CN106188583B

CN106188583B - A kind of silk gum conductive hydrogel and preparation method thereof and holder prepared therefrom

Info

Publication number: CN106188583B
Application number: CN201610549724.0A
Authority: CN
Inventors: 王琳; 王征; 王健; 李晓麟; 杨文�; 张磊
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2016-07-13
Filing date: 2016-07-13
Publication date: 2018-11-09
Anticipated expiration: 2036-07-13
Also published as: CN106188583A

Abstract

The present invention relates to a kind of silk gum conductive hydrogels, are crosslinked by sericin, the carbon nanometer micro-pipe of sericin and 0.01-0.1mg/mL comprising 0.0125-0.0833g/mL in the silk gum conductive hydrogel.The invention also discloses a kind of preparation method of silk gum conductive hydrogel and holder and its applications made of the silk gum conductive hydrogel.The silk gum conductive hydrogel and holder prepared therefrom of the present invention is provided simultaneously with excellent conductive characteristic, syringeability and memory deformation behavior, and the ability of inducing cell differentiation, therefore the wound repair for the tissue closely related with bioelectrical activity such as be particularly suitable for nerve, heart, muscle and treatment.The silk gum conductive hydrogel and its prepared holder and sustained release pharmaceutical formulation of the present invention can be transplanted in vivo by way of injection or Minimally Invasive Surgery, utmostly reduce the wound and pain that patient brings by transfer operation, the infection risk thus brought is also reduced, and does not generate serious immune response after implanting.

Description

A kind of silk gum conductive hydrogel and preparation method thereof and holder prepared therefrom

Technical field

The present invention relates to technical field of biological material, more specifically it relates to a kind of silk gum conductive hydrogel and preparation method thereof with And holder prepared therefrom.

Background technology

Hydrogel (Hydrogel) is the gel using water as decentralized medium, the water soluble polymer with cross-linked network In there are hydrophobic grouping and hydrophilic radical, hydrone is connected to netted inside by hydrophilic radical and water molecules, and hydrophobic group The cross-linked polymer of group's water-swellable.

Sericin (Silk Sericin) is wrapped around a kind of natural macromolecular viscous protein on fibroin fiber surface layer, about The 20-30% for accounting for silk cocoon content, by molecular weight be 24-400kDa polypeptide form, molecule by serine, asparatate and 18 kinds of amino acid compositions such as glycine.In recent years, there is adherency due to the good biocompatibility of sericin, to cell and protect The biology performances such as shield effect, become the emerging material of field of biomedical materials.

Carbon nanometer micro-pipe (Carbon Nanotubes, CNTs) is a diameter of Nano grade rolled by one layer of graphite linings Micro-pipe.The diameter of carbon nanometer micro-pipe is in the several nanometers of zero between tens nanometers, and draw ratio is big, about 100-1000.Carbon The special structure of nanometer micro-pipe, makes it have unique performance, answers the fields such as mechanics, electronics, calorifics, energy storage are great With value.In terms of electric property, carbon nanometer micro-pipe different-diameter and helicity can make it that metallic conductivity be presented and partly lead Bulk properties.Using characteristics such as carbon nanometer micro-pipe fabulous electric conductivity, electroluminescent, carbon nanometer micro-pipe can be made to be led in composite material Domain plays new effect.

In the physiological function of human body, many physiological activities are all closely related with bioelectrical activity.For example, in nerve, flesh In the tissues such as meat, height precisely and quickly generate with propagate electric signal at a very rapid rate same cell membrane surface and carefully It is transmitted between born of the same parents.The functional activity of the tissues such as these bioelectrical activities and nerve, muscle is closely related.

At present in organizational project and regenerative medicine field, a variety of biomaterials have been studied and have been manufactured that, but these Material does not often have syringeability and memory deformability, close in nerve fiber, cardiac muscular tissue etc. and bioelectrical activity The tissue repair in relevant field then also requires material conductive with regeneration.However, the existing hydrogel material in this field Which kind of is still provided simultaneously with good electric conductivity, memory deformability and bioactivity without.

