CN114573839B - A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin - Google Patents

Abstract

The invention relates to a preparation method of curcumin-loaded human hair keratin/chitosan hydrogel, which belongs to the field of hydrogel technology, and comprises the following steps: human hair keratin extraction: human hair pretreatment and degreasing, and then Carry out crude extraction of human hair keratin, then purify human hair keratin, and finally concentrate human hair keratin solution to obtain human hair keratin solution; preparation of composite hydrogel: blend human hair keratin solution with chitosan The blend solution is obtained, the curcumin is added to obtain a suspension, and the suspension is freeze-dried; cross-linked to obtain a composite hydrogel. The present invention can prepare the curcumin-loaded human hair keratin/chitosan hydrogel by casting, freeze-drying and sodium tripolyphosphate cross-linking techniques, and the human hair keratin/chitosan hydrogel loaded curcumin can promote large The recovery of rat nerve crush injury is better than that of simple hydrogel, and human hair keratin, chitosan and curcumin can synergistically play a role in promoting nerve repair.

Description

A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin

technical field

The invention relates to the field of hydrogel technology, in particular to a preparation method of curcumin-loaded human hair keratin/chitosan hydrogel.

Background technique

Peripheral nerve injury (PNI) is a common clinical trauma, and the causes of trauma include cutting, stretching, compression and other special reasons. Among patients with limb trauma, about 2-3% of patients have nerve damage, among which crush injuries account for a relatively high proportion, and about 83% of patients with nerve damage under the age of 55 have brought huge losses to society. Although the peripheral nervous system has some ability to regenerate after injury compared with the central nervous system, its regeneration is about 1-3mm/day slower. This also means that injuries far away from the target organ will have a longer recovery process, and long-term denervation will cause target organ degeneration, including muscle atrophy and fibrosis, sensory impairment, motor impairment, and even loss of function leading to permanent disability, and then It has a serious impact on the patient's mental health and daily life.

Timely and targeted treatment will improve the prognosis of peripheral nerve injury. When injury involves axons, the nerve undergoes three major processes: Wallerian degeneration (clearing of the distal stump), axonal regeneration, and target organ reinnervation. Nerve anastomosis and autologous nerve transplantation are still the main treatment methods when nerve transection and nerve defect occur; while nerve release surgery is a beneficial treatment method when severe compression and chronic compression occur. Although the treatment method is effective, the nerve recovery period is still long, and the recovery effect of patients is not satisfactory. In addition to conventional surgical treatment, more abundant treatment options still need to be studied.

Drug treatment is one of the reliable means, and many drugs have been developed, such as dexamethasone, tacrolimus, vitamin B12, resveratrol, curcumin, etc. Studies have shown that these drugs have the ability to promote the repair of nerve damage. Curcumin from turmeric rhizomes has been widely studied as a drug for PNI treatment. The main mechanism of action includes promoting the proliferation and migration of SCs, reducing the apoptosis of SCs, protecting the root ganglion, inhibiting oxidative stress and resisting inflammation. These findings reveal the application prospect of curcumin in PNI. However, the characteristics of curcumin such as low bioavailability, malabsorption, and high metabolic rate make it difficult to exert a targeted and lasting effect on the damaged nerves.

Hydrogels are a common form of biomedical materials in the field of tissue engineering due to their inherent physicochemical and biological properties, including high water content, structure similar to natural ECM, porous structure, tunable biodegradability, and biocompatibility These excellent features support their use as carriers to provide localized drug delivery. Basic requirements for biomaterials include biocompatibility, biodegradability, and appropriate mechanical properties. Commonly used materials include natural polymers (keratin, collagen, gelatin, cellulose, chitosan, etc.) and synthetic polymers (polylactic acid, polyglycolic acid, polycaprolactone, etc.). Human hair keratin is a natural polymer extracted from human hair. Compared with synthetic polymers, human hair keratin has excellent biocompatibility and biodegradability, and studies have confirmed its ability to promote nerve regeneration. However, the fragility, low strength and low flexibility of keratin itself limit the application of keratin in the field of biomedicine. The blending of polymers can develop materials with enhanced properties/functions/activity, new materials can retain some of the advantages of raw materials, and new polymers with more advantages can be synthesized by changing the ratio of raw materials. Chitosan is also a widely used biomedical material. Compared with keratin, it has stronger mechanical properties. Therefore, the blending of chitosan and keratin is expected to improve the shortcomings of keratin's mechanical properties.

Contents of the invention

The object of the present invention is to provide a kind of preparation method of the human hair keratin/chitosan hydrogel of loading curcumin, to solve the problem proposed in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin, comprising the following steps:

Extraction of human hair keratin: first pretreatment and degreasing of human hair, then rough extraction of human hair keratin, purification of human hair keratin, and concentration of human hair keratin solution to obtain human hair keratin solution;

Preparation of the composite hydrogel: blending human hair keratin solution and chitosan to obtain a blend, adding curcumin to obtain a suspension, and freeze-drying the suspension; cross-linking to obtain a composite hydrogel.

As a further technical solution of the present invention, the human hair pretreatment and degreasing are as follows: human hair is cut into 1-2cm segments, then the impurities on the hair surface are washed away with soap and water, and then chloroform with a volume ratio of 2:1 Soak and degrease human hair with a mixed solution of methanol for 24 hours. During this period, stir the solution with a glass rod every 8 hours to ensure full contact between human hair and the mixed solution. After degreasing, wash the hair with ethanol for 3-4 times to wash off the residual mixed solution. , and then dry the human hair for later use.

