CN113101007A - Preparation method of drug-loaded nanofiber nerve conduit - Google Patents
Preparation method of drug-loaded nanofiber nerve conduit Download PDFInfo
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Abstract
The invention discloses a preparation method of a drug-loaded nanofiber nerve conduit, which comprises the following steps: (1) preparing astragalus polysaccharide nanospheres by using chitosan and astragalus polysaccharide as raw materials and sodium tripolyphosphate as a cross-linking agent by using a chemical cross-linking method; (2) mixing the astragalus polysaccharide nanospheres with a gelatin solution, and then carrying out vacuum freeze drying to obtain the inner core of the nerve conduit; (3) dissolving gelatin and polylactic acid in hexafluoroisopropanol to prepare spinning solution, and preparing the fiber nerve conduit shell by using an electrostatic spinning method; (4) and filling the nerve conduit inner core into the cavity of the fiber nerve conduit shell, then adding the nerve conduit inner core into a genipin solution for crosslinking, washing, and freeze-drying to obtain the nerve conduit inner core. The nerve conduit prepared by the method has excellent biocompatibility, mechanical property and degradation property, can provide a porous channel for nerve fiber regeneration, and can slowly release astragalus polysaccharide to further accelerate the nerve repair process, thereby better treating peripheral nerve injury.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a preparation method of a drug-loaded nanofiber nerve conduit.
Background
Repair of peripheral nerve injury is a challenging medical problem, autologous nerve transplantation is the "gold standard" in peripheral nerve repair due to its non-immunogenic role, but there are also problems with limited supply of donor tissue, risk of neuroma formation, size mismatch and axonal distribution between the donor nerve and the injury site. The nerve conduit is an artificial tissue engineering tube and is used for bridging nerve endings as a substitute treatment method of autologous nerve transplantation. However, the lack of effective biocompatibility and bioactivity is a major drawback of most artificial nerve conduits.
Common preparation methods for nerve conduits include casting molding, solution impregnation, melt extrusion, membrane laying, electrostatic spinning, and the like. The catheter prepared by the electrostatic spinning method has nano-scale fibers, is high in specific surface area and porosity, can simulate the structure of natural extracellular matrix, and can keep the activity of growth factors in the processing process because the electrospinning is carried out at normal temperature and normal pressure.
The nerve conduit material can be divided into two categories of natural material and artificial synthetic material according to the source, and the two categories have advantages and disadvantages respectively. The natural material has high bionic property and inherent bioactivity, but the natural structure of the material is easy to change in the extraction and utilization processes. Synthetic materials are flexible in structure and performance and have better custom properties, but lack the biomimetic and biocompatible properties of natural materials. The natural polymer material mainly comprises protein and polysaccharide, Chitosan (CS) is a natural biological material, is the only known alkaline polysaccharide with positive charge, has good biocompatibility, biodegradability and antibacterial property, can support the adhesion and growth of Schwann cells, can inhibit the growth of fibrocytes and the formation of scars, and can promote the growth of endothelial cells. Polylactic acid (PLA) is a synthetic degradable material, which is non-toxic, strong in plasticity, good in mechanical property and biocompatibility, but slow in degradation rate, acidic in degradation product, and easy to cause inflammation of surrounding tissues, and generally needs to be compounded with other materials.
Researches show that astragalus polysaccharide can increase the expression of NGF protein, thereby promoting nerve regeneration. Angiogenesis refers to the process of formation of new capillary vessels by microvascular endothelial cells through sprouting, migration, proliferation, matrix remodeling and the like during the growth and development process of an organism or during the wound repair process. The astragalus polysaccharide can directly or indirectly activate CD34+ cells, promote angiogenesis in nerves, improve microcirculation of injured nerves, increase supply of nerve regeneration substances, and accelerate recovery of axial flow. Studies of Chentugang and the like find that the astragalus flavenoides can increase the number of peripheral blood endothelial tissue cells, promote the proliferation capacity, the migration capacity and the in vitro angiogenesis capacity of the endothelial tissue cells and increase along with the increase of astragalus polysaccharide and action time.
