CN107261209A - A kind of method of use phyllosilicate/chitosan self-assembled modified micro/nano-fibre film layer by layer - Google Patents

Abstract

The invention discloses a method for self-assembling and modifying micro/nano fiber membranes with layered silicate/chitosan layer by layer, and belongs to the field of material technology. In the present invention, a charged micro/nano fiber membrane is first prepared as a substrate, and oppositely charged chitosan or layered silicate is alternately assembled on the substrate according to the charging condition of the substrate, and the obtained micro/nano fiber membrane is dried, A composite micro/nanofibrous membrane with a desired number of layers is obtained. The modified micro/nanofibrous membrane has greatly improved mechanical properties, increased hydrophilicity, antibacterial properties, and is more suitable for the adhesion, growth and differentiation of osteoblasts on its surface. The present invention selects layered silicate alone as the assembly material for the first time, and has many advantages such as mild conditions, simple process, no restriction on the size and shape of the bottom plate material, and no other impurities and by-products are produced during the modification process.

Description

A self-assembled layer-by-layer self-assembly of layered silicate/chitosan modified micro/nanofibrous membranes method

technical field

The invention belongs to the technical field of materials, and in particular relates to a method for self-assembling and modifying micro/nano fiber membranes with layered silicate/chitosan layer by layer.

Background technique

For decades, the development of bone tissue engineering has saved millions of patients suffering from bone defects and bone trauma from the ravages of diseases all over the world. Therefore, the research on bone tissue engineering scaffolds has gained a lot of attention. An ideal scaffold for bone tissue engineering should meet two basic requirements. First, the scaffold should have a certain mechanical strength, be able to resist external forces, and keep its physical structure stable, thereby ensuring stable cell growth and tissue development. Furthermore, suitable surface properties and excellent biocompatibility are crucial for their promotion of osteoblast adhesion, proliferation, and differentiation.

As a micro/nano material with a special three-dimensional structure, the micro/nanofibrous membrane is widely used in drug sustained release, adsorption filtration, and other fields. In addition, because it has a three-dimensional structure similar to the extracellular matrix, it can simulate the extracellular matrix. Matrix structure and provide a scaffold for cell growth and adhesion, so it is also an important tissue engineering material. Most micro/nanofibers have weak mechanical strength and smooth surface, which is not conducive to the stable growth of cells, so they need to be modified before they are applied in tissue engineering. As the only positively charged natural alkaline polysaccharide, chitosan has excellent biocompatibility and antibacterial activity, which can ensure that the biocompatibility of the material is not destroyed while introducing antibacterial properties. Layered silicate can enhance the mechanical strength of the material, control the roughness of the micro/nanofibrous membrane surface, and at the same time promote the antibacterial properties of chitosan. These two materials are ideal for the improvement of micro/nanofiber membranes. sexual material.

The Chinese patent "A Modified Micro/Nanofiber Membrane with Orderly Arrangement and Its Preparation and Application" (publication number CN106283399A) discloses an orderedly arranged modified micro/nanofiber membrane and its preparation method and application prospect. The specific method is: blending collagen, silk fibroin and polycaprolactone in a certain proportion to prepare a spinning solution, and performing electrostatic spinning, drying, crosslinking and other steps. Ordered micro/nanofibers were prepared by blending and altering the collector of electrospinning, and achieved the purpose of modification. Although the modification in this patent is carried out on the micro/nano fiber membrane, it is modified by blending and changing the collector. In the field of tissue engineering, due to the adhesion and growth of cells, the surface characteristics of the material have an important impact on its performance. The modification method of blending will not only change the surface of the material, but also change the main body of the material, so that the Structure and performance bring about unknown changes. The modification method of layer-by-layer self-assembly is only to modify the surface of the micro/nano fiber without changing the original components of the micro/nano fiber.

