WO2023108469A1

WO2023108469A1 - Tubular nanofiber material and preparation method therefor

Info

Publication number: WO2023108469A1
Application number: PCT/CN2021/138267
Authority: WO
Inventors: 潘浩波; 吴桐
Original assignee: 深圳先进技术研究院
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2023-06-22

Abstract

Provide in the present invention is a preparation method for a tubular nanofiber material, comprising the following steps: 1) subjecting a water-soluble substance to electrostatic spinning by using an electrostatic spinning device, and collecting a tubular material 1 by using a collecting device, the collecting device comprising a cylindrical metal rod; 2) subjecting a spinning solution to electrostatic spinning by using the electrostatic spinning device, and collecting a tubular material 2 by using the collecting device in which the tubular material 1 is collected; 3) dissolving the tubular material 1 with an organic solvent, and performing collecting to obtain the tubular material 2; and 4) treating the tubular material with a crystalline material to obtain a tubular nanofiber material. The present invention provides a solution for simulating the micro-environment morphology of a vascular extracellular matrix and promoting an in-vivo induced endothelium formation by using a fiber material having a shish-kebab structure. Compared with a traditional chemical small-molecule modification mode, the physical crystallization mode used in the present patent has the advantages of the operation being simple and convenient, the structure being stable, and failure caused by the influence of an external environment being not prone to occur.

Description

A kind of tubular nanofiber material and preparation method thereof

technical field

The invention relates to the field of biomedicine, in particular to a tubular nanofiber material and a preparation method thereof.

Background technique

In situ induction of angiogenesis in vivo is by constructing a functional material that mimics the microenvironment of the natural extracellular matrix structurally and functionally. This scaffold can induce cell-directed differentiation in vivo. With the gradual degradation of the scaffold, New tissue grows back in the damaged area. Specifically, in situ induction of angiogenesis is to emphasize the activity and functional modification of scaffold materials, induce angiogenesis, promote rapid tissue repair and regeneration, and complete tissue construction in vivo. The difference from traditional tissue engineering research methods is that there is no cell inoculation and in vitro culture, relying on materials to mobilize the body's self-healing ability, and guide or induce the regeneration of damaged tissues. For small-diameter artificial blood vessels, the structural design of scaffold materials mainly considers the microscopic morphology, mechanical strength and biodegradability of the material.

The thickness of electrospun fibers is similar to that of collagen fibers. Recently, researchers have discovered a new polymer fiber hybrid string crystal structure. This periodically distributed string crystal structure is very similar to the microstructure of collagen fibers in the human body. , in its staggered microstructure, contains gaps formed between consecutive collagen molecular ends, and forms gaps and overlapping regions with periodic distribution. The string crystal microstructure can significantly improve cell compatibility, which is beneficial to promote the regeneration of cells and tissues. In addition to the shape of individual fibers, the arrangement of fibers also has an impact on cell behavior. There are many specific tissues with highly ordered microstructures in the human body, such as nerves, tendons, ligaments, skeletal muscles, and blood vessels. The ordered arrangement of cells and extracellular matrix within these tissues endows them with specific functions. This unique histological structure plays a pivotal role in the functioning of the tissue. The ordered arrangement of the multi-level (fibrous) components of natural blood vessels is an essential feature of their structural specificity.

Contents of the invention

Therefore, the technical problem to be solved by the present invention is to overcome the current lack of small-diameter artificial blood vessel materials that can meet the requirements of transplantation in clinical practice. While satisfying good mechanics and cell compatibility, the design and construction of small-caliber artificial blood vessel materials is also The endothelializing ability of the material needs to be considered synergistically. The invention provides a method for preparing a tubular nanofiber material (artificial blood vessel material) with simple technology, low cost, high biological safety, and large-scale industrial production. This method constructs a small-caliber artificial vascular material that can promote endothelialization through the controllable preparation of the micromorphology of the string crystal fiber material, which provides a new idea for the research on the endothelialization strategy and anticoagulant properties of artificial vascular materials.

The invention provides a method for preparing a tubular nanofiber material, comprising the steps of:

1) The electrospinning device uses a water-soluble substance to perform electrospinning, and uses a collection device to collect the tubular material 1; the collection device includes a cylindrical metal rod;

2) The electrospinning device uses a spinning solution to perform electrospinning, and collects the tubular material 2 by using a collection device that collects the tubular material 1;

3) dissolving the tubular material 1 with an organic solvent, and collecting the tubular material 2;

4) Treating the tubular material 2 with a crystalline material to obtain a tubular nanofibrous material.

