CN103446617A - Gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold and preparation method - Google Patents
Gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold and preparation method Download PDFInfo
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Abstract
The invention discloses a gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold and a preparation method, belonging to the technical field of high molecular materials. The preparation method comprises the following steps: dropwise adding gentamicin sulfate solution with concentration of 10-50 mg/ml into gelatin microspheres, placing overnight at the temperature of 4-25 DEG C and then performing freeze drying to obtain a gentamicin sulfate/gelatin microsphere complex; adding the gentamicin sulfate/gelatin microsphere complex into 2-10 percent by weight of silk fibroin solution, uniformly stirring and then performing freeze drying to obtain the gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin porous composite scaffold, wherein the porosity of the gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold is 75-95 percent, the aperture of the gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold is 50-200 mum, and the gentamicin sulfate/gelatin microsphere complex-loaded silk fibroin scaffold has long-acting antibacterial and drug slow-release functions and can be applied to the fields of skin wound healing and wound dressing.
Description
Technical field
The invention belongs to technical field of polymer materials, particularly fibroin albumen support and the preparation method of a kind of load gentamycin sulfate/gelatine microsphere complex.
Background technology
Skin is the organ of human body maximum, mainly is divided into epidermal area, skin corium and subcutaneous tissue, human body is had to the function of important barrier, machinery, perception and regulation and control.But skin is one of human body most fragile and organ of the most easily coming to harm, and skin ulceration, wound, surgical injury and burn are the major reasons of skin damage, wherein burn is the main cause of skin damage.According to statistics, the number that burn is died from the whole world every year is about 300,000, and the fire victim in China every year is about 1,000 ten thousand, and wherein 3,200,000 need skin transplantation.Infection is the key factor of restriction burned skin healing, because infect the normal processes that can change wound repair, all very harmful to burn and other injury types.In the fire victim, infect the microenvironment of not only having destroyed wound healing, sometimes even can cause serious whole body complication.Burn according to estimates in fatality rate and approximately have 75% to be by due to infecting.In order to address this problem, research worker has turned to sight on research and development dressing for skin or substitute, thereby hope can reduce the problem that skin wound is brought or can infection of infecting, and promotes skin wound healing.
Fibroin albumen is a kind of hydrophobic protein, the mechanical property excellence, and Stability Analysis of Structures, have good biocompatibility, and source is abundant, with low cost, is a kind of good natural macromolecular material.Based on these advantages of fibroin albumen, it is widely used on field of tissue engineering technology always, aspects such as repair of cartilage, reticular connective tissue reparation, bone reparation and neural reparation.There are some researches show, can promote fibroblastic increment with fibroin albumen, differentiation, skin wound healing is had to facilitation, so fibroin albumen can be used as dressing for skin, but, because fibroin albumen itself does not have anti-microbial property, this use to fibroin albumen limits to some extent, therefore, prepare a kind of fibroin albumen support with antibacterial ability and become problem demanding prompt solution.
Gentamycin sulfate is a kind of broad-spectrum antibiotic, all very effective to gram positive bacteria and negative bacterium, being widely used in responsive microbial system infects or local infection, but long-term taking or blood drug level in vivo are too high, can bring a series of toxic and side effects, for example ear is poisoning, nephrotoxicity, hematuria, even after anaphylactic shock, causes death, has reduced clinical therapeutic efficacy.For the dosage that reduces gentamycin sulfate with in the toxic and side effects of human body, but guarantee its drug effect, to reaching the purpose of efficient, low toxicity, low-residual, must develop a kind of novel pharmaceutical carrier system.
Gelatin, have good biocompatibility, extensively has been used on the aspects such as biomedical and pharmaceutical carrier, and the microsphere particularly prepared by gelatin, become a kind of very ripe pharmaceutical carrier system.
In recent years, utilize fibroin albumen as three-dimensional rack, the application and research on dressing for skin becomes focus, there are some researches show, with fibroin albumen, as dressing for skin, in the reparative experiment of the holostrome skin injury of mice, obtains good effect; And gelatine microsphere extensively utilizes already on biomedical and pharmaceutical carrier; On the other hand, how improving the dosing mode of gentamycin sulfate at human body, reduce blood drug level and the effect that reaches slow release, is also the problem that research worker is paid close attention to.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides the preparation method of the fibroin albumen support of a kind of load gentamycin sulfate/gelatine microsphere complex.Preparation method of the present invention be by gentamycin sulfate and the compound back loading of gelatine microsphere on silk fibroin porous scaffold, through cryodesiccated method, form compound rest.
