CN106283399A - A kind of arrange orderly modified nano fiber film and preparation thereof and application - Google Patents

Abstract

The invention relates to an orderly arranged modified nanofiber membrane and its preparation and application. The nanofiber membrane is a collagen/silk fibroin fiber membrane modified by polycaprolactone, and the pore diameter of the nanofiber membrane is 10-25 μm. Preparation: Dissolve collagen, silk fibroin, and polycaprolactone in a solvent, stir and mix to obtain a spinning solution; at room temperature, the spinning solution is electrospun, dried, then cross-linked, washed with water, Vacuum drying, that is. Application as a cell culture scaffold. The method of the present invention is simple and convenient to operate, and the fiber membrane with ordered fibers is obtained by blending modification and changing the collector, which improves the porosity of the fiber membrane, and its mechanical properties and biocompatibility are good, which is beneficial for cells to enter The growth inside the scaffold has a good application prospect in cell culture.

Description

An ordered modified nanofiber membrane and its preparation and application

technical field

The invention belongs to the field of nanomaterials and their preparation and application, and in particular relates to an ordered modified nanofiber membrane and its preparation and application.

Background technique

Electrospinning technology is currently the most common method of producing nanofibers in the world. Electrospinning has a nanometer or micrometer fiber scale, small pore size, high porosity, and uniform distribution. It is used in nanofiltration materials, sensing materials, and especially It is widely used in the modification of biological scaffolds, medical materials, tissue engineering, etc. Now, electrospinning technology has become the most important method for preparing tissue engineering scaffolds.

Collagen and silk fibroin are natural polymers with good biocompatibility. They have very broad application prospects in the field of biomedicine. They can be used to prepare electrospun nanofiber membranes, but their mechanical properties are poor. There is a problem with later application.

An electrospinning device generally consists of a syringe pump, a high-voltage power supply, and a receiving device. After the spinning starts, the fibers are continuously dispersed and deposited on the flat receiver. Due to the potential gradient between the injection needle and the receiving plate, the fibers are preferentially accumulated at the position closest to the injection point and deposited in a layered form. Finally, the fibers are tightly arranged. The two-dimensional structure of the fiber, the dense arrangement of the fibers results in a smaller pore size of the spun fiber, and when applied to the tissue engineering scaffold, it will prevent cells from entering the scaffold to grow.

The pore size and order of the electrospun scaffolds play an important role in the cell culture performance of the scaffolds. Orderly spun fibers can promote cell adhesion, survival and proliferation, guide cell migration, increase the pore size of the scaffold on the basis of ordered spun fibers, facilitate the growth of cells into the scaffold, and promote the transmission of nutrients and excretion of metabolites. The methods of obtaining ordered nanofibers by changing the receiving device mainly include roller collection, turntable collection, conductive plate collection with insulating ports, etc. Most of the collection methods obtain ordered fibers arranged in parallel, and cannot obtain organic fibers with large aperture order fibers.

Therefore, in view of the above problems, it is necessary to provide a method capable of preparing ordered modified collagen/silk fibroin fiber membranes, so as to solve the problems existing in the prior art.

Contents of the invention

The technical problem to be solved by the present invention is to provide an orderly arranged modified nanofiber membrane and its preparation and application. The fibrous membrane improves the porosity of the fibrous membrane, and has good mechanical properties and biocompatibility, which is beneficial for cells to grow inside the scaffold, and has a good application prospect in cell culture.

An ordered modified nanofiber membrane of the present invention, the nanofiber membrane is polycaprolactone modified collagen/

Silk fibroin fiber membrane, nanofiber membrane pore size is 10-25μm.

A method for preparing an ordered modified nanofiber membrane of the present invention, comprising:

(1) dissolving collagen, silk fibroin, and polycaprolactone in a solvent, stirring and mixing to obtain a spinning solution;

(2) Under room temperature conditions, the spinning solution in step (1) is electrospun and dried to obtain a white ordered fiber film; wherein the receiving device used for the electrospinning is a probe collector;

(3) Cross-linking the white ordered fiber membrane obtained in step (2), washing with water, and drying in vacuum to obtain an ordered modified composite fiber membrane.

In the step (1), the mass ratio of collagen, silk fibroin and polycaprolactone is 0.5-5:0.5-5:1-10.

The solvent in the step (1) is hexafluoroisopropanol; the concentration of the spinning solution is 8-10 wt%.

The specific process of electrospinning in the step (2) is as follows: the electrospinning voltage is 12-18kV, the distance between the spinning needle and the receiver is 10-15cm, and the advancing speed of the syringe pump is 1-1.5mL/h .

In the described step (2), the probe collector is made by embedding the probe into an aluminum foil plate receiver to form a probe array, as shown in Figure 1, the probe array is 4 * 4 ~ 8 * 8; The distance between the probes is 2-4 cm.

The drying in the step (2) is drying in a vacuum oven for 2 to 3 days.

The crosslinking in the step (3) is: glutaraldehyde steam crosslinking, and the crosslinking time is 4-12h.