As a further technical solution of the present invention, the crude extraction of human hair keratin is as follows: Weigh 20g of dry human hair and place it in a 500ml three-neck round bottom flask, then add 0.125M sodium sulfide solution prepared with distilled water to 500ml, and mix well Finally, fix the flask in a constant temperature water bath and react at 40°C for 5 hours. During the reaction, use a stirrer to stir moderately. After the reaction, filter the reaction solution with a 200-mesh screen to remove extra human hair fibers, and then dissolve the solution The impurities in the solution are further removed by centrifugation, and the obtained supernatant is the crude human hair keratin extraction solution.

As a further technical solution of the present invention, the human hair keratin is purified as follows: the human hair keratin crude extraction solution is dialyzed with a 10kDa dialysis bag to remove impurities such as sodium sulfide in the reaction solution, and the dialysate buffer is distilled water , the dialysis time is 72 hours, the water is changed every 8 hours in the first 24 hours, and then every 12 hours after that, the initial volume of the crude extraction solution in the dialysis bag should be less than 1/4 of the volume of the dialysis bag to prevent dialysis During the process, the volume of the solution is too large to burst the dialysis bag.

As a further technical solution of the present invention, the human hair keratin solution is concentrated as follows: pour the dialyzed human hair keratin solution into a round bottom flask, concentrate the solution with a rotary evaporator, and slowly heat up to a heating temperature of 55°C, with a rotation speed of 5-20rpm, the human hair keratin solution after the initial concentration was centrifuged again to remove the flocs produced in the vacuum concentration process, and finally a pure human hair keratin solution was obtained, and the protein concentration was determined by Bradford protein concentration, and Store in a 4°C refrigerator for later use.

As a further technical solution of the present invention, the human hair keratin solution is blended with chitosan as follows: take chitosan powder and place it in a 50ml centrifuge tube, dilute the human hair keratin solution, and dilute the diluted Add the keratin solution into the centrifuge tube, shake slightly to mix the chitosan powder and the human hair keratin solution, and quickly add 1% acetic acid of the human hair keratin/chitosan blend to the test tube with a pipette The chitosan powder is protonated and dissolved, and the human hair keratin/chitosan suspension is obtained after vigorously shaking the test tube, so that the total mass concentration of chitosan and human hair keratin is 20mg/ml, then curcumin is added to the test tube and Shake evenly, place the test tube in an ultrasonic cleaner for 20 minutes to remove air bubbles generated during the shaking process, and then use a syringe to draw the suspension and add it to molds of different shapes.

As a further technical solution of the present invention, the mass ratio of chitosan to human hair keratin is 1:1.

As a further technical solution of the present invention, the curcumin mass concentration is 6 mg/ml.

As a further technical solution of the present invention, the freeze-drying of the suspension is as follows: put the mold in the refrigerator, freeze the suspension rapidly at -20°C, and transfer the mold to a freeze dryer to freeze-dry overnight .

As a further technical solution of the present invention, the cross-linking is: preparing 3% sodium tripolyphosphate and 1.5M sodium chloride solution with pH=5 as a cross-linking agent, adding the cross-linking agent to immerse the lyophilized hydrogel statically After cross-linking for 20 minutes, the hydrogel was repeatedly washed with distilled water to remove excess sodium tripolyphosphate and sodium chloride to obtain the final composite hydrogel.

Compared with the prior art, the beneficial effects of the present invention are: the curcumin-loaded human hair keratin/chitosan hydrogel can be prepared by casting, freeze-drying and sodium tripolyphosphate cross-linking techniques, and the curcumin-loaded human hair keratin/chitosan hydrogel Hair keratin/chitosan hydrogel can promote the recovery of nerve crush injury in rats, and the hydrogel loaded with curcumin has a better promoting effect than pure hydrogel. Human hair keratin, chitosan and curcumin can synergistically Play a role in promoting nerve repair.

Description of drawings

Fig. 1 is the RSC cell scratch experiment diagram of different concentrations of curcumin;

A of Fig. 2 is the structural representation of human hair keratin/chitosan hydrogel and the human hair keratin/chitosan hydrogel of loading curcumin;

B of Fig. 2 is the standard curve figure of curcumin concentration-absorbance;

C of Fig. 2 is the figure of curcumin release rate;

A of Fig. 3 is a diagram of the degradation of the hydrogel implanted around the rat nerve for 8 weeks;

Figure 3 B is the HE staining image of the important organs of rats at the 4th week of hydrogel implantation;

Figure 3 C is a graph of the body weight change of rats within 4 weeks after hydrogel implantation;

D of Fig. 3 is the expression level map of TNT-α in rat blood within 4 weeks after hydrogel implantation;

E of Fig. 3 is the expression level figure of IL-10 in rat blood within 4 weeks after hydrogel implantation;

A of Fig. 4 is the change figure of rat's footprint in 4 weeks;

B of Figure 4 is a schematic diagram of SFI measurement and calculation;

C of Fig. 4 is the SFI statistical analysis diagram;

A of Fig. 5 is a recording diagram of compound muscle action potentials in different experimental groups;

B of Figure 5 is the conduction velocity diagram of the regenerated nerve;

Figure 5 C is the CMPA peak amplitude map of regenerated nerves (*, P<0.05; **, P<0.01);

A of Fig. 6 is a toluidine blue staining figure (1000 × observation);

B of Fig. 6 is transmission electron micrograph;

C of Fig. 6 is axon count map;

D in Figure 6 is a diagram of the inner diameter of the axon;

E of Fig. 6 is axon inner diameter/total stop diameter (G-ratio) figure;

F in Fig. 6 is the map of myelin sheath thickness (*, P<0.05; **, P<0.01);

A of Fig. 7 is the exterior view of the gastrocnemius muscle;

B of Fig. 7 is the Masson staining picture of gastrocnemius muscle cross section;

C of Fig. 7 is the gastrocnemius relative wet weight map;

D in Figure 7 is a map of the proportion of muscle fiber area in the gastrocnemius muscle cross section (*, P<0.05; **, P<0.01).

