electrospinning is 15kV, and it is 15cm to receive distance, and electrospinning 3h obtains 200 μ m-thicks
The electrospun fiber membrane of degree is made of the fiber of a diameter of 500~600nm.Middle strata (glycolide-co- lactides) lotion later
Electrospinning is carried out, electrospinning condition is：Injection rate is 0.2mL/h, and the voltage of electrospinning is 10kV, and it is 15cm, electrospinning 3h to receive distance
The tunica fibrosa of 200 μm of thickness is obtained, is made of the fiber of a diameter of 500~600nm.Electrospinning, electricity finally are carried out to outer layer solution
Spinning condition is：Injection rate is 0.2mL/h, and the voltage of electrospinning is 15kV, and it is 15cm to receive distance, and electrospinning 3h obtains 200 μ m-thicks
The tunica fibrosa of degree, 1~2 μm of diameter.
Embodiment 3：
Polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones)And PELCL-REDV
It is 3 according to mass ratio:1 is dissolved in chloroform and n,N-Dimethylformamide mixed solution, and solvent volume ratio is 7:1, it is configured to dense
It spends for 200mg/mL solution as oil phase, with carrier TMC-g-PEG-REDV by N/P ratios for 16:1 load miRNA-126 conducts
Water phase, stirring, prepares stable lotion.By poly- (glycolide-co- lactides)It is dissolved in chloroform and N, N- diformazans
In base formamide mixed solution, solvent volume ratio is 7:1, a concentration of 250mg/mL solution is configured to as oil phase, with carrier
TMC-g-PEG-VAPG is 16 by N/P ratios:1 load miRNA-145 prepares stable lotion as water phase, stirring.By poly- ε-
CaprolactoneIt is dissolved in chloroform and n,N-Dimethylformamide mixed solution, solvent volume ratio is 7:1, it is configured to
A concentration of 300mg/mL solution.
Acquired solution is subjected to electrospinning, poly- (L- lactide-co- 6-caprolactones) the lotion electricity of internal layer polyethylene glycol-b- successively
It spins, condition is：Injection rate is 0.7mL/h, and the voltage of electrospinning is 10kV, and it is 12cm to receive distance, and electrospinning 1h obtains 300 μ m-thicks
The electrospun fiber membrane of degree is made of the fiber of a diameter of 700~800nm.Middle strata (glycolide-co- lactides) lotion later
Electrospinning is carried out, electrospinning condition is：Injection rate is 0.7mL/h, and the voltage of electrospinning is 15kV, and it is 10cm, electrospinning 1h to receive distance
The tunica fibrosa of 300 μm of thickness is obtained, is made of the fiber of a diameter of 700~800nm.Electrospinning, electricity finally are carried out to outer layer solution
Spinning condition is：Injection rate is 0.8mL/h, and the voltage of electrospinning is 10kV, and it is 10cm to receive distance, and electrospinning 2h obtains 300 μ m-thicks
The undulation degree film of degree, 1.5~2 μm of diameter
Embodiment 4：
Poly- (L- lactide-co- the 6-caprolactones) (M of polyethylene glycol-b-n=7 × 104) and PELCL-REDV
It is 4 according to mass ratio:1 is dissolved in chloroform and n,N-Dimethylformamide mixed solution, and solvent volume ratio is 8:1, it is configured to dense
It spends for 100mg/mL solution as oil phase, with carrier TMC-g-PEG-REDV by N/P ratios for 18:1 load miRNA-126 conducts
Water phase, stirring, prepares stable lotion.By poly- (glycolide-co- lactides)It is dissolved in chloroform and N, N- diformazans
In base formamide mixed solution, solvent volume ratio is 8:1, a concentration of 250mg/mL solution is configured to as oil phase, with carrier
TMC-g-PEG-VAPG is 18 by N/P ratios:1 load miRNA-145 prepares stable lotion as water phase, stirring.By poly- ε-oneself
LactoneIt is dissolved in chloroform and n,N-Dimethylformamide mixed solution, solvent volume ratio is 8:1, it is configured to concentration
For 250mg/mL solution.
Acquired solution is subjected to electrospinning, poly- (L- lactide-co- 6-caprolactones) the lotion electricity of internal layer polyethylene glycol-b- successively
It spins, condition is：Injection rate is 0.4mL/h, and the voltage of electrospinning is 13kV, and it is 10cm to receive distance, and electrospinning 2h obtains 200 μ m-thicks
The electrospun fiber membrane of degree is made of the fiber of a diameter of 500~700nm.Middle strata (glycolide-co- lactides) lotion later
Electrospinning is carried out, electrospinning condition is：Injection rate is 0.5mL/h, and the voltage of electrospinning is 12kV, and it is 10cm, electrospinning to receive distance
1.5h obtains the tunica fibrosa of 200 μ m thicks, is made of the fiber of a diameter of 600~800nm.Electricity finally is carried out to outer layer solution
It spins, electrospinning condition is：Injection rate is 0.6mL/h, and the voltage of electrospinning is 15kV, and it is 13cm to receive distance, and electrospinning 1h obtains 250
The tunica fibrosa of μ m thick, 1~2 μm of diameter.
