CN106334192A - Sericin hydrogel, preparation method and application thereof - Google Patents

Abstract

The invention provides a sericin hydrogel, a preparation method and an application thereof. The preparation method comprises the following steps: 1) extracting sericin and preparing the sericin to powder, 2) preparing hydrazide-modified sericin and preparing the sericin to powder, 3) preparing oxidized glucan and preparing the glucan to powder, 4) preparing a hydrazide-modified sericin solution and an oxidative glucan solution, and 5) mixing the hydrazide-modified sericin solution and the oxidative glucan solution to prepare the sericin hydrogel. The sericin hydrogel has good biodegradability and biocompatibility, a plurality of macromolecular and micromolecular drugs are carried by taking sericin hydrogel as a drug carrier, and the sericin hydrogel can be injected into body for releasing the drugs through hypodermic injection or intramuscular injection. The hydrogel has fluorescence characteristic, can be degraded through fluorescence monitoring gel and can be realized. The sericin hydrogel can be used for skin restoration. The sericin hydrogel scaffold can be obtained through freeze drying, and can be used as a biomedical material.

Description

A kind of sericin hydrogel and its preparation method and application

Technical field

The present invention relates to bio-medical composition field, more particularly, to a kind of sericin hydrogel and its Preparation method and application.

Background technology

Sericin (silk sericin) is wrapped around a kind of natural macromolecular albumen on fibroin fiber top layer. For a long time due to the people deficiency that sericin is recognized and the limitation studied, lead to there are about every year 50,000 tons of sericin are taken as waste to process in filature industry, waste natural resources valuable in a large number, And environment is caused with serious pollution.If discarded sericin is reclaimed as green material application In organizational project with regenerative medicine field it would be possible to huge economic results in society and environmental benefit can be brought.

In recent years, in field of tissue engineering technology, sericin is because of its good biological activity such as biodegradable Property, low immunogenicity, non-oxidizability, cell adhesion and more and more widely paid close attention to.At present, There are the sericin that high temperature alkaline extraction extracts by research team and other macromolecular materials such as gelatin, gathered Vinyl alcohol, hydroxymethyl cellulose, polyacrylamide etc. are simply mixed and are prepared into thin film, biological support, mix Close gel etc. and be applied to field of tissue engineering technology.However, current sericin to be prepared into form of hydrogels topmost Method still simply fills it in other polymers network rather than is autonomously formed crosslinking.This is main It is because being kept completely separate the sericin in Bombyx bombycis and fibroin and need the processing methods such as high temperature or alkali liquor, this The serious degraded of sericin will certainly be led to.This may be exactly that sericin can not be by way of crosslinked The reason form gel.

So far, also there is no the sericin that high temperature or alkaline extraction extract by way of independently crosslinking It is prepared into the report of injection hydrogel.

Content of the invention

The present invention provides a kind of preparation method of sericin hydrogel, and the method is passed through to modify through hydrazides Sericin solution mix to prepare with oxidized dextran solution there is good stability and mechanicalness The sericin hydrogel of energy, operational approach is simple and easy to do.

Present invention also offers a kind of sericin hydrogel, there are good biological degradability and bio-compatible Property, carry multiple macromole and small-molecule drug as pharmaceutical carrier, and subcutaneous injection or flesh can be passed through The mode of meat injection is injected in vivo release medicine.

Present invention also offers described sericin hydrogel is in fluorescent probe, pharmaceutical carrier, and preparation Application in tissue engineering material.

Present invention also offers a kind of sericin hydrogel lyophilizing support, by by described sericin Hydrogel is placed in subzero and freezes and be vacuum dried and obtains.

Present invention also offers application in organizational project for the described sericin hydrogel lyophilizing support.

A kind of preparation method of sericin hydrogel that the present invention provides, comprises the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare oxidized dextran:

Weigh glucosan, sodium metaperiodate respectively with 0.1-0.8g/ml, 0.01-0.2g/ml ratio is dissolved in Glucan aqueous solution and sodium metaperiodate aqueous solution is obtained in 10-50ml water；

The sodium periodate solution of 10-50ml is slowly dropped to the glucosan water of 10-50ml under ice bath In solution, under the conditions of 4 DEG C, lucifuge is reacted 1～24 hour；

By reactant liquor dialyse 12～72 hours, more freeze-dried obtain oxidizability be 10-40% oxidized dextran Powder；

4) sericin solution and the oxidized dextran solution of hydrazides modification are prepared

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；By step 3) the oxidized dextran powder that obtains is molten In distilled water, making concentration is 5-500g/l oxidized dextran solution；

5) by 1:1-5 volume ratio by 4) in the sericin solution modified of the hydrazides that obtains and oxidation Portugal Polysaccharide solution is mixed to get described sericin hydrogel.

Further, in the scheme of the application, the concentration of the sericin solution that described hydrazides is modified is 0.2g/l.

Further, the concentration of described oxidized dextran solution is 100-350g/l.

Further, the oxidizability of described oxidized dextran is 10%-35%.

In the scheme of the application, hydrazides modify sericin solution and oxidized dextran solution dense Degree is easily controlled, and can control the multifrequency nature of gel by regulating and controlling concentration, such as imbibition, degraded, Drug release etc..

Further, in the solution of the present invention, the molecular weight of described glucosan is 40-120kda.

Further, step 1) in dialysis the use of molecular cut off is 8～14kda bag filter Carry out.

Further, step 2) and 3) in dialysis use molecular cut off be 3.5kda saturating Analysis bag is carried out.

Present invention also offers a kind of preparation method of sericin hydrogel, comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

4) by 6:1-3:1 volume by 3) in the sericin solution modified of the hydrazides that obtains with concentration be The genipin aqueous solution of 10g/l is obtained described sericin hydrogel.

Present invention also offers a kind of preparation method of sericin hydrogel, comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

7) by 100:1-5 volume by 3) in the sericin solution modified of the hydrazides that obtains and 250g/l Glutaraldehyde water solution be mixed to prepare described sericin hydrogel.