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention.

Example 1

A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin, comprising the following steps:

S1. Human hair keratin extraction:

1. Human hair pretreatment and degreasing: cut human hair into 1-2cm sections, then wash off the impurities on the hair surface with soap and water, and then soak human hair in a mixed solution of chloroform and methanol with a volume ratio of 2:1 Degrease for 24 hours. During this period, use a glass rod to stir the solution every 8 hours to ensure full contact between the human hair and the mixed solution. After degreasing, wash the hair with ethanol for 3-4 times to wash off the residual mixed solution, and then dry the human hair for later use;

2. Crude extraction of human hair keratin: Weigh 20g of dry human hair and place it in a 500ml three-necked round bottom flask, then add 0.125M sodium sulfide solution prepared with distilled water to 500ml, mix well and fix the flask in a constant temperature water bath. React at 40°C for 5 hours, moderately stir with a stirrer during the reaction, filter the reaction solution with a 200-mesh screen to remove extra human hair fibers after the reaction, and then centrifuge the solution to further remove impurities in the solution, the above obtained The serum is the human hair keratin crude extraction solution;

3. Purification of human hair keratin: Dialyze the crude human hair keratin extraction solution with a 10kDa dialysis bag to remove impurities such as sodium sulfide in the reaction solution. The dialysate buffer is distilled water, and the dialysis time is 72 hours, the first 24 hours Change the water every 8 hours, and then change the water every 12 hours. The initial volume of the crude extraction solution in the dialysis bag should be less than 1/4 of the volume of the dialysis bag to prevent the solution from breaking the dialysis bag during dialysis. ;

4. Concentrate the human hair keratin solution. Pour the dialyzed human hair keratin solution into a round bottom flask, concentrate the solution with a rotary evaporator, and slowly heat up to a heating temperature of 55°C with a rotation speed of 5-20rpm. The concentrated human hair keratin solution was centrifuged again to remove the flocs produced in the vacuum concentration process, and finally a pure human hair keratin solution was obtained. The protein concentration was determined by Bradford protein concentration, and stored in a refrigerator at 4°C for use;

S2. Preparation of composite hydrogel

1. Suspension preparation: Weigh 0.12g chitosan powder and place it in a 50ml centrifuge tube, dilute the human hair keratin solution, add 4ml diluted keratin solution (20mg/ml) into the centrifuge tube, Shake slightly to make the chitosan powder and human hair keratin solution fully mixed, and quickly add 1% acetic acid of human hair keratin/chitosan blend solution to the test tube with a pipette to protonate and dissolve the chitosan powder, Vigorously shake the test tube to obtain a human hair keratin/chitosan suspension, then add curcumin to the test tube and shake evenly (6mg curcumin is added to 1ml blend), put the test tube in an ultrasonic cleaner for 20min to remove the shaking process The bubbles generated in the medium, and then use the syringe to draw the suspension and add it to different shapes of molds;

2. Freeze-drying of the suspension: put the mold in the refrigerator, freeze the suspension quickly at -20°C, and transfer the mold to a freeze dryer to freeze-dry overnight;

3. Cross-linking: prepare 3% sodium tripolyphosphate and 1.5M sodium chloride solution with pH=5 as a cross-linking agent, add the cross-linking agent to immerse the freeze-dried hydrogel and let stand for cross-linking for 20 minutes, then wash it repeatedly with distilled water The hydrogel removes excess sodium tripolyphosphate and sodium chloride to obtain the final composite hydrogel.

Example 2

A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin, comprising the following steps:

S1. Human hair keratin extraction:

1. Human hair pretreatment and degreasing: cut human hair into 1-2cm sections, then wash off the impurities on the hair surface with soap and water, and then soak human hair in a mixed solution of chloroform and methanol with a volume ratio of 2:1 Degrease for 24 hours. During this period, use a glass rod to stir the solution every 8 hours to ensure full contact between the human hair and the mixed solution. After degreasing, wash the hair with ethanol for 3-4 times to wash off the residual mixed solution, and then dry the human hair for later use;

2. Crude extraction of human hair keratin: Weigh 20g of dry human hair and place it in a 500ml three-necked round bottom flask, then add 0.125M sodium sulfide solution prepared with distilled water to 500ml, mix well and fix the flask in a constant temperature water bath. React at 40°C for 5 hours, moderately stir with a stirrer during the reaction, filter the reaction solution with a 200-mesh screen to remove extra human hair fibers after the reaction, and then centrifuge the solution to further remove impurities in the solution, the above obtained The serum is the human hair keratin crude extraction solution;

3. Purification of human hair keratin: Dialyze the crude human hair keratin extraction solution with a 10kDa dialysis bag to remove impurities such as sodium sulfide in the reaction solution. The dialysate buffer is distilled water, and the dialysis time is 72 hours, the first 24 hours Change the water every 8 hours, and then change the water every 12 hours. The initial volume of the crude extraction solution in the dialysis bag should be less than 1/4 of the volume of the dialysis bag to prevent the solution from breaking the dialysis bag during dialysis. ;

4. Concentrate the human hair keratin solution. Pour the dialyzed human hair keratin solution into a round bottom flask, concentrate the solution with a rotary evaporator, and slowly heat up to a heating temperature of 55°C with a rotation speed of 5-20rpm. The concentrated human hair keratin solution was centrifuged again to remove the flocs produced in the vacuum concentration process, and finally a pure human hair keratin solution was obtained. The protein concentration was determined by Bradford protein concentration, and stored in a refrigerator at 4°C for use;