Said glutaraldehyde vapor crosslinking specifically includes: putting the fiber film into a vacuum drier, putting glutaraldehyde solution into the drier, and carrying out glutaraldehyde vapor crosslinking, wherein the concentration of the glutaraldehyde solution is 25-50wt% .

The water washing in the step (3) is: water washing for 2 to 4 days, and the vacuum drying is: vacuum oven drying for 1 to 2 days.

An application of the ordered modified nanofiber membrane of the present invention, the application of the ordered nanofiber membrane as a support for cell culture in vitro.

Beneficial effect

(1) The method of the present invention is simple and easy to operate, and the mechanical properties of the electrospun fiber membrane are enhanced by blending natural polymer collagen, silk fibroin and synthetic polymer polycaprolactone;

(2) The present invention uses the probe collector formed by the probe array to prepare ordered fibers by changing the shape of the collector. The needle tip of the probe is a tip, and there is a tip effect on the same probe, and the charge density of the tip part is relatively large , the strength of the electric field near it is stronger. During the electrospinning process, the fiber falls near the needle tip under the action of the electric field force, and the charged fiber is attracted the most by the electric field of the tip, guiding the fiber to drift toward the needle tip, so that the fiber follows the shape between the needle tips Orderly overlapped with the direction to form a nanofibrous membrane with an orderly arrangement and a large pore size;

(3) When the scaffold prepared by the present invention is used for cell culture in the later stage, it has a certain mechanical strength and can better support the growth of cells on the fibrous membrane, and the fibrous membrane with larger pore size makes the cells have a three-dimensional growth tendency, which can promote cell adhesion and proliferation. And promote the transmission of nutrients and the discharge of metabolites.

Description of drawings

Fig. 1 is the schematic diagram of probe collector in the preparation method;

Fig. 2 is the optical micrograph of the ordered fiber film that 6 * 6 probe collectors prepare in embodiment 1;

Fig. 3 is the optical micrograph of the ordered fiber membrane that 7 * 7 probe collectors prepare in embodiment 2;

Fig. 4 is the SEM photograph of the ordered fiber membrane that 6 * 6 probe collectors prepare in embodiment 1;

Fig. 5 is the SEM photograph of the ordered fiber membrane that 7 * 7 probe collectors prepare in embodiment 2;

FIG. 6 is a diagram of the mechanical properties of ordered fiber membranes prepared in Examples 1, 2, and 3.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Collagen, silk fibroin, and polycaprolactone are added in the hexafluoroisopropanol solvent with a mass ratio of 3.5:3.5:3, and the concentration of the spinning solution is 8wt%. After fully stirring, a uniform spinning solution.

(2) Put the prepared spinning solution into the syringe and put it into the syringe pump, connect the needle to the anode of the high voltage generator, use the probe receiver as the receiving device, the probe array is 6×6, the probe The distance between them is 3cm, the probe receiver is connected to the cathode, the voltage is 15kV, the distance between the spinning needle and the tip of the probe receiver is 12cm, and the advancing speed of the syringe pump is 1mL/h. After electrospinning, an apparently uniform and ordered white fiber film was collected on the probe collector. The resulting fibrous film was dried in a vacuum oven for 2 days.

(3) Put the fiber membrane obtained by electrospinning into a vacuum desiccator, put the petri dish at the bottom of the desiccator, and fill the petri dish with a concentration of 25% glutaraldehyde solution, vacuumize and carry out glutaraldehyde vapor crosslinking After 12 hours, it was taken out, washed with ultrapure water for 2 days, and dried in a vacuum oven for 1 day. Figure 2 is an optical microscope photo of the electrospun fiber membrane before crosslinking. Figure 4 is the SEM photo of the electrospun fiber membrane, which shows that the fibers are arranged in an orderly manner, and a large pore size is formed between the fibers, and the average pore size of the fiber membrane is 14.7 μm. Fig. 6 is a diagram of the mechanical properties of the fiber membrane, which shows that the breaking strength of the crosslinked fiber membrane is 3.1 MPa.

Example 2

(1) Collagen, silk fibroin, and polycaprolactone are added in the hexafluoroisopropanol solvent with a mass ratio of 3.5:3.5:3, and the concentration of the spinning solution is 8wt%. After fully stirring, a uniform spinning solution.

(2) Put the prepared spinning solution into the syringe and put it into the syringe pump, connect the needle to the anode of the high-voltage generator, use the probe receiver as the receiving device, the probe array is 7×7, and the probe The distance between them is 2.5cm, the probe receiver is connected to the cathode, the voltage is 15kV, the distance between the spinning needle and the tip of the probe receiver is 12cm, and the advancing speed of the syringe pump is 1mL/h. After electrospinning, an apparently uniform and ordered white fiber film was collected on the probe collector. The resulting fibrous film was dried in a vacuum oven for 2 days.