Claims (10)
1. a kind of three layers of artificial blood vessel's electrospun fiber membrane of the small nucleic acid of load, it is characterised in that artificial blood vessel is by three layers of electrospinning film
It constitutes, internal layer is poly- (L- lactide-co- 6-caprolactones) the electricity spinning fibre films of polyethylene glycol-b-, and internal load has tri-methylated
The particle and PELCL-REDV that the compound miRNA-126 of chitosan-grafting-polyethylene glycol-REDV are formed；Middle level is poly- (glycolide-
Co- lactides) electricity spinning fibre film, internal load has trimethyl chitosan chloride-grafting-compound miRNA- of polyethylene glycol-VAPG
145 particles formed；Outer layer is poly-epsilon-caprolactone electricity spinning fibre film.
2. tunica fibrosa as described in claim 1, it is characterised in that internal layer thickness is 100~300 μm.
3. tunica fibrosa as described in claim 1, it is characterised in that intima-media thickness is 100~300 μm.
4. tunica fibrosa as described in claim 1, it is characterised in that outer layer is made of the fiber of 1~2 μm of diameter.
5. the preparation method of three layers of artificial blood vessel's electrospun fiber membrane of the small nucleic acid of load of claim 1, it is characterized in that including
Following procedure：
(1) it is (1~5) according to mass ratio by polyethylene glycol-b- poly- (L- lactide-co- 6-caprolactones) and PELCL-REDV:1
It is dissolved in chloroform and n,N-Dimethylformamide (DMF) mixed solution, chloroform：DMF volume ratios are (4~8):1, it is configured to concentration
It is 100~200mg/mL solution as oil phase；With carrier TMC-g-PEG-REDV by the N/P ratios of P in N and miRNA in chitosan
For (12~18):1 load miRNA-126 prepares stable internal layer electrospinning lotion as water phase, stirring；
(2) poly- (glycolide-co- lactides) is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform:DMF bodies
Product is than being (4~8):1, a concentration of 150~250mg/mL solution is configured to as oil phase, and N/ is pressed with carrier TMC-g-PEG-VAPG
P ratios are (12~18):1 load miRNA-145 is as water phase；Stirring, prepares stable middle level electrospinning lotion；
(3) poly-epsilon-caprolactone is dissolved in chloroform and n,N-Dimethylformamide mixed solution, chloroform：DMF volume ratios be (4~
8):1, it is configured to a concentration of 200~300mg/mL solution；
(4) electrospinning will be carried out by the lotion obtained by step (1), electrospinning condition is：Injection rate is 0.2~0.7mL/h, electrospinning
Voltage is 10~15kV, and it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the electrospun fiber membrane of 100~300 μ m thicks；It
Afterwards, electrospinning will be carried out by the lotion obtained by step (2), electrospinning condition is：Injection rate is 0.2~0.8mL/h, the voltage of electrospinning
For 10~15kV, it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the electrospun fiber membrane of 100~300 μ m thicks；Finally, will
Electrospinning is carried out by the solution obtained by step (3), electrospinning condition is：Injection rate is 0.2~0.8mL/h, and the voltage of electrospinning is 10
~15kV, it is 10~15cm to receive distance, and 1~3h of electrospinning obtains the electrospun fiber membrane of 100~300 μ m thicks.
6. method as claimed in claim 5, it is characterized in that inner fiber film polyethylene glycol-b- it is poly- (L- lactide-co- ε-oneself
Lactone) number-average molecular weight be (5~9) × 104, lactide and caprolactone molar ratio are 3:1.
7. method as claimed in claim 5, it is characterized in that the number of the mean fiber film poly- (glycolide-co- lactides) is equal
Molecular weight is (4~8) × 104, lactide and glycolide molar ratio are 3:1.
8. method as claimed in claim 5, it is characterized in that the number-average molecular weight of the outer layer fiber film poly-epsilon-caprolactone is
(5~10) × 104。
9. three layers of artificial blood vessel's electrospun fiber membrane application vasotransplantation field of the small nucleic acid of load.
10. three layers of electrospun fiber membrane of the small nucleic acid of load are applied to small-caliber artificial blood vessel material.
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