Disclosure of Invention
The invention aims to provide a preparation method of a drug-loaded nanofiber nerve conduit, the nerve conduit prepared by the method has excellent biocompatibility, mechanical property and degradation property, can provide a porous channel for nerve fiber regeneration, and can slowly release astragalus polysaccharide to further accelerate the nerve repair process, thereby better treating peripheral nerve injury.
In order to solve the technical problems, the invention adopts the following technical scheme:
a preparation method of a drug-loaded nanofiber nerve conduit comprises the following steps:
(1) preparing astragalus polysaccharide nanospheres by using chitosan and astragalus polysaccharide as raw materials and sodium tripolyphosphate as a cross-linking agent by using a chemical cross-linking method;
(2) mixing the astragalus polysaccharide nanospheres with a gelatin solution, and then carrying out vacuum freeze drying to obtain the inner core of the nerve conduit;
(3) dissolving gelatin and polylactic acid in hexafluoroisopropanol to prepare spinning solution, and preparing the fiber nerve conduit shell by using an electrostatic spinning method;
(4) and filling the nerve conduit inner core into the cavity of the fiber nerve conduit shell, then adding the nerve conduit inner core into 0.1-2% genipin solution for crosslinking, washing, and freeze-drying to obtain the nerve conduit inner core.
Preferably, in the step (1), the weight ratio of the chitosan to the astragalus polysaccharide is 2-5: 1, preferably 4: 1.
preferably, the diameter of the astragalus polysaccharide nanosphere is 80-100 nm.
Preferably, in the step (2), the weight ratio of the astragalus polysaccharide nanospheres to the gelatin is 0.001-0.01: 1, preferably 0.002: 1.
preferably, in the step (3), the weight ratio of the gelatin to the polylactic acid is 0.5-5: 1, preferably 1: 1.
preferably, the concentration of the genipin solution is 1.5%.
Preferably, in the step (4), the crosslinking temperature is 35-45 ℃ and the crosslinking time is 10-60 min.
Compared with the prior art, the invention has the following beneficial effects:
1. the nerve conduit provided by the invention is designed with a double structure, and comprises an inner astragalus polysaccharide nanosphere and an outer directional nanofiber mold conduit shell, wherein the inner core of the nerve conduit ensures the biocompatibility of the conduit, also prolongs the release period of the astragalus polysaccharide, and further provides a multi-channel three-dimensional structure, thereby being beneficial to promoting the growth of nerves and repairing nerve injury; and the externally oriented nanofibers can also provide the correct directional guidance for nerve regeneration. The invention realizes the synergistic effect of the medicament on promoting nerve regeneration, greatly improves the speed of nerve repair, wherein the astragalus polysaccharide can also provide a good microenvironment for the growth, proliferation and differentiation of nerve cells, and reduces the occurrence of inflammatory reaction.
2. The preparation method is simple, the operability is strong, and the prepared nerve conduit has excellent biocompatibility, mechanical property and degradation property and has good application prospect.
Drawings
FIG. 1 is a diagram of LMS of prepared astragalus polysaccharide nanospheres.
Fig. 2 is a particle size distribution diagram of the prepared astragalus polysaccharide nanospheres.
Fig. 3 is an SEM image of the prepared fibrous nerve conduit shell.
Fig. 4 is a diagram of a finished nerve conduit prepared and a cross-sectional SEM image of the conduit.
FIG. 5 shows the results of cytotoxicity tests on RSC96 cells.
FIG. 6 shows the results of cytotoxicity test of PC12 cells.
FIG. 7 is a graph showing the release profile of Astragalus polysaccharides from nerve conduits.