S2. Preparation of composite hydrogel

1. Suspension preparation: Weigh 0.10g chitosan powder and place it in a 50ml centrifuge tube, dilute the human hair keratin solution, add 5ml diluted keratin solution (20mg/ml) into the centrifuge tube, Shake slightly to make the chitosan powder and human hair keratin solution fully mixed, and quickly add 1% acetic acid of human hair keratin/chitosan blend solution to the test tube with a pipette to protonate and dissolve the chitosan powder, Vigorously shake the test tube to obtain a human hair keratin/chitosan suspension, then add curcumin to the test tube and shake evenly (6mg curcumin is added to 1ml blend), put the test tube in an ultrasonic cleaner for 20min to remove the shaking process The bubbles generated in the medium, and then use the syringe to draw the suspension and add it to different shapes of molds;

2. Freeze-drying of the suspension: put the mold in the refrigerator, freeze the suspension quickly at -20°C, and transfer the mold to a freeze dryer to freeze-dry overnight;

3. Cross-linking: prepare 3% sodium tripolyphosphate and 1.5M sodium chloride solution with pH=5 as a cross-linking agent, add the cross-linking agent to immerse the freeze-dried hydrogel and let stand for cross-linking for 20 minutes, then wash it repeatedly with distilled water The hydrogel removes excess sodium tripolyphosphate and sodium chloride to obtain the final composite hydrogel.

Example 3

A preparation method of human hair keratin/chitosan hydrogel loaded with curcumin, comprising the following steps:

S1. Human hair keratin extraction:

1. Human hair pretreatment and degreasing: cut human hair into 1-2cm sections, then wash off the impurities on the hair surface with soap and water, and then soak human hair in a mixed solution of chloroform and methanol with a volume ratio of 2:1 Degrease for 24 hours. During this period, use a glass rod to stir the solution every 8 hours to ensure full contact between the human hair and the mixed solution. After degreasing, wash the hair with ethanol for 3-4 times to wash off the residual mixed solution, and then dry the human hair for later use;

2. Crude extraction of human hair keratin: Weigh 20g of dry human hair and place it in a 500ml three-necked round bottom flask, then add 0.125M sodium sulfide solution prepared with distilled water to 500ml, mix well and fix the flask in a constant temperature water bath. React at 40°C for 5 hours, moderately stir with a stirrer during the reaction, filter the reaction solution with a 200-mesh screen to remove extra human hair fibers after the reaction, and then centrifuge the solution to further remove impurities in the solution, the above obtained The serum is the human hair keratin crude extraction solution;

3. Purification of human hair keratin: Dialyze the crude human hair keratin extraction solution with a 10kDa dialysis bag to remove impurities such as sodium sulfide in the reaction solution. The dialysate buffer is distilled water, and the dialysis time is 72 hours, the first 24 hours Change the water every 8 hours, and then change the water every 12 hours. The initial volume of the crude extraction solution in the dialysis bag should be less than 1/4 of the volume of the dialysis bag to prevent the solution from breaking the dialysis bag during dialysis. ;

4. Concentrate the human hair keratin solution. Pour the dialyzed human hair keratin solution into a round bottom flask, concentrate the solution with a rotary evaporator, and slowly heat up to a heating temperature of 55°C with a rotation speed of 5-20rpm. The concentrated human hair keratin solution was centrifuged again to remove the flocs produced in the vacuum concentration process, and finally a pure human hair keratin solution was obtained. The protein concentration was determined by Bradford protein concentration, and stored in a refrigerator at 4°C for use;

S2. Preparation of composite hydrogel

1. Suspension preparation: Weigh 0.08g chitosan powder and place it in a 50ml centrifuge tube, dilute the human hair keratin solution, add 6ml diluted keratin solution (20mg/ml) into the centrifuge tube, Shake slightly to make the chitosan powder and human hair keratin solution fully mixed, and quickly add 1% acetic acid of human hair keratin/chitosan blend solution to the test tube with a pipette to protonate and dissolve the chitosan powder, Vigorously shake the test tube to obtain a human hair keratin/chitosan suspension, then add curcumin to the test tube and shake evenly (6mg curcumin is added to 1ml blend), put the test tube in an ultrasonic cleaner for 20min to remove the shaking process The bubbles generated in the medium, and then use the syringe to draw the suspension and add it to different shapes of molds;

2. Freeze-drying of the suspension: put the mold in the refrigerator, freeze the suspension quickly at -20°C, and transfer the mold to a freeze dryer to freeze-dry overnight;

3. Cross-linking: prepare 3% sodium tripolyphosphate and 1.5M sodium chloride solution with pH=5 as a cross-linking agent, add the cross-linking agent to immerse the freeze-dried hydrogel and let stand for cross-linking for 20 minutes, then wash it repeatedly with distilled water The hydrogel removes excess sodium tripolyphosphate and sodium chloride to obtain the final composite hydrogel.

The raw materials used in the above examples are shown in Table 1 below.

Table 1

Comparative example 1

Install the preparation method of the above-mentioned embodiment 1, except that curcumin is not added, other steps refer to embodiment 2 to make human hair keratin/chitosan hydrogel.

Experimental example

Preparation of curcumin release buffer: add a certain amount of PBS to the beaker, then slowly add 0.5% Tween-80 under rotating stirring, after Tween-80 is completely dissolved, the curcumin release buffer is obtained as a blank control, and the preparation The curcumin release buffer was stored at 4 °C for later use.