Another object of the present invention is to provide the fibroin albumen support of the load gentamycin sulfate that obtained by above-mentioned preparation method/gelatine microsphere complex.Compound rest prepared by the present invention has drug slow release function, can reduce the injury to human body brought due to high blood drug level, and this support antibacterial effect is obvious.
A further object of the present invention is the application of the fibroin albumen support of the load gentamycin sulfate that provides above-mentioned/gelatine microsphere complex.
Purpose of the present invention is achieved through the following technical solutions: the preparation method of the fibroin albumen support of a kind of load gentamycin sulfate/gelatine microsphere complex comprises the steps:
(1) be that 10～50mg/ml gentamicin sulfate solution is added drop-wise in gelatine microsphere by concentration, spend the night in 4～25 ℃ of placements, then lyophilization, obtain gentamycin sulfate/gelatine microsphere complex; Wherein to add the amount of gentamicin sulfate solution be 2～10ml to every gram gelatine microsphere;
(2) gentamycin sulfate of step (1)/gelatine microsphere complex is added in 2～10wt% silk fibroin protein solution, stir, then lyophilization, obtain the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex; Every milliliter of silk fibroin protein solution adds 10mg gentamycin sulfate/gelatine microsphere complex;
In step (1):
Described 10～50mg/ml gentamicin sulfate solution preferably adopts following methods to be prepared: gentamycin sulfate is dissolved in deionized water, stirs, obtaining concentration is 10～50mg/ml gentamicin sulfate solution;
The particle diameter of described gelatine microsphere is preferably 5～30 μ m, and expansion rate is preferably 300～500%;
Described gelatine microsphere preferably adopts following methods to be prepared: by 0.1g sorbitol anhydride oleate (Span-80, Span80) be added in the flask that fills the 100ml vegetable oil, after 60 ℃ of stirring 0.5～5h, by the mode dripped, by 10ml, concentration is that the 10wt% aqueous gelatin solution joins in vegetable oil, after continuing to stir 180min, solution is transferred to ice bath, continue to stir 30min, then add 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 30ml, stir 30min, standing, filter, obtain microsphere; The microsphere bubble, in 10ml acetone, is soaked to 30min with the 1mol/L glycine after 4 ℃ of curing 24h, centrifugal, get precipitation, with ethanol and isopropyl alcohol, replace washing precipitate, the precipitate after cleaning is placed in to the deionized water soaked overnight, lyophilization, obtain gelatine microsphere;
The speed of described stirring is preferably 300～400r/min;
Described centrifugal condition optimization is in 25 ℃, the centrifugal 10min of 10000r/min;
Described cryodesiccated condition optimization is-60 ℃ of lyophilization 24h;
In step (2):
Described 2～10wt% silk fibroin protein solution preferably adopts following methods to be prepared: Bombyx bombycis is shredded, then add 100 ℃, concentration is 0.2wt%NaCO
3boil 2h in aqueous solution, NaCO
3the ratio of aqueous solution and Bombyx bombycis is: the 0.2wt%NaCO of 100ml
3aqueous solution adds 5g Bombyx bombycis; Come unstuck rear extremely neutral with the washed with de-ionized water fibroin, in 60 ℃ of oven dry, obtain fibroin albumen; Get fibroin albumen, add in the 9.8mol/L lithium bromide water solution, with deionized water dialysis 2～4 days, centrifugal after 40～60 ℃ of heating in water bath 3～5h, obtain 2～10wt% silk fibroin protein solution;
Described Bombyx bombycis is preferably the Bombyx bombycis of pupa;
Described dialysis preferably adopts the bag filter that molecular cut off is 8000～12000 to be dialysed;
Described centrifugal condition optimization is in 25 ℃, the centrifugal 10min of 10000r/min;
Described silk fibroin protein solution concentration is 3～7wt% silk fibroin protein solution more preferably;
Described cryodesiccated condition optimization is-60 ℃ of lyophilization 24h;
The fibroin albumen support of a kind of load gentamycin sulfate/gelatine microsphere complex, obtained by above-mentioned preparation method; The fibroin albumen brace aperture rate of described load gentamycin sulfate/gelatine microsphere complex is preferably 75～95%, and aperture is preferably 50～200 μ m;
The fibroin albumen support of described load gentamycin sulfate/gelatine microsphere complex should be applied in fields such as skin wound healing and wound dressings.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention on silk fibroin porous scaffold, can reach medicament slow release by gentamycin sulfate and the compound back loading of gelatine microsphere, can reduce the injury to human body brought due to high blood drug level, and this support antibacterial range is wide, successful.The gelatine microsphere particle diameter prepared is 5～30 μ m, and expansion rate is 300～500%; The fibroin albumen brace aperture rate prepared is 75～95%, and aperture is 50～200 μ m.