Fig. 8 is a graph comparing the degradation weight loss of the nerve conduit prepared by the present invention before and after genipin cross-linking.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1
A preparation method of a drug-loaded nanofiber nerve conduit comprises the following steps:
1) preparation of astragalus polysaccharide nanospheres
The astragalus polysaccharide nanospheres are prepared using chemical cross-linking methods well known in the art. Dissolving chitosan 40mg in acetic acid solution 20m 11%, dissolving Astragalus polysaccharides 10mg in water, and filtering with 0.22 μm microporous membrane respectively; dropwise adding 1mg/ml sodium tripolyphosphate solution into chitosan acetic acid solution (the dropwise adding amount is 20ml) under high-speed stirring, continuously stirring for 10min after the dropwise adding is finished, then adding the astragalus polysaccharide aqueous solution, stirring overnight at room temperature, ultracentrifuging (16000r/min, 60min), removing supernatant, washing the precipitate with 75% ethanol and distilled water for 3 times respectively, freezing the obtained precipitate at-80 ℃ for 24h, and drying the precipitate into powder by using a vacuum drier for later use.
FIG. 1 is a diagram of LMS of prepared astragalus polysaccharide nanospheres. The nanosphere has monodispersity and good shape.
Fig. 2 is a particle size distribution diagram of the prepared astragalus polysaccharide nanospheres. Only one single peak, indicating a homogeneous morphology, with an average particle size of 80-100 nm.
2) Preparation of inner core of nerve conduit
And preparing the catheter core layer by using a vacuum freeze drying method. Mixing 20mg of astragalus polysaccharide nanospheres with 10g of gelatin, dissolving with water, filling into a special mold, freezing at-20 ℃ for 5-8h in a refrigerator, freeze-drying for 12h in a vacuum drier, taking out the material, and preparing into the inner core of the nerve conduit.
3) Preparation of fibrous nerve conduit shell
And preparing the fiber nerve conduit shell layer by adopting an electrostatic spinning technology. Mixing 0.3g of gelatin and polylactic acid according to the mass ratio of 1:1, dissolving in 10ml of hexafluoroisopropanol to prepare a spinning solution, magnetically stirring for 0.5-2h, and ultrasonically degassing for 10 min; adding the spinning solution into a 10ml syringe, connecting with a 21G stainless steel nozzle, setting the distance between the nozzle and a receiving plate to be 15cm, and carrying out electrospinning under the conditions of voltage of 9-12kV, negative voltage of 3KV and positive voltage flow rate of 1.0 ml/h. The prepared sample was vacuum dried for 24 hours to discharge the residual organic solvent.
FIG. 3 is an SEM image of the prepared fibrous nerve conduit shell, wherein the fibers are arranged orderly and oriented, and the size of the fibers is nano-scale and can induce nerve oriented growth.
4) Synthesis and crosslinking of nerve conduits
Segmenting the outer shell and the inner core of the fiber nerve conduit, wherein the length of the outer shell is 17mm, the length of the inner core is 15mm, filling the inner core of the conduit into the outer shell, preheating 1.5% genipin cross-linking solution to 37 ℃, putting the prepared nerve conduit into absolute ethyl alcohol, immediately taking out, putting the nerve conduit into the cross-linking solution, cross-linking for 30min at 37 ℃, and turning over once every 10 min. Taking out, washing with anhydrous ethanol for 5min, washing with distilled water for 3 times, each for 10min, taking out, and freeze drying. The materials are subpackaged in self-sealing bags, sterilized and stored at normal temperature for later use.
FIG. 4 is a diagram of a finished nerve conduit prepared according to the present invention, which has a length of 17mm, an inner diameter of about 2mm and an outer diameter of about 2.2mm, and a SEM image of a cross section of the conduit. The right picture shows that the inner core is completely wrapped by the electrostatic spinning shell, and the inner core of the catheter has larger pores and more channels.