Curcumin standard preparation: Weigh 10mg curcumin, dissolve it with curcumin release buffer, transfer to a 100mL volumetric flask to obtain a 100 μg/ml curcumin solution; use curcumin release buffer to dissolve 100 μg/ml curcumin The curcumin solution was diluted to a curcumin standard solution with a concentration gradient of 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, and 10 μg/ml. Measure the absorbance of 2μg/ml, 3μg/ml, 4μg/ml, 5μg/ml, 6μg/ml, 10μg/ml curcumin standard solution at 427nm and draw the concentration-absorbance standard curve.

Cell scratch assay is a common method to analyze cell migration ability. Its principle is to artificially create a blank area on the fused monolayer cells, called "scratch", when the cells grow to fuse into a monolayer state. Cells at the edge of the scratch will gradually migrate into the blank area to heal the "scratch", and the speed of healing represents the strength of cell migration. As shown in Figure 1, as the concentration of curcumin increased, the scratches of RSC96 cells had a faster healing area. Compared with the blank control group, DMSO as a solvent for curcumin had no effect on the healing area of RSC96 scratches. When the concentration of curcumin is greater than or equal to 0.1 μg/mL, curcumin can promote the scratch healing of RSC96 cells. It can be seen that curcumin needs to reach a certain concentration to promote the migration of RSC96 cells.

As shown in B of Figure 2, the maximum absorption wavelength of the curcumin release buffer is 427nm, and the regression equation of the curcumin solution at 427nm is y=0.1495x+0.012, R ² =0.9996, as can be seen from the standard curve, the curcumin concentration is between 2- The linear relationship is good in the range of 10ug/mL.

Using PBS buffer to simulate the liquid environment in the human body, it was observed that the curcumin-loaded human hair keratin/chitosan hydrogel could continuously release curcumin for 12 days (Fig. 2C). Compared with pure chitosan hydrogel, curcumin was released faster in the composite gel with added human hair protein. In addition, with the increase of the proportion of human hair keratin in the human hair keratin/chitosan hydrogel, the release rate of curcumin increased. SEM showed that as the hydrogel with a large proportion of human hair keratin in the human hair keratin/chitosan hydrogel had a larger pore density, the decrease in the proportion of chitosan meant less cross-linking network, water The molecules can diffuse quickly into the hydrogel, so there is a faster release of curcumin. This difference is more significant in the first 6 days, and the difference becomes smaller after 6 days. This may be due to the reduction of the total amount of curcumin in the hydrogel with the release time, curcumin has a concentration requirement for the promotion of RSC96 cell proliferation and migration, and the drug release of human hair keratin/chitosan hydrogel can be Ensure that the effective drug concentration is quickly reached in the early stage of local nerve damage, which may be beneficial to better promote the repair of nerve damage.

Animal experiment

Hydrogel preparation: According to the comprehensive consideration of the characterization of the composite hydrogel, the cytocompatibility and the release experiment of curcumin, the hydrogel with the human hair keratin chitosan ratio of 1:1 was selected for animal experiments. The preparation steps of hydrogel and curcumin-loaded hydrogel are the same as the previous experiments, and the human hair keratin/chitosan blend and the human hair keratin/chitosan/curcumin suspension are cast at 2ml/well Put it in a 6-well plate, then freeze-dry, cross-link, wash, freeze-dry again, and store in a refrigerator at 4°C in the dark after sterilization. During the animal experiment, the hydrogel was cut to a size of 0.8×0.5cm ² , which contained about 500 μg of curcumin, and was balanced with sterile physiological saline swelling water during the operation.

Animal grouping: 190-210g male SD rats were selected for animal experiments. Sixteen SD rats were randomly divided into 4 groups with 4 rats in each group, namely the sham operation group (Sham), the simple crush injury control group (Control), and the human hair keratin/chitosan hydrogel implantation group (CK). ) and loaded curcumin composite hydrogel implant group (CKC).

Surgical procedures: The rats were fasted for water one day before the operation. After isoflurane inhalation anesthesia, the right hind limbs of the rats were shaved and sterilized with 70% ethanol. Sciatic nerve, about 5mm above the bifurcation of the sciatic nerve, use a hemostat to completely close the sciatic nerve for 30 seconds. Use 9-0 nylon suture to mark approximately 1 mm from the proximal end of the injury, and suture the skin with 4-0 nylon suture. The rats in the Sham group were not subjected to nerve extrusion after the nerve was exposed, and the rats in the CK group and CKC group were marked and wrapped with hydrogel around the injured nerve.

The same human hair keratin hydrogel was implanted around nerves in four additional rats to study hydrogel degradation. All rats were reared in the same environment after the operation, the living status and body weight changes of the rats were regularly observed and recorded, and samples were collected under anesthesia at the specified time.

On the 7th, 14th, 21st and 28th day after operation, the sciatic nerve indexes of all experimental rats were measured. After smearing ink evenly on both hind feet of the rat, put the rat on one end of a rectangular box of 80×10×10 cm, and let it walk to the other end by itself. The bottom of the box is covered with flat white paper, and 5 -6 footprints. Before the experiment, the rats have been trained repeatedly to ensure that the rats pass through the box quickly without staying. Rat footprint data were measured and the sciatic nerve index was calculated. Select clear footprints to measure three indexes of normal foot (N) and injured foot (E): A, PL (footprint length); B, TS (toe width); C, IT (middle toe width) width). Substitute the above data into the sciatic nerve index (Sciatic Function Index) formula, calculate and record the sciatic nerve function index. Sciatic nerve function index SFI=0 is normal, and -100 is complete damage.