A kind of sericin hydrogel that the present invention provides, is made up of described preparation method.

Further, present invention also offers described sericin hydrogel is in preparing fluorescent probe Application.

Further, present invention also offers described sericin hydrogel is in preparing pharmaceutical carrier Application.

Further, present invention also offers tissue engineering material prepared by described sericin hydrogel In application.Described tissue engineering material for example could be for the gel of skin repair, biological glue.

Present invention also offers a kind of preparation method of sericin hydrogel lyophilizing support, by will be described Sericin hydrogel be placed in subzero and freeze and be vacuum dried and obtain.The sericin that the method is made Protein hydrogel lyophilizing support can use as organizational project biological support.

In the solution of the present invention, described from silkworm be wild type silkworm.

The present invention also provided a kind of sericin pharmaceutical hydrogel preparation, including described sericin Hydrogel, and for killing the chemotherapeutic of tumor.

Further, described chemotherapeutic is amycin.Further, described sericin hydrogel medicine Thing preparation is unit preparation.In described unit formulation, the amount of sericin hydrogel is 200 μ l, described Ah The amount of mycin is 100 μ g.In the scheme of the application, described unit formulation is to meet to a mice one The preparation of effective ingredient, such as a unit (pin) injection etc. needed for secondary administration.

The present invention also provided a kind of pharmaceutical preparation for wound healing, including described sericin Hydrogel, and wound healing dressing.Further, described wound healing dressing is many love skins.

The solution of the present invention has the advantage that

1) present invention, using wild type silkworm Bombyx bombycis as raw material, is extracted using high temperature alkaline solution method and passes through hydrazides Modify and obtain the sericin solution that wherein sericin remains good natural characteristic, this solution can Sterilized by way of simple filtration；And powdered long-term guarantor is prepared by cryodesiccated mode Deposit.

2) sericin that hydrazides is modified by the present invention first and the natural polymers such as oxidized dextran Crosslinked making hydrogel, operational approach is simple and easy to do.And obtain there is good biological degradability and life The thing compatibility, can carry the hydrogel of multiple macromole and small-molecule drug as pharmaceutical carrier.

3) present invention provide sericin hydrogel can by adjust hydrazides modify sericin solution, The concentration of oxidized dextran solution and temperature adjusting gelation rate (gel in-situ can be realized as needed).

4) the sericin hydrogel of the present invention, can be by subcutaneous injection or muscle note used as pharmaceutical carrier The mode penetrated is injected in vivo release medicine, and can be by adjusting sericin solution, the oxidation that hydrazides is modified The concentration of dextran solution controls the rate of release of effective control macromole and small-molecule drug, effectively solves Determine the problem of medicine violent release, and the growth that antitumor drug effectively suppresses tumor can have been carried.

5) sericin hydrogel of the present invention has fluorescent characteristic, can pass through the degraded of fluorescence monitoring gel And the release of medicine.

6) the sericin hydrogel of the present invention has good skin repair function.

7) the sericin hydrogel of the present invention is through freeze-dried obtaining the sericin of different pore size and size Hydrogel scaffold, can be used for preparing Various Tissues injury repairing material.

Brief description

Fibroin deficiency sericin (2) that Fig. 1 a extracts for lithium bromide, sodium carbonate extraction sericin egg The sds-page electrophoresis pattern of the sericin (4) that (3), hydrazides are modified in vain.

Fig. 1 b be sericin, hydrazides modify sericin and sericin hydrogel sdh-2 red External spectrum.

Fig. 1 c is glucosan and oxidized dextran dex-al-3 infrared spectrogram.

Fig. 1 d is glucosan and oxidized dextran dex-al-3 nuclear magnetic resonance map.

Fig. 2 a is sericin hydrogel (sdh-1, sdh-2, sdh-3) in condition of different temperatures Lower gel time block diagram.

Fig. 2 b is sericin hydrogel (sdh-1, sdh-2, sdh-3) in different ph environment Under maximum expansion rate of water absorption (37 DEG C) block diagram.

Fig. 2 c is sericin hydrogel (sdh-1, sdh-2, sdh-3) rheological property figure.

Fig. 3 is the variable concentrations sericin hydrogel lyophilized products observed under scanning electron microscope Cut-away view.

Fig. 4 is sericin hydrogel (sdh-1, sdh-2, sdh-3) in different ph values pbs In degradation rate (37 DEG C) curve chart

Fig. 5 is Human normal hepatocyte (hl7702), mice myocyte (c2c12) sericin water Gel cytotoxicity detection statistics block diagram.

Fig. 6 a is sericin hydrogel (sdh-1, sdh-2, sdh-3) external hrp release Curve chart.

Fig. 6 b is the external amycin dox of sericin hydrogel (sdh-1, sdh-2, sdh-3) Releasing curve diagram.

Fig. 7 a is the sericin fluorescence spectrum of sodium carbonate high temperature extraction.Fig. 7 b modifies for hydrazides Sericin fluorescence spectrum.

Fig. 7 c, 7d, 7e are sericin hydrogel fluorescence spectrum.

Fig. 7 f, 7g be laser confocal microscope under observe sericin hydrogel (sdh-1, Sdh-2, sdh-3) lyophilized products figure.

Fig. 8 a is injected into the subcutaneous small animal imaging of c57bl/6j mouse back for sericin hydrogel Figure.

It is subcutaneous not that Fig. 8 b is injected into c57bl/6j mouse back for sericin hydrogel carrying amycin With time point small animal imaging figure (green-sericin hydrogel (sdh-2), redness-amycin (dox)).

It is subcutaneous not that Fig. 8 c is injected into c57bl/6j mouse back for sericin hydrogel carrying amycin With time point amycin (dox) fluorescence intensity change curve chart.

Fig. 8 d is different time points degraded and change in fluorescence figure in sericin water gel.

Fig. 8 e is degradation rate and fluorescence intensity correlation analysiss figure in sericin water gel.