The maximum load of the nerve conduit before genipin cross-linking is 23.5-26.8N, and the breaking load is 14.3-17.8N. The maximum load after crosslinking is 42.9-43.4N, the breaking load is 28.7-35.4N, and the difference before and after crosslinking is obvious (P < O.05). The nerve conduit prepared by the invention has certain mechanical strength and can play a good supporting role when being used for a nerve conduit bracket.
Through detection, the air permeability of the nerve conduit before genipin cross-linking is (58.2 +/-2.6)%, the air permeability of the nerve conduit after cross-linking is (76.2 +/-3.6)%, and the air permeability after cross-linking is slightly increased. The good air permeability can enable the nerve conduit to exchange with surrounding tissue fluid, and a good microenvironment is created in the conduit.
Before the crosslinking, the water absorption rate of the nerve conduit is (658+ 46)%, and after the crosslinking of 1% genipin, the water absorption rate reaches (786+ 45)%. The increase of water absorption rate is beneficial to the exchange of nutrients by the nerve cell matrix.
Test examples
1. Cell biocompatibility test
1.1 test methods
1.1.1 culture of cells
RSC96 cell culture fluid: high glucose DMEM (89%), diabody (1%), fetal bovine serum (10%);
PC12 cell culture fluid: RPMI1640 (81.5%), diabody (1%), fetal bovine serum (2.5%), horse serum (15%);
1.1.2 subculture of cells
Cell recovery: first, the cells were removed from the liquid nitrogen tank, thawed at 37 ℃, placed in 5mL of cell culture medium on a cell platform, centrifuged, the supernatant was aspirated, 5mL of fresh culture medium was added, gently blown repeatedly, transferred to a cell culture flask, and placed in CO2Culturing in a cell culture box with saturation humidity of 5%, and changing fresh culture solution every other day.
Cell passage: observing the growth condition of the cells under an inverted microscope, adding pancreatin for digestion for 30s-l minutes when the cell bottle bottom is approximately full, transferring the cell sap in the cell culture bottle into a 15mL centrifuge tube, centrifuging, sucking the supernatant, counting by using a cell counter, diluting, and placing the cell culture bottle in a cell culture box for continuous culture.
1.1.3 cytotoxicity assays
The principle of CCK-8 is that WST-8 is reduced by dehydroreductase in cell mitochondria under the action of electronic coupling to generate an orange yellow substance, the darker the color is, the better the cell proliferation effect is, the more the number of living cells is, and conversely, the lighter the color is, the less the number of living cells is, and the higher the toxicity is.
Selecting RSC96 and PC12 cells to perform toxicity evaluation on nerve conduits, wherein the total 4 groups comprise an astragalus polysaccharide group (APS), an astragalus polysaccharide nanosphere group (APS-CS NPS), a drug-loaded nanofiber nerve conduit group (APS-CS NPS cather) and a blank Control group (Control).
The cytotoxicity test of the material is carried out according to national standard, each test material is placed on an ultra-clean workbench and is irradiated by ultraviolet light for 12 hours for sterilization, the test material is soaked in normal saline for 10 minutes, then the cells cultured to 2-3 generations are digested by pancreatin, and the concentration of the diluted cells is lxlO4mL, 100 μ L of cell suspension was added to each well, 6 duplicate wells per group, and the culture was incubated for 5 days with fresh medium changed every other day.
Respectively carrying out toxicity detection on 1 day, 3 days and 5 days, firstly observing the growth state of cells under an inverted fluorescence microscope, then adding 10 mu LCCK-8 into each hole, slightly shaking to uniformly mix the cells, culturing for 4 hours in a cell culture box, taking out a culture plate, removing a supernatant, detecting the OD value of the supernatant on a microplate reader, and drawing a cytotoxicity graph according to the OD value.
1.2 results of the experiment
Fig. 5 shows the results of the cytotoxicity test of RSC96 cells, and fig. 6 shows the results of the cytotoxicity test of PC12 cells. From the figure, the OD value of the drug-loaded nanofiber nerve conduit group is the highest, which indicates that the drug-loaded nanofiber nerve conduit group has the strongest promotion effect on the proliferation of the PC12 cells.