Invention content

In order to solve the above technical problems, the present invention provides a kind of silk gum conductive hydrogel, by sericin crosslinking At, and include the sericin of 0.0125-0.0833g/mL and the carbon nanometer micro-pipe of 0.01-0.1mg/mL.

The present invention also provides the preparation methods of the silk gum conductive hydrogel, include the following steps：

1) sericin aqueous solution is prepared using the silk without bombyx mori silk fibroin；

2) carbon nanometer micro-pipe solution is prepared；

3) the carbon nanometer micro-pipe solution is added in the sericin aqueous solution, mixing obtains mixed solution；

4) crosslinking agent is added in the mixed solution obtained to step 3), is stood after mixing, waits for that its crosslinking obtains the silk Glue conductive hydrogel.

Preferably, the sericin aqueous solution is obtained by dissolving out sericin from silk with LiBr aqueous solutions, and And a concentration of 1.5-10% of the sericin aqueous solution.

Preferably, the silk is the silk that is produced by bombyx mori silk fibroin deletion form silkworm.

Preferably, the Geniposide of 0.01g/mL, the carbon nanometer micro-pipe solution are also included in the carbon nanometer micro-pipe solution Make a concentration of 10mg/mL and Geniposide of carbon nanometer micro-pipe in PBS solution by the way that carbon nanometer micro-pipe and Geniposide to be dissolved in A concentration of 0.01g/mL be then ultrasonically treated and obtain.

Preferably, the crosslinking agent is the genipin solution of 0.01g/mL.

Preferably, it the described method comprises the following steps：

1) sericin aqueous solution is prepared：The silk cocoon for taking bombyx mori silk fibroin deletion form silkworm to be produced, shreds, cleaning；It will cleaning Silk cocoon be soaked in the LiBr aqueous solutions of 6mol/L with the ratio of every gram of 55mL, fully impregnate that be placed on water-bath 24 in 35 DEG C small When, to dissolve sericin；It obtains liquid to centrifuge under 3500rpm, removes insoluble substance, obtain clear solution；Xiang Qi It is middle that the Tris-HCl buffer solutions of the 1mol/L pH 9.0 by 1/4 part of the clear solution stereometer are added, and it is packed into retention molecule It is subsequently placed in the 0.001mol/L Tris-HCl buffer solutions of pH 9.0 and dialyses in the bag filter of amount 3500, obtain the silk gum Protein solution；

2) carbon nanometer micro-pipe solution is prepared：The PBS phosphoric acid that carbon nanometer micro-pipe and Geniposide powder are dissolved in pH 7.4 delays It rushes in salting liquid, makes the final concentration of 10mg/mL of carbon nanometer micro-pipe and the final concentration of 0.01g/mL of Geniposide；Then ultrasound 5 is used Minute mixes well, and is spaced 2 seconds within 3 seconds per ultrasound, obtains the carbon nanometer micro-pipe solution；

3) the carbon nanometer micro-pipe solution is added in the sericin aqueous solution dense to the carbon nanometer micro-pipe Degree is 0.1-1.0mg/mL, and mixing obtains mixed solution；

4) genipin solution of 0.01g/mL is added in the mixed solution obtained to step 3), is stood extremely in 37 DEG C after mixing It is 30 minutes few, wait for that its crosslinking obtains the silk gum conductive hydrogel.

The silk gum conductive hydrogel can be used for preparing medical material, such as prepare medicament slow release preparation and cell therapy load Body, or prepare the material regenerated for injury tissue with reparation.

The present invention also provides a kind of holders prepared by above-mentioned silk gum conductive hydrogel.The holder can be used for preparing doctor Drug material, such as medicament slow release preparation and cell therapy carrier are prepared, or prepare the material regenerated for injury tissue with reparation.