Preferably, the crystalline material is at least one of the following: polycaprolactone, polylactic acid, polylactide-caprolactone copolymer and polyethylene glycol-caprolactone copolymer.

Preferably, the water-soluble substance is polyethylene oxide, polyvinyl alcohol or polyethylene glycol or carboxymethyl cellulose;

The spinning solution is polycaprolactone solution or polylactic acid solution.

Preferably, the solvent of the spinning solution is a mixture of chloroform and N,N-dimethylformamide; Preferably, the volume ratio of chloroform to N,N-dimethylformamide (5-7): 3.5; More preferably, the volume ratio of chloroform to N,N-dimethylformamide is 6.5:3.5;

The solvent of the eluting solution is deionized water;

The mass fraction of polycaprolactone in the spinning solution is 5%-15%;

The mass fraction of the solute in the crystallization solution is 0.1%-2%;

The organic solvent is an alcohol solution; the volume percentage of the alcohol solution is 0-50%.

Preferably, the outer diameter of the cylindrical metal rod is 1-4 mm.

Preferably, the collecting device further includes two conductive plates, the length direction of which is parallel to the axial direction of the metal round rod; the two conductive plates are respectively located on both sides of the metal round rod; the motor is connected to one end of the metal round rod.

Preferably, in step 1):

The needle model is No. 20, the vertical distance between the needle and the cylindrical metal rod below is 12cm, the solution flow rate is 0.5ml/h, the voltage is 13 kV, and the spinning time is 2 hours;

In step 2): the needle type is No. 18, the vertical distance between the needle and the cylindrical metal rod below is 15 cm, the solution flow rate is 1 ml/h, the voltage is 18 kV, and the spinning time is 3 hours.

The tubular nanofiber material prepared by the above method.

Preferably, the inner diameter of the tubular nanofibrous material is 1.5mm-4mm; preferably 2-3mm.

An artificial organ or tissue prepared by using the above-mentioned tubular nanofibrous material; the tissue is a blood vessel.

The molecular weight of polycaprolactone is 50,000 to 150,000;

The molecular weight of polyethylene oxide is 600,000 to 1,000,000;

The present invention has the following advantages:

(1) The present invention proposes the scheme of using the fiber material with a string crystal structure to imitate the microenvironmental morphology of the extracellular matrix of blood vessels and promote the induction of endothelial formation in vivo. Compared with the traditional chemical small molecule modification method, the physical crystallization method used in this patent is easy to operate and stable in structure, and is not easily affected by the external environment.

(2) The fiber material with skew crystal structure imitates the microstructure morphology of extracellular matrix to a certain extent, and its cytocompatibility has been significantly improved compared with ordinary smooth fibers.

(3) The tubular nanofibrous material was used in rabbit carotid artery transplantation to verify its role in inducing vascular endothelial regeneration in vivo, which provided a new direction for the construction of small-caliber artificial vascular materials.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is a schematic diagram of a method for preparing a tubular nanofiber material according to an embodiment of the present invention;

1 is an electrospinning device, 2 is a fiber filament, 3 is a motor, 4 is a conductive plate, 5 is a metal round rod; 6 is a bearing;

Fig. 2 is an outline view of a tubular nanofiber material according to an embodiment of the present invention.

Fig. 3 is a microscope photo of a skewered fiber structure material according to an embodiment of the present invention.

Fig. 4 is a mechanical test result of a tubular nanofiber material according to an embodiment of the present invention, 200 and 400 indicate that the wall thickness of the tubular nanofiber material is 200 and 400 μm.

Figure 5 is the results of cell live/dead staining and cytoskeleton staining in one embodiment of the present invention, PCL represents the tubular nanofiber material without crystallization treatment, and PCL-SK represents the tubular fiber material after crystallization solution treatment.

Fig. 6 is the endothelial cell (a) endocytosis to low-density lipoprotein and (b) nitric oxide release of an embodiment of the present invention; TCPS is not put into the nanofiber material, is used as a control; PCL indicates that there is no crystallization treatment The tubular nanofibrous material of PCL-SK represents the tubular fibrous material treated with the crystallization solution.