2. Drug release detection
2.1 test methods
Placing 15mm nerve conduit in distilled water, and placing on a constant temperature oscillator at 37 deg.C; sampling at time points of 0.5h, lh, 2h, 6h, 12h, 24h, 48h, 72h and 96h, measuring the absorbance at 278nm, calculating the content of the astragalus polysaccharide at each time point according to a standard curve, and drawing a slow release curve of the astragalus polysaccharide released from the astragalus polysaccharide.
2.2 results of the experiment
FIG. 7 is a graph showing the release profile of Astragalus polysaccharides from nerve conduits. The astragalus polysaccharide has a faster release speed in the first 2 days and tends to be stable in the 3 rd day, and the cumulative sustained release rate is 28.37%.
3. Degradation test of nerve conduits
3.1 test methods
The material is brought to pH7.4, simulating an in vivo temperature environment in the phosphate buffer solution for constant temperature degradation, inspecting the degradation performance of the nerve conduit before and after genipin crosslinking, and inspecting the indexes: the quality of the nerve conduit changes at each time point. The equal-size nerve conduit before and after cross-linking is sliced, dried and weighed, the mass is recorded as Wo, the slices are placed into a centrifuge tube, an equal amount of PBS (pH 7.4) solution is added, and the constant-temperature degradation in a simulated body fluid environment is carried out. Taking out each sample at a fixed time point, washing the surface of the material with distilled water, drying the excess water on the surface with filter paper, drying to constant weight, and weighing as W1And the calculated mass weight loss ratio is as follows: (W)0-W1)/W0×l00％。
3.2 results of the experiment
Fig. 8 is a graph comparing degradation weight loss of the nerve conduit prepared by the invention before and after genipin cross-linking, and the weight loss change phenomenon in the graph indicates that after the 4 th week degradation experiment is finished, the weight loss rate of a cross-linking group can only reach 40%, and the degradation rate of the non-cross-linked nerve conduit reaches 62%, which indicates that genipin reduces the degradation speed of the nerve conduit.
Example 2
A preparation method of a drug-loaded nanofiber nerve conduit comprises the following steps:
1) preparation of astragalus polysaccharide nanospheres
Dissolving 25mg of chitosan in 20m of 11% acetic acid solution, dissolving 10mg of astragalus polysaccharide in water, and respectively filtering with 0.22 μm microporous filter membrane; dropwise adding 1mg/ml sodium tripolyphosphate solution into chitosan acetic acid solution under high-speed stirring, continuously stirring for 10min after dropwise adding, then adding the above astragalus polysaccharide aqueous solution, stirring overnight at room temperature, ultracentrifuging, removing supernatant, washing precipitate, and freeze-drying to obtain powder for use.
2) Preparation of inner core of nerve conduit
Mixing 100mg of astragalus polysaccharide nanospheres with 10g of gelatin, dissolving with water, filling into a special mold, freeze-drying, and taking out to obtain the nerve conduit inner core.
3) Preparation of fibrous nerve conduit shell
0.3g of gelatin and polylactic acid are mixed according to the mass ratio of 0.5:1, dissolved in 10ml of hexafluoroisopropanol to prepare spinning solution, and subjected to electrospinning and vacuum drying to discharge residual organic solvent.
4) Synthesis and crosslinking of nerve conduits
Segmenting a fiber nerve conduit shell and a conduit inner core, filling the conduit inner core into the shell, preheating 0.5% genipin cross-linking liquid to 35 ℃, putting the prepared nerve conduit into absolute ethyl alcohol, immediately taking out the nerve conduit, putting the nerve conduit into the cross-linking liquid, cross-linking for 45min at 35 ℃, washing the nerve conduit with the absolute ethyl alcohol after taking out, washing the nerve conduit with distilled water, freeze-drying the nerve conduit, packaging the nerve conduit into self-sealing bags, and storing the nerve conduit at normal temperature for later use after sterilization.