Fig. 9 a carries amycin suppression melanoma c57bl/6j mouse tumor for sericin hydrogel Size figure.

Fig. 9 b carries amycin suppression melanoma c57bl/6j Mouse Weight for sericin hydrogel Variation diagram.

Fig. 9 c carries amycin suppression melanoma c57bl/6j mouse survival for sericin hydrogel Phase schemes.

Fig. 9 d carries amycin suppression melanoma c57bl/6j mouse tumor for sericin hydrogel Size pictorial diagram (the 14th day after treatment).

Fig. 9 e carries amycin suppression melanoma c57bl/6j mouse tumor for sericin hydrogel Paraffin embedding h＆e colored graph (the 14th day after treatment).

Fig. 9 f carries amycin suppression melanoma c57bl/6j mouse tumor for sericin hydrogel Paraffin embedding tunel fluorescence staining figure (the 14th day after treatment).

Figure 10 repairs c57bl/6j mice for sericin hydrogel and scalds different time points wound size Cartogram, likes that skin is commercialization wound dressing, as comparison more.

Specific embodiment

The preparation of the sericin hydrogel that embodiment 1 present invention provides

The selection of Bombyx bombycis:

From wild type silkworm Bombyx bombycis (Inst. of Silkworm, Chinese Academy of Agricultural Sciences's offer), main chemistry Composition is: sericin and fibroin.

Step 1), extract sericin, and be made into powder

1. (100g is cut into 1cm to weigh wild type silkworm Bombyx bombycis²Fragment, after be placed in cleaning beaker In, cleaned with ultra-pure water 3 times, 3500rpm centrifugation removes moisture removal in 5 minutes；

2. the molar concentration adding 3～4l in the Bombyx bombycis fragment obtaining in step 1 is 0.02mol/l Na₂co₃Solution or the deionized water of 3～4l, this beaker is put into 100 DEG C of water in thermostat water bath Bath 1h, makes sericin dissolve, obtains sericin solution；

3. step 2 is obtained solution and proceed to 3500rpm centrifugation 5 minutes in centrifuge tube, remove insoluble Property material, obtains the solution of centrifugation；

4. the solution of the centrifugation in step 3 is transferred to the good bag filter of pretreatment (retention molecule Amount: 8-14kda) in, will be equipped with the bag filter two ends bag filter clamp of the sericin liquid of centrifugation, Placement contains ddh₂In the beaker of o；This beaker is placed in low rate mixing dialysis on agitator, every 3 Hour changes a water, dialyses 72 hours altogether, obtains clarification sericin solution；

5. optional, then clarification sericin liquid is gone in centrifuge tube, 3500rpm is centrifuged 5 minutes, goes Except precipitation；

6. sericin liquid is placed in freezer dryer and is lyophilized into sericin powder；

7. sericin powder is placed in -20 DEG C of Refrigerator stores standby.

Step 2), prepare hydrazides modification sericin

1. weigh sericin powder 6g to be dissolved in 30ml dimethyl sulfoxide under the conditions of 35 DEG C；

2., again by 4.3gn, n- carbonyl dimidazoles are added to 1) in, react 1～3 hour, obtain sericin Dimethyl sulphoxide solution；

3. weigh adipic dihydrazide 54g and be dissolved in 100ml dimethyl sulfoxide under the conditions of 45 DEG C, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

4. and by every gram of sericin add 6-10g adipic dihydrazide, by described adipic dihydrazide Dimethyl sulphoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in and fills at 20～45 DEG C Divide reaction 0.5～36 hour；

5. the reactant liquor in step 4 is transferred to pretreatment good bag filter (molecular cut off: 3500 Da in), will be equipped with the bag filter two ends bag filter clamp of sericin liquid, place and contain ddh₂o Beaker in；This beaker is placed in low rate mixing dialysis on agitator, changes a water every 3 hours, Dialyse 48 hours altogether；

6. the sericin solution modified obtained hydrazides in step 5 is placed in freezer dryer and freezes Dry become hydrazides modify sericin powder, be placed in -20 DEG C of Refrigerator stores standby.

Step 3), prepare oxidized dextran

1. weigh the glucosan that 10g relative molecular weight is 40kda and be dissolved in 50mlddh₂In o；

2. weigh the naio of 2.64g₄It is dissolved in 20mlddh₂In o；

3. by naio₄Aqueous solution is slowly dropped in oxidized dextran aqueous solution under condition of ice bath, after It is placed in -4 DEG C of refrigerators abundant reaction 24 hours；

4. the solution in step 3 is transferred in bag filter (3500da), this bag filter is placed In containing ddh₂In the beaker of o；This beaker is placed in low rate mixing dialysis on agitator, every 3 Hour changes a water, dialyses 48 hours altogether；

5. oxidized dextran aqueous solution is placed in freezer dryer and is lyophilized into oxidized dextran powder, put In -20 DEG C of Refrigerator stores；

6. the oxidizability recording the oxidized dextran made using oxidimetry is 35%.

Step 4) prepare sericin solution and the oxidized dextran solution that hydrazides is modified

1. the fibroin powder that hydrazides is modified is dissolved in ddh₂In o, it is left that concentration is adjusted to 200g/l The right side, the sericin solution that the hydrazides obtaining is modified；

2. oxidized dextran powder is dissolved in ddh₂In o, concentration is adjusted to 100g/l, obtains To oxidized dextran solution.

Step 5) sericin hydrogel preparation

By 4) in the sericin solution modified of described hydrazides of every 1ml in add the oxygen of 1ml Change dextran solution；Be respectively placed in after being sufficiently stirred for mixing 4 DEG C, 25 DEG C, at 37 DEG C, obtain sericin Protein hydrogel sdh-1.