As a multifunctional and potential modification technology, layer-by-layer self-assembly technology has the advantages of simple operation, low cost, strong applicability, and no requirement on the size and shape of the substrate. Control modification, and the choice of modified materials is very wide. During the layer-by-layer self-assembly process, polyelectrolytes with opposite charges are adsorbed step by step and alternately, which is easy to operate and does not produce other by-products. In addition, compared with the layer-by-layer self-assembly process on the flat plate, the research on the layer-by-layer self-assembly modification on the micro/nanofiber membrane is relatively rare, which includes two levels of modification: (1) in each (2) modify the macroscopic surface of the micro/nanofiber membrane. After the layer-by-layer self-assembly process, a multi-level modification of the multi-level structure of the micro/nanofibrous membrane will be obtained.

Based on the above considerations, micro/nanofibers were selected as the base plate, chitosan was used as the positive layer component and layered silicate was used as the negative layer component, and the micro/nanofiber membrane was modified by layer-by-layer self-assembly method. A micro/nanofibrous membrane with stronger mechanical properties, antibacterial properties and good biocompatibility has been obtained, which has a very broad application prospect in the field of tissue engineering.

Contents of the invention

The purpose of the present invention is to provide a method for self-assembly modification of micro/nano fiber membrane layer by layer by using chitosan and layered silicate. The method can modify the micro/nano fiber membrane, improve its physical and biological properties, and prepare the micro/nano fiber membrane more suitable for bone tissue engineering.

The object of the present invention is achieved through the following technical solutions:

A method for modifying micro/nano fiber membranes by layer-by-layer self-assembly of layered silicate/chitosan, using charged micro/nano fiber membranes as the substrate, when the charge of the substrate is positive, sequentially on the substrate Assemble layered silicate and chitosan on top until the desired number of double layers is reached; when the charge on the substrate is negative, assemble chitosan and layered silicate on the substrate in turn until the desired number of double layers is reached number, the assembled micro/nanofibrous membrane was dried to obtain a modified micro/nanofibrous membrane.

Preferably, the absolute value of the zeta potential of the substrate is not less than 10mV.

Preferably, the required number of double layers is 1-50 layers.

Preferably, the charged micro/nano fiber membrane substrate is made of silk fibroin, collagen, cellulose acetate, chitosan and its derivatives, poly-L-lactic acid, polycaprolactone, polylactic-glycolic acid One or more of the copolymers are prepared.

Preferably, the micro/nano fiber membrane substrate is prepared by any one of electrospinning method, wet spinning method, dry spinning method, centrifugal spinning method, phase separation method or composite spinning method .

Preferably, the chitosan is assembled according to the following method: configure the chitosan acetic acid solution, soak the substrate in the positively charged chitosan acetic acid solution, and after a certain period of time, take out the membrane and wash it with a cleaning solution to remove it. Chitosan was not successfully assembled on the membrane surface.

Preferably, the phyllosilicate is assembled according to the following method: configure the phyllosilicate suspension, soak the substrate in the negatively charged phyllosilicate suspension, and take out the film after a certain period of time , wash with cleaning solution to remove the unassembled phyllosilicate on the surface of the membrane.

Preferably, the concentration of the chitosan acetic acid solution is 0.1-10 mg/mL.

Preferably, the layered silicate is montmorillonite or rectorite.

Preferably, the concentration of the phyllosilicate assembly suspension is 0.1-10 mg/mL.

Preferably, the cleaning solution is deionized water or 0.01-1 mol/L sodium chloride solution.

Preferably, the soaking assembly time is 10-60 minutes.

Preferably, the drying method is natural drying, vacuum freeze drying or drying in a vacuum oven, and the drying is complete drying.

A micro/nano fiber membrane self-assembled and modified layer by layer silicate/chitosan is prepared by the above method.

The invention uses the micro/nano fiber film as an assembly base, and alternately assembles chitosan and layered silicate on the surface of the multi-level structure through the action of electrostatic force. The introduction of chitosan endowed the nanofibrous membrane with antibacterial properties and maintained its good biocompatibility. However, layered silicate alone was introduced into the composite nanofibrous membrane as a negatively charged assembly material, which greatly enhanced the mechanical properties and surface properties of the fibrous membrane. The invention has many advantages, including mild conditions, simple process, no restriction on the size and shape of the bottom plate material, and no other impurities or other by-products are introduced during the modification process.

