WO2022188399A1 - 具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用 - Google Patents

具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用 Download PDF

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WO2022188399A1
WO2022188399A1 PCT/CN2021/122111 CN2021122111W WO2022188399A1 WO 2022188399 A1 WO2022188399 A1 WO 2022188399A1 CN 2021122111 W CN2021122111 W CN 2021122111W WO 2022188399 A1 WO2022188399 A1 WO 2022188399A1
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silk protein
protein nanofiber
gel
nanofiber gel
mixing
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PCT/CN2021/122111
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English (en)
French (fr)
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伍丽君
吕强
高素玥
丁召召
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

Definitions

  • the invention belongs to the technical field of wound repair, and in particular relates to a silk protein nanofiber gel dressing with pressure spreadability, a preparation method and application thereof.
  • Flap transplantation is a commonly used treatment method for plastic surgery, burns, trauma, etc. It can provide effective protection for wounds and restore skin function.
  • the key to successful flap transplantation is the rapid remodeling of the flap and the vascularized network of the wound base, which can provide nutrition to the flap and avoid flap necrosis. Flap necrosis due to insufficient blood supply is an important factor in the failure of flap transplantation, and it is also the core problem that restricts the size, shape and application fields of flaps. Therefore, how to improve the vascularization-promoting ability of the flap and promote the rapid and effective reconstruction of the vascularization network of the flap and the wound-base are the key issues in flap transplantation, and related technologies and products are of great significance and broad application prospects in the field of wound repair.
  • the main reasons include: (1) it is difficult for traditional gels to form a uniform coating to provide uniform vascularization stimulation to the flap, and this non-uniformity naturally leads to partial necrosis of the flap; (2) although the contained drugs can promote vascularization vascularization, but the gel itself creates a physical barrier between the flap and the wound base, limiting the rapid formation of a vascularized network between the two. Therefore, an ideal drug-loaded gel dressing should have good spreadability, be able to spread evenly over the flap and wound base, and at the same time, the formed coating should be thin enough to avoid physical obstruction of the gel itself. According to other research results, Preferably the thickness of the coating can be below 200 microns.
  • the pressure-spreadable gel will not change the routine operation of the existing clinicians, and is an ideal choice for drug-loaded vascularization gel dressings.
  • the biomaterial gel with pressure spreadability and the formed coating meets the requirement of thickness below 200 ⁇ m.
  • Silk protein has been widely used in the field of wound repair because of its excellent biocompatibility, low inflammatory response and absorbability.
  • researchers have prepared silk protein nanofiber gels and sponges with different conformations and sizes to further improve their ability to repair tissues.
  • nanofibers can also be loaded with different drugs at the same time, and achieve different functions through the controlled release of drugs. Therefore, considering the significant advantages of silk protein in different tissue repair, gel preparation, and different drug loading and controlled release, using silk protein nanofibers as the matrix to develop and prepare gel dressings that can meet the special requirements of flap vascularization has the advantages of It is feasible to a certain extent, but similar to other biological materials, silk protein nanofiber gels with the above-mentioned pressure spreadability have not been reported.
  • the technical problem to be solved by the present invention is to provide a silk protein nanofiber gel dressing with pressure spreadability, its preparation method and application, and the silk protein nanofiber gel dressing can be spread and shaped under mild pressure. Forms ultra-thin coatings.
  • the invention provides a preparation method of a silk protein nanofiber gel dressing with pressure spreadability, comprising the following steps:
  • the concentration of the first silk protein nanofiber gel is 2-5wt%; the concentration of the second silk protein nanofiber gel is 0.3-2wt%; the rheologically changed silk protein nanofiber gel
  • the concentration of the glue is 0.6 to 1.5 wt %.
  • the fibroin nanofibers in the first fibroin nanofiber gel are high-crystalline fibroin nanofibers; the crystallinity of the high-crystalline fibroin nanofibers is greater than or equal to 40%; the high-crystalline fibroin nanofibers
  • the diameter of the fiber is 10-30 nm; the length is 0.1-3 ⁇ m.
  • the speed of mixing and stirring in the step S1) is 200-5000rpm; the time of mixing and stirring is 1-48h;
  • the mixing speed is 10 ⁇ 800rpm; the mixing time is 10min ⁇ 12h;
  • the speed of the centrifugation is 2000-15000rpm; the time of the centrifugation is 5min-2h.
  • the volume ratio of the rheologically changed silk protein nanofiber gel to the drug-loaded silk protein nanofiber gel in the step S2) is (8-24):1.