The preparation of the sericin hydrogel that embodiment 2 present invention provides

In the present embodiment, sericin liquid and preparation method thereof is identical with the preparation method of embodiment 1, and difference is, Step 4) and step 5) carry out according to following:

1. the fibroin powder that hydrazides is modified is dissolved in ddh₂In o, it is left that concentration is adjusted to 200g/l The right side, the sericin solution that the hydrazides obtaining is modified；

2. oxidized dextran powder is dissolved in ddh₂In o, concentration is adjusted to 200g/l, obtains To oxidized dextran solution；

3. in every 1ml sericin solution, add 5ml oxidized dextran solution；

4. be respectively placed in after being sufficiently stirred for mixing 4 DEG C, 25 DEG C, at 37 DEG C, obtain sericin water-setting Glue sdh-2.

The preparation of the sericin hydrogel that embodiment 3 present invention provides

The preparation method difference from preparation of Example 1 of sericin hydrogel in the present embodiment It is, step 4) and step 5) carry out according to following:

1. the fibroin powder that hydrazides is modified is dissolved in ddh₂In o, concentration is adjusted to 200g/l, The sericin solution that the hydrazides obtaining is modified；

2. oxidized dextran powder is dissolved in ddh₂In o, concentration is adjusted to 350g/l, obtains Oxidized dextran solution；

3. in every 1ml sericin solution, add 1ml oxidized dextran solution；

4. be respectively placed in after being sufficiently stirred for mixing 4 DEG C, 25 DEG C, at 37 DEG C, obtain sericin hydrogel sdh-3.

Embodiment 3-1

The preparation of sericin hydrogel lyophilizing support

By 25 DEG C of acquisitions to sericin hydrogel sdh-1, sdh-2, sdh-3 are immediately placed in -80 DEG C, take out after freezing 24h；Sample is placed in drying in frozen vacuum dryer (true according to sample size Fixed suitable drying time), obtain three kinds of different pore size sericin lyophilizing supports, aperture respectively 128nm, 47.1nm、10.4nm.

The preparation of the sericin hydrogel that embodiment 4 present invention provides

The preparation method difference from preparation of Example 1 of sericin hydrogel in the present embodiment It is, step 3) carry out according to following:

1. weigh the glucosan that 10g relative molecular weight is 40kda to be dissolved in 50mlddh2o；

2. weigh the naio of 0.66g₄It is dissolved in 10mlddh₂In o；

3. by naio₄Aqueous solution is slowly dropped in oxidized dextran aqueous solution under condition of ice bath, after be placed in Fully react 24 hours in -40 DEG C of refrigerators；

4. the solution in step 3 is transferred in bag filter (3500da), this bag filter is positioned over and contains There is ddh₂In the beaker of o；This beaker is placed in low rate mixing dialysis on agitator, changes one every 3 hours Secondary water, dialyses 48 hours altogether；

5. oxidized dextran aqueous solution is placed in freezer dryer and is lyophilized into oxidized dextran powder, be placed in - 200 DEG C of Refrigerator stores；It is 10% that the oxidized dextran made records oxidizability using oxidimetry.

Make hydrogel according still further to embodiment 1 method, its performance parameter after testing with sdh-1, sdh-2, Sdh-3 is similar.

Applicant also makes oxidizability 10-40%'s using different glucosans and sodium metaperiodate proportioning Oxidized dextran and its parameter:

Table 1

^aCommercialization dextran molecule amount is (mw) is 40kda.

The preparation of the sericin hydrogel that embodiment 5 present invention provides

The preparation method difference from preparation of Example 1 of sericin hydrogel in the present embodiment It is, step 2) carry out according to following:

1. weighing sericin powder 1g and being dissolved in 1l mass percent under the conditions of 35 DEG C is 2% In MES solution；

2. in step 1 solution add 8g 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 4-40g n- N-Hydroxysuccinimide, is stirred at room temperature 24 hours, obtains sericin reactant liquor；

3. the hydrazine hydrate being 85% to Deca 2.5ml mass percent in the sericin reactant liquor of step 2 Solution, fully reacts 24 hours under room temperature.

4. the solution in step 3 is transferred in the good bag filter of pretreatment (3500da), will be equipped with The bag filter two ends bag filter clamp of sericin liquid, places and contains ddh₂In the beaker of o；Should Beaker was placed in low rate mixing dialysis on agitator, changed a water every 3 hours, dialysed 48 hours altogether；

6. the sericin solution modified obtained hydrazides in step 4 is placed in freezer dryer and freezes Dry become hydrazides modify sericin powder, be placed in -20 DEG C of Refrigerator stores standby.

Make hydrogel according still further to embodiment 1 method, its performance parameter after testing with sdh-1, sdh-2, Sdh-3 is similar.

The preparation of the sericin hydrogel that embodiment 6 present invention provides

The preparation method difference from preparation of Example 1 of sericin hydrogel in the present embodiment It is, step 4) and step 5) carry out according to following:

1. the fibroin powder that hydrazides is modified is dissolved in ddh₂In o, concentration is adjusted to 200g/l, The sericin solution that the hydrazides obtaining is modified；

2. in every 1ml sericin solution, add 10～50 μ l glutaraldehyde water solution (250g/l)；

3. it is respectively placed at 25 DEG C after being sufficiently stirred for mixing, obtain faint yellow sericin hydrogel.

The preparation of the sericin hydrogel that embodiment 7 present invention provides

The preparation method difference from preparation of Example 1 of sericin hydrogel in the present embodiment It is, step 4) and step 5) carry out according to following:

1. the fibroin powder that hydrazides is modified is dissolved in ddh₂In o, concentration is adjusted to 200g/l, The sericin solution that the hydrazides obtaining is modified；

2. genipin powder is dissolved in ddh₂In o, concentration is adjusted to 10g/l, obtains genipin Solution；

2. in every 1ml～1.8ml sericin solution, add 300 μ l genipin aqueous solutions；

3. it is respectively placed at 4 DEG C after being sufficiently stirred for mixing, obtain navy blue sericin hydrogel.

Embodiment 6 and the sericin hydrogel of embodiment 7 offer, have preferable biocompatibility, can For organizational project and regenerative medicine field, and not modified sericin can not form hydrogel.