SFI=109.5(ETS-NTS)/NTS-38.3(EPL-NPL)/NPL+13.3(EIT-NIT)/NIT-8.8.

On the 28th day after operation, neurophysiological detection was performed on the experimental rats. After the rats were fully anesthetized with 0.4ml/100g chloral hydrate, the right hind limbs of the rats were skinned, disinfected, and the injured segmental nerves were exposed as described above. The recording electrodes were inserted into the abdomen of the gastrocnemius muscle to record signals, the stimulating electrodes were respectively stimulated at the proximal end and the distal end of the injured nerve, and the distance between the two stimulation points was recorded. The compound muscle action potential (CMAP) and amplitude latency (latency) were recorded by computer, and the nerve conduction velocity (NCV) was calculated.

Nerve conduction velocity = distance between stimulation points/(difference in latency between stimulation points).

After the nerve electrophysiological examination, the distal end of the sciatic nerve on the injured side was cut according to the marked points and fixed with 2.5% glutaraldehyde for 24 hours. After an additional 1 h of fixation in 1% osmium tetroxide, dehydrate with an ethanol gradient and embed the nerves with epoxy resin. The nerves were then sliced into 1 μm thick slices using an ultramicrotome. The sections were dipped in 0.5% toluidine blue and stained for 30 min, then separated with acetic acid and washed three times with water. After the slices were sealed with a mounting medium, the myelin sheath structure of the nerves was observed and photographed under an optical microscope. And use ImageJ software to count the axons.

The process of collecting and fixing nerve tissue was the same as that of toluidine blue staining. Nerve tissues fixed with osmium tetroxide were rinsed three times with pre-cooled PBS for 15 min each, then dehydrated with gradient ethanol and embedded in epoxy resin. The embedded tissue was ultra-thin sectioned at 70 nm, stained with uranyl acetate for 30 min and lead citrate for 10 min, respectively. After the staining was completed, the cross-sectional photos of the nerve tissue were observed and recorded under a transmission electron microscope. The inner diameter, outer diameter and myelin sheath thickness of nerve axons were analyzed using ImageJ software.

In order to evaluate the biocompatibility of human hair keratin shell/glycan hydrogel in nerve tissue, we detected the expression levels of two inflammatory factors (TNF-α, IL-10) in the blood of experimental rats in each group within 4 weeks , measured the body weight changes of the rats, and carried out HE staining on the important organs of the rats in each group to observe the main structural changes of the organs. In addition, we also implanted the hydrogel around the nerve tissue of healthy rats to evaluate the hydrogel biodegradability.

After neurophysiological testing, the bilateral gastrocnemius muscles of rats were isolated, weighed and fixed with 4% paraformaldehyde for 24 hours. The fixed gastrocnemius muscle was dehydrated by ethanol gradient, embedded in paraffin, and sectioned, and then stained with Ehrlich hematoxylin staining solution, Ponceau red-acid fuchsin solution and aniline blue acetate staining solution for 5 min respectively, and then dehydrated by ethanol gradient and sealed. slice and dry at room temperature. The morphological changes of gastrocnemius muscle fibers were observed and recorded under an optical microscope, and the cross-sectional area of gastrocnemius muscle fibers was measured with ImageJ software.

On the 0th, 1st, 3rd, 7th, 14th, and 28th days after the operation, the blood of rats in different experimental groups was collected by the inner canthus blood collection method, about 0.8ml each time, and the samples were collected with coagulation-promoting tubes, and the samples were placed at room temperature for 30 minute. Then centrifuge (1000rpm, 15min), absorb the upper serum and store it in a -80°C refrigerator for later use. The inflammatory cytokines in rat serum: IL-10 and TNF-α were determined by ELISA kit. The detection process was carried out according to the instructions of the kit. In short, the absorbance of each sample at 450 nm was detected with a microplate reader. A standard curve (concentration-absorbance) was made through cytokine standards of different concentrations, and the concentration of the cytokine in the sample was calculated using the standard curve.

On the 28th day, the rats were euthanized after the nerve and muscle samples were taken, and the heart, liver, spleen, lung, and kidney organs were removed respectively, and fixed in 4% paraformaldehyde for 24 hours. The fixed tissue was dehydrated by ethanol gradient, embedded in paraffin, sectioned, then deparaffinized in the section with xylene, hydrated with ethanol (from high concentration to low concentration), and then stained with hematoxylin and eosin dyes. Dyeing time is 2-3min. After staining, the slides were sealed with neutral gum, air-dried at room temperature, and the main structural changes of the organs were observed under an optical microscope and recorded by taking pictures.

ImajeJ software was used for image processing, and Graphpad prism7.0 and Origin2021 were used for data processing and analysis. All data are expressed as mean ± standard error of the mean. Differences between groups were compared by one-way analysis of variance (ANOVA). A P value <0.05 was considered statistically significant.

In order to evaluate the biocompatibility of human hair keratin chitosan hydrogel in nerve tissue, we detected the expression levels of two inflammatory factors (TNF-α, IL-10) in the blood of experimental rats in each group within 4 weeks, The weight changes of the rats were measured, and HE staining was performed on the vital organs of the rats in each group to observe the main structural changes of the organs. In addition, we also implanted the hydrogel around the nerve tissue of healthy rats to evaluate the hydrogel biodegradability.