The silk gum conductive hydrogel and holder prepared therefrom of the present invention, is a kind of completely new Biocomposite material, has Good biocompatibility has conductive characteristic, syringeability, memory deformation behavior, fluorescent tracing characteristic and promotes cell point The characteristics such as change.Based on these good characteristics, it can be used for following purposes：

1. preparing tissue repair and regenerated material, and since it is provided simultaneously with excellent conductive characteristic, syringeability With memory deformation behavior, therefore the wound for the tissue closely related with bioelectrical activity such as be particularly suitable for nerve, heart, muscle It repairs and treats；

2. preparing drug delivery vehicle, carries drug or the other treatment factor, small molecule wait until in vivo and controllably to delay On The Drug Release, and then play desired effect；

3. being used as cell carrier, cell is carried, is transplanted in vivo with injection system or minimally invasive, non-invasive manner, is played and make With；

4. being used as internal fluorescent tracing substance, the tracing in vivo being applied in clinical diagnosis and treatment.

The silk gum conductive hydrogel and its prepared holder and sustained release pharmaceutical formulation of the present invention can be by injection or micro- The mode of invasive procedures is transplanted in vivo, is utmostly reduced the wound and pain that patient brings by transfer operation, is also reduced Thus the infection risk brought, and do not generate serious immune response after implanting.

Description of the drawings

Fig. 1 is the preparation flow figure of silk gum conductive hydrogel；

Fig. 2 is the microstructure comparison diagram under the light microscope of holder before and after injection；

Fig. 3 is the fluorescence micrograph dyed through Live&Dead after the holder of carrying cell is injected；

Fig. 4 is the streaming statistical chart of the life or death cell after the holder of carrying cell is injected thereon；

Fig. 5 is the resistance measurement figure of holder；

Fig. 6 is the resistivity measurements comparison diagram of sterilizing front and back support；

Fig. 7 is BMSC cell inoculations in latter 7th day on holder and the expression of the 14th day GFAP, Tuj1, MAP2 gene；

Fig. 8 is the microphoto for being inoculated in the BMSC cells of holder after calcein dyes；

Fig. 9 is the stereoscan photograph of the holder and crt bracket of the carbon nanometer micro-pipe containing various concentration；

Figure 10 is the holder and crt bracket of the nanometer micro-pipe of carbon containing 0.5mg/mL calculated according to the SEM electromicroscopic photographs of Fig. 9 Aperture statistical chart；

Figure 11 is the statistical chart of the elasticity modulus of holder；

Figure 12 is the expansion rate figure of holder at any time；

Figure 13 is the activity analysis figure that mesenchymal stem cell is inoculated in cell after 48 hours on holder；

Figure 14 is the streaming statistical chart that mesenchymal stem cell is inoculated in life or death cell after 48 hours on holder；

Figure 15 is the X-ray carried out after holder is injected into Mice Body and fluorescent scanning image；

Figure 16 is the statistical chart that HRP sustained release agents discharge HRP at any time.

Specific implementation mode

Principles and features of the present invention are described below in conjunction with example and attached drawing, example is served only for explaining this hair It is bright, it is not intended to limit the scope of the present invention.

The preparation of embodiment 1-4 silk gum conductive hydrogels

The preparation flow of the silk gum conductive hydrogel of the present embodiment is as shown in Figure 1.

1. preparing silk gum aqueous solution

Silk cocoon of the silk used in the present embodiment from bombyx mori silk fibroin deletion mutation kind (is purchased from the Chinese Academy of Agricultural Sciences Sericulture research institute).By the following method silk gum aqueous solution is prepared from the silk cocoon：

1) it weighs silkworm mutating variety silk cocoon 1g and is cut into 1cm²Fragment, be placed in clean beaker, use ultra-pure water Cleaning 3 times, 3500rpm centrifuge 5 minutes removal moisture content；

2) the LiBr aqueous solutions of a concentration of 6mol/L of 55mL are added in the silk cocoon fragment obtained into step 1), by the burning Cup is put into thermostat water bath 35 DEG C of water-baths 24 hours, dissolves sericin；

3) step 2) is obtained into solution and is transferred to 3500rpm centrifugations 5 minutes in centrifuge tube, insoluble substance is removed with filter, Obtain clear solution；