Sericin that embodiment 8 sericin, hydrazides are modified, sericin hydrogel and described The characterization test of sericin hydrogel lyophilizing support

Sericin that Fig. 1 a is extracted from fibroin deficiency Bombyx bombycis using lithium bromide for prior art, this Shen Please embodiment 1 step 1) sericin of sodium carbonate extraction made, embodiment 1 step 2) make Hydrazides modify sericin sds-page electrophoresis pattern.

Respectively by step 1 in embodiment 1), 2) sericin of sodium carbonate extraction made, and The sericin that hydrazides is modified, the sericin being extracted from fibroin deficiency Bombyx bombycis using lithium bromide with prior art Albumen is analyzed by sds-page electrophoresis, and result is shown in Fig. 1 a.Wherein 1 is index zone (marker), 2 Extract the sericin that fibroin deficiency Bombyx bombycis extract for lithium bromide, 3 is the sericin of sodium carbonate extraction, 4 sericins modified for hydrazides.Compared with the fibroin deficiency sericin extracting with lithium bromide, carbonic acid The sericin that sodium extracts, the sericin of hydrazides modification are all mainly diffusion-type, but modify through hydrazides The sericin of diffusion-type also can be prepared into hydrogel afterwards.

Fig. 1 b is embodiment 1 step 2) sericin (sericin-adh) modified of the hydrazides made, Step 1) sericin (sericin) made and embodiment 2 step 5) the sericin water made The infrared spectrogram of gel (sdh-2).

Measure sericin using Fourier transform infrared spectrometer (nexus, thermal nicolet, usa) Albumen, the sericin of hydrazides modified and sericin hydrogel are in 4000 650cm^-1Feature Peak.

As shown in Figure 1 b: can be seen that sericin egg from characteristic peak amide, amide, amide The sericin (sericin-adh) that (sericin), hydrazides are modified in vain, sericin hydrogel (sdh-2) Infrared spectrum collection of illustrative plates is essentially identical, show polypeptide secondary structure in sericin hydrogel (sdh-2) with Pure silk glue protein is similar, and sericin hydrogel (sdh-2) can maintain the natural structure of sericin well As.

Fig. 1 c is step 3 in embodiment 1) glucosan (dextran) made and oxidized dextran (dex-al-3) nuclear magnetic resonance map.

Be can be seen that the peak occurring between displacement is for 5.0-6.0 by Fig. 1 c be oxidized dextran (dex-al-3) carbonyl peak being connect on is it was demonstrated that successfully prepared oxidized dextran.

Fig. 1 d is glucosan and oxidized dextran infrared spectrogram.

Measure sericin using Fourier transform infrared spectrometer (nexus, thermal nicolet, usa) Protein hydrogel is in 4000 650cm^-1Characteristic peak.

As shown in Figure 1 d: there is a peak at 1729 on oxidized dextran (dex-al-3) infared spectrum, This peak is the-c=o- stretching vibration peak on aldehyde radical, and on the infared spectrum of glucosan no this peak, Illustrate that the hydroxyl on glucosan its molecule oxidized becomes aldehyde radical, glucosan becomes oxidized dextran； In addition can also be seen that from collection of illustrative plates, oxidized dextran removes the-c=o- stretching vibration on above-mentioned aldehyde radical Outside peak, other peaks are substantially consistent with the infrared absorption peak of glucosan, and the hydroxyl on dextran molecule is described Only some becomes aldehyde radical to base.

Fig. 2 a exists for sericin hydrogel sdh-1, sdh-2, sdh-3 that embodiment 1-3 is made Gel time block diagram under condition of different temperatures.

The sericin that this sericin hydrogel is modified by hydrazides and oxidized dextran (oxidizability is 35%, Concentration is respectively 100g/l, 200g/l, 350g/l) at 4 DEG C, 25 DEG C and 37 DEG C by volume (1 1) Preparation, observes and records its crosslinking time.

As shown in Figure 2 a, with the rising of oxidized dextran concentration, required crosslinking time is shorter；With The rising of temperature, required crosslinking time is shorter.Illustrate by controlling oxidized dextran concentration and temperature Degree adjusts gelation time, can be utilized this property that this hydrogel is prepared into injectable hydrogel.And should Injection-type hydrogel can overcome most of injection-type hydrogels to be injected in vivo easy diffusion really as pharmaceutical carrier Fixed, and the violent release at medicine initial stage can be reduced.

Fig. 2 b exists for sericin hydrogel sdh-1, sdh-2, sdh-3 that embodiment 1-3 is made Maximum expansion rate of water absorption (37 DEG C) block diagram under different ph environment.

Above-mentioned expansion rate of water absorption passes through sericin hydrogel lyophilizing, weighs, is soaked in three kinds without ph In the pbs solution of value (ph 6.0, ph 7.4, ph 11.0), in different time points, take out by following Formula measures.(wherein ws is the weight under swelling state, and wd is dry weight).

$swelling\left(\%\right)=\frac{ws-wd}{wd}\×100$

As shown in Figure 2 b: sdh-1 sericin hydrogel is in the ring of ph 6.0, ph 7.4 and ph 11.0 Under border, maximum expansion rate can respectively reach 5.3 times, 9.1 times, 9.3 times；Sdh-2 sericin water In the environment of ph 6.0, ph 7.4 and ph 11.0, maximum expansion rate can respectively reach 4.2 to gel Again, 7.3 times, 7.7 times；Sdh-3 sericin hydrogel is in ph 6.0, ph 7.4 and ph 11.0 Under environment, maximum expansion rate can respectively reach 3.7 times, 6.2 times, 6.5 times.Show this sericin Hydrogel has good water absorbing properties, and the water absorbing properties of this hydrogel can be by controlling oxidized dextran Concentration adjust.

Fig. 2 c flows for sericin hydrogel sdh-1, sdh-2, sdh-3 that embodiment 1-3 is made Become performance map.