Description of drawings

Fig. 1 is the morphological diagram of the silk fibroin nanofiber membrane and the modified nanofiber membrane prepared in Example 1. The left side of the figure is the scanning electron microscope image of the surface of the silk fibroin nanofiber membrane, and the right side is the scanning electron microscope image of the layered silicate/chitosan layer-by-layer self-assembled modified nanofiber membrane with a double layer number of 15.5.

Fig. 2 is a graph of fiber diameter distribution of the silk fibroin nanofiber membrane and the modified nanofiber membrane prepared in Example 1. a is the fiber diameter distribution diagram of the silk fibroin nanofiber membrane, and b is the fiber diameter distribution diagram of the layer-by-layer silicate/chitosan self-assembled modified nanofiber membrane with a bilayer number of 15.5.

Fig. 3 is a time-dependent curve of the contact angle of the silk fibroin nanofiber membrane and the modified nanofiber membrane prepared in Example 1. a is the contact angle variation curve of silk fibroin nanofiber membrane with time, b is the contact angle variation curve of layer-by-layer silicate/chitosan self-assembled modified nanofiber membrane with a bilayer number of 15.5.

Fig. 4 is a comparison chart of tensile strength and elongation at break of the silk fibroin nanofiber membrane prepared in Example 1 and the modified nanofiber membrane. In the figure, a represents the silk fibroin nanofiber membrane, b represents the layer-by-layer silicate/chitosan layer-by-layer self-assembled modified nanofiber membrane with a double-layer number of 5, and c represents the layered silica with a double-layer number of 10. Salt/chitosan layer-by-layer self-assembled modified nanofibrous membrane, d represents the surface scanning electron microscope image of the layered silicate/chitosan layer-by-layer self-assembled modified nanofiber membrane with a double layer number of 15, e represents the bilayer Layer-by-layer self-assembly of layered silicate/chitosan modified nanofibrous membrane with 15.5 layers.

Fig. 5 is a scanning electron microscope image of the growth of osteoblasts on the surface of the silk fibroin nanofibrous membrane prepared in Example 1 and the surface of the modified nanofibrous membrane cultured for 72 h. a represents the silk fibroin nanofiber membrane, and b represents the layer-by-layer silicate/chitosan self-assembled modified nanofiber membrane with a bilayer number of 15.5.

detailed description

The technical solutions of the present invention are further described below through specific examples, the purpose of which is to help better understand the content of the present invention, but these specific embodiments do not limit the protection scope of the present invention in any way.

1. Preparation of Charged Micro/Nanofibrous Membranes

Take silk fibroin as an example:

The silk fibroin was dissolved in a hexafluoroisopropanol solvent, and magnetically stirred for 24 hours to obtain a 7wt% silk fibroin spinning solution. Then, the silk fibroin nanofiber membrane was prepared by electrospinning technology. The relevant parameters of electrospinning were: the voltage was 16kV, the spinning solution advancing speed was 1mL/h, the distance between the spinning needle and the receiver was 12cm, the relative temperature and The relative humidity was 25°C and 40%, respectively. Subsequently, the obtained electrospun fibroin nanofiber membrane was vacuum-dried at 55° C., so that the residual solvent was fully volatilized.

Those skilled in the art can also choose collagen, cellulose acetate, chitosan and its derivatives, chitosan oligosaccharide, polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, polystyrene, poly One or more of methyl methacrylate, polyvinyl alcohol, methacrylated polyethyleneimine, polyacrylonitrile, polyurethane, and polyisobutylene are used as raw materials for micro/nano fiber membranes, and are selected according to the selected raw materials Appropriate solvents and preparation methods are used for preparation.