4) the Tris-HCl buffer solutions (1mol/L, pH 9.0) of 1/4 volume are added in the clear solution obtained to step 3)；

5) solution in step 4) is transferred in the bag filter (MWCO 3500) pre-processed, then by bag filter two End clamp is positioned in the beaker of the 0.001mol/L Tris-HCl buffer solutions containing pH 9.0；The beaker is placed in Dialysis is mixed slowly on blender, a water was changed every 3 hours, is dialysed 48 hours altogether, wherein last time is saturating using ultra-pure water Analysis；

6) 5) the sericin aqueous solution after middle dialysis is gone in centrifuge tube, 4000rpm is centrifuged 5 minutes, removal precipitation；

7) sericin aqueous solution is reloaded into bag filter and by bag filter both ends clamp, then by bag filter It is placed in the PEG6000 solution that mass percentage concentration is 20% and concentrates；Sericin aqueous solution, which is concentrated to concentration, is about Until 0.0274g/mL；

8) 4 DEG C of refrigerators are placed in save backup.

2. preparing carbon nanometer micro-pipe solution

1) carbon nanometer micro-pipe and Geniposide powder are dissolved in the PBS phosphate buffered saline solutions of pH 7.4, make carbon nanometer The final concentration of 10mg/mL of micro-pipe and the final concentration of 0.01g/mL of Geniposide；

2) the ready carbon nanometer micro-pipe solution of step 1) is mixed well into (the U.S. in 5 minutes with 30% amplitude ultrasound 125 ultrasonic cell disintegration instrument of qsonica), it is spaced 2 seconds within 3 seconds per ultrasound.

3. the composite synthesis of silk gum conductive hydrogel

1) the carbon nanometer micro-pipe solution is added by the proportioning in table 1 in above-mentioned sericin aqueous solution, makes gained A concentration of 0.1mg/mL (embodiment 1), 0.25mg/mL (embodiment 2), the 0.5mg/mL of carbon nanotube described in mixed solution (embodiment 3) or 1mg/mL (embodiment 4), mixes well, to be not added with the mixed solution of the carbon nanometer micro-pipe and Geniposide Silk gum aqueous solution be control；

2) genipin solution of 0.01g/mL is added in the mixed solution obtained to step 1) by the proportioning in table 1；

3) mixed solution that step 2) obtains is mixed well, is placed in 37 DEG C of incubators at least 30 minutes or more, waits for its friendship Connection forms hydrogel.

The amount of sericin solution, carbon nanometer micro-pipe solution, genipin solution added by the every milliliter of hydrogel of table 1

The preparation of embodiment 5-8 holders and performance

1. the preparation of holder

The silk gum conductive hydrogel of embodiment 1-4 is put into mold, the cylindrical of thickness 1cm diameters 1cm is made, at Glue is placed on -80 DEG C, and freezing is taken out afterwards for 24 hours；Sample is placed in frozen vacuum dryer dry (determined according to sample size suitable Suitable drying time), respectively obtain the holder of embodiment 5-8, that is, the holder of embodiment 5-8, which separately includes 0.1mg/mL, (to be implemented Example 5), 0.25mg/mL (embodiment 6), 0.5mg/mL (embodiment 7) or 1mg/mL (embodiment 8) carbon nanometer micro-pipe, with being not added with carbon Hydrogel method described above prepared by nanometer micro-pipe solution prepares crt bracket.

2. the memory deformability of holder

Hydrogel of different shapes is prepared according to the method described above, and holder is made, and holder is resuspended in ultra-pure water or PBS In, the holder is squeezed, folds and can make the holder that arbitrary deformation occur, or dwindles into minimum volume, but is worked as Again after aquation, holder restores original shapes and sizes, shows that the holder of the present invention has extraordinary memory deformability.