Fig. 7 is (a) schematic diagram of blood vessel transplantation, (b) color Doppler and (c) CD31 staining results of an embodiment of the present invention.

Detailed ways

Example 1

(1) Preparation of electrospinning solution: firstly, polycaprolactone (Sigma-Aldrich, product number 440744, molecular weight 80000) was dissolved in a mixture of chloroform and N,N-dimethylformamide with a volume ratio of 6.5:3.5 Among them, its mass fraction is 13%. Stir overnight to fully dissolve the polycaprolactone in the chloroform and N,N-dimethylformamide solution.

(2) Preparation of elution solution: Polyethylene oxide (Sigma-Aldrich company, product number 182028, molecular weight 600000) powder was dissolved in deionized water at room temperature to prepare a polyethylene oxide solution with a mass fraction of 5%. Stir overnight to fully hydrolyze polyethylene oxide to form a uniform and stable solution.

(3) Preparation of crystallization solution: polycaprolactone was added into amyl acetate, and the mass fraction of polycaprolactone was 1%. Heated to 63°C to fully dissolve it. And heat preservation at 60 ℃, standby.

(4) Preparation of tubular electrospun nanofiber material:

Using the experimental method shown in Figure 1 (reference number 1 in Figure 1 is an electrospinning device, and reference number 2 is a fiber filament), a tubular electrospinning fiber material is prepared by using a collection device comprising a cylindrical metal rod. The collection device also includes two conductive flat plates 4, the length direction of the conductive flat plates is parallel to the axial direction of the cylindrical metal rod 5, and the two conductive flat plates are respectively located on both sides of the cylindrical metal rod 5 for enhancing the orientation degree of spinning fibers; The motor 3 is connected with one end of the cylindrical metal rod 5; the bearing 6 is connected with the other end of the cylindrical metal rod 5, so that the cylindrical metal rod 5 rotates with the motor. The outer diameter of the cylindrical metal rod 5 used by the collecting device is 2.5 mm.

Specifically include the following steps:

(a) On the electrospinning device 1, the eluting solution is used for electrospinning to prepare fiber filaments. The needle type of the electrospinning device 1 is No. 20, and the vertical distance between the needle head and the cylindrical metal rod below is 12 cm , a solution flow rate of 0.5ml/h, a voltage of 13 kV, and a spinning time of 2 hours to obtain a tubular material 1, which was then left to stand for more than 1 hour.

(b) Use the spinning solution to carry out electrospinning, the needle model is No. 18, and the distance between the cylindrical metal rod below (the cylindrical metal rod used in this step is the cylindrical metal rod that collects the tubular material 1) 15 cm, the solution flow rate is 1 ml/h, the voltage is 18 kV, and the spinning time is 3 hours, and the tubular material 2 is prepared. After the end, remove the cylindrical metal rod together with the tubular material 1 and the tubular material 2, and let it stand at room temperature for more than 24 hours to fully volatilize the solvent.

(c) Immerse the cylindrical metal rod together with the tubular material in 0, 25% (v/v) and 50% (v/v) alcohol solutions successively, and use the magnetic rotor to rotate the cylindrical metal rod and the tubular material, each For more than 3 hours, the eluting solution can be fully hydrolyzed, thereby creating a gap between the metal rod and the tubular material, so that the tubular material 2 can be easily slipped and removed from the cylindrical metal rod.

(d) Let the tubular material 2 stand at room temperature for more than 48 hours to fully evaporate the solvent and water.

(e) Cut the tubular material 2 into a length of 1 cm, wait for the crystallization solution to cool down to 35°C, use a syringe to draw 100 μl of the crystallization solution, and evenly pour it on the inner surface of the tubular material 2 to obtain a tubular nanofiber material. The fiber material is left to stand at room temperature for more than 48 hours to fully evaporate the solvent.

Experimental results: the present invention has been verified through preliminary experiments, and the results are as follows.

Observation of shape:

The actual appearance of the prepared tubular nanofibrous material is shown in FIG. 2 . Mandrels with different diameters were used in the experiment, and the inner diameters of tubular nanofibrous materials were about 1.5, 2, 2.5, 3 and 4 mm, respectively. The surface of the entire material is smooth, the overall thickness is uniform, and the length can reach more than 5cm, which can meet the requirements of subsequent animal experiments (inner diameter 2-3mm, length 2-3cm). The wall thickness of the tubular nanofiber material is relatively uniform, there are no obvious defects and collapses, and the overall roundness is good. In addition, for human blood vessels with a diameter of 2-3 mm, due to different types of blood vessels, the thickness between 100-1000 μm is in line with the actual physiological conditions.