As shown in Figure 2 c, as g ' > g " when, illustrate that sericin has changed into gel.Oxidation Portugal gathers Sugared concentration is 350g/l, and the gel time of the sericin hydrogel of 200g/l and 100g/l is respectively as follows: 5 Second, 145 seconds and 618 seconds about.Maximum elastance is respectively as follows: 2218pa, 1355pa, 369pa. Greatest viscosity modulus is respectively as follows: 487.5pa, 351.2pa, 62.29pa.Show this sericin hydrogel There is good elastomeric viscous performance, and the elastomeric viscous performance of this hydrogel can be gathered by controlling oxidation Portugal The concentration of sugar is adjusted.

Fig. 3 is in the variable concentrations sericin hydrogel lyophilized products observed under scanning electron microscope Portion's structure chart.

As shown in Figure 3: there is the silk of different oxidized dextran concentration (100g/l, 200g/l, 350g/l) Glue protein hydrogel sdh-1 (a), sdh-2 (b), the lyophilized products of sdh-3 (c) are being swept Retouch and inside observed under electron microscope, there are a large amount of pore space structures.

Table 3 is variable concentrations sericin hydrogel lyophilized products aperture and porosity statistical table

As shown in table 3: there are the different oxidized dextran concentration (sericin of 100g/l, 200g/l, 350g/l Hydrogel lyophilized products aperture respectively may be about 128.83 ± 34.88 μm, 47.13 ± 27.77 μm, and 10.41 ±4.83μm.Additionally, through three kinds of statistical computation have different oxidized dextran concentration (100g/l, 200g/l, The porosity of sericin hydrogel lyophilized products 350g/l) respectively 81.06 ± 0.46, 76.58±4.00、73.82±3.28.Show that sericin hydrogel lyophilized products aperture and porosity can be led to Overregulate the regulation and control of oxidized dextran concentration.And this pore space structure within sericin hydrogel is conduct The favourable conditions of pharmaceutical carrier.

Fig. 4 is sericin hydrogel (sdh-1, sdh-2, sdh-3) in different ph values pbs In degraded (37 DEG C) curve chart.

For the impact to degraded for the test ph environment, sericin hydrogel is soaked in different ph In the pbs solution of value (ph 6.0, ph 7.4, ph 11.0), in different time points, take out pbs Drying is weighed, and result is shown in Fig. 4.

As shown in figure 4, sericin hydrogel was degraded rapidly in the 1st day, sericin egg after 7 days Plain boiled water gel gradually starts slowly to degrade, and hydrogel degraded has ph response.Wherein in ph 11.0 Under the conditions of, sericin hydrogel (sdh-1, sdh-2, sdh-3) degradation rate is the fastest, 65 It can substantially completely be degraded；And degradation rate the slowest for ph 6.0, sericin hydrogel (sdh-1, sdh-2, sdh-3) 65 days degradation rates are respectively 40.58%, 43.45%, 38.14%.

Embodiment 9 sericin hydrogel biocompatibility in vitro is tested

First, experimentation

1. first by dmem high glucose medium, at 37 DEG C, in 5% co₂, 100% humidity In the case of people liver cell hl7702, mouse muscle-forming cell c2c12 are cultivated 24 hours, will be from People liver cell hl7702, mouse muscle-forming cell c2c12 that Tissue Culture Flask is collected use culture medium Eddy diffusion, dispel after, plant in 96 orifice plates, every hole cell number is about 8000；

2. then sericin hydrogel (sdh-1, sdh-2, sdh-3) is laid on containing cell 96 orifice plates in, setting sericin hydrogel addition compared to cell culture medium be respectively 4g/l, 8g/l, 16g/l, 32g/l, 64g/l, the sericin hydrogel of the every kind of quality of every kind of concentration is various Three hole Duplicate Samples；

3. following incubation 48 hours；

In 4.48 hours backward 3, every hole adds 10 μ l cck-8 to be incubated 1 hour altogether；

5. measure its absorbance in 490nm and 630nm using microplate reader.

2nd, experimental analysiss

1. sericin hydrogel (sdh-1, sdh-2, sdh-3) is thin to hl7702, c2c12 The test of cellular toxicity: as shown in figure 5, sericin hydrogel (sdh-1, sdh-2, sdh-3) Substantially there is no toxicity to hl7702, c2c12 cell, cell survival rate (i.e. cell viability) is basic It is held at 90% about；

2. the results show, this sericin hydrogel has preferable biocompatibility.

Embodiment 10 sericin hydrogel is as macromolecular drug hrp controlled release carrier

First, experimentation

1. take 4 μ l horseradish peroxidase (hrp) solution (2 μ g/ μ l) to modify with 150 μ l hydrazides Sericin liquid (200g/l) mixing；

2. in 1 add volume ratio 150 μ l oxidized dextran (concentration is respectively 100g/l, 200g/l, 350g/l), room temperature is placed 0.5 hour, after put into 4 DEG C of refrigerators and place 24 hours；

3. add pbs (ph 7.4) 2ml to be placed in 37 DEG C in the sample；

4. at the 0.5th, 1,2,3,4,6,8,12,19,24,30,40,50 days, carefully Take out clear liquid, and rejoin 1ml pbs (ph 7.4)；

5. adopt euzymelinked immunosorbent assay (ELISA) (elisa) to measure the content of hrp in supernatant, by calculating each In time point supernatant, accumulative hrp content and the ratio of actual drug loading obtain accumulative release rate.

2nd, experimental analysiss

Sericin hydrogel hrp release analysis

The sericin liquid that horseradish peroxidase (hrp) solution and hydrazides are modified by volume 2/75 Mixing, adds the oxidized dextran that the isopyknic oxidizability of sericin liquid modified with hydrazides is 35% Solution (concentration 100g/l, 200g/l, 350g/l), room temperature place 0.5 hour, after put into 4 DEG C of refrigerators Place 24 hours, add pbs (ph 7.4) to be placed in 37 DEG C in the sample.In corresponding time point, carefully Collect supernatant, and rejoin pbs (ph 7.4), measured using euzymelinked immunosorbent assay (ELISA) (elisa) and collect To supernatant in hrp content, by calculating accumulative hrp in Each point in time supernatant Content obtains accumulative release rate with the ratio of actual drug loading.