  • the speed of mixing and stirring in the S2) is 200-5000 rpm; the time of mixing and stirring is 1-48 h.
  • vacuum treatment is also performed to obtain a silk protein nanofiber gel dressing with pressure spreadability.
  • the vacuum degree of the vacuum treatment is 0.01-100 Pa; the time of the vacuum treatment is 10-60 min.
  • the present invention also provides a silk protein nanofiber gel dressing with pressure spreadability, the silk protein nanofiber gel dressing can be spread into a thin film with a thickness of less than or equal to 200 microns under pressure, and the pressure remains after the pressure is removed. Maintain the spread state.
  • the present invention also provides an application of the above-mentioned silk protein nanofiber gel dressing as a gel dressing for skin flap transplantation.
  • the present invention provides a method for preparing a silk protein nanofiber gel dressing with pressure spreadability, comprising the following steps: S1) mixing and stirring a first silk protein nanofiber gel with water to obtain a silk protein with changed rheology Nanofiber gel; mixing the drug for promoting angiogenesis with the second silk protein nanofiber gel to obtain a drug-loaded silk protein nanofiber gel; S2) mixing the rheology-modified silk protein nanofiber gel with drug loading The silk protein nanofiber gel is mixed and stirred to obtain a silk protein nanofiber gel dressing with pressure spreadability.
  • the invention realizes the special function of forming an ultra-thin coating by spreading and shaping the gel under mild pressure by regulating the rheological properties of the silk protein nanofiber gel, and simultaneously uses the silk protein nanofiber as a carrier to load and promote blood vessels.
  • the regenerative medicine can meet the special needs of uniform vascularization of the skin flap, and the prepared silk protein nanofiber gel dressing can spread evenly under mild pressure, and can still maintain the spread state after the pressure is removed, and the obtained coating thickness is as low as Below 200 microns, thus eliminating the obstruction of the gel to the reconstruction of the flap and the vascular network of the wound; in addition, the drug loaded by the dressing can be released slowly, providing a uniform and stable stimulating environment for the vascularization of the flap, combined with silk protein nanofibers Its own biocompatibility and the ability to promote tissue repair can effectively improve the vascularization level of the skin flap and improve the survival efficiency of the skin flap; and the preparation method is simple, the dressing performance obtained is stable, easy to scale preparation, and the ability to promote vascularization is remarkable. , which not only meets the needs of flap vascularization, but also has good application value for vascularization of other wounds, and has a broad market prospect.
  • Fig. 1 is a schematic diagram of a method for using the silk protein nanofiber gel dressing with pressure spreadability provided by the present invention as a wound repair gel dressing;
  • Fig. 2 is the test chart of the pressure spreading effect of the drug-loaded silk protein nanofiber gel dressing obtained in Example 1 of the present invention and the silk protein nanofiber gel with different concentrations of rheological changes;
  • Example 3 is a morphological diagram of the silk protein nanofiber gel with a concentration of 2.5wt% diluted to 0.8wt% without shearing in Example 1 of the present invention
  • Example 4 is a graph showing the results of an animal experiment of the drug-loaded silk protein nanofiber gel dressing obtained in Example 1 of the present invention.
  • Figure 5 is a morphological diagram of the 1wt% silk protein nanofiber gel before and after stirring in Example 2 of the present invention
  • Figure 6 is a diagram of the rheological properties of the silk protein nanofiber gel with a concentration of 1wt% and the silk protein nanofiber hydrogel loaded with 60uM DFO in step (3) of Example 2 of the present invention;
  • Fig. 7 is the Raman spectrogram of 1wt% silk protein nanofiber hydrogel before and after loading deferoxamine in Example 2 of the present invention.
  • FIG. 8 is the images of the silk protein nanofiber hydrogel loaded with endothelial cell growth factor and the silk protein nanofiber hydrogel with a concentration of 1.1 wt % in Example 3 of the present invention before and after removing bubbles under vacuum;
  • Figure 9 shows the results of mixing the silk protein nanofiber hydrogel with a concentration of 2 wt % and the silk protein nanofiber hydrogel with a concentration of 1.3 wt % in Example 4 of the present invention loaded with endothelial cell growth factor before and after removing bubbles under vacuum image;
  • FIG. 10 is a test chart of the swelling property of the drug-loaded silk protein nanofiber gel obtained in Example 5 of the present invention.
