CN114533953A

CN114533953A - Nanoparticle composite hydrogel nerve conduit and preparation method thereof

Info

Publication number: CN114533953A
Application number: CN202210080592.7A
Authority: CN
Inventors: 高会场; 曹晓东; 王浩
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-05-27
Anticipated expiration: 2042-01-24
Also published as: CN114533953B

Abstract

The invention discloses a nanoparticle composite hydrogel nerve conduit and a preparation method thereof, 1) miRNA-29a @ ZIF-8 nanoparticles, a silk fibroin solution and tyramine modified gelatin are prepared; 2) dispersing miRNA-29a @ ZIF-8 nanoparticles in a silk fibroin solution, dissolving tyramine modified gelatin in the silk fibroin solution, mixing and stirring the two solutions uniformly, and adding horseradish peroxidase to obtain a mixed solution; injecting the mixed solution into a tubular mold, and placing at a specific temperatureAfter the thermoreversible hydrogel is formed in the refrigerator, the catheter is pushed out and soaked in hydrogen peroxide, the catheter is taken out after crosslinking for a period of time, two ends of the catheter are cut off, and the part which is not crosslinked is washed away, so that the nanoparticle composite hydrogel nerve catheter is obtained. The nerve conduit prepared by the method has excellent biocompatibility, meets the mechanical requirement of the nerve conduit, and releases miRNA-29a and Zn²⁺Can accelerate the nerve repair process, thereby better treating peripheral nerve injury.

Description

Nanoparticle composite hydrogel nerve conduit and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a nanoparticle composite hydrogel nerve conduit and a preparation method thereof.

Background

Tissue damage caused by accidents and the like is often accompanied by peripheral nerve defects due to the wide distribution of peripheral nerves in the human body. Once the peripheral nerve defect occurs, self-repair is difficult to realize, and clinically common treatment methods such as severed suture, autologous or allogeneic nerve transplantation and the like face various problems at present, such as difficulty in repairing long-distance nerve defects, insufficient donor sources, ethical limitations and the like. In recent years, research and development of artificial nerve conduits provide a new idea for repairing peripheral nerve defects. The artificial nerve conduit has biocompatibility and bioactivity, can be completely degraded in vivo, can protect damaged nerve tissues, guide and promote nerve regeneration, does not need to be taken out in a secondary operation, and greatly lightens the burden of patients.

At present, the following performance requirements are mainly required for the artificial nerve conduit: 1. suitable biodegradability; 2. good biocompatibility; 3. matched mechanical properties; 4. has tissue regeneration promoting effect. However, it is difficult for a single material to simultaneously satisfy all the above characteristics, and it is a current development trend to use multiple materials with different advantages to jointly construct a composite artificial nerve conduit.

Gelatin is a further hydrolysate of collagen, retains some collagen signal sequences such as RGD, can better support cell adhesion and growth, but is difficult to apply to nerve tissue engineering due to the defects of high degradation rate and poor mechanical property. The silk fibroin is a biomaterial approved and approved by FDA, is mainly extracted from silkworm silk, is cheap and safe, and has sufficient mechanical properties to meet the requirement of the nerve conduit on extrusion and kinking resistance. The gelatin and silk fibroin materials have the advantages of both the gelatin and silk fibroin materials, and the artificial nerve conduit which has good biocompatibility and meets the requirement of mechanical property is prepared.

In order to improve the nerve regeneration promoting capacity of the artificial nerve conduit, the artificial nerve conduit is arranged on a bracketThe corresponding active factors such as load cells, nerve growth factor, platelet-rich plasma and the like are common methods, and the bioactive substances can nourish neuronal cells and promote the growth of neurite. MicroRNA is a rich small non-coding RNA, and plays an important role in neural development and neural regeneration as an important regulatory factor in gene expression and transcription processes. In peripheral nervous system injury, miRNA-29a can regulate PTEN gene expression and promote elongation of neuron axons. The ZIF-8 nano-particles are used as a carrier of miRNA-29a and compounded with the hydrogel catheter, so that the effective delivery of genes can be realized, and the tissue repair function of the catheter can be enhanced. Zn in ZIF-8 degradation process²⁺It also has nerve regeneration promoting effect. In addition Zn²⁺And miRNA-29a can also guide macrophage to change from M1 phenotype to M2 phenotype, promote Schwann cell maturation through immune regulation, secrete nerve growth factor, and further act on nerve regeneration.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a nanoparticle composite hydrogel nerve conduit and a preparation method thereof, the nanoparticle composite hydrogel nerve conduit prepared by the method has good biocompatibility and meets the mechanical property required by peripheral nerve repair, and the released miRNA-29a @ ZIF-8 can further accelerate the nerve repair process, so that peripheral nerve injury can be better treated.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a preparation method of a nanoparticle composite hydrogel nerve conduit comprises the following steps:

1) dissolving miRNA-29a in DEPC water, mixing with ZIF-8 suspension, placing on a shaking table for incubation to obtain solution A, and centrifuging, collecting and precipitating the solution A to obtain miRNA-29a @ ZIF-8 nanoparticles; fully dissolving the twice degummed silk in a lithium bromide solution, dialyzing for a period of time by using deionized water, and centrifuging to remove impurities to obtain a silk fibroin solution; dissolving gelatin in 2-equilin ethanesulfonic acid buffer aqueous solution, adding tyramine hydrochloride for full dissolution, finally adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for reaction, dialyzing for a period of time in a deionized water environment, and freeze-drying to obtain spongy tyramine modified gelatin;

2) dispersing miRNA-29a @ ZIF-8 nanoparticles in a silk fibroin solution to obtain a solution B; dissolving tyramine modified gelatin in a silk fibroin solution to obtain a solution C; mixing and stirring the solution B and the solution C uniformly, and adding horseradish peroxidase to obtain a mixed solution; and injecting the mixed solution into a tube-mounted mold, placing the tube-mounted mold in a refrigerator at a specific temperature, after the thermo-reversible hydrogel is formed, pushing out and soaking the tube-mounted mold in hydrogen peroxide, taking out the tube-mounted mold after crosslinking for a period of time, cutting off two ends of the catheter, and washing off the part which is not crosslinked to obtain the nanoparticle composite hydrogel nerve catheter.

Furthermore, the concentration of the ZIF-8 suspension is 1-3 mg/ml, wherein the optimal concentration is 1.5 mg/ml.

Further, the weight ratio of the miRNA-29a to the ZIF-8 nanoparticles is 2-6: 100, and the optimal ratio is 4: 100.

Further, the concentration of the lithium bromide solution was 9.3M.

Further, the concentration of tyramine hydrochloride was 0.01g/ml, and the concentrations of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were 0.74mg/ml and 0.22mg/ml, respectively.

Further, the concentration of the miRNA-29a @ ZIF-8 nano-particles in the silk fibroin solution is 0.12-0.2% by mass, wherein the optimal concentration is 0.16%.

Further, the concentration of tyramine modified gelatin in the silk fibroin solution was 30 w/v%.

Further, the concentration of the horseradish peroxidase is 60-240 Unit/ml, and the optimal concentration is 60 Unit/ml; the concentration of the hydrogen peroxide is 5-20 mM, and the optimal concentration is 10 mM.

Further, in the step 2), the time for soaking the thermoreversible hydrogel in hydrogen peroxide for crosslinking is 5-20min, wherein the optimal time is 10 min.

The invention also provides a nanoparticle composite hydrogel nerve conduit prepared by the method, which is used for peripheral nerve tissue regeneration and repair.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the nanoparticle composite hydrogel nerve conduit provided by the invention is a composite hydrogel artificial nerve conduit loaded with miRNA-29a @ ZIF-8, and can well solve the problems of the existing artificial nerve conduit.

2. The gelatin material adopted by the invention has good biocompatibility, in-vivo degradation performance and cell adhesion capacity, the silk fibroin is cheap and safe, the mechanical property is good, and the mechanical requirement of the artificial nerve conduit can be met.

3. The composite hydrogel nerve conduit with the advantages of both horseradish peroxidase catalysis and hydrogen peroxide soaking crosslinking can be obtained, and the inner diameter of the nerve conduit can be conveniently and rapidly regulated and controlled by controlling the time for soaking hydrogen peroxide.

4. The ZIF-8 nanoparticles with positive surface Zeta potentials are used, negatively charged miRNA-29a can be adsorbed through electrostatic interaction and compounded with a hydrogel nerve conduit, the extension of neuron axons is promoted through the regulation and control effect of the miRNA-29a, the nerve repair process is accelerated, in addition, the miRNA-29a @ ZIF-8 can also regulate the immune response of damaged nerves, and the maturation and myelination of Schwann cells are promoted through the guide of the phenotype transformation of macrophages, so that the miRNA-29a @ ZIF-8 further acts on nerve regeneration.

Drawings

Fig. 1 is an overall topography of the nanoparticle composite hydrogel nerve conduit (i.e., artificial nerve conduit) in example 1.