As shown in Figure 6 a: sericin hydrogel has well to macro-molecular protein medicine (hrp) Controlled-release function, drug release rate can be regulated and controled by adjusting the concentration of oxidized dextran, be good big point Sub- pharmaceutical grade protein carrier material.

Embodiment 11 sericin hydrogel is as small molecule, anti-tumor drug amycin controlled release carrier

First, experimentation

1. take the sericin egg that 6 μ l amycin (dox) solution (20 μ g/ μ l) are modified with 150 μ l hydrazides White liquor (20%, g/l) mixes；

2. in 1 add volume ratio 150 μ l oxidized dextran (concentration is respectively 100g/l, 200g/l, 350g/l), room temperature is placed 0.5 hour, after put into 4 DEG C of refrigerators and place 24 hours；

3. add pbs (ph 7.4) 1ml to be placed in 37 DEG C in the sample；

4., at the 0.5th, 1,2,3,4,7,10,15,22,32,42 days, carefully take out clear liquid, And rejoin 1ml pbs (ph 7.4)；

5. use nanodrop to measure supernatant absorbance, Each point in time is calculated by standard curve In supernatant, accumulative doxorubicin content, the ratio of the numerical value obtaining and actual drug loading is tired out Meter release rate.

2nd, experimental analysiss

The release analysis of sericin hydrogel amycin

The sericin liquid that amycin (dox) solution is modified with hydrazides 1/50 mixes by volume, Add the oxidized dextran solution that the isopyknic oxidizability of sericin liquid modified with hydrazides is 35% (concentration 100g/l, 200g/l, 350g/l), room temperature is placed 0.5 hour, after put into 4 DEG C of refrigerators and place 24 hours, pbs (ph 7.4) is added to be placed in 37 DEG C in the sample.In corresponding time point, careful collection Supernatant, and rejoin pbs (ph 7.4), measure supernatant absorbance using nanodrop, Accumulative doxorubicin content in Each point in time supernatant is calculated by standard curve, by the numerical value obtaining Obtain accumulative release rate with the ratio of actual drug loading

As shown in Figure 6 b: the sericin hydrogel controlled-release function good to small-molecule chemical medicine, The rate of release of small-molecule chemical medicine can be regulated and controled by adjusting the concentration of oxidized dextran, be good Small-molecule chemical drug carrier material.

The outer fluorescent characteristic of embodiment 12 sericin water gel

First, sericin fluorescent characteristic test

1. take the sericin powder that the sericin powder of the sodium carbonate extraction of lyophilizing and hydrazides are modified respectively The each 100 μ g water in end are configured to the sericin solution that concentration is 100 μ g/ml；

2. utilize in fluorescence spectrum tester test 1 sericin solution from the fluorescence of 320nm～600nm Spectrum.

2nd, the test of sericin hydrogel (sdh-1, sdh-2, sdh-3) fluorescent characteristic

1. take each 100 μ l of sericin hydrogel (sdh-1, sdh-2, sdh-3) respectively, preparation Become sericin aquagel membrane；

2. utilize fluorescence spectrum tester test 1 in sericin hydrogel (sdh-1, sdh-2, Sdh-3) from the fluorescence spectrum of 320nm～600nm.

3rd, sericin hydrogel (sdh-1, sdh-2, sdh-3) lyophilized products laser copolymerization Burnt test

1. prepare sericin hydrogel (sdh-1, sdh-2, sdh-3) freeze-dried after obtain lyophilizing Product；

2. utilize confocal laser scanning microscope sericin hydrogel (sdh-1, sdh-2, Sdh-3 lyophilized products).

5th, experimental analysiss

1. as shown in Fig. 7 a, 7b, the sericin of sericin after hydrazides modified and sodium carbonate extraction Albumen is compared, and fluorescence spectrum does not have generation and significantly changes, and illustrates that hydrazides is modified to sericin Fluorescent characteristic does not have a significant impact；

2. as shown in Fig. 7 c, 7d, 7e, sericin hydrogel and sericin and hydrazides modified Sericin compare fluorescence spectrum and do not have and occur significantly to change, sericin hydrogel is described still So maintain the fluorescent characteristic of sericin；

3., as shown in Fig. 7 f, 7g, laser co-focusing result shows sericin hydrogel lyophilized products Remain in that the fluorescent characteristic of sericin.

Fluorescent characteristic in embodiment 13 sericin water gel

First, fluorometric investigation in sericin hydrogel (sdh-1, sdh-2, sdh-3) mice body

1. each for sericin hydrogel (sdh-1, sdh-2, sdh-3) 200 μ l are passed through injection Mode be expelled to the back of c57bl/6j mice；

2. observing it in exciting light using small animal imaging instrument is 420nm, and launching light is the glimmering of 520nm Light.

2nd, release doxorubicin fluorescence tracer flow in sericin hydrogel mice body

1. take the sericin egg that 2 μ l amycin (dox) solution (20 μ g/ μ l) are modified with 100 μ l hydrazides White liquor (200g/l) mixes；

2. add the oxidized dextran (concentration is 200g/l) of volume ratio 100 μ l in 1, by note It is subcutaneous that the mode penetrated is expelled to c57bl/6j Mice Body；

3. use small animal imaging instrument to this c57bl/6j mice the 1st the 0th, 2,4,8,12, Tested within 18 days.

3rd, experimental analysiss

1. as shown in Figure 8 a, small animal imaging result show sericin hydrogel (sdh-1, Sdh-2, sdh-3) remain on the characteristic of its fluorescence in c57bl/6j mice body, can be utilized The characteristic of its fluorescence carries out the spike of drug disposition controlled release.

2., as shown in Fig. 8 b, 8c, medicine can be positioned by the fluorescent characteristic of this sericin hydrogel Carrier concrete orientation in vivo, and medicine is monitored in vivo by the fluorescence intensity detecting amycin Release conditions.