  • the present invention provides a method for preparing a silk protein nanofiber gel dressing with pressure spreadability, comprising the following steps: S1) mixing and stirring a first silk protein nanofiber gel with water to obtain a silk protein with changed rheology Nanofiber gel; mixing the drug for promoting angiogenesis with the second silk protein nanofiber gel to obtain a drug-loaded silk protein nanofiber gel; S2) mixing the rheology-modified silk protein nanofiber gel with drug loading The silk protein nanofiber gel is mixed and stirred to obtain a silk protein nanofiber gel dressing with pressure spreadability.
  • the invention firstly regulates the rheology of the silk protein nanofiber gel to a suitable range by adjusting the concentration of the silk protein nanofibers, and then further regulates the rheology through suitable shearing action, finally obtains the ideal rheological properties, and realizes the coagulation. Mild pressure of the glue spreads. Subsequently, the drug-loaded silk protein nanofibers were compounded with the above system, and the effect of adding the drug-loaded nanofibers on the rheology of the system was reduced by adjusting the ratio of the two, and through the shearing treatment of stirring again, the drug was uniformly dispersed at the same time. , to obtain the desired performance.
  • the key to the above technology is to optimize the synergy between the concentration of silk protein nanofibers and the shearing effect on the basis of a deep understanding of silk protein nanofibers, and finally achieve a breakthrough in material properties, obtain pressure-spreadable properties, and low coating thickness after spreading.
  • Silk protein nanofiber drug-loaded gel dressing at 200 ⁇ m.
  • the preparation methods adopted in the present invention all adopt easy-to-control physical methods and are realized in the aqueous phase.
  • the method is simple and controllable, has good repeatability, is conducive to maintaining the activity of active substances, is easy to scale production, and has broad clinical application prospects. .
  • the concentration of the first silk protein nanofiber gel is preferably 2-5 wt %, more preferably 2-5 wt % 4 wt %, more preferably 2-3 wt %; in the embodiments provided by the present invention, the concentration of the first silk protein nanofiber gel is specifically 2 wt %, 2.5 wt % or 3 wt %; the first silk protein
  • the nanofiber gel can be prepared according to the preparation method well-known to those skilled in the art, and there is no special limitation. In the present invention, it is preferable to concentrate the silk protein nanofiber aqueous solution at 40° C. to 60° C.
  • a first silk protein nanofiber aqueous solution the first silk protein nanofiber aqueous solution is concentrated at 20° C. to 35° C. to a second silk protein nanofiber aqueous solution with a concentration of 18 to 24 wt %; water is added to the second silk protein nanofiber aqueous solution Dilute to the third silk protein nanofiber aqueous solution; seal and incubate the third silk protein nanofiber aqueous solution at 50°C to 70°C to obtain the first silk protein nanofiber gel.
  • the fibroin nanofibers in the first fibroin nanofiber gel are preferably high crystalline fibroin nanofibers; the high crystalline fibroin nanofibers have high crystallinity and high charge density, so that there is a hydrophobicity between the fibroin nanofibers.
  • the dynamic balance of action and charge action so based on the change of concentration, silk protein nanofibers will gradually transform from solution state to gel state in water, so there is an intermediate state between gel and solution, which can spread under pressure to form a coating.
  • the high negative charge density of silk protein nanofibers endows the gel with shear thinning properties, and due to the negative charge, the gap between silk protein nanofibers is The repulsive force can maintain the rheological properties after shearing treatment, so that the shearing gel can significantly improve the spreading performance under the premise of maintaining the solid state of the gel.
  • the ideal state of silk protein nanofiber gel is obtained through the synergistic effect of concentration and shearing treatment. Combined with the drug-loading characteristics of silk protein nanofibers, it is expected to break through the existing technology.
  • the crystallinity of the highly crystalline silk protein nanofibers is preferably greater than or equal to 40%, more preferably 40% to 80%, and more preferably 40% ⁇ 60%, most preferably 40% to 50%;
  • the diameter of the high crystalline fibroin nanofibers is preferably 10-30 nm, more preferably 15-25 nm, and more preferably 20 nm; the length is preferably 0.1-3 ⁇ m, more preferably is 0.5-2.5 ⁇ m, more preferably 0.5-1.5 ⁇ m, and most preferably 1 ⁇ m;
  • the first silk protein nanofiber gel is mixed and stirred with water; the water is preferably deionized water;
  • the rate of mixing and stirring is preferably 200 to 5000 rpm, more preferably 500 to 4000 rpm, and still more preferably 600 to 3000 rpm; in the embodiments provided by the present invention, the mixing rate is specifically 800 rpm, 900 rpm, 600 rpm, 3000
  • the invention makes full use of its hydrophobicity and high negative charge, and regulates the effect of hydrophobicity and high negative charge on the physical and chemical properties of the gel through the synergistic effect of nanofiber concentration change and shearing. Finally, a silk protein nanofiber gel with a thickness of less than 200 microns that can be spread under pressure is obtained, which meets the special requirements of skin flap regeneration.