3. the syringeability of holder is analyzed

The syringeability of the holder of testing example 5-8 by the following method：

1) holder of embodiment 5-8 and crt bracket are impregnated 24 hours in 75% ethyl alcohol and is sterilized, then use ultra-pure water It washes twice, is resuspended in the PBS of 2mL pH 7.4；

2) use 16gauge syringes that the holder is subcutaneously injected in Mice Body.

The both macro and micro form of holder is observed in operation.The results show that in injection process, holder keeps macro See structure, form it is complete, and holder subcutaneously restore complete structure stretching, extension.Under ordinary optical microscope, either 6mm The holder of × 6mm or the holder of 4mm × 4mm keep microstructure complete (Fig. 2).

After the holder of embodiment 5-8 is injected in Mice Body, before the holder can restore injection in Mice Body Shapes and sizes, this enables the holder to reach target area by injection.

It dyes and shows through Live&Dead, it was demonstrated that after injection, the cell carried on holder can keep survival (Fig. 3). Fig. 4 by Live&Dead staining for flow analysis shows that, the cell that the process of injection carries conductive hydrogel is almost without damage Wound, 92.3% or more cell survival can be kept, it is confirmed that injection process to the cell of carrying almost without damage.

4. the electric conductivity of holder

Measure the electric conductivity of holder by the following method

1) between the holder after aquation being connected to light emitting diode, it is connected to power supply；As a result the holder of embodiment 5-8 is shown It can make lumination of light emitting diode, illustrate that its electric conductivity is good.

2) copper wire is used to be inserted into holder as electrode, the distance between positive and negative anodes of keeping parallelism are 1cm, use work It stands to measure resistance；As a result show that the electric conductivity of these holders is good, and can be by changing the concentration of carbon nanometer micro-pipe Change its electric conductivity (Fig. 5).

3) holder is impregnated 24 hours in 75% ethyl alcohol and is sterilized, then washed twice with ultrapure, before its sterilizing of re-test Whether resistance afterwards, the process for studying sterilizing have an impact the electric conductivity of silk gum conductive hydrogel；As a result show that the sterilizing is grasped Work will not have an impact (Fig. 6) electric conductivity of the holder of embodiment 5-8.

5. the rush cell differentiation of holder

Carry out the neural cellular differentiation ability of detection support by the following method

1) by after the sterilizing of the holder of embodiment 5-8 and aquation, mesenchymal stem cell (BMSC) is inoculated with by preceding method；

2) cell sample is collected respectively the 7th day and the 14th day, pass through q-RT-PCR Real_time quantitative detections and nerve cell point Change related gene GFAP, Tuj1, MAP2 and Nes.

3, the cellular morphology of the BMSC on silk gum conductive hydrogel is seeded in by calcein dyeing display.

As a result it shows：The BMSC cells on holder are seeded in the 7th day and the 14th day, it can be seen that neuron differentiation is related Gene GFAP, Tuj1, MAP2 have significantly up-regulation (Fig. 7)；Calcein dyeing shows the form of living cells by undifferentiated Elliptical deformation is the similar spindle shape (Fig. 8) of neuron；Thus the oriented neuron direction of cell point that prompt is seeded on holder The trend of change illustrates that conductive filament hydrogel has the ability for promoting differentiation.

6. the porosity of holder

Pass through the holder of scanning electron microscopic observation embodiment 5-8 and the inside result of crt bracket.Fig. 9 is scanning electron microscopy The internal stent structure chart that arrives under the microscope arrives the holder added with carbon nanometer micro-pipe of this either embodiment as seen from the figure Or the inside of the holder of the not carbon containing nanometer micro-pipe of control all has a large amount of pore space structures.

It is calculated by the following formula porosity：

V_wIt is the volume of holder, M₀It is the initial weight of holder, M_tBe impregnate ethyl alcohol after quality (absolute ethyl alcohol density p= 0.789g/cm³).The porosity difference of the holder of embodiment 5 (0.1mg/mL) and embodiment 8 (1.0mg/mL) is calculated through statistics For 94.40 ± 2.42% and 95.13 ± 3.61%, show that holder is multiple hole structure.It is calculated according to the SEM electromicroscopic photographs of Fig. 9 Embodiment 7 holder (nanometer of carbon containing 0.5mg/mL micro-pipe) and crt bracket (not carbon containing nanometer micro-pipe) aperture, as a result such as Shown in Figure 10, either the hole diameter of the inside of 7 holder of embodiment or crt bracket is all mainly fallen in 60-110 μm of section It is interior.