Example 3
A preparation method of a drug-loaded nanofiber nerve conduit comprises the following steps:
1) preparation of astragalus polysaccharide nanospheres
Dissolving 50mg of chitosan in 20m of 11% acetic acid solution, dissolving 10mg of astragalus polysaccharide in water, and respectively filtering with 0.22 μm microporous filter membrane; dropwise adding 1mg/ml sodium tripolyphosphate solution into chitosan acetic acid solution under high-speed stirring, continuously stirring for 10min after dropwise adding, then adding the above astragalus polysaccharide aqueous solution, stirring overnight at room temperature, ultracentrifuging, removing supernatant, washing precipitate, and freeze-drying to obtain powder for use.
3) Preparation of inner core of nerve conduit
Mixing 10mg of astragalus polysaccharide nanospheres with 10g of gelatin, dissolving with water, filling into a special mold, freeze-drying, and taking out to obtain the nerve conduit inner core.
3) Preparation of fibrous nerve conduit shell
0.3g of gelatin and polylactic acid are mixed according to the mass ratio of 3:1, dissolved in 10ml of hexafluoroisopropanol to prepare spinning solution, and subjected to electrospinning and vacuum drying to discharge residual organic solvent.
4) Synthesis and crosslinking of nerve conduits
Segmenting a fiber nerve conduit shell and a conduit inner core, filling the conduit inner core into the shell, then preheating 2% genipin cross-linking liquid to 45 ℃, putting the prepared nerve conduit into absolute ethyl alcohol, immediately taking out the nerve conduit, putting the nerve conduit into the cross-linking liquid, carrying out cross-linking for 15min at 45 ℃, taking out the nerve conduit, washing the nerve conduit with the absolute ethyl alcohol, then washing the nerve conduit with distilled water, carrying out freeze drying, then subpackaging the nerve conduit in self-sealing bags, and storing the nerve conduit at normal temperature for later use after sterilization.
The cell compatibility of examples 2 and 3 was measured according to the method of test example 1, using RSC96 cells as the cells, and measuring time at 5 days after co-culture, and the OD values were measured as follows:
as can be seen from comparison of the test results, the drug-loaded nanofiber nerve conduit prepared in example 1 has higher biocompatibility with cells than those of examples 2 and 3.
Claims (7)
1. A preparation method of a drug-loaded nanofiber nerve conduit is characterized by comprising the following steps:
(1) preparing astragalus polysaccharide nanospheres by using chitosan and astragalus polysaccharide as raw materials and sodium tripolyphosphate as a cross-linking agent by using a chemical cross-linking method;
(2) mixing the astragalus polysaccharide nanospheres with a gelatin solution, and then carrying out vacuum freeze drying to obtain the inner core of the nerve conduit;
(3) dissolving gelatin and polylactic acid in hexafluoroisopropanol to prepare spinning solution, and preparing the fiber nerve conduit shell by using an electrostatic spinning method;
(4) and filling the nerve conduit inner core into the cavity of the fiber nerve conduit shell, then adding the nerve conduit inner core into 0.1-2% genipin solution for crosslinking, washing, and freeze-drying to obtain the nerve conduit inner core.
2. The method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the weight ratio of the chitosan to the astragalus polysaccharide is (2-5): 1.
3. the method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: the diameter of the astragalus polysaccharide nanosphere is 80-100 nm.
4. The method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: in the step (2), the weight ratio of the astragalus polysaccharide nanospheres to the gelatin is 0.001-0.01: 1.
5. the method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: in the step (3), the weight ratio of the gelatin to the polylactic acid is 0.5-5: 1.
6. the method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: the concentration of the genipin solution is 1.5%.
7. The method for preparing the drug-loaded nanofiber nerve conduit as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the crosslinking temperature is 35-45 ℃, and the crosslinking time is 10-60 min.
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