FIG. 2 is a graph comparing the depth of the electrophoretic bands of miRNA-29a @ ZIF-8 and miRNA-29a in fetal bovine serum at different time points in example 3.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

1) Dissolving miRNA-29a in DEPC water at a concentration of 110 mu g/ml, and then adding a ZIF-8 suspension until the concentration of ZIF-8 is 1mg/ml, wherein the mass ratio of miRNA-29a to ZIF-8 is 2: 100. the mixture was then incubated on a shaker at 500rpm/min for 30 min. And finally, centrifuging the solution at 12000rpm for 10min, and collecting the precipitate to obtain the miRNA-29a @ ZIF-8 nano-particles.

2) Gelatin is dissolved in 50mM 2-equilin ethanesulfonic acid buffer aqueous solution at the concentration of 0.02g/mL, then 0.01g/mL tyramine hydrochloride is added, the solution is fully dissolved at the temperature of 50 ℃, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (0.37g/0.11 g) are added after the solution is cooled to the room temperature, the stirring reaction is carried out for 12 hours, and after dialysis for 4 days in a deionized water environment, the sponge gelatin modified product is obtained by freeze-drying, namely the tyramine modified gelatin.

3) Weighing 4g of twice degummed silk, fully dissolving in 20mL of 9.3M lithium bromide solution, dialyzing for three days, and centrifuging the solution twice at 11000rpm for 30min to remove impurities to obtain a silk fibroin solution with the concentration of about 6 wt%.

4) The tyramine modified gelatin is dissolved in silk fibroin solution with the mass fraction of 5% wt at the temperature of 60 ℃ in the concentration of 30 w/v%, and horseradish peroxidase is added to the solution until the concentration is 60 Unit/ml. miR-29a @ ZIF-8 is dissolved in silk fibroin solution with the same volume at the concentration of 0.12%, and ultrasonic treatment is carried out for 30 minutes to ensure that the silk fibroin solution is uniformly dispersed. Then, the two solutions were mixed, stirred, and poured into a tubular mold having a height of 14mM, an outer diameter of 5mM, and an inner diameter of 4mM, and placed in a refrigerator at 4 ℃ for 3 hours, after the thermoreversible hydrogel was formed, the mold was pushed out, and the mold was immersed in 5mM hydrogen peroxide. And (3) taking out after crosslinking for 20 minutes, cutting 1mm of both ends of the catheter, and washing off the uncrosslinked part by using hot water at 40 ℃ to obtain the artificial nerve catheter with the height of 10mm, the outer diameter of 3mm and the inner diameter of 2.5mm, wherein the macroscopic form of the artificial nerve catheter is shown in figure 1.

Example 2

1) Dissolving miRNA-29a in DEPC water at a concentration of 110 mu g/ml, and then adding a ZIF-8 suspension until the concentration of ZIF-8 is 3mg/ml, wherein the mass ratio of miRNA-29a to ZIF-8 is 6: 100. the mixture was then incubated on a shaker at 500rpm for 30 min. And finally, centrifuging the solution at 12000rpm for 10min, and collecting the precipitate to obtain the miRNA-29a @ ZIF-8 nano-particles.

2) Dissolving gelatin in a concentration of 0.02g/mL in a 50mM 2-equilin ethanesulfonic acid buffer aqueous solution, then adding 0.01g/mL tyramine hydrochloride, fully dissolving at 50 ℃, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (0.37g/0.11 g) after the solution is cooled to room temperature, stirring and reacting for 12h, dialyzing for 4 days in a deionized water environment, and freeze-drying to obtain a spongy gelatin modified product, namely the tyramine modified gelatin.

3) Weighing 4g of twice degummed silk, fully dissolving in 20mL of 9.3M lithium bromide solution, dialyzing for three days, and centrifuging the solution twice for 30min at 11000rpm to remove impurities to obtain a silk fibroin solution with the concentration of about 6 wt%.

4) The tyramine modified gelatin is dissolved in silk fibroin solution with the mass fraction of 5 wt% at the temperature of 60 ℃ at the concentration of 30 w/v%, and horseradish peroxidase is added to the solution to the concentration of 240 Unit/ml. miR-29a @ ZIF-8 is dissolved in silk fibroin solution with the same volume at the concentration of 0.16%, and ultrasonic treatment is carried out for 30 minutes to ensure that the silk fibroin solution is uniformly dispersed. Then, the two solutions were mixed and stirred uniformly, poured into a tubular mold having a height of 14mM, an outer diameter of 5mM and an inner diameter of 3mM, and placed in a refrigerator at 4 ℃ for 3 hours, after the thermoreversible hydrogel was formed, the mold was pushed out, and the hydrogel was soaked in 20mM hydrogen peroxide. And (3) taking out after crosslinking for 5 minutes, cutting off 1mm from two ends of the catheter, and washing off the uncrosslinked part by using hot water at 40 ℃ to obtain the artificial nerve catheter which is 10mm in height, 3mm in outer diameter and 2.5mm in inner diameter and is loaded with 0.08% miRNA-29a @ ZIF-8.