The field emission scanning electron microscopy and atomic force microscopy images of the string crystal fiber structure material are shown in Figure 3. In the figure, obvious polycaprolactone crystals can be seen on the fiber, and these crystals grow periodically on the fiber, and almost all wafers can be epitaxially grown on the surface of the fiber, presenting a typical string crystal structure. In addition, the fibers can be oriented and arranged, the surface of the fibers is smooth, the thickness is uniform, and the pore size can also be suitable for the growth and reproduction of cells.

Mechanical properties:

The tensile mechanical properties of the tubular nanofibrous material are tested by stretching, and the tests are carried out in the circumferential direction and the longitudinal direction respectively. The two ends of the artificial blood vessel with a length of 2.5 cm are clamped with instrument clamps. The stretching speed along the length direction (axial direction) is 5 mm/min until breaking. During the mechanical test in the circumferential direction, use two "L"-shaped metal rods to clamp on the clamps at both ends of the instrument, cut off the artificial blood vessel to a length of 0.5cm, and insert the artificial blood vessel into the two "L"-shaped metal rods from above at the same time And pre-tighten, stretch to break at a speed of 5mm/min, and test the maximum stress at break. The stress-strain curves of the test method and mechanical test results are shown in Figure 4, respectively. It can be seen from the figure that the strength of the blood vessel material is about 4MPa, which can already meet the requirements for human blood vessels. Furthermore, the material exhibits greater strength and lower stiffness in the circumferential direction compared to the length direction, which is also likely a result of the orientation of the spun fibers in their circumferential direction.

Live/dead staining of cells was carried out using cytotoxicity test reagents (Invitrogen, USA): red and green dyes were added to PBS buffer according to the ratio in the instructions. Then suck out the cell culture medium in the well plate, and then re-add the buffer solution to wash 2 times. After aspirating the buffer solution, add the stain solution, cover and wait for 45 minutes in shading. The analysis of the cytoskeleton is mainly achieved by staining the cell actin. Add 4% paraformaldehyde (PFA)/PBS solution (Beiyuntian Company) to fix the cells for 15 minutes. Then it was aspirated again and then washed 2 times with buffer and then aspirated. Subsequently, 0.1% polyethylene glycol octylphenyl ether (Triton-X 100, Biyuntian Company) was added to puncture the cell membrane for 5 minutes. Then remove the solution and wash it twice with buffer solution, and finally add staining solution and stain in light for 1 hour at room temperature. Observations were performed using a Nikon inverted fluorescence microscope.

The results of endothelial cell survival and skeleton morphology are shown in Figure 5. It can be seen that there are more cells adsorbed on the sample of string crystal structure, and it can also be seen that the cells show a more spread state, and many cells have generated pseudopodia, which indicates that the cells have The state of growth on the sample with skew crystal structure is better. The survival rate of all sample cells is also relatively ideal, and there is almost no red spot, so it can be proved that the material has good cytocompatibility and has not produced obvious cytotoxicity. The results of cytoskeleton staining showed that the cells grown on the sample with string crystal structure showed a more spread shape, and many filopodia had appeared in the cells on it. At 7 days, on the sample modified polycaprolactone string crystals, the state of the cells was very good. Especially on those specimens with larger diameters of string crystals, bundles of actin microfilaments can already be seen. In addition, the oriented arrangement of fibers can obviously induce the oriented growth of cells.

Functional expression of endothelial cells:

When the cells were cultured for a specific number of days, fluorescently labeled acetylated low-density lipoprotein (Shanghai Mokang Biology) was added to the culture medium and incubated for 4 hours. The culture solution was aspirated and washed three times, and then 4% paraformaldehyde (PFA)/PBS solution (Beiyuntian Company) was added to fix the cells for 15 minutes. Photographs were taken with a Nikon inverted fluorescence microscope and the fluorescence intensity was calculated. Nitric oxide release was measured using a nitric oxide detection kit (Biyuntian Company). When the cells were cultured for a specific number of days, 50 μl of the culture supernatant was drawn and added to a 96-well plate, and then 50 μl each of Griess Reagent (Griess Reagent) I and II in the kit were added sequentially. Absorbance was then measured at 540 nm using a microplate reader.