  • the angiogenesis-promoting drug may be a vascularization-promoting drug well-known to those skilled in the art, and there is no special limitation, and it can be a small molecule Drugs, macromolecular drugs or other functional molecules with the ability to promote vascularization, preferably deferoxamine or growth factor VEGF in the present invention
  • the concentration of the second silk protein nanofiber gel is preferably 0.3-2wt%, more Preferably, it is 0.6-2wt%; in the embodiments provided by the present invention, the concentration of the second silk protein nanofiber gel is specifically 1.2wt%, 1wt%, 2wt% or 0.6wt%;
  • the nanofiber gel can be prepared according to the method of the first silk protein nanofiber gel; the silk protein nanofibers in the second silk protein nanofiber gel are also preferably high crystalline silk protein nanofibers;
  • the crystallinity of the crystalline fibroin nanofibers is preferably greater than or equal to 40%, more preferably 40%
  • the preparation of the above-mentioned rheology-modified silk protein nanofiber gel and drug-loaded silk protein nanofiber gel has no order.
  • the rheologically modified silk protein nanofiber gel and the drug-loaded silk protein nanofiber gel are mixed and stirred;
  • the volume ratio of the rheologically modified silk protein nanofiber gel to the drug-loaded silk protein nanofiber gel is preferably (8-24): 1; in the examples provided by the present invention, the volume ratio of the rheologically changed silk protein nanofiber gel to the drug-loaded silk protein nanofiber gel is specifically 8:1, 15:1, 24:1 or 12:1;
  • the speed of the mixing and stirring is preferably 200-5000 rpm, more preferably 500-4000 rpm, and more preferably 500-2000 rpm; in the embodiments provided by the present invention, the speed of the mixing and stirring is specific is 1000rpm, 600rpm, 2000rpm or 500rpm;
  • the mixing time is preferably 1-48h, more preferably 2-40h, more preferably 2-30h, most preferably 2-24h; in the embodiments provided by the present invention, all The mixing and stirring time is specifically 6h, 14h, 4h, 2h
  • vascularization-promoting drugs By loading vascularization-promoting drugs on silk protein nanofibers, the same silk protein nanofibers are easily dispersed to each other to achieve uniform dispersion of drugs in the gel, and combined with ultra-thin coating to achieve uniform effect on flap vascularization, solving the problem of current
  • the technical system is difficult to function uniformly and the physical hindering effect of the gel, and the performance is significantly improved; at the same time, the drug is loaded on the nanofibers, and then compounded with the treated nanofiber gel, and the ratio of the two is adjusted to reduce the load.
  • the influence of drug nanofibers on the bulk rheology of the gel system makes the system more flexible, and suitable drugs can be selected according to different requirements to meet different application needs.
  • vacuuming is also performed to obtain a silk protein nanofiber gel dressing with pressure spreadability;
  • the vacuum degree of the vacuuming is preferably 0.01-100Pa, more preferably 1-100Pa, and more preferably 5 ⁇ 100Pa, most preferably 10 ⁇ 100Pa;
  • the time of vacuum treatment is preferably 10 ⁇ 60min, more preferably 20 ⁇ 60min. Air bubbles can be removed by vacuuming.
  • the invention realizes the special function of forming an ultra-wave coating by spreading and shaping the gel under mild pressure, and at the same time, the silk protein nanofiber is used as a carrier to load angiogenesis-promoting drugs, so as to meet the requirements of the skin flap.
  • Special requirements for uniform vascularization, and the prepared silk protein nanofiber gel dressing can spread evenly under mild pressure, and can still maintain the spread state after the pressure is removed, and the thickness of the obtained coating is as low as 200 microns, thereby eliminating coagulation.
  • the present invention also provides a pressure-spreadable silk protein nanofiber gel dressing prepared by the above method, the silk protein nanofiber gel dressing can be spread into a film with a thickness of less than or equal to 200 microns under pressure, and The spreading state is maintained even after the pressure is removed.
  • the pressure is preferably 500g.
  • FIG. 1 is a schematic diagram of the method of using the silk protein nanofiber gel dressing with pressure spreadability provided by the present invention as a wound repair gel dressing.
  • deionized water was added to the silk protein nanofiber gel with a concentration of 2.5wt% (the crystallinity of silk protein nanofibers was 40%, the diameter was 20nm, and the length was 1 ⁇ m), and the concentration was adjusted to 0.3-2wt