The experimental results showed that the hydrogel could be degraded within 8 weeks (Fig. 3A). All the rats in the experiments did not die, and the rats could eat normally after the operation, and there was no significant increase in body weight on the first 1-3 days after the operation, which may be related to the stress of the operation, after which the body weight showed a normal growth trend. There was no significant difference in body weight of the rats (Figure 3C). As far as the body is concerned, a series of host reactions will occur after the implantation of exogenous foreign bodies, mainly including local reactions, systemic toxic reactions, acute inflammation, chronic inflammatory reactions, and foreign body reactions. When inflammation occurs, many cytokines in the body will be overexpressed to activate inflammatory pathways, so the expression level of inflammation-related cytokines in the blood can reflect the degree of inflammation in the body. These inflammatory factors include: TNF-α, IL-6, IL-8 and IL-10 etc. The experimental results showed that there was no significant difference in the expression levels of TNF-α and IL-10 among the rats in each group (D in FIG. 3 and E in FIG. 3 ). The implantation of the hydrogel did not cause a strong inflammatory response. The results of HE staining showed that compared with the rats in the sham operation group, there was no significant difference between the CK group and the CKC group. All tissues and organs had good cell morphology, no inflammatory cell aggregation, regular arrangement of the myocardium, normal liver lobule, white pulp of the spleen and red Marrow, alveoli, glomerulus and other tissues, without obvious pathological tissue. The above results show that the human hair keratin chitosan hydrogel has good biocompatibility and degradability.

The sciatic nerve innervates the movement of the hind limbs. After the injury, the hind feet of the rats curled up, and the stretching of the five toes was impaired while the feet dragged when walking. SFI quantifies hindfoot functional recovery by comprehensive analysis of footprints, with SFI = 0 as normal and -100 as complete injury. All crush-injured rats showed foot dragging after operation, which indicated that the motor function of the rear feet of the rats was impaired after nerve injury. As shown in Figure 4, at the 2nd week after surgery, compared with the control group, the SFI of rats in the CKC group was significantly increased (P<0.05), while there was no significant difference in the CK group (P>0.05), which may be related to the combination of water in the early stage of injury. The curcumin in the gel is related to the release of curcumin, and curcumin in the damaged part promotes nerve repair. At the 3rd and 4th week, compared with the control group, the SFI of the rats in the CKC group and the CK group was significantly increased (P<0.05), indicating that the composite hydrogel itself also has the function of promoting nerve repair. The promotion effect of the composite hydrogel on nerve injury repair may be enhanced due to the loading of curcumin.

The characteristic pathological changes after peripheral nerve crush injury, that is, Wallerian degeneration, will cause the myelin sheath disintegration axon degeneration at the distal end of the damage, and the nerve conduction will also be lost; while the new axon will be from the proximal end to the distal end Growth, reinnervation of target organs, and gradual restoration of nerve conduction function. This injury and recovery process can be detected by the compound muscle action potential (CMAP). As shown in Figure 5, at the 4th week after surgery, compared with the control group, the peak amplitude of CMAP in the CK and CKC groups was greater (P<0.01); and the peak amplitude of CMAP in the CKC group was also greater than that in the CK group (P<0.05) . Similarly, the regenerated nerve conduction velocity was faster in the CK and CKC groups (P<0.01); the nerve conduction velocity in the CKC group was greater than that in the CK group (P<0.01). The experimental results show that both the composite hydrogel and the curcumin-loaded hydrogel can promote the recovery of damaged nerve conduction function, and the curcumin-loaded hydrogel is better than the simple hydrogel. This may be related to the local release of curcumin in the hydrogel, and curcumin at the damaged site promotes nerve repair.

In order to study the histological recovery of nerve regeneration, we cross-sectioned the distal end of the injured nerve, observed the changes in the number of axons by staining with toluidine blue, and observed the changes in the diameter of axons and the thickness of myelin sheath by transmission electron microscopy. As shown in Figure 6 A (toluidine blue-stained nerve), compared with the sham operation group, the damaged nerve fibers showed uneven distribution of myelinated nerve fibers, different sizes, and irregular shape of axons; but compared with the control group These performances were improved in nerve fibers compared to CK and CKC groups. Statistical analysis of the number of axons showed (C in Figure 6), the CK group and CKC group were significantly more than the control group (P<0.01; P<0.01), and the CKC group was more than the CK group (P<0.01); as in Figure 6B , observed by transmission electron microscope, the inner diameter of axons and the thickness of myelin in the control group were significantly reduced, while the changes in the CK group and CKC group were significantly improved. Statistical analysis of the inner diameter of the axons showed (D in Figure 6) that the CK group and CKC group were significantly larger than the control group (P<0.05; P<0.01). The statistical analysis of axon inner diameter/total fiber diameter (G-ratio) showed (Figure 6E), the CK group and CKC group were significantly smaller than the control group (P<0.05; P<0.01), and the CKC group was smaller than the CK group (P<0.05 ), which is closer to the G-ratio of the sham group. Statistical analysis of myelin sheath thickness showed (Figure 6F), the CK group and CKC group were significantly larger than the control group (P<0.05; P<0.01), and the CKC group was larger than the CK group (P<0.01). The experimental results show that both the composite hydrogel and the composite hydrogel loaded with curcumin can promote nerve recovery after nerve crush injury in rats, and the hydrogel loaded with curcumin has a better promoting effect than that of the simple composite hydrogel, among which significantly Increased myelin sheath thickness of injured nerves.