Example 3

1) Dissolving miRNA-29a in DEPC water at a concentration of 110 mu g/ml, and then adding a ZIF-8 suspension until the concentration of ZIF-8 is 1.5mg/ml, wherein the mass ratio of miRNA-29a to ZIF-8 is 4: 100. the mixture was then incubated on a shaker at 500rpm for 30 min. And finally, centrifuging the solution at 12000rpm for 10min, and collecting the precipitate to obtain the miRNA-29a @ ZIF-8 nano-particles. The stability of miRNA-29a @ ZIF-8 was judged by the change of its electrophoretic band with the incubation time in serum, as shown in FIG. 2.

4) The tyramine modified gelatin is dissolved in silk fibroin solution with the mass fraction of 5 wt% at the temperature of 60 ℃ in the concentration of 30 w/v%, and horseradish peroxidase is added to the solution until the concentration is 60 Unit/ml. miRNA-29a @ ZIF-8 is dissolved in silk fibroin solution with the same volume at the concentration of 0.2%, and is subjected to ultrasonic treatment for 30 minutes to be uniformly dispersed. Then, the two solutions were mixed and stirred uniformly, poured into a tubular mold having a height of 14mM, an outer diameter of 5mM and an inner diameter of 4mM, and placed in a refrigerator at 4 ℃ for 3 hours, after the thermoreversible hydrogel was formed, the mold was pushed out, and immersed in 10mM hydrogen peroxide. And (3) taking out after crosslinking for 10 minutes, cutting 1mm from two ends of the catheter, and washing off the uncrosslinked part by using hot water at 40 ℃ to obtain the artificial nerve catheter which is 10mm in height, 3mm in outer diameter and 2.5mm in inner diameter and is loaded with 0.1 percent of ZIF-8.

The examples of the present invention are given for clarity of illustration only, and are not intended to limit the embodiments of the present invention. Other variants and modifications of the above-described embodiments will be obvious to those skilled in the art, and it is not necessary or necessary to exhaustively enumerate all embodiments herein. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a nanoparticle composite hydrogel nerve conduit is characterized by comprising the following steps:

2) dispersing miRNA-29a @ ZIF-8 nanoparticles in a silk fibroin solution to obtain a solution B; dissolving tyramine modified gelatin in a silk fibroin solution to obtain a solution C; uniformly mixing and stirring the solution B and the solution C, and adding horseradish peroxidase to obtain a mixed solution; and injecting the mixed solution into a tube-mounted mold, placing the tube-mounted mold in a refrigerator at a specific temperature, after the thermo-reversible hydrogel is formed, pushing out and soaking the tube-mounted mold in hydrogen peroxide, taking out the tube-mounted mold after crosslinking for a period of time, cutting off two ends of the catheter, and washing off the part which is not crosslinked to obtain the nanoparticle composite hydrogel nerve catheter.

2. The method of claim 1, wherein: in the step 1), the concentration of the ZIF-8 suspension is 1-3 mg/ml.

3. The method of claim 1, wherein: in the step 1), the weight ratio of miRNA-29a to ZIF-8 nanoparticles is 2-6: 100.

4. the method of claim 1, wherein: in step 1), the concentration of the lithium bromide solution was 9.3M.

5. The method of claim 1, wherein: in step 1), the concentration of tyramine hydrochloride was 0.01g/ml, and the concentrations of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were 0.74mg/ml, 0.22 mg/ml.

6. The method of claim 1, wherein: in the step 2), the concentration of the miRNA-29a @ ZIF-8 nano-particles in the silk fibroin solution is 0.12-0.2% by mass.

7. The method of claim 1, wherein: in step 2), the concentration of tyramine-modified gelatin in the silk fibroin solution is 30 w/v%.

8. The method of claim 1, wherein: in the step 2), the concentration of the horseradish peroxidase is 60-240 Unit/ml, and the concentration of hydrogen peroxide is 5-20 mM.

9. The method of claim 1, wherein: in the step 2), the time for soaking the thermally reversible hydrogel in hydrogen peroxide for crosslinking is 5-20 min.

10. The nanoparticle composite hydrogel nerve conduit prepared by the method of any one of claims 1 to 9, which is used for peripheral nerve tissue regeneration and repair.