(2) preparation technology of the present invention is simple, material source is extensive, and production efficiency is high, and cost is low, can be applicable to industrialized great production.
The accompanying drawing explanation
Fig. 1 is comparative example 1 silk fibroin porous scaffold scanning electron microscope (SEM) photograph.
Fig. 2 is the scanning electron microscope (SEM) photograph of the gelatine microsphere of embodiment 1.
Fig. 3 is the scanning electron microscope (SEM) photograph of fibroin albumen support of the load gentamycin sulfate/gelatine microsphere complex of embodiment 1.
Fig. 4 is the gentamycin sulfate release graphics of the fibroin albumen support of comparative example 2 the silk fibroin porous compound rest of load gentamycin sulfate and the load gentamycin sulfate of embodiment 1/gelatine microsphere complex; Wherein: the gentamycin sulfate release graphics of the silk fibroin porous compound rest of the load gentamycin sulfate that a is comparative example 2; The gentamycin sulfate release graphics of the fibroin albumen support of the load gentamycin sulfate that b is embodiment 1/gelatine microsphere complex.
Fig. 5 is that the fibroin albumen support of comparative example 1 silk fibroin porous scaffold, comparative example's 2 the silk fibroin porous compound rest of load gentamycin sulfate and the load gentamycin sulfate of embodiment 1/gelatine microsphere complex is to colibacillary antibiotic ring design sketch; Wherein: the antibacterial effect figure of the silk fibroin porous scaffold that a is comparative example 1; The antibacterial effect figure of the fibroin albumen support of the load gentamycin sulfate that b is embodiment 1/gelatine microsphere complex; The antibacterial effect figure of the silk fibroin porous compound rest of the load gentamycin sulfate that c is comparative example 2.
Fig. 6 is the antibiotic ring design sketch of the fibroin albumen support of comparative example 1 silk fibroin porous scaffold, comparative example's 2 the silk fibroin porous compound rest of load gentamycin sulfate and the load gentamycin sulfate of embodiment 1/gelatine microsphere complex to staphylococcus aureus; Wherein: the antibacterial effect figure of the silk fibroin porous scaffold that a is comparative example 1; The antibacterial effect figure of the fibroin albumen support of the load gentamycin sulfate that b is embodiment 1/gelatine microsphere complex to staphylococcus aureus; The antibacterial effect figure of the silk fibroin porous compound rest of the load gentamycin sulfate that c is comparative example 2.
Fig. 7 is comparative example 1 silk fibroin porous scaffold, comparative example's 3 silk fibroin porous compound rest, comparative example's 3 the fibroin albumen support of load vancomycin/gelatine microsphere complex of load vancomycin; Wherein: the antibacterial effect figure of the silk fibroin porous scaffold that a is comparative example 1; The silk fibroin porous scaffold of the load vancomycin that d is comparative example 3/gelatine microsphere complex is to colibacillary antibacterial effect figure; The silk fibroin porous compound rest of the load vancomycin that e is comparative example 3 is to colibacillary antibacterial effect figure.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) the Bombyx bombycis 5g that will remove pupa is at the 0.2wt%NaCO of 100 ℃
3boil 2h in aqueous solution 100ml, come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven that is placed in 60 ℃ is dried, and obtains fibroin albumen; Get in the lithium bromide water solution that the 2.5g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 5h, with the deionized water dialysis, (molecular cut off of bag filter is 8000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 3.5wt% silk fibroin protein solution;