The pore space structure of holder is the condition that nutrition and gas exchanges are carried out as pharmaceutical carrier and cell carrier.

7. the mechanical performance of holder

The elasticity modulus of holder is measured, obtained result is as shown in table 2 and Figure 11

The elasticity modulus of 2 holder of table

By the measurement result of elasticity modulus it is found that the holder of above example all has good mechanical performance, and It can carry out the mechanical performance of adjusting bracket as desired by the concentration for changing used nanometer micro-pipe in the preparation.

8. 37 DEG C of expansion rate of water absorption of holder

The holder of embodiment 5-8 and crt bracket are weighed, and are soaked in respectively in the PBS of pH 7.4, in different time Point takes out, and measures as follows：

Wherein, Ws is the weight under swelling state, and Wd is dry weight.

As a result as shown in figure 12, the water absorbing properties of the holder of embodiment 5-8 are moderate, after impregnating a few days ago rapid expansion, branch Frame manages stabilization substantially, is continued until that experiment terminates, this shows that these holders can be used for implanting without to surrounding tissue Generate larger compression.

9. passing through the biocompatibility of cell viability detection support

The biocompatibility of holder is measured by the following method：

1) use DMEM high glucose mediums in 37 DEG C, 5%CO₂Mesenchymal stem cell is cultivated under 100% humidity (BMSC) 24 hours, the BMSC of culture is collected, is suspended again using culture medium, after dispelling, with the quantity of 8000 cells in every hole It is inoculated in 96 orifice plates；

2) by the aforementioned method for preparing hydrogel and holder, directly hydrogel is prepared as mold using 96 orifice plates and branch is made Frame is rinsed 3 times with PBS after sterilizing, then the cell after resuspension is laid in corresponding hole, if three hole Duplicate Samples, it is small to be incubated 48 When；

3) 10 μ L CCK-8 are added into every hole, are incubated 1 hour, are then sucked out, are placed in new hole；

4) its absorbance at 490nm is measured using microplate reader.

Measurement result is as shown in figure 13, and the holder and crt bracket of embodiment 5-8 all have good biocompatibility.

10. the biocompatibility by flow cytometer showed holder is tested

The biocompatibility of above-mentioned silk gum conductive hydrogel is measured by the following method：

1) by mesenchymal stem cell 2000cells/mm²It is seeded on the holder and crt bracket of embodiment 5-8；

2) Live&Dead dyeing and flow cytometer showed are carried out after 48 hours, dead cell and living cells account for the ratio of total cell number Example.

As a result as shown in figure 14, the survival rate of cell is more than 94% on the holder of embodiment 5-7, on the holder of embodiment 8 Cell survival rate is more than 90%.

11. the internal fluorescent tracing specificity analysis of holder

It after holder sterilizing and aquation by embodiment 5-8, is injected into Mice Body, fluorescence and X is carried out in mouse imager Optical scanning is imaged.Imaging results are as shown in figure 15, and holder has clear and special fluorescent characteristic in animal body, this proof, real The holder for applying a 5-8 can be used for internal fluorescent tracing.

The preparation of embodiment 9-12 horseradish peroxidases (HRP) sustained release agent

HRP solution is added in the preparation process of embodiment 1-4 so that the content of HRP is 8 μ in the holder being finally made g；Remaining operation prepares identical with the hydrogel in embodiment 1-4, thus obtains four kinds of silk gum conductive hydrogels for including HRP, The silk gum conductive hydrogel that these four include HRP is lyophilized with the holder preparation method in embodiment 5-8, forms embodiment 9-12 HRP sustained release agents.