Embodiment 14

First, sericin hydrogel fluorescent characteristic monitoring vivo degradation

1. prepare each 200 μ l of sericin hydrogel (sdh-2) to be expelled to by way of injection The back of c57bl/6j mice；

2. the careful gel taking out mouse back at the 4th, 8,12,16,22,40,70 days, will This gel lyophilizing is weighed.

2nd, experimental analysiss

As shown in Fig. 8 d, 8e, degraded situation in vivo has and closes more by force this sericin hydrogel with it Connection property, can understand its degraded feelings in vivo by monitoring the fluorescence intensity of this sericin hydrogel Condition.

Embodiment 15

First, sericin hydrogel carries amycin (dox) suppression melanoma

1. by way of injection, 0.2ml is comprised 2 × 10⁵The pbs of b16f10 mouse melanin tumor cell Solution is expelled to c57bl/6j mouse back；

2. when tumor grows to 30～40mm²When, mice (every body weight 25g) is randomly divided into four groups, respectively For: sdh-2+dox group；Sdh-2 group；Dox group；Pbs group；

3. by sdh-2 (200 μ l/ Mouse Weight)+dox (4mg/kg Mouse Weight) by way of injection； sdh-2(200μl)；dox(4mg/kg)；Pbs (200 μ l) is expelled to tumor vicinity；

4. every the tumor size of 1 day detection mice, record life cycle and body weight；

5. in the 15th day, four groups randomly select mice one, cut out tumor and observe size, and by this tumor group Knit paraffin embedding, section carries out h＆e, tunel dyeing.

2nd, experimental analysiss

1. as illustrated in fig. 9, sdh-2+dox group tumor size is significantly less than other three groups, and this sericin is described Protein hydrogel carries amycin and can substantially suppress melanoma；

2. as shown in figure 9b, four groups of Mouse Weights do not change significantly, and this sericin hydrogel is described Carry amycin no obvious toxic-side effects；

3. as is shown in fig. 9 c, sdh-2+dox group life cycle is considerably longer than other three groups, and this sericin egg is described Plain boiled water gel carries amycin and can substantially suppress melanoma；

4. as shown in figure 9d, the 15th day when, sdh-2+dox group tumor size is significantly less than other three groups, Illustrate that this sericin hydrogel carries amycin and can substantially suppress melanoma；

5. as shown in figure 9e, sdh-2+dox group apoptosis degree is significantly greater than other three groups, and this is described Sericin hydrogel carries amycin and can substantially suppress melanoma；

6. as shown in figure 9f, sdh-2+dox group apoptosis degree is significantly greater than other three groups, and this is described Sericin hydrogel carries amycin and can substantially suppress melanoma.

To sum up, compared to dox group is administered alone, or sdh-2 group is administered alone, the silk of the application Glue protein hydrogel carries amycin (sdh-2+dox) group and has the suppression melanoma significantly increasing Ability

Embodiment 16

First, sericin hydrogel is applied to tissue repair

1. basal diameter is 1cm²Cylindrical stainless steel column immerses 10 minutes in 100 DEG C of water；

2. cylinder in 1 is taken out and be pressed into c57bl/6j mouse back 5 seconds；

3. c57bl/6j mice in 2 is randomly divided into two groups: like skin (duoderm) group more, like skin+sdh-2 more Group；

4. in the 3rd day, c57bl/6j mouse back scalded skin in 2 is cut off, apply many love skin+sdh-2；

5., using step 4 as treatment starting point, observe wound recovery situation within the 7th, 14,21 days after treatment.

2nd, experimental analysiss

1. as shown in Figure 10, like skin+sdh-2 group c57bl/6j mice wound size the 7th, 14 more Its more love skin (duoderm) is organized little.Illustrate that the sericin hydrogel of the application and many love skins make simultaneously With more business-like Moist ganze dressing, there is the wound repair effect significantly improving.

Claims (10)

1. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare oxidized dextran:

Weigh glucosan, sodium metaperiodate respectively with 0.1-0.8g/ml, 0.01-0.2g/ml ratio is dissolved in Glucan aqueous solution and sodium metaperiodate aqueous solution is obtained in 10-50ml water；

The sodium periodate solution of 10-50ml is slowly dropped to the glucosan water of 10-50ml under ice bath In solution, under the conditions of 4 DEG C, lucifuge is reacted 1～24 hour；

By reactant liquor dialyse 12～72 hours, more freeze-dried obtain oxidizability be 10-40% oxidized dextran Powder；

4) sericin solution and the oxidized dextran solution of hydrazides modification are prepared

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；By step 3) the oxidized dextran powder that obtains is dissolved in In distilled water, making concentration is 5-500g/l oxidized dextran solution；

5) by 1:1-5 volume ratio by 4) in the sericin solution modified of the hydrazides that obtains and oxidation Portugal Polysaccharide solution is mixed to get described sericin hydrogel.

2. preparation method according to claim 1 is it is characterised in that the molecular weight of described glucosan For 40-120kda.

3. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

4) by 6:1-3:1 volume by 3) in the sericin solution modified of the hydrazides that obtains with concentration be The genipin aqueous solution of 10g/l is obtained described sericin hydrogel.

4. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

4) by 100:1-5 volume by 3) in the sericin solution modified of the hydrazides that obtains and 250g/l Glutaraldehyde water solution be mixed to prepare described sericin hydrogel.

5. a kind of sericin hydrogel is it is characterised in that prepare described in any one of claim 1-4 Method obtains.

6. application in fluorescent probe for the sericin hydrogel described in claim 5.

7. application in pharmaceutical carrier for the sericin hydrogel described in claim 5.

8. application in preparing tissue engineering material for the sericin hydrogel described in claim 5.

9. a kind of sericin hydrogel lyophilizing support is it is characterised in that by will be according to claim 5 Described sericin hydrogel is placed in subzero and freezes and be vacuum dried and obtains.

10. application in organizational project for the sericin hydrogel lyophilizing support described in claim 9.