CN102078636A - Hydrogel dressing containing recombinant human epidermal growth factor and preparation method and application thereof - Google Patents
Hydrogel dressing containing recombinant human epidermal growth factor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a hydrogel dressing containing a recombinant human epidermal growth factor. The hydrogel dressing consists of 100 volume parts of mixed solution A and 2 to 8 volume parts of cross-linking agent; the mixed solution A is prepared by mixing 2.0 to 5.0 mass percent of polyvinyl alcohol aqueous solution and 0.5 to 2.0 mass percent of sodium alginate aqueous solution according to the mass ratio of (2-5): 1, and adding 0 to 0.1 mass percent of sodium benzoate, 5 to 20ug/g of recombinant human epidermal growth factor and 2.0 to 10 mass percent of glycerol; and the gel dressing is prepared by taking the 2 to 8 volume parts of cross-linking agent prepared by mixing sodium borate solution and calcium chloride solution, dripping the cross-linking agent into the 100 volume parts of mixed solution A, standing and defoaming to obtain the gel dressing. The dressing has good biocompatibility and mild and simple preparation condition, can be formed at room temperature, can effectively keep the activity of the recombinant epidermal growth factor and has controlled release effect on the recombinant epidermal growth factor. The dressing has good effect of treating the wound of rats suffering from diabetes mellitus, and can accelerate healing of the wound.
Description
Technical field
The invention belongs to the bio-medical engineering material field, particularly a kind of aerogel dressing that contains recombinant human epidermal growth factor (rh-EGF) and preparation method thereof, with and as the application of treatment diabetic foot chronic ulcer medicine.
Background technology
Diabetes foot chronic ulcer has a strong impact on the life security and the quality of life of diabetics, and its sickness rate height, hazardness is big, medical expense is expensive, is the diabetes lethal major reasons that disable, and also is the main cause that causes the diabetic amputation.Ulcer of foot is in case very difficulty is treated in generation.Studies show that it is the major reason that the diabetes wound healing postpones that growth factor levels descends.Because recombinant growth factors can accelerating wound healing, so the topical application exogenous growth factor is quickened diabetic foot ulcer one of the clinical method commonly used that heals.Yet,, degraded by matrix metalloproteinase (MMPs) easily when being directly used in wound because the somatomedin half-life is short; The spray-type carrier easily causes the loss of somatomedin, and lacks slow release and continuous action function, makes the topical application of the exogenous growth factor can not get ideal effect.
On the basis of traditional treatment, a series ofly in recent years confirm to promote ulcer healing, avoid the new medical dressing of amputation to come out through clinical observation, further improved the prevention and the treatment of diabetic foot.
According to the different wound type and the different phase of ulcer healing, the dressing type and the function that are used for diabetic foot ulcer are different.Mainly comprise thin film (protection wound surface), foam (protection, fill, absorb), hydrogel (debridement, rehydration, preserve moisture), hydrocolloid (debridement, protection), alginate (hemostasis, absorb), dosing dressing (band antibacterium function) etc.Wherein hydrogel adds that by processing of high molecular material glue-linked polymer forms on polymer liner, can absorb wound exudate, sepage partly can be retained in the dressing again, makes wound surface produce little wet, little acid and low-oxygen environment, promotes tissue repair and re-epithelialization; Be proved the healing of chronic ulcer evident in efficacy in recent years.
(RadiationPhysics and Chemistry such as Chinese patent CN1121876C (at 2003 Granted publication day JIUYUE 24 days) and Yoshii, 1999,55 (2): 1331) adopt the method for crosslinking electron beam irradiation to prepare the dressing of polyvinyl alcohol composite aquogel.But radiative process can cause biological agent degeneration inactivation, and this method is restricted when preparation contains somatomedin dressing.Chinese patent application 200410051638.4 (open day on July 6th, 2005) and Chinese patent application 200510036465.3 (open day on February 15th, 2006) are carried on basic fibroblast growth factor and prepare gel preparation in the carbomer.But the main component of gel-type vehicle is an acrylic resin, has shortcomings such as release is rapid, degraded is difficult, biocompatibility is relatively poor.Therefore, develop that a kind of low price, preparation technology are simple, the diabetic foot chronic ulcer special gel dressing of good biocompatibility is significant.At present, domestic as yet relevant for patent, the article report of the special-purpose dressing of diabetic foot chronic ulcer.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the shortcoming of prior art with not enough, providing a kind of is primary raw material with sodium alginate and polyvinyl alcohol, with calcium chloride and sodium borate is cross-linking agent, adds the diabetic foot chronic ulcer dedicated water gel dressing that recombinant human epidermal growth factor (rh-EGF) and preservative sodium benzoate are prepared from.