Available solvents include hexafluoroisopropanol, chloroform, dichloromethane, trifluoroacetic acid, tetrahydrofuran, benzene, toluene, phenetole, chlorobenzene, N,N dimethylformamide, N,N-dimethylformamide One or more of acetamide, ethyl formate, ethyl acetate, acetic acid, phosphoric acid, methanol, formic acid, amyl alcohol, and water are used as solvents, and the spinning solution with a concentration of 3-30wt% is prepared.

The available preparation methods include electrostatic spinning method, wet spinning method, dry spinning method, centrifugal spinning method, phase separation method and composite spinning method.

It should be pointed out that the selection of these raw materials, solvents, and preparation methods by those skilled in the art in combination with common knowledge in the art does not affect the modification method of the micro/nano fiber membrane and the final product obtained by the present invention. The modification of the micro/nano fiber membranes obtained by these raw materials, solvents and preparation methods by the above methods all belong to the protection scope of the present invention.

In addition, when the selected micro/nano fiber membrane itself has no charge or less charge (the absolute value of zeta potential is less than 10mV), it can be processed by sol-gel technology, liquid deposition, vapor deposition, wet chemical method, plasma Treatment, graft copolymerization, and in-situ polymerization can enhance the surface charge.

Take polycaprolactone as an example:

Polycaprolactone was dissolved in hexafluoroisopropanol, and magnetically stirred for 24 hours to obtain a 10 wt% polycaprolactone spinning solution. Then, the polycaprolactone nanofiber membrane was prepared by electrospinning technology, and the relevant parameters of electrospinning were: the voltage was 15kV, the spinning solution propulsion speed was 2mL/h, and the distance between the spinning needle and the receiver was 10cm. The temperature and relative humidity were 25°C and 40%, respectively. The resulting electrospun polycaprolactone nanofiber membrane was then vacuum-dried at 55 °C to allow the residual solvent to fully evaporate. Then using wet chemical method, soak polycaprolactone in 50% ethanol solution for 2 hours, then place it in 0.15mol/L sodium chloride solution of 1.0mg/mL polyvinylamine, after 10 minutes Take it out and rinse it three times with deionized water to obtain a positively charged polycaprolactone nanofiber membrane.

Take cellulose acetate as an example:

Acetone and N,N-dimethylacetamide were mixed at a ratio of 2:1 (w/w), and cellulose acetate was dissolved in the mixed solution to obtain a 16 wt% cellulose acetate spinning solution. Then, the cellulose acetate nanofiber membrane was prepared by electrospinning technology, and the parameters related to electrospinning were: the voltage was 16kV, the spinning solution propulsion speed was 1mL/h, the distance between the spinning needle and the receiver was 15cm, and the relative temperature and relative humidity were 25°C and 45%, respectively. The resulting electrospun polycaprolactone nanofiber membrane was then vacuum-dried at 55 °C to allow the residual solvent to fully evaporate. Then soak the obtained cellulose acetate nanofiber membrane in 0.05 mol/L sodium hydroxide solution for 7 days, take it out and rinse it with deionized water to obtain a negatively charged cellulose nanofiber membrane.

Take silk fibroin as an example:

Mix phosphoric acid and formic acid in different proportions, dissolve silk fibroin in the mixed solution, filter and defoam the mixed solution to obtain a silk fibroin spinning solution with a concentration of 15%; use formic acid as the coagulation Liquid, using wet spinning technology to prepare micro/nanofibrous membranes. The parameters related to wet spinning are: the advancing speed of spinning solution is 30mL/h, and the relative temperature and relative humidity are 25°C and 40%, respectively. The resulting wet-spun fibroin micro/nanofiber membrane was then soaked in methanol for 24 h to completely solidify and crystallize and remove residual formic acid. Then the silk fibroin micro/nanofibers were immersed in 60°C distilled water for 15 minutes, followed by 5 times stretching. Finally, tension drying is carried out to prevent shrinkage during drying.