Above-mentioned sustained release agent is placed in the PBS of 1mL pH 7.4, is placed in 37 DEG C；The 1st, 2,4,8,12,24,48,72, 96,120,144,168,192,216,240,264,288,323 hours careful taking-up clear liquids, and rejoin 1mL PBS (pH 7.4)；The content that HRP in supernatant is measured using enzyme-linked immunization (ELISA) is tired out by calculating in Each point in time supernatant The HRP contents of meter obtain accumulative release rate, as a result as shown in figure 16, the HRP of embodiment 9-12 with the ratio of practical drugloading rate Sustained release agent has good slow releasing function to HRP, and release time-histories is long, and release rate is high.

The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.

Claims

1. a kind of preparation method of silk gum conductive hydrogel, which is characterized in that include the following steps：

2) carbon nanometer micro-pipe solution is prepared；

4) crosslinking agent is added in the mixed solution obtained to step 3), is stood after mixing, waits for that its crosslinking obtains the silk gum and leads Electric hydrogel, the silk gum conductive hydrogel include the carbon of the sericin and 0.01-0.1mg/mL of 0.0125-0.0833g/mL Nanometer micro-pipe.

2. preparation method according to claim 1, which is characterized in that the sericin aqueous solution passes through water-soluble with LiBr Liquid dissolves out sericin to obtain from the silk, also, a concentration of 0.015-0.1g/mL of the sericin aqueous solution.

3. preparation method according to claim 2, which is characterized in that the silk is is given birth to by bombyx mori silk fibroin deletion form silkworm The silk of production.

4. preparation method according to claim 1, which is characterized in that also include 0.01g/ in the carbon nanometer micro-pipe solution The Geniposide of mL, the carbon nanometer micro-pipe solution make carbon nanometer by the way that carbon nanometer micro-pipe and Geniposide to be dissolved in PBS solution Then a concentration of 10mg/mL of micro-pipe and a concentration of 0.01g/mL of Geniposide are ultrasonically treated to obtain.

5. preparation method according to claim 1, which is characterized in that the crosslinking agent is that the Geniposide of 0.01g/mL is molten Liquid.

6. preparation method according to claim 1, which is characterized in that include the following steps：

1) sericin aqueous solution is prepared：The silk cocoon for taking bombyx mori silk fibroin deletion form silkworm to be produced, shreds, cleaning；By the silkworm of cleaning Cocoon is soaked in the ratio of every gram of 55mL in the LiBr aqueous solutions of 6mol/L, is fully impregnated and is placed on water-bath 24 hours in 35 DEG C, To dissolve sericin；It obtains liquid to centrifuge under 3500rpm, removes insoluble substance, obtain clear solution；It is added thereto By the Tris-HCl buffer solutions of 1/4 part of 1mol/L pH 9.0 of the clear solution stereometer, and it is packed into molecular cut off 3500 Bag filter in dialyse, obtain the sericin aqueous solution；

2) carbon nanometer micro-pipe solution is prepared：Carbon nanometer micro-pipe and Geniposide powder are dissolved in the PBS phosphate-buffered salts of pH 7.4 In solution, make the final concentration of 10mg/mL of carbon nanometer micro-pipe and the final concentration of 0.01g/mL of Geniposide；Then ultrasound is filled for 5 minutes Divide mixing, is spaced 2 seconds within 3 seconds per ultrasound, obtains the carbon nanometer micro-pipe solution；

3) the carbon nanometer micro-pipe solution is added in the sericin aqueous solution a concentration of to the carbon nanometer micro-pipe 0.1-1.0mg/mL, mixing obtain mixed solution；

4) genipin solution that 0.01g/mL is added in the mixed solution obtained to step 3), stands at least 30 after mixing in 37 DEG C Minute, wait for that its crosslinking obtains the silk gum conductive hydrogel.

7. application of the silk gum conductive hydrogel described in claim 1 in medical material.

8. a kind of holder, which is characterized in that by the way that silk gum conductive hydrogel described in claim 1 to be freeze-dried to obtain.

9. application of the holder according to any one of claims 8 in medical material.