Another object of the present invention is to provide the above-mentioned preparation method that contains the aerogel dressing of recombinant human epidermal growth factor.
Another object of the present invention is to provide the above-mentioned application that contains the aerogel dressing of recombinant human epidermal growth factor.
Purpose of the present invention is achieved through the following technical solutions:
The aerogel dressing that contains recombinant human epidermal growth factor is prepared by the composition of following volume parts:
Mixed solution A: 100 parts by volume
Cross-linking agent: 2～8 parts by volume;
Described mixed solution A, prepare by the following method: under 10～40 ℃, polyvinyl alcohol (PVA), sodium alginate (SA) are mixed with aqueous solution, with mass percent 2.0～5.0% polyvinyl alcohol water solutions and mass percent 0.5～2.0% sodium alginate aqueous solution according to (2～5): 1 mixes, stir, add the sodium benzoate of mass percent 0～0.1%, the recombinant human epidermal growth factor (rh-EGF) of 5～20 μ g/g and the glycerol of mass percent 2.0%～10% then, obtain mixed solution A;
Described cross-linking agent is mixed by isocyatic sodium borate aqueous solution and calcium chloride water equal-volume ratio.
The mass fraction of described sodium borate aqueous solution and calcium chloride water is 1.0～2.0%.
The weight average molecular weight of described polyvinyl alcohol (PVA) is 12～150,000.
Described sodium alginate 1% solution viscosity is 200～500mPas.
Described recombinant human epidermal growth factor is commercial, buys the ASIA in CYTOLAB/PEPROTECH.
The above-mentioned preparation method that contains the aerogel dressing of recombinant human epidermal growth factor may further comprise the steps:
Under (1) 10～40 ℃, polyvinyl alcohol, sodium alginate are mixed with aqueous solution, polyvinyl alcohol water solution and sodium alginate aqueous solution were mixed according to mass ratio in 1: 4～4: 1, stir, add sodium benzoate, rh-EGF and glycerol according to proportioning then, obtain mixed solution A;
Under (2) 10～40 ℃, the cross-linking agent of getting 2～8 parts by volume under stirring condition slowly splashes in the mixed solution A of 100 parts by volume, leaves standstill de-bubble, promptly gets the aerogel dressing that contains recombinant human epidermal growth factor.
The gained aerogel dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity scope 1.2～10Pas (37 ℃) of the dressing of different proportionings; Elastic modelling quantity scope 20～50Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its modulus change 0～6%, and viscosity changes 0～10%.
Outward appearance, character, abnormal smells from the patient, pH value have no significant change the rh-EGF aerogel dressing down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
The above-mentioned application that contains the aerogel dressing of recombinant human epidermal growth factor, the application of described aerogel dressing in preparation treatment diabetic foot chronic ulcer medicine.
The present invention has following advantage and beneficial effect with respect to prior art:
(1) the present invention is a primary raw material with sodium alginate and the good macromolecular material of two kinds of biocompatibility of polyvinyl alcohol, adopt in-situ cross-linked technology to prepare the novel diabetic foot chronic ulcer special gel dressing of a class, this dressing has excellent biological compatibility.
(2) aerogel dressing of the present invention molding at ambient temperature, the preparation condition gentleness, technology is simple.
(3) the aerogel dressing of the present invention activity of epidermal growth factor that can effectively keep recombinating, and it is had controlled-release effect.
(4) zoopery shows, aerogel dressing of the present invention has excellent curative to the diabetes rat wound, healing that can accelerated in wounds.
Description of drawings
Fig. 1 is the heart, liver, the kidney segment comparison diagram of high dose group and rats in normal control group, wherein, A is the section of high dose group rat heart, B is the section of high dose group rat liver, C is the section of high dose group rat kidney, D is the section of blank group rat heart, E: the section of blank group rat liver, F: blank group rat kidney section.
Fig. 2 is an intact skin group rat skin irritant reaction photo, and wherein, A is to before the gel, and B is to 48h behind the gel, and C is to 72h behind the gel, and D is to 7d behind the gel, and E is to 14d behind the gel.
Fig. 3 is a damaged skin group rat skin irritant reaction photo, and wherein, A is to before the gel, and B is to 48h behind the gel, and C is to 72h behind the gel, and D is to 7d behind the gel, and E is to 14d behind the gel.
Fig. 4 is a rats in test groups sensitivity response photo, and before wherein A was sensitization, B was 24h after the sensitization, and C is 48h after the sensitization, and D is 72h after the sensitization.
Fig. 5 is the activity detection figure after rh-EGF discharges from dressing.
Fig. 6 is that different pharmaceutical is handled the figure that influences to the rat wound healing effect, and wherein, A is the blank group, and B is the hydrogel matrix group, and C is a rh-EGF hydrogel group, and D is a rh-EGF aqueous solution group.
The specific embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1
Contain the preparation method of the aerogel dressing of recombinant human epidermal growth factor, may further comprise the steps:
Under (1) 40 ℃, get polyvinyl alcohol (PVA) (Shantou Xilong Chemical Factory, Guangdong, molecular weight 12～150,000) be mixed with 5.0% aqueous solution, (east, Wenzhou City, Zhejiang Province rises chemical reagent factory to get sodium alginate, medium viscosity) is mixed with 2.0% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed the polyvinyl alcohol/mixed solution of sodium alginate that is made into 89.95g by 4: 1 (mass ratio), stir, add sodium benzoate (SB) 0.05g then, 1mg rh-EGF (buying ASIA) in CYTOLAB/PEPROTECH, glycerol 10g, mixed dissolution obtains mixed solution A;
Under (2) 40 ℃, sodium borate and calcium chloride are mixed with 1% solution respectively, mixed in 1: 1 by volume and cross-linking agent, getting the 4.0mL cross-linking agent slowly splashes in the 100mL mixed solution A, stir, leave standstill de-bubble, promptly get the aerogel dressing that contains recombinant human epidermal growth factor.
Gained dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity 5.6Pas that the shear viscosity of dressing is (37 ℃); Elastic modelling quantity 38Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its elastic modulus change 4.2 ± 0.3%, and viscosity changes 6.5 ± 0.5%.
The senior rheology expanding system of the ARES/RFS type that adopts U.S. TA Instrument company to produce is measured the steady-state flow variable element (apparent viscosity, shear rate and shear stress) and the dynamic rheological property parameter (elastic modelling quantity, viscous modulus etc.) of aerogel dressing substrate.Select for use the cylinder type mould to test, outside diameter of cylinder is 34.0mm, and internal diameter is 32.0mm, and cylinder heights is 33.0mm.
After adding rh-EGF, outward appearance, character, abnormal smells from the patient, pH value have no significant change hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Embodiment 2
Contain the preparation method of the aerogel dressing of recombinant human epidermal growth factor, may further comprise the steps:
Under (1) 10 ℃, get polyvinyl alcohol (PVA) (Shantou Xilong Chemical Factory, Guangdong, molecular weight 12～150,000) be mixed with 2.0% aqueous solution, (east, Wenzhou City, Zhejiang Province rises chemical reagent factory to get sodium alginate, medium viscosity) is mixed with 0.5% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed the polyvinyl alcohol/mixed solution of sodium alginate that is made into 89.95g by 5: 1 (mass ratio), stir, add sodium benzoate 0.03g then, 1mg rh-EGF (buying ASIA) in CYTOLAB/PEPROTECH, glycerol 2.0g, mixed dissolution obtains mixed solution A;
Under (2) 10 ℃, sodium borate and calcium chloride are mixed with 2% solution respectively, mixed in 1: 1 by volume and cross-linking agent, getting the 2.0mL cross-linking agent slowly splashes in the 100mL mixed solution A, stir, leave standstill de-bubble, promptly get the aerogel dressing that contains recombinant human epidermal growth factor.
Gained dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity of dressing is 2.4Pas (37 ℃); Elastic modelling quantity 30Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its elastic modulus change 2.8 ± 0.4%, and viscosity changes 3.8 ± 0.7%.
After adding rh-EGF, outward appearance, character, abnormal smells from the patient, pH value have no significant change hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Embodiment 3
Contain the preparation method of the aerogel dressing of recombinant human epidermal growth factor, may further comprise the steps:
Under (1) 25 ℃, get polyvinyl alcohol (PVA) and be mixed with 3.0% aqueous solution, get sodium alginate and be mixed with 1.0% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed the polyvinyl alcohol/mixed solution of sodium alginate that is made into 89.95g by 2: 1 (mass ratio), stir, add sodium benzoate 0.04g, 1mgrh-EGF (buying ASIA) then, glycerol 8.0g in CYTOLAB/PEPROTECH, mixed dissolution obtains mixed solution A;
Under (2) 25 ℃, sodium borate and calcium chloride are mixed with 1.5% solution respectively, mixed in 1: 1 by volume and cross-linking agent, getting the 8.0mL cross-linking agent slowly splashes in the 100mL mixed solution A, stir, leave standstill de-bubble, promptly get the aerogel dressing that contains recombinant human epidermal growth factor.
Gained dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity 6.8Pas of the dressing of different proportionings (37 ℃); Elastic modelling quantity 43Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its elastic modulus change 2.9 ± 0.5%, and viscosity changes 4.3 ± 0.4%.
After adding rh-EGF, outward appearance, character, abnormal smells from the patient, pH value have no significant change hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Embodiment 4
Contain the preparation method of the aerogel dressing of recombinant human epidermal growth factor, may further comprise the steps:
Under (1) 10 ℃, get polyvinyl alcohol (PVA) (Shantou Xilong Chemical Factory, Guangdong, molecular weight 12～150,000) be mixed with 2.0% aqueous solution, (east, Wenzhou City, Zhejiang Province rises chemical reagent factory to get sodium alginate, medium viscosity) is mixed with 0.5% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed the polyvinyl alcohol/mixed solution of sodium alginate that is made into 97.9g by 5: 1 (mass ratio), stir, add sodium benzoate 0.1g then, 0.5mg rh-EGF (buying ASIA) in CYTOLAB/PEPROTECH, glycerol 2.0g, mixed dissolution obtains mixed solution A;
Under (2) 10 ℃, sodium borate and calcium chloride are mixed with 2% solution respectively, mixed in 1: 1 by volume and cross-linking agent, getting the 2.0mL cross-linking agent slowly splashes in the 100mL mixed solution A, stir, leave standstill de-bubble, promptly get the aerogel dressing that contains recombinant human epidermal growth factor.
Gained dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity of dressing is 2.4Pas (37 ℃); Elastic modelling quantity 30Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its elastic modulus change 2.8 ± 0.4%, and viscosity changes 3.8 ± 0.7%.
After adding rh-EGF, outward appearance, character, abnormal smells from the patient, pH value have no significant change hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Embodiment 5
Contain the preparation method of the aerogel dressing of recombinant human epidermal growth factor, may further comprise the steps:
Under (1) 25 ℃, get polyvinyl alcohol (PVA) and be mixed with 3.0% aqueous solution, get sodium alginate and be mixed with 1.0% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed the polyvinyl alcohol/mixed solution of sodium alginate that is made into 92g by 2: 1 (mass ratio), stir, add 2mg rh-EGF (buying ASIA) then, glycerol 8.0g in CYTOLAB/PEPROTECH, mixed dissolution obtains mixed solution A;
Under (2) 25 ℃, sodium borate and calcium chloride are mixed with 1.5% solution respectively, mixed in 1: 1 by volume and cross-linking agent, getting the 8.0mL cross-linking agent slowly splashes in the 100mL mixed solution A, stir, leave standstill de-bubble, promptly get the aerogel dressing that contains recombinant human epidermal growth factor.
Gained dressing has the characteristic of shear shinning, at 1.0s
-1Under the shear rate condition, the shear viscosity 6.8Pas of the dressing of different proportionings (37 ℃); Elastic modelling quantity 43Pa (37 ℃); 2 weeks of room temperature are placed in dressing, its elastic modulus change 2.9 ± 0.5%, and viscosity changes 4.3 ± 0.4%.
After adding rh-EGF, outward appearance, character, abnormal smells from the patient, pH value have no significant change hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Embodiment 6 safety testings
Test method: hank year, body weight healthy, skin zero damage is the rat of 200～300g (Zhongshan University, North School Park zoopery center provides, down with) 20, male and female half and half, female should not produce and not have pregnant.Estimate that according to the data of related compound on trial test and the structure aerogel dressing of the present invention can toxigenicity, thus 20 rats are divided into 2 groups, 10 every group, male and female half and half.The 1st group is high dose group, the aerogel dressing of making for embodiment 1.The 2nd group is the blank group, does not give and any medicine.Administration was lost hair or feathers rat back with Mechanical Method in preceding 24 hours, the about 20cm of depilation scope
2Lose hair or feathers and get the aerogel dressing that embodiment 1 makes after 24 hours and evenly be coated with by 5g/kg dosage and high dose group rat back depilation district, cover with gauze and cellophane then, reuse nonirritant adhesive plaster and binder are fixed.Matched group is not smeared, as blank.With warm water or the residual thing that tried of non-stimulated removal of solvents, observe every day, continuous 7～14 days after 24 hours.To note animal whole body poisoning manifestations and death condition after the administration, comprise the variation of the weight of animals, skin, hair, eyes and mucosa.Variations such as breathing, circulation, central nervous system, extremity activity.If meeting dead animal then need perform an autopsy on sb and perusal, when the visible pathological changes of naked eyes, then need carry out pathologic finding.After the off-test, whole rats are put to death core, liver, nephridial tissue carry out pathologic finding.
Result of the test: according to the heart, liver, the kidney segment comparison diagram of Fig. 1 high dose group and rats in normal control group, the high dose group and the matched group heart, liver, nephropathy reason are checked and be there is no unusually as can be known, illustrate that prepared dressing has good biological safety.
Embodiment 7 rat skin irritant tests
Test method: hank year, body weight healthy, skin zero damage is 30 of the rats of 200～300g (it is the same to originate), male and female half and half, female should not produce and not have pregnant.30 rats are divided into intact skin group, damaged skin group and matched group, every group each 10, male and female half and half.Test and its spinal column both sides, back were lost hair or feathers the about 20cm of depilation scope with Mechanical Method in preceding 24 hours
2The mice of damaged skin group is scratched with the skin of disinfectant injection needle with its depilation district, so that oozing of blood degree of being to be arranged.
Lose hair or feathers get after 24 hours aerogel dressing that embodiment 1 makes by 5g/kg dosage evenly in depilation district, two rats in test groups backs, cover with gauze and preservative film then, reuse nonirritant adhesive plaster and binder are fixed.Matched group is not smeared, as blank.With warm water or the residual thing that tried of non-stimulated removal of solvents, observe at once after 24 hours.Behind 1h, 24h, 48h, 72h, observe medicine-feeding part irritant reaction situation then once more.Observe every day, continuous 7～14 days.
Every rat observed result is carried out the irritant reaction scoring by table 1, calculates mean scores and carries out the stimulus intensity evaluation by table 2 again.
Table 1 skin irritation reaction scoring
Reaction meansigma methods=erythema forms total points+edema and forms total points/total number of animals
Table 2 skin irritation intensity evaluation
Result of the test (see Table 3, Fig. 2, Fig. 3):
Table 3 intact skin group and damaged skin group rat skin irritant reaction scoring meansigma methods
Embodiment 8 rat skin sensitization test (STT)s
Hank year, body weight healthy, skin zero damage is 20 of the rats of 200～300g (it is the same to originate), male and female half and half, female should not produce and not have pregnant.Rat is divided into 2 groups at random by body weight, 10 every group, male and female half and half.First group is test group, the aerogel dressing of making for embodiment 1, and second group is the blank group, does not give and any medicine.
1. sensitization contact: the about 3cm * 3cm of depilation scope is lost hair or feathers in both sides, rats in test groups back with Mechanical Method in preceding 24 hours of test.Get the aerogel dressing 0.1g that embodiment 1 makes and be coated in depilation district, back part of animal right side, cover with gauze and preservative film, the immobilization with adhesive tape of reuse nonirritant continues 6 hours.The 7th day and fortnight in kind repeat once.
2. excite contact: after last is given aerogel dressing sensitization 14 days, will be tried thing 0.1g and be applied to depilation district, left side, rats in test groups back, remove behind the 6h and tried thing, observe at once, observe the skin allergy situation once more in 24h, 48h, 72h then.Press table 3 scoring, judge and tried thing skin allergy character.For reaction is tried the sensitization intensity of thing, can judge its sensitization rate by the classification of table 4.Can judge its sensitization rate by the classification of table 4.
The standards of grading of table 4 skin allergy degree
Table 5 sensitization of skin evaluation mark
Result of the test (see Table 6, Fig. 4):
Table 6 rats in test groups sensitization of skin reaction meansigma methods
Release mode and the stability observing of embodiment 9rh-EGF in aerogel dressing
1, the aerogel dressing that makes according to every gram embodiment 1 ratio that adds 10 μ g rh-EGF prepares aerogel dressing.
2, adopt the inversion method that 5g rh-EGF aerogel dressing is put into and contain 100ml PBS liquid beaker, respectively at 5min, 10min, 20min, 30min, 1hr, 1.5hr, 2hr, 4hr, 6hr, 8hr, 10hr, 12hr, 24hr are at the same position 0.5ml that takes a sample, and each sampling back replenishes commensurability equality of temperature PBS.
3, adopt double-antibody sandwich ABC-ELISA method carry out concentration detect (test kit source: Senxiong Science ﹠ Technology Industry Co., Ltd., Shanghai):
(1) sets up standard curve: establish gauge orifice 8 holes, adding sample diluting liquid in every hole (refers in above-mentioned experiment, the releasing product of gel dressing in medium (got a certain amount of release liquid, dilution then)) 100 μ l, first hole adds the rh-EGF 100 μ l of concentration known, with sample injector sucking-off 100 μ l, move to second hole behind the mixing.Oppose so repeatedly and doubly be diluted to seven apertures in the human head, last, sucking-off 100 μ l discard from seven apertures in the human head, and making it volume is 100 μ l.Octal is a blank.
(2) application of sample: every hole, product to be tested hole adds sample diluting liquid and respectively at 5min, 10min, 20min, 30min, 1hr, 1.5hr, 2hr, 4hr, 6hr, 8hr, 10hr, each 50 μ l of the sample that 12hr, 24hr obtain.
(3) will place 120 minutes down at 37 ℃ behind the abundant mixing of Sptting plate.
(4) wash plate: with Sptting plate thorough washing 4-6 time, seal is dried on filter paper with cleaning mixture (mass fraction 0.05%Tween20-PBS).
(5) every hole adds first antibody working solution (biotinylated anti-people EGF antibody) 50 μ l, and Sptting plate was placed 60 minutes down at 37 ℃.
(6) wash plate: the same.
(7) every hole adds enzyme labelled antibody working solution (the Streptavidin 100 μ l of horseradish peroxidase-labeled.
(8) Sptting plate was placed 60 minutes down at 37 ℃.
(9) wash plate: the same.
(10) every hole adds substrate working solution (horseradish peroxidase substrate OPD) 100 μ l, puts 37 ℃ of dark place reactions 5-10 minute.
(11) every hole adds 1 termination liquid (2mol/L sulphuric acid) mixing.
(12) survey light absorption value at the 492nm place.
4, calculate the accumulative total release rate.The concentration * 100% that accumulative total release rate=actual concentration that records/in theory discharges fully
5, making afterwards respectively at the rh-EGF hydrogel, 1d, 7d, 14d detect, and outward appearance, character, abnormal smells from the patient, pH value change the stability of assessment rh-EGF hydrogel down in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d) to observe the rh-EGF hydrogel at duration of test.
6, repeated experiments is 2 times.
Result of the test (seeing Table 7):
24 hours accumulative total release rates (%) behind the table 7rh-EGF hydrogel storage different time
Outward appearance, character, abnormal smells from the patient, pH value have no significant change down 2.rh-EGF hydrogel is in different temperatures (4 ℃, 25 ℃, 37 ℃), different storage times (1d, 7d, 14d).
Activity after embodiment 10rh-EGF discharges in aerogel dressing detects
Test method:
1. get each 13 time point of 1d, 7d in the foregoing description 7,14d respectively, discharge liquid 0.4ml add 3.6ml contain in the DMEM culture medium (Sigma-AldrichCompany) of 10% (volume fraction) calf serum (contain rh-EGF 1～10ng/ml), standby with the filtration of 0.22 μ m filter.
2.Balb/c3T3 cell (deriving from Chinese Academy of Sciences's cell bank) is inoculated in 96 orifice plates after going down to posterity.
3. get above-mentioned standby release liquid (promptly respectively at 5min, 10min, 20min, 30min, 1hr, 1.5hr, 2hr, 4hr, 6hr, 8hr, 10hr, the release liquid that 12hr, 24hr obtain) each 200 μ l adding has been inoculated in 96 orifice plates of Balb/c3T3 cell, establishes positive controls simultaneously and (contains rh-EGF standard substance 1～64ng/ml in the culture medium, be respectively 1ng/ml, 4ng/ml, 16ng/ml, 64ng/ml, each concentration is established 3 holes) each 12 hole of negative control group (not containing rh-EGF in the culture medium).In 37 ℃ of 5%CO
2Environment was cultivated 48 hours down.
4.MTT method is in 492nm wavelength place detection of active: cell culture added MTT (5g/L) 20 μ l after 48 hours, continued to cultivate 4 hours, removed supernatant, added DMSO 150 μ l, detected absorbance in 492nm wavelength place.
Result of the test is seen Fig. 5, Fig. 5 is the active testing result after rh-EGF discharges in embodiment 1 dressing, and the average activity (absorbance) of experimental group (the release liquid of the rh-EGF hydrogel behind storage 1d, 7d, the 14d is respectively 1 day group, 7 days groups, 14 days groups), positive controls, negative control group is respectively 0.90 ± 0.10,0.86 ± 0.10,0.80 ± 0.14,0.87 ± 0.15,0.55 ± 0.10.The activity of the short BALB/c3T3 propagation of experimental group and positive controls all is higher than negative control group (p＜0.05).Illustrate that dressing substrate can effectively keep the activity of rh-EGF.
Embodiment 11 contains rh-EGF dressing to diabetes rat Wound healing and bone regeneration effect test
Test method:
Get 56 250～300 gram cleaning level SD rats (available from Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center), after adaptability raised for 1 week, 3d intraperitoneal injection 35mg/kg STZ induced diabetes continuously.Tail vein blood detects the random blood sugar level behind the injection 3d, if rat random blood sugar level 〉=16.7mmol/L is considered as diabetes model and sets up successfully.
The diabetes rat of choosing the modeling success continues to feed, and ad lib, drinking-water during the nursing are monitored body weight and blood glucose weekly, and according to blood sugar level subcutaneous injection isophane insulin (NPH) 1～2U/d, blood glucose maintains 16.7mmol/L～28mmol/L.
Diabetes rat becomes mould after 6 weeks, pentobarbital 30mg/kg intraperitoneal injection of anesthesia, the back cropping, 75% alcohol disinfecting, the sterilization template that with the length of side is 1.3cm is as a token of with the square wound of scalpel formation 1.3cm * 1.3cm in 1cm place behind the positive middle distance skull in Mus back, excision extension reaches fascia deeply, and wound forms the single cage of every the rat in back and raises.
The difference that laboratory animal is handled factor by wound surface divides 4 to organize (seeing Table 8) greatly.Blank group: disregard; Hydrogel matrix group: exterior-applied liquid medicine gel-type vehicle every day (" hydrogel matrix " is meant that the aerogel dressing that embodiment 1 makes does not comprise rh-EGF) each wound surface of 0.5g/; Rh-EGF hydrogel group: each wound surface of rh-EGF hydrogel 0.5g/ that external every day embodiment 1 makes (containing rh-EGF 5 μ g); Rh-EGF aqueous solution group: each wound surface of external every day rh-EGF aqueous solution 0.5ml/ (containing rh-EGF 5 μ g).
Table 8 becomes 28 groupings of the male SD diabetes rat of mould
Form back (the wound size is measured in the treatment of medication on one side on one side) 0d, 3d, 5d, 7d, 10d, 14d at wound respectively and wound is taken that (A610 Canon), places a number that the scale ruler is arranged and indicate rat simultaneously with digital camera.Wound area calculates with Image J (1.36b version).
The each measurement repeated 2 times, gets its average.
Result of the test compares the wound healing rate and finds that the difference of 4 groups of healing rates has statistical significance (p＜0.05) as shown in Figure 6.It is all the highest at each time point healing rate to contain rh-EGF hydrogel group.Wound forms back the 3rd, 5d hydrogel processed group (B, C) healing rate is higher than blank group (p＜0.05); 10d hydrogel processed group (B, C) is better than non-aqueous gel processed group (A, D) (p＜0.05); Three treatment groups of 14d all are better than blank group (p＜0.05).The dressing that contains rh-EGF can fast and effeciently promote the wound healing (seeing Table 9) of diabetes rat.
4 groups of healing rates of table 9 different time (
%)
Annotate: * and A group compare p＜0.05; △ and D group compare p＜0.05.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. contain the aerogel dressing of recombinant human epidermal growth factor, it is characterized in that: the composition by following volume parts prepares:
Mixed solution A: 100 parts by volume
Cross-linking agent: 2～8 parts by volume;
Described mixed solution A, prepare by the following method: under 10～40 ℃, with mass percent 2.0～5.0% polyvinyl alcohol water solutions and mass percent 0.5～2.0% sodium alginate aqueous solution according to mass ratio (2～5): 1 mixes, stir, add the sodium benzoate of mass percent 0～0.1%, the recombinant human epidermal growth factor of 5～20 μ g/g and the glycerol of mass percent 2.0～10% then, obtain mixed solution A.
2. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: described cross-linking agent is mixed by isocyatic sodium borate aqueous solution and calcium chloride water equal-volume ratio;
The mass fraction of described sodium borate aqueous solution and calcium chloride water is 1.0～2.0%.
3. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: the weight average molecular weight of described polyvinyl alcohol is 12～150,000.
4. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: described sodium alginate 1% viscosity in aqueous solution is 200～500mPas.
5. the described preparation method that contains the aerogel dressing of recombinant human epidermal growth factor of claim 1 is characterized in that may further comprise the steps:
Under (1) 10～40 ℃, polyvinyl alcohol, sodium alginate are mixed with aqueous solution, with mass percent 2.0～5.0% polyvinyl alcohol water solutions and mass percent 0.5～2.0% sodium alginate aqueous solution according to mass ratio (2～5): 1 mixes, stir, add sodium benzoate, rh-EGF and glycerol according to proportioning then, obtain mixed solution A;
Under (2) 10～40 ℃, the cross-linking agent of getting 2～8 parts by volume under stirring condition slowly splashes in the mixed solution A of 100 parts by volume, leaves standstill de-bubble, promptly gets the aerogel dressing that contains recombinant human epidermal growth factor.
6. the described application of aerogel dressing in preparation treatment diabetic foot chronic ulcer medicine that contains recombinant human epidermal growth factor of claim 1.
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| CN105287364A (en) * | 2015-11-24 | 2016-02-03 | 无锡中科光远生物材料有限公司 | Drug-loaded hydrogel coating, and making method and application thereof |
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| CN106188350A (en) * | 2016-07-21 | 2016-12-07 | 广东工业大学 | Polyvinylalcohol graft polymer, its preparation method and hydrogel and application thereof |
| CN106492260A (en) * | 2016-12-22 | 2017-03-15 | 青岛琛蓝海洋生物工程有限公司 | A kind of alginate based aquagel dressing and preparation method thereof |
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| CN106188350A (en) * | 2016-07-21 | 2016-12-07 | 广东工业大学 | Polyvinylalcohol graft polymer, its preparation method and hydrogel and application thereof |
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