CN113476656A - Preparation method and application of nano material of collagen and graphene oxide - Google Patents

Abstract

The invention discloses a preparation method of a nano material of collagen and graphene oxide, which is used for obtaining the nano composite material with the functions of collagen and graphene oxide, wherein the particle size of the nano material is in a nano level, the particles are uniform, the separation and the collection are easy, the dispersibility is good, and the solution stability is strong; the preparation method has no environmental pollution and no harm to the health of operators. The collagen composite graphene oxide composite material loaded acellular scaffold is applied to repairing long-distance sciatic nerve injury in a rat body, has strong biocompatibility, promotes the repair of the long-distance sciatic nerve injury, and has good application prospect in the field of biological tissue repair.

Description

Preparation method and application of nano material of collagen and graphene oxide

Technical Field

The invention relates to a preparation method and application of a nano material of collagen and graphene oxide.

Background

Emerging and rapidly developing nanotechnology is a science which intersects with many disciplines, nano biomedicine is an important branch of the science, and a nano structure has a series of unique properties and functions and plays an important role in developing life science and promoting human health. The tissue engineering scaffold prepared from the nano material can promote cell differentiation, tissue regeneration, peripheral nerve regeneration and central nerve injury repair.

Graphene is a famous functional nano material discovered in 2004, and has good adsorbability, no toxicity and a bacteriostatic function. The application is wide. Graphene oxide is a graphene derivative, so that the characteristic of super-strong adsorption performance of the graphene oxide is continued, and the defects of easy agglomeration and difficult hydrolysis of graphene are overcome due to the fact that the graphene oxide has a large number of functional groups such as hydroxyl, epoxy, carboxyl and the like, so that the graphene oxide has good biocompatibility and aqueous solution stability, and can be subjected to chemical functional modification. Collagen is a biological macromolecular substance, exists in connective tissues in a large amount, can have better biocompatibility and biodegradability after being properly treated, is a good biological material, and is widely applied to the field of biological medicines. The collagen is obtained from Jinshi biological company by enzymolysis of tissues and organs in animal body. The graphene oxide nano material has super strong adsorption capacity, and can be modified into a nano material with better performance.

Disclosure of Invention

The invention aims to provide a preparation method of a collagen and graphene oxide composite nano material, so as to obtain a nano composite material with both collagen and graphene oxide functions.

The technical purpose of the invention is realized by the following technical scheme: a preparation method of a nano material of collagen and graphene oxide comprises a preparation step of a carboxylated graphene oxide solution and a preparation step of a nano material of collagen and carboxylated graphene oxide; the preparation method of the carboxylated graphene oxide solution comprises the following steps: firstly, weighing 50mg of carboxylated graphene oxide, dissolving the carboxylated graphene oxide in a beaker, ultrasonically dispersing for 1h, transferring the dispersion liquid into a 50mL volumetric flask, washing the flask with deionized water for multiple times, and fixing the volume to obtain 1mg/mL of carboxylated graphene oxide solution; the preparation method of the collagen composite carboxylated graphene oxide nano material comprises the following steps: placing collagen in a beaker, placing a carboxylated graphene oxide solution in the beaker, performing magnetic stirring, fully stirring, then performing centrifugal separation on the mixture by using a centrifugal machine, collecting precipitates in a centrifugal tube, washing with deionized water, then placing the obtained product in an evaporation dish, and finally performing vacuum drying at the temperature of 25 ℃ for about 4 hours to obtain the collagen composite carboxylated graphene oxide nano material.

By adopting the technical scheme, (1) the scanning range is from 4500cm through infrared spectrogram analysis of the nano material of the collagen compounded carboxylated graphene oxide^-1To 500cm^-1，3400cm^-1The treatment band is collagen amide A band, 2994cm^-1The band is collagen amide B band and 1619cm^-1Is collagen protein amide I band, 1492cm^-1At 1365cm in the collagen II band^-1The site should be the amide III band of collagen, wherein the amide I band is related to the secondary structure, so 1619cm^-1The peak is actually the characteristic peak of collagen, 3400cm^-1The peak of the spectrum should belong to the peak generated by the stretching vibration of O-H, 1714cm^-1The peak at (A) is a peak generated by C = O stretching resonance, 1619cm^-1Should be an O — H bond or a C = C bond. Collagen has therefore been successfully complexed with carboxylated graphene oxide. (2) The scanning electron microscope image analysis of the nano material of the collagen and the carboxylated graphene oxide under different magnification respectively shows that the surfaces of the collagen and the carboxylated graphene oxide composite material are very smooth and have no granular substances. It was thus demonstrated that collagen and carboxylated graphene oxide have successfully been complexed together to form a thin film. (3) By analyzing the energy spectrum diagram of the nano material of collagen compounded with the carboxylated graphene oxide, the final product contains elements such as C, O, Fe, Na, Mg, Al, Ca, K, S, Si, Cl, Au and the like. Wherein the existing collagen is containedThe elements also comprise elements contained in the carboxylated graphene oxide, so that the collagen and the carboxylated graphene oxide can be successfully compounded.