(2) 0.1g Span80 is added in the flask of the vegetable oil that fills 100ml, after stirring 0.5h, 60 ℃, 400r/min add 10wt% aqueous gelatin solution 10ml, after continuing to stir 180min, ice bath stirs 15min, then adds 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 40ml, stir 15min, standing, filter, obtain microsphere; Microsphere is steeped in 10ml acetone, soak 30min with the 1mol/L glycine after 4 ℃ of curing 24h, then in 25 ℃, the centrifugal 10min of 10000r/min, get precipitation, the precipitation of alternately washing with ethanol and isopropyl alcohol, precipitation is placed in to the deionized water soaked overnight, and-60 ℃ of lyophilization 24h, obtain gelatine microsphere; As shown in Figure 2, the gelatine microsphere particle diameter is 5～30 μ m, and expansion rate is 400～500%;
(3) gentamycin sulfate of 40 μ l, 25mg/ml is dripped in the gelatine microsphere of 0.1g step (2), spend the night in 4 ℃ of placements ,-60 ℃ of lyophilization 24h, obtain gentamycin sulfate/gelatine microsphere complex;
(4) gentamycin sulfate of 10mg step (3)/gelatine microsphere complex is added in the 3.5wt% silk fibroin protein solution of 1ml step (1), stir,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex; The scanning electron microscope (SEM) photograph of the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex as shown in Figure 3;
(5) the silk fibroin porous compound rest of the load gentamycin sulfate of step (4)/gelatine microsphere complex is placed in to the PBS solution of pH=7.4, while just starting, every 4h, sample once, after 12h, every 12h, sample once, after 24h, every 24h, sample once, every sub-sampling 2ml, the releasing effect of test gentamycin sulfate; As shown in Figure 4, as can be seen from Figure 4, load gentamycin sulfate/gelatine microsphere complex has the medicament slow release effect to result, and pharmaceutical release time reaches 144h;
(6) the silk fibroin porous compound rest of the load gentamycin sulfate of step (4)/gelatine microsphere complex is placed in respectively on the solid medium that scribbles escherichia coli and staphylococcus aureus, 37 ℃ of placements are spent the night, carry out antibiotic ring experiment, observe the antibacterial effect of the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex; Result as shown in Figure 5 and Figure 6; The difference diameter of antibiotic ring is: 23.1mm and 24.1mm, from Fig. 5 and Fig. 6, can find out, and the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex has antibacterial effect preferably.
Embodiment 2
(1) will go the Bombyx bombycis 5g of pupa to boil 2h in the 0.2wt%NaCO3 aqueous solution 100ml of 100 ℃, and come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven oven dry that is placed in 60 ℃ obtains fibroin albumen; Get in the lithium bromide water solution that the 1g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 3h, with the deionized water dialysis, (molecular cut off of bag filter is 10000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 2wt% silk fibroin protein solution;
(2) 0.1g Span80 is added in the flask of the vegetable oil that fills 100ml, after stirring 2.5h, 60 ℃, 400r/min add 10wt% aqueous gelatin solution 10ml, after continuing to stir 180min, ice bath stirs 15min, then add 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 40ml, stir 15min, standing, filter, obtain microsphere; Microsphere is steeped in 10ml acetone, soak 30min with the 1mol/L glycine after 4 ℃ of curing 24h, then in 25 ℃, the centrifugal 10min of 10000r/min, get precipitation, the precipitation of alternately washing with ethanol and isopropyl alcohol, precipitation is placed in to the deionized water soaked overnight, and-60 ℃ of lyophilization 24h, obtain gelatine microsphere;
(3) gentamycin sulfate of 20 μ l, 50mg/ml is dripped in the gelatine microsphere of 0.1g step (2), spend the night in 4 ℃ of placements ,-60 ℃ of lyophilization 24h, obtain gentamycin sulfate/gelatine microsphere complex;
(4) gentamycin sulfate of 10mg step (3)/gelatine microsphere complex is added in the 2wt% silk fibroin protein solution of 1ml step (1), stir,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex.