2. Layer-by-layer self-assembly modification of the obtained micro/nanofibrous membrane

Example 1

(1) obtain the silk fibroin micro/nanofibrous film that zeta potential is less than-10mV;

(2) Add chitosan powder into 0.5wt% acetic acid solution, stir magnetically for 3-5 hours until the solution is clear and transparent, fully dissolve chitosan, and prepare a chitosan solution with a concentration of 1 mg/mL. Soak the silk fibroin micro/nanofiber membrane in the obtained chitosan solution, make the membrane fully contact with the solution, take out the membrane after 20 minutes, wash it with deionized water three times to remove the unassembled chitosan on the membrane surface;

(3) The rectorite is added into deionized water, dispersed by an ultrasonic instrument, and the rectorite suspension with a concentration of 1mg/mL is prepared, and the silk fibroin nanofiber membrane assembled with chitosan is immersed in the obtained pump Put the membrane in the suspension of the stalactite, make the membrane fully contact with the solution, take out the membrane after 20 minutes, wash it with deionized water three times to remove the unassembled rectorite on the surface of the membrane, and a double layer has been successfully assembled so far;

(4) Repeat step (2) and step (3) several times in sequence, and dry the obtained micro/nanofiber membrane to obtain composite micro/nanofiber membranes with double layers of 5, 10, 15, and 15.5, respectively.

The morphology of the resulting silk fibroin nanofiber membrane and the modified nanofiber membrane is shown in Figure 1, the left side of Figure 1 is a scanning electron microscope image of the surface of the silk fibroin nanofiber membrane, and the right side of Figure 1 is the rectorite with a double layer number of 15.5 /Chitosan layer-by-layer self-assembled modified nanofiber membrane surface scanning electron microscope image. It can be seen that the microscopic morphology of the modified nanofibers has changed significantly, and the surface has become rough. Moreover, the average diameter of the modified fibers increased from 594±112 nm to 886±143 nm (Fig. 2). At the same time, the mechanical properties of the modified nanofibrous membrane were enhanced to 5.64Mpa (Figure 4), and the water contact angle was reduced to 60.3° (Figure 3), and the water contact angle would be 0 within 6.4s after contacting the membrane, showing a hydrophilic sex increased. Compared with the modified silk fibroin nanofiber membrane, the modified nanofiber membrane has increased biocompatibility and is more suitable for the adhesion, growth and migration of osteoblasts (Fig. 5).

Example 2

(1) Obtain a polycaprolactone micro/nanofiber membrane with a zeta potential greater than +10mV;

(2) Montmorillonite is added to deionized water, dispersed by an ultrasonic instrument, and a suspension of montmorillonite with a concentration of 1 mg/mL is prepared, and the positively charged polycaprolactone nanofiber membrane is immersed in the obtained montmorillonite In the soil suspension, the membrane was fully contacted with the solution, and the membrane was taken out after 20 minutes, and washed three times with deionized water to remove the unassembled montmorillonite on the membrane surface;

(3) Add chitosan powder into 0.5wt% acetic acid solution, stir magnetically for 3-5 hours until the solution is clear and transparent, fully dissolve chitosan, and prepare a chitosan solution with a concentration of 1 mg/mL. Soak the polycaprolactone nanofiber membrane assembled with rectorite in the obtained chitosan solution, make the membrane fully contact with the solution, take out the membrane after 20 minutes, and wash it three times with deionized water to remove the unassembled surface of the membrane. Chitosan, so far assembled a double layer;

(4) Step (2) and step (3) are repeated several times in sequence, and the obtained micro/nanofiber membrane is dried to obtain a composite nanofiber membrane with the required number of layers.

Example 3

(1) Obtain a cellulose acetate micro/nano fiber membrane with zeta potential less than -10mV;

(2) Add chitosan powder into 0.5wt% acetic acid solution, stir magnetically for 3-5 hours until the solution is clear and transparent, fully dissolve chitosan, and prepare a chitosan solution with a concentration of 10 mg/mL. Soak the cellulose nanofiber membrane in the obtained chitosan solution, make the membrane fully contact with the solution, take out the membrane after 20 minutes, and wash it three times with deionized water to remove the unassembled chitosan on the membrane surface;

(3) The rectorite is added to deionized water, dispersed by an ultrasonic instrument, and a rectorite suspension with a concentration of 10 mg/mL is prepared, and the cellulose nanofiber membrane assembled with chitosan is immersed in the obtained rectorite In the rock suspension, make the film fully contact with the solution, take out the film after 20 minutes, wash it with deionized water three times to remove the unassembled rectorite on the film surface, and a double layer has been successfully assembled so far;

(4) Step (2) and step (3) are repeated several times in sequence, and the obtained micro/nanofiber membrane is dried to obtain a composite nanofiber membrane with the required number of layers.