The gastrocnemius muscle is the target organ of the sciatic nerve, and the gastrocnemius muscle that loses its innervation after nerve injury will atrophy, manifested by smaller muscle fibers, increased collagen, and reduced muscle weight. We tested the gastrocnemius muscle weight, muscle fiber cross-sectional area to evaluate the atrophy of the gastrocnemius muscle. As shown in Figure 7A, the appearance of the gastrocnemius muscle showed obvious atrophy on the side of the nerve injury, while the atrophy was milder in the CK group and CKC group. Statistical analysis of the relative wet weight of gastrocnemius muscle showed that the CKC group was significantly larger than the control group (P<0.05), but there was no significant difference between the CK group and the control group. Through Masson staining of the gastrocnemius cross section, it can be seen that the gastrocnemius muscle fibers were significantly reduced and the muscle fiber gap was significantly increased after nerve injury, while the CK group and CKC group were improved (Figure 7B). Statistical analysis of muscle fiber area ratio showed that the CK group and CKC group were significantly larger than the control group (P<0.01; P<0.01), and the CKC group was significantly larger than the CK group. The experimental results showed that the hydrogel and curcumin-loaded hydrogel alleviated the atrophy of the gastrocnemius muscle after nerve injury, which was due to the fact that the hydrogel and the curcumin-loaded hydrogel promoted nerve regeneration after injury and shortened the gastrocnemius optic nerve Control time, thereby reducing muscle atrophy. Curcumin-loaded hydrogels showed better results than pure hydrogels.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention.

In addition, it should be understood that although this specification is described according to implementation modes, not each implementation mode only includes an independent technical solution, and this description in the specification is only for clarity, and those skilled in the art should take the specification as a whole , the technical solutions in the various embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art.

Claims (4)

1. a preparation method of human hair keratin/chitosan hydrogel loaded with curcumin, is characterized in that, comprises the following steps: Extraction of human hair keratin: first pretreatment and degreasing of human hair, then rough extraction of human hair keratin, purification of human hair keratin, and concentration of human hair keratin solution to obtain human hair keratin solution; Composite hydrogel preparation: blending human hair keratin solution and chitosan to obtain a blend, adding curcumin to obtain a suspension, and freeze-drying the suspension; cross-linking to obtain a composite hydrogel; The human hair pretreatment and degreasing are as follows: cut the human hair into 1-2cm segments, then wash off the impurities on the hair surface with soap and water, and then treat the human hair with a mixed solution of chloroform and methanol with a volume ratio of 2:1. Soak and degrease for 24 hours. During this period, stir the solution with a glass rod every 8 hours to ensure full contact between the human hair and the mixed solution. After degreasing, wash the hair with ethanol for 3-4 times to wash off the residual mixed solution, and then dry the human hair for later use. ; The crude extraction of human hair keratin is as follows: Weigh 20g of dry human hair and place it in a 500ml three-necked round bottom flask, then add 0.125M sodium sulfide solution prepared with distilled water to 500ml, mix and fix the flask in a constant temperature water bath , reacted at 40°C for 5 hours, stirred moderately with a stirrer during the reaction, filtered the reaction solution with a 200-mesh screen to remove extra human hair fibers after the reaction, and then centrifuged the solution to further remove impurities in the solution, and obtained The supernatant is the human hair keratin crude extraction solution; The human hair keratin is purified as follows: the human hair keratin crude extraction solution is dialyzed with a 10kDa dialysis bag to remove the sodium sulfide impurity in the reaction solution, the dialysate buffer is distilled water, and the dialyzing time is 72 hours, the first 24 hours Change the water every 8 hours, and then change the water every 12 hours. The initial volume of the crude extraction solution in the dialysis bag should be less than 1/4 of the volume of the dialysis bag to prevent the solution from breaking the dialysis bag during dialysis. ; The human hair keratin solution is concentrated as follows: pour the dialyzed human hair keratin solution into a round bottom flask, concentrate the solution with a rotary evaporator, slowly heat up to a heating temperature of 55°C, and a rotation speed of 5-20rpm, The human hair keratin solution after preliminary concentration was centrifuged again to remove the flocs produced in the vacuum concentration process, and finally a pure human hair keratin solution was obtained, and the protein concentration was determined by Bradford protein concentration, and stored in a refrigerator at 4°C for later use; The human hair keratin solution is blended with chitosan as follows: take chitosan powder and place it in a 50ml centrifuge tube, dilute the human hair keratin solution, add the diluted keratin solution into the centrifuge tube, Shake slightly to make the chitosan powder and human hair keratin solution fully mixed, and quickly add 1% acetic acid of human hair keratin/chitosan blend solution to the test tube with a pipette to protonate and dissolve the chitosan powder, Shake the test tube vigorously to obtain a human hair keratin/chitosan suspension, so that the total mass concentration of chitosan and human hair keratin is 20 mg/ml, then add curcumin to the test tube and shake evenly, and place the test tube in ultrasonic cleaning Ultrasound in the instrument for 20 minutes to remove the bubbles generated during the shaking process, and then use a syringe to draw the suspension and add it to molds of different shapes; The cross-linking is as follows: prepare 3% sodium tripolyphosphate and 1.5M sodium chloride solution with pH=5 as a cross-linking agent, add the cross-linking agent to immerse the lyophilized hydrogel and let it stand for cross-linking for 20 minutes, then use distilled water repeatedly The hydrogel was washed to remove excess sodium tripolyphosphate and sodium chloride to obtain the final composite hydrogel. 2. the preparation method of the human hair keratin/chitosan hydrogel of load curcumin according to claim 1, is characterized in that, the mass ratio of described chitosan and human hair keratin is 1:1. 3. the preparation method of the human hair keratin/chitosan hydrogel of load curcumin according to claim 1, is characterized in that, described curcumin mass concentration is 6mg/ml. 4. the preparation method of the human hair keratin/chitosan hydrogel of load curcumin according to claim 1 is characterized in that, described suspension freeze-drying is: mold is put in refrigerator, at-20 The suspension was quickly frozen at ℃, and after freezing, the mold was transferred to a lyophilizer for lyophilization overnight.

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