Embodiment 3
(1) the Bombyx bombycis 5g that will remove pupa is at the 0.2wt%NaCO of 100 ℃
3boil 2h in aqueous solution 100ml, come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven that is placed in 60 ℃ is dried, and obtains fibroin albumen; Get in the lithium bromide water solution that the 7.5g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 4h, with the deionized water dialysis, (molecular cut off of bag filter is 12000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 10wt% silk fibroin protein solution;
(2) 0.1g Span80 is added in the flask of the vegetable oil that fills 100ml, after stirring 5h, 60 ℃, 400r/min add 10wt% aqueous gelatin solution 10ml, after continuing to stir 180min, ice bath stirs 15min, then add 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 40ml, stir 15min, standing, filter, obtain microsphere; Microsphere is steeped in 10ml acetone, soak 30min with the 1mol/L glycine after 4 ℃ of curing 24h, then in 25 ℃, the centrifugal 10min of 10000r/min, get precipitation, precipitation by ethanol and washed with isopropyl alcohol, precipitation is placed in to the deionized water soaked overnight, and-60 ℃ of lyophilization 24h, obtain gelatine microsphere;
(3) gentamycin sulfate of 100 μ l, 10mg/ml is dripped in the gelatine microsphere of 0.1g step (2), spend the night in 4 ℃ of placements ,-60 ℃ of lyophilization 24h, obtain gentamycin sulfate/gelatine microsphere complex;
(4) gentamycin sulfate of 10mg step (3)/gelatine microsphere complex is added in the 10wt% silk fibroin protein solution of 1ml step (1), stir,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous compound rest of load gentamycin sulfate/gelatine microsphere complex.
The comparative example 1
(1) will go the Bombyx bombycis 5g of pupa to boil 2h in the 0.2wt%NaCO3 aqueous solution 100ml of 100 ℃, and come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven that is placed in 60 ℃ is dried, and obtains fibroin albumen; Get in the lithium bromide water solution that the 2.5g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 5h, with the deionized water dialysis, (molecular cut off of bag filter is 10000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 3.5wt% silk fibroin protein solution;
(2) get the 3.5wt% silk fibroin protein solution of step (1) ,-60 ℃ of lyophilization 24h, obtain silk fibroin porous scaffold; The porosity of silk fibroin porous scaffold is 75～95%, and aperture is 50～200 μ m; The scanning electron microscope (SEM) photograph of silk fibroin porous scaffold as shown in Figure 1;
(3) silk fibroin porous scaffold of step (2) is put and is placed in respectively on the solid medium that scribbles escherichia coli and staphylococcus aureus, 37 ℃ of placements are spent the night, and carry out antibiotic ring experiment, observe the antibacterial effect of fibroin albumen support; From antibiotic ring experiment, can see, silk fibroin porous scaffold does not have antibacterial effect, and result is as Fig. 5 a, shown in Fig. 6 a, Fig. 7 a.
The comparative example 2
(1) the Bombyx bombycis 5g that will remove pupa is at the 0.2wt%NaCO of 100 ℃
3boil 2h in aqueous solution 100ml, come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven that is placed in 60 ℃ is dried, and obtains fibroin albumen; Get in the lithium bromide water solution that the 2.5g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 5h, with the deionized water dialysis, (molecular cut off of bag filter is 8000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 3.5wt% silk fibroin protein solution;
(2) 40 μ l, 25mg/ml gentamycin sulfate are mixed with the silk fibroin protein solution of the step (1) of 1ml ,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous compound rest that load has gentamycin sulfate; The porosity of silk fibroin porous scaffold is 75～95%, and aperture is 50～200 μ m;
(3) the silk fibroin porous compound rest of the load gentamycin sulfate of step (2) is put to the PBS solution that is placed in respectively pH=7.4, at set intervals, the releasing effect of sampling 2ml test gentamycin sulfate; Result as shown in Figure 4, can be seen by Fig. 4, and be 48h the release time of the gentamycin sulfate of the silk fibroin porous compound rest of load gentamycin sulfate; Due to gentamycin sulfate by directly being added drop-wise on the fibroin albumen support, so the silk fibroin porous compound rest of load gentamycin sulfate does not have slow-release function, slow release effect is very weak in other words, so, in 4h, release rate approaches 80%, and discharge fully after 48h.
(5) the silk fibroin porous compound rest of the load gentamycin sulfate of step (2) is positioned over respectively on the solid medium that scribbles escherichia coli and staphylococcus aureus, 37 ℃ of placements are spent the night, carry out antibiotic ring experiment, observe the antibacterial effect of the silk fibroin porous compound rest of load gentamycin sulfate; The diameter of antibiotic ring is respectively 24.1mm and 25.1mm, and result as shown in Figure 5 and Figure 6; From Fig. 5 and Fig. 6, can see, the antibacterial effect of the silk fibroin porous compound rest of load gentamycin sulfate has certain antibacterial effect.Because gentamycin sulfate directly mixes with silk fibroin protein solution, through lyophilization, be prepared from, therefore the burst size of gentamycin sulfate is large, so its antibacterial effect is more superior than the silk fibroin porous compound rest of gentamycin sulfate/gelatine microsphere complex, but both reach antibiotic requirement, and the gentamycin sulfate/silk fibroin porous compound rest of gelatine microsphere complex has the function of slow releasing pharmaceutical, so its long-term antibacterial effect can be more superior.