Example 4

(1) obtain the silk fibroin micro/nanofibrous film that zeta potential is less than-10mV;

(2) Add chitosan powder into 0.5wt% acetic acid solution, stir magnetically for 3-5 hours until the solution is clear and transparent, fully dissolve chitosan, and prepare a chitosan solution with a concentration of 1 mg/mL. Soak the silk fibroin micro/nanofiber membrane in the obtained chitosan solution, make the membrane fully contact with the solution, take out the membrane after 20 minutes, wash it with deionized water three times to remove the unassembled chitosan on the membrane surface;

(3) Montmorillonite is added to deionized water, dispersed by an ultrasonic instrument, and a suspension of montmorillonite with a concentration of 1mg/mL is prepared, and the silk fibroin nanofiber membrane assembled with chitosan is immersed in the obtained montmorillonite suspension. In the desoil suspension, make the membrane fully contact with the solution, take out the membrane after 20 minutes, wash it with deionized water three times to remove the unassembled montmorillonite on the surface of the membrane, and a double layer has been successfully assembled so far;

(4) Step (2) and step (3) are repeated several times in sequence, and the obtained micro/nanofiber membrane is dried to obtain a composite nanofiber membrane with the required number of layers.

Claims (10)

1. A method for modifying micro/nano fiber membranes with layered silicate/chitosan layer by layer self-assembly, is characterized in that: with charged micro/nano fiber membranes as substrate, when the charge of substrate is When the timing is positive, assemble layered silicate and chitosan on the substrate in sequence until the required number of double layers is reached; when the charge on the substrate is negative, assemble chitosan and layered silicate on the substrate in sequence, Until the desired number of double layers is reached, the assembled micro/nanofibrous membrane is dried to obtain a modified micro/nanofibrous membrane. 2. The method according to claim 1, characterized in that: the absolute value of the zeta potential of the substrate is not less than 10 mV. 3. The method according to claim 1, characterized in that: the required number of double layers is 1-50 layers. 4. The method according to claim 1, characterized in that: the micro/nano fiber membrane substrate is made of silk fibroin, collagen, cellulose acetate, chitosan and derivatives thereof, poly-L-lactic acid, poly It is prepared by one or more of caprolactone and polylactic acid-glycolic acid copolymer. 5. The method according to claim 1 or 4, characterized in that: the micro/nano fiber membrane substrate is formed by electrospinning, wet spinning, dry spinning, centrifugal spinning, phase spinning, etc. It can be prepared by any one of separation method or composite spinning method. 6. method according to claim 1, is characterized in that: described chitosan is assembled according to following method: configuration chitosan acetic acid solution, substrate is soaked in the positively charged chitosan acetic acid solution, certain After a period of time, the membrane is taken out, and the unassembled chitosan on the surface of the membrane is removed by cleaning with a cleaning solution. 7. The method according to claim 1, characterized in that: the phyllosilicate is assembled according to the following method: configure the phyllosilicate suspension, and soak the substrate in negatively charged phyllosilicate In the salt suspension, after a certain period of time, the membrane is taken out and washed with a cleaning solution to remove the unassembled phyllosilicate on the surface of the membrane. 8. The method according to claim 1, characterized in that: the layered silicate is montmorillonite or rectorite. 9. The method according to claim 6 or 7, characterized in that: the cleaning solution is deionized water or 0.01-1mol/L sodium chloride solution. 10. A micro/nano fiber membrane self-assembled and modified layer by layer silicate/chitosan, characterized in that it is prepared by the method according to any one of claims 1-9.