LDL uptake and nitric oxide release are important functional indicators of endothelial cells. The results of quantitative analysis of the fluorescence intensity of LDL showed (as shown in Figure 6) that compared with other groups, the skew crystal morphology led to a significant increase in the uptake of LDL by endothelial cells. In addition, it was observed that endothelial cells released a greater amount of nitric oxide on the surface with the large-sized kebab structure than on the other two surfaces. It shows that the string crystal structure is beneficial to the maturation of endothelial cells and the expression of endothelial function.

In Vivo Transplantation Research:

The artificial blood vessel sample used has an inner diameter of about 2.5 mm and a length of 1 cm. New Zealand rabbits were anesthetized and fixed on the operating table in a supine position, and the front of the neck was shaved and disinfected with povidone iodine. An anterior median incision was made to separate the tracheal fascia and expose the carotid arteries on both sides. After the arterial clip was placed, the arterial vessel was cut off, and the grafted vessel was anastomosed end-to-side with the carotid artery with a thin 8-0 thread, and repaired properly when there was a lot of bleeding. After the anastomosis, the blood flow only passes through the graft vessel, and the graft vessel can be seen to be full and pulsating.

New Zealand rabbit carotid artery transplantation experiments showed good patency after one month of transplantation, as shown in Figure 7, three months after transplantation, section staining showed that artificial blood vessels with string crystal materials can promote the spreading of endothelial cells to a certain extent, which may also be As a result of the acceleration of the endothelialization process.

Claims

A method for preparing a tubular nanofibrous material, comprising the steps of:

1) The electrospinning device uses a water-soluble substance to perform electrospinning, and uses a collection device to collect the tubular material 1; the collection device includes a cylindrical metal rod;

2) The electrospinning device uses a spinning solution to perform electrospinning, and collects the tubular material 2 by using a collection device that collects the tubular material 1;

3) dissolving the tubular material 1 on the collection device with an organic solvent, and collecting the tubular material 2;

4) Treating the tubular material 2 with a crystalline material to obtain a tubular nanofibrous material.
The method according to claim 1, wherein the crystalline material is at least one of the following:

Polycaprolactone, polylactic acid, polylactide-caprolactone copolymer, and polyethylene glycol-caprolactone copolymer.
The method according to claim 1 or 2, wherein the water-soluble substance is polyethylene oxide, polyvinyl alcohol or polyethylene glycol or carboxymethyl cellulose;

The spinning solution is polycaprolactone solution or polylactic acid solution.
The method according to any one of claims 1-3, wherein the solvent of the spinning solution is a mixture of chloroform and N,N-dimethylformamide; preferably, chloroform and N,N-dimethylformamide Methylformamide volume ratio (5-7): 3.5; more preferably, the volume ratio of chloroform and N,N-dimethylformamide is 6.5:3.5;

The mass fraction of polycaprolactone in the spinning solution is 5%-15%;

The mass fraction of the solute in the crystallization solution is 0.1%-2%;

The organic solvent is an alcohol solution; the volume percentage of the alcohol solution is 0-50%.
The method according to any one of claims 1-4, characterized in that, the outer diameter of the cylindrical metal rod is 1-4mm.
The method according to any one of claims 1-5, wherein the collecting device further comprises two conductive plates, the length direction of the conductive plates is parallel to the axial direction of the metal round bar; On both sides of the rod; the motor, connected to one end of the metal round rod.
According to the arbitrary described method of claim 1-6, it is characterized in that, in step 1):

The needle model is No. 20, the vertical distance between the needle and the cylindrical metal rod below is 12cm, the solution flow rate is 0.5ml/h, the voltage is 13 kV, and the spinning time is 2 hours;

In step 2): the needle type is No. 18, the vertical distance between the needle and the cylindrical metal rod below is 15 cm, the solution flow rate is 1 ml/h, the voltage is 18 kV, and the spinning time is 3 hours.
The tubular nanofibrous material prepared by the method according to any one of claims 1-7.
The tubular nanofiber material according to claim 8, characterized in that, the inner diameter of the tubular nanofiber material is 1.5mm-4mm; preferably 2-3mm.
The artificial organ or tissue prepared by using the tubular nanofibrous material according to claim 7; the tissue is a blood vessel.