The comparative example 3
(1) the Bombyx bombycis 5g that will remove pupa is at the 0.2wt%NaCO of 100 ℃
3boil 2h in aqueous solution 100ml, come unstuck rear extremely neutral with the washed with de-ionized water fibroin, the baking oven that is placed in 60 ℃ is dried, and obtains fibroin albumen; Get in the lithium bromide water solution that the 2.5g fibroin albumen adds 25ml, 9.8mol/L, after 60 ℃ of heating in water bath 5h, with the deionized water dialysis, (molecular cut off of bag filter is 8000, dialysis time is 3 days, change water every day once), then in 25 ℃, the centrifugal 10min of 10000r/min, obtain the 3.5wt% silk fibroin protein solution;
(2) 0.1g Span80 is added in the flask of the vegetable oil that fills 100ml, after stirring 2.5h, 60 ℃, 400r/min add 10wt% aqueous gelatin solution 10ml, after continuing to stir 180min, ice bath stirs 15min, then add 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 40ml, stir 15min, standing, filter, obtain microsphere; Microsphere is steeped in 10ml acetone, soak 30min with the 1mol/L glycine after 4 ℃ of curing 24h, then in 25 ℃, the centrifugal 10min of 10000r/min, get precipitation, the precipitation of alternately washing with ethanol and isopropyl alcohol, precipitation is placed in to the deionized water soaked overnight, and-60 ℃ of lyophilization 24h, obtain gelatine microsphere;
(3) vancomycin of 20 μ l, 50mg/ml is dripped in the gelatine microsphere of 0.1g step (2), spend the night in 4 ℃ of placements ,-60 ℃ of lyophilization 24h, obtain vancomycin/gelatine microsphere complex;
(4) 40 μ l, 25mg/ml vancomycin are mixed with the silk fibroin protein solution of the step (1) of 1ml ,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous compound rest that load has vancomycin;
(5) vancomycin of 10mg step (3)/gelatine microsphere complex is added in the 10wt% silk fibroin protein solution of 1ml step (1), stir,-60 ℃ of lyophilization 24h, obtain the silk fibroin porous scaffold of load vancomycin/gelatine microsphere complex.
(6) silk fibroin porous scaffold of the load vancomycin that the silk fibroin porous compound rest of load vancomycin step (4) obtained and step (5) obtain/gelatine microsphere complex is positioned over respectively and scribbles on colibacillary solid medium, 37 ℃ of placements are spent the night, carry out antibiotic ring experiment, observe the antibacterial effect of these two kinds of compound rests.From antibiotic ring experiment, can see, these two kinds of supports all do not have antibacterial effect to escherichia coli, and result as shown in Figure 7.Due to vancomycin, only to gram positive bacteria, for example staphylococcus aureus, have antibacterial effect, but, for gram negative bacteria, for example escherichia coli, do not have antibacterial effect, and this has limited the use of this compound rest.And gentamycin sulfate has obvious antibacterial effect to Gram-positive and negative bacterium, this can find out from comparative example 2.So the fibroin albumen support of gentamycin sulfate prepared by the present invention/gelatine microsphere complex than the silk fibroin porous scaffold of vancomycin/gelatine microsphere complex, has better, more fully antibacterial ability and effect, the scope of use is advantage more comprehensively.
Above-described embodiment is preferably embodiment 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 principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. the preparation method of the fibroin albumen support of load gentamycin sulfate/gelatine microsphere complex, is characterized in that comprising the steps:
(1) be that 10～50mg/ml gentamicin sulfate solution is added drop-wise in gelatine microsphere by concentration, spend the night in 4～25 ℃ of placements, then lyophilization, obtain gentamycin sulfate/gelatine microsphere complex; Wherein to add the amount of gentamicin sulfate solution be 2～10ml to every gram gelatine microsphere;
(2) gentamycin sulfate of step (1)/gelatine microsphere complex is added in 2～10wt% silk fibroin protein solution, stir, then lyophilization, obtain the fibroin albumen support of load gentamycin sulfate/gelatine microsphere complex; Every milliliter of silk fibroin protein solution adds 10mg gentamycin sulfate/gelatine microsphere complex.
2. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 1/gelatine microsphere complex, it is characterized in that: the 10～50mg/ml gentamicin sulfate solution described in step (1) adopts following methods to be prepared: gentamycin sulfate is dissolved in deionized water, stir, obtaining concentration is 10～50mg/ml gentamicin sulfate solution.
3. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 1/gelatine microsphere complex, it is characterized in that: the particle diameter of the gelatine microsphere described in step (1) is 5～30 μ m, and expansion rate is 300～500%.
4. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 1/gelatine microsphere complex, it is characterized in that: the gelatine microsphere described in step (1) adopts following methods to be prepared: the 0.1g sorbitol anhydride oleate is added in the flask that fills the 100ml vegetable oil, after 60 ℃ of stirring 0.5～5h, by the mode dripped, by 10ml concentration, be that the 10wt% aqueous gelatin solution joins in vegetable oil, after continuing to stir 180min, solution is transferred to ice bath, continue to stir 30min, then add 25wt% glutaraldehyde water solution 0.1ml, continue to stir 2h, add 4 ℃ of acetone 30ml, stir 30min, standing, filter, obtain microsphere, the microsphere bubble, in 10ml acetone, is soaked to 30min with the 1mol/L glycine after 4 ℃ of curing 24h, centrifugal, get precipitation, with ethanol and isopropyl alcohol, replace washing precipitate, the precipitate after cleaning is placed in to the deionized water soaked overnight, lyophilization, obtain gelatine microsphere.
5. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 4/gelatine microsphere complex, it is characterized in that: the speed of described stirring is 300～400r/min;
Described centrifugal condition is in 25 ℃, the centrifugal 10min of 10000r/min;
Described cryodesiccated condition is-60 ℃ of lyophilization 24h.
6. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 1/gelatine microsphere complex, it is characterized in that: the 2～10wt% silk fibroin protein solution described in step (2) adopts following methods to be prepared: Bombyx bombycis is shredded, then add 100 ℃, concentration is 0.2wt%NaCO
3boil 2h in aqueous solution, NaCO
3the ratio of aqueous solution and Bombyx bombycis is: the 0.2wt%NaCO of 100ml
3aqueous solution adds 5g Bombyx bombycis; Come unstuck rear extremely neutral with the washed with de-ionized water fibroin, in 60 ℃ of oven dry, obtain fibroin albumen; Get fibroin albumen, add in the 9.8mol/L lithium bromide water solution, with deionized water dialysis 2～4 days, centrifugal after 40～60 ℃ of heating in water bath 3～5h, obtain 2～10wt% silk fibroin protein solution.
7. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 6/gelatine microsphere complex, is characterized in that: the Bombyx bombycis that described Bombyx bombycis is pupa;
The bag filter that described dialysis is 8000～12000 with molecular cut off is dialysed;
Described centrifugal condition is in 25 ℃, the centrifugal 10min of 10000r/min.
8. the preparation method of the fibroin albumen support of load gentamycin sulfate according to claim 1/gelatine microsphere complex is characterized in that: the cryodesiccated condition described in step (2) is-60 ℃ of lyophilization 24h.
9. the fibroin albumen support of the load gentamycin sulfate obtained by the described preparation method of claim 1～8 any one/gelatine microsphere complex, is characterized in that porosity is 75～95%, and aperture is 50～200 μ m.
10. the fibroin albumen support of load gentamycin sulfate claimed in claim 9/gelatine microsphere complex is in skin wound healing and art of wound dressings application.
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| CN107050509A (en) * | 2017-05-08 | 2017-08-18 | 吉林大学 | It is a kind of that there is plasticity sustained-release micro-spheres type timbering material of carrying multi-medicament function and preparation method thereof |
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| CN109833509A (en) * | 2019-01-18 | 2019-06-04 | 太阳雨林（厦门）生物医药有限公司 | A kind of multiple sustained release blood vessel embolism load drug composition |
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| CN108159485A (en) * | 2017-12-28 | 2018-06-15 | 广州贝奥吉因生物科技有限公司 | A kind of chitosan/silk fibroin bracket, preparation method and its application for loading curcumin/gelatine microsphere compound |
| CN109833509A (en) * | 2019-01-18 | 2019-06-04 | 太阳雨林（厦门）生物医药有限公司 | A kind of multiple sustained release blood vessel embolism load drug composition |
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