(3) Put the fiber membrane obtained by electrospinning into a vacuum desiccator, put the petri dish at the bottom of the desiccator, and fill the petri dish with a concentration of 25% glutaraldehyde solution, vacuumize and carry out glutaraldehyde vapor crosslinking After 12 hours, it was taken out, washed with ultrapure water for 2 days, and dried in a vacuum oven for 1 day. Figure 3 is an optical microscope photo of the electrospun fiber membrane before crosslinking. Fig. 5 is the SEM photograph of electrospun fiber membrane and shows that fiber is in orderly arrangement, and the fiber membrane prepared by 6 * 6 probe collectors in comparative example 1 is more orderly, and the pore size formed between fibers is larger, The average pore size of the fiber membrane was 15.9 μm. Figure 6 is a diagram of the mechanical properties of the fiber membrane, showing that the breaking strength of the fiber membrane after crosslinking is 6.1 MPa, compared with the fiber membrane prepared by the 6 × 6 probe collector in Comparative Example 1, and the fiber prepared by the 7 × 7 probe collector Membrane mechanical properties are better.

Example 3

(1) Collagen, silk fibroin, and polycaprolactone are added in the hexafluoroisopropanol solvent with a mass ratio of 3.5:3.5:3, and the concentration of the spinning solution is 8wt%. After fully stirring, a uniform spinning solution.

(2) Put the prepared spinning solution into the syringe and put it into the syringe pump, connect the needle to the anode of the high voltage generator, use the probe receiver as the receiving device, the probe array is 8×8, the probe The distance between them is 2cm, the probe receiver is connected to the cathode, the voltage is 15kV, the distance between the spinning needle and the tip of the probe receiver is 12cm, and the advancing speed of the syringe pump is 1mL/h. After electrospinning, an apparently uniform and ordered white fiber film was collected on the probe collector. The resulting fibrous film was dried in a vacuum oven for 2 days.

(3) Put the fiber membrane obtained by electrospinning into a vacuum desiccator, put the petri dish at the bottom of the desiccator, and fill the petri dish with a concentration of 25% glutaraldehyde solution, vacuumize and carry out glutaraldehyde vapor crosslinking After 12 hours, it was taken out, washed with ultrapure water for 2 days, and dried in a vacuum oven for 1 day. The SEM photos of the electrospun fiber membranes showed that the fibers were arranged in an orderly manner, and large pores were formed between the fibers, and the average pore size of the fiber membrane was 13.2 μm. Fig. 6 is a diagram of the mechanical properties of the fiber membrane. The test of the mechanical properties of the fiber membrane shows that the breaking strength of the crosslinked fiber membrane is 4.1 MPa.

Claims (10)

1. the modified nano fiber film that an arrangement is orderly, it is characterised in that: described nano fibrous membrane is Polycaprolactone modified Collagen protein/fibroin fiber film, nanofiber membrane aperture is 10-25 μm.

2. a preparation method for the modified nano fiber film that arrangement as claimed in claim 1 is orderly, including:

(1) collagen protein, fibroin albumen, polycaprolactone are dissolved in solvent, stirring and evenly mixing, obtain spinning liquid；

(2) under room temperature condition, in step (1), spinning liquid carries out electrostatic spinning, is dried, obtains fibrous membrane；Wherein electrostatic spinning institute It is probe catcher with reception device；

(3) fibrous membrane that step (2) obtains is cross-linked, washing, vacuum drying, orderly modified composite fiber must be arranged Film.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute State collagen protein in step (1), fibroin albumen, the mass ratio of polycaprolactone are 0.5～5:0.5～5:1～10.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute Stating solvent in step (1) is hexafluoroisopropanol；The concentration of spinning liquid is 8～10wt%.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute State electrostatic spinning in step (2) specifically comprises the processes of: electrostatic spinning voltage is 12～18kV, between spinning syringe needle and receptor away from From for 10～15cm, the fltting speed of syringe pump is 1～1.5mL/h；Probe catcher is to be embedded aluminium foil flat board by probe to receive It is made that device forms probe array, and probe array is 4 × 4～8 × 8；Between probe catcher middle probe, spacing is 2～4cm.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute State and step (2) is dried into vacuum drying oven is dried 2～3 days.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute Stating crosslinking in step (3) is: glutaraldehyde vapor crosslinking, crosslinking time is 4-12h.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 7, it is characterised in that: institute State glutaraldehyde vapor crosslinking particularly as follows: fibrous membrane is put in vacuum desiccator, exsiccator is put into glutaraldehyde solution, carry out penta 2 Aldehyde vapor crosslinking, wherein the concentration of glutaraldehyde solution is 25～50wt%.

A kind of preparation method arranging orderly modified nano fiber film the most according to claim 2, it is characterised in that: institute Stating washing in step (3) is: wash 2～4 days, is vacuum dried and is: vacuum drying oven is dried 1～2 day.

10. the application of the modified nano fiber film that an arrangement as claimed in claim 1 is orderly, it is characterised in that: described row Show the nano fibrous membrane application as cell injuring model support of sequence.