The invention aims to provide application of a collagen and graphene oxide composite material loaded acellular scaffold in repairing rat sciatic nerve loss.

The technical purpose of the invention is realized by the following technical scheme: an application of a nano material loaded acellular scaffold of collagen composite graphene oxide in rat sciatic nerve loss repair comprises the following steps: step 1: ultrasonically dissolving 2mg of collagen composite carboxylated graphene oxide nano material in 100mL of distilled water to prepare 20 mu g/mL of collagen loaded graphene oxide solution, and placing the solution in a shaking table for 40 revolutions per minute; placing the acellular sciatic nerve scaffold obtained by a chemical washing method into a collagen-loaded graphene oxide solution, mixing at 25 ℃ for 24 hours to obtain a collagen-compounded carboxylated graphene oxide-loaded acellular scaffold; step 2: the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide is utilized to carry out surgical bridging on sciatic nerve injury of a rat with a long distance of 10mm, and the nerve repair condition of the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide in a living body is observed after 12 weeks of postoperative observation.

By adopting the scheme, the composite material of the collagen composite graphene oxide is in a nano level, the particles are uniform, and the stability of the collagen loaded graphene oxide solution is strong. Meanwhile, compared with an electron microscope image of a nano material loaded acellular scaffold of preoperative collagen and carboxylated graphene oxide and an electron microscope image of a nano material loaded acellular scaffold of postoperative collagen and carboxylated graphene oxide, the material is transplanted into a rat body through an operation to repair sciatic nerve defects of a long distance of 10mm, the skin of the rat is well healed after the operation, the appetite and the activity are normal, the material is good in histocompatibility, and meanwhile, after 12 weeks of operation, a plurality of nerve axons can be regenerated through scanning electron microscope detection, blood vessels and Schwann cells can be regenerated, and nerve repair is promoted.

In conclusion, the invention has the following beneficial effects: (1) the method is simple to operate and low in cost, and the prepared nano material of the collagen composite carboxylated graphene oxide nano has the advantages of nano-grade particle size, uniform particles, easiness in separation and collection, good dispersibility and strong solution stability; meanwhile, the preparation method does not cause environmental pollution and is harmless to the health of operators; (2) the nano material of the collagen composite carboxylated graphene oxide nano should be in a living body, has strong biocompatibility, shows the promotion of sciatic nerve repair of long-distance injury, and has good application prospect in the field of biological tissue repair.

Drawings

Fig. 1 is an infrared spectrum of a nano material of collagen-compounded carboxylated graphene oxide;

FIG. 2 is a scanning electron microscope image of the nano material of collagen composite carboxylated graphene oxide under different magnifications respectively;

fig. 3 is an energy spectrum of a nano material of collagen-compounded carboxylated graphene oxide;

fig. 4 is an electron microscope image of a nanomaterial-loaded decellularized scaffold of preoperative collagen composite carboxylated graphene oxide;

fig. 5 is an electron microscope image of a nanomaterial-loaded decellularized scaffold of post-operative collagen-complexed carboxylated graphene oxide.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

Example 1: a preparation method of a nano material of collagen and graphene oxide comprises a preparation step of a carboxylated graphene oxide solution and a preparation step of a nano material of collagen and carboxylated graphene oxide.

The preparation method of the carboxylated graphene oxide solution comprises the following steps: firstly, 50mg of carboxylated graphene oxide is weighed and dissolved in a beaker, ultrasonic dispersion is carried out for 1 hour, then the dispersion liquid is transferred to a 50mL volumetric flask, and deionized water is used for washing for multiple times to realize constant volume, so that 1mg/mL of carboxylated graphene oxide solution is obtained. The preparation method of the collagen composite carboxylated graphene oxide nano material comprises the following steps: placing collagen in a beaker, placing a carboxylated graphene oxide solution in the beaker, performing magnetic stirring, fully stirring, then performing centrifugal separation on the mixture by using a centrifugal machine, collecting precipitates in a centrifugal tube, washing with deionized water, then placing the obtained product in an evaporation dish, and finally performing vacuum drying at the temperature of 25 ℃ for about 4 hours to obtain the collagen composite carboxylated graphene oxide nano material.

FIG. 1 is an infrared spectrum of a nano material of collagen and carboxylated graphene oxide, with a scanning range of 4500cm^-1To 500cm^-1，3400cm^-1The treatment band is collagen amide A band, 2994cm^-1The band is collagen amide B band and 1619cm^-1Is collagen protein amide I band, 1492cm^-1At 1365cm in the collagen II band^-1The site should be the amide III band of collagen, wherein the amide I band is related to the secondary structure, so 1619cm^-1The peak is actually the characteristic peak of collagen, 3400cm^-1The peak of the spectrum should belong to the peak generated by the stretching vibration of O-H, 1714cm^-1The peak at (A) is a peak generated by C = O stretching resonance, 1619cm^-1Should be an O — H bond or a C = C bond. Collagen has therefore been successfully complexed with carboxylated graphene oxide.

Fig. 2 is a scanning electron microscope image of the nano material of collagen and carboxylated graphene oxide at different magnifications, and observation shows that the surfaces of the collagen and carboxylated graphene oxide composite material are very smooth and have no granular substances. It was thus demonstrated that collagen and carboxylated graphene oxide have successfully been complexed together to form a thin film.

Fig. 3 is an energy spectrum of the collagen-carboxylated graphene oxide composite nanomaterial, and the final product contains elements such as C, O, Fe, Na, Mg, Al, Ca, K, S, Si, Cl, and Au by analysis. The collagen and the carboxylated graphene oxide are successfully compounded.

Example 2: an application of a nano material loaded acellular scaffold of collagen composite graphene oxide in rat sciatic nerve loss repair comprises the following steps: step 1: ultrasonically dissolving 2mg of collagen composite carboxylated graphene oxide nano material in 100mL of distilled water to prepare 20 mu g/mL of collagen loaded graphene oxide solution, and placing the solution in a shaking table for 40 revolutions per minute; placing the acellular sciatic nerve scaffold obtained by a chemical washing method into a collagen-loaded graphene oxide solution, mixing at 25 ℃ for 24 hours to obtain a collagen-compounded carboxylated graphene oxide-loaded acellular scaffold; step 2: the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide is utilized to carry out surgical bridging on sciatic nerve injury of a rat with a long distance of 10mm, and the nerve repair condition of the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide in a living body is observed after 12 weeks of postoperative observation.

The implementation effect is as follows: the composite material of the collagen and the graphene oxide is in a nano level, the particles are uniform, and the stability of the collagen loaded graphene oxide solution is strong. Meanwhile, comparing fig. 4 (electron microscope image of the nano material loaded acellular scaffold of preoperative collagen and carboxylated graphene oxide) and fig. 5 (electron microscope image of the nano material loaded acellular scaffold of postoperative collagen and carboxylated graphene oxide), the collagen and carboxylated graphene oxide composite material is transplanted into a rat body through an operation, sciatic nerve defects of a long distance of 10mm are repaired, after the operation, the skin of the rat is well healed, appetite and activities are normal, the tissue compatibility of the material is good, meanwhile, after 12 weeks, after the operation, a plurality of nerve axons can be regenerated through scanning electron microscope detection, blood vessels and Schwann cells are regenerated, and nerve repair is promoted.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims (2)

1. A preparation method of a nano material of collagen composite graphene oxide is characterized by comprising the following steps: the preparation method comprises the steps of preparing a carboxylated graphene oxide solution and preparing a nano material of collagen and carboxylated graphene oxide;

the preparation method of the carboxylated graphene oxide solution comprises the following steps: firstly, weighing 50mg of carboxylated graphene oxide, dissolving the carboxylated graphene oxide in a beaker, ultrasonically dispersing for 1h, transferring the dispersion liquid into a 50mL volumetric flask, washing the flask with deionized water for multiple times, and fixing the volume to obtain 1mg/mL of carboxylated graphene oxide solution;

the preparation method of the collagen composite carboxylated graphene oxide nano material comprises the following steps: placing collagen in a beaker, placing a carboxylated graphene oxide solution in the beaker, performing magnetic stirring, fully stirring, then performing centrifugal separation on the mixture by using a centrifugal machine, collecting precipitates in a centrifugal tube, washing with deionized water, then placing the obtained product in an evaporation dish, and finally performing vacuum drying at the temperature of 25 ℃ for about 4 hours to obtain the collagen composite carboxylated graphene oxide nano material.

2. The application of the collagen composite graphene oxide nanomaterial-loaded decellularized scaffold according to claim 1 in rat sciatic nerve loss repair is characterized in that: the method comprises the following steps:

step 1: ultrasonically dissolving 2mg of collagen composite carboxylated graphene oxide nano material in 100mL of distilled water to prepare 20 mu g/mL of collagen loaded graphene oxide solution, and placing the solution in a shaking table for 40 revolutions per minute; placing the acellular sciatic nerve scaffold obtained by a chemical washing method into a collagen-loaded graphene oxide solution, mixing at 25 ℃ for 24 hours to obtain a collagen-compounded carboxylated graphene oxide-loaded acellular scaffold;

step 2: the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide is utilized to carry out surgical bridging on sciatic nerve injury of a rat with a long distance of 10mm, and the nerve repair condition of the nano material loaded acellular scaffold of the collagen and the carboxylated graphene oxide in a living body is observed after 12 weeks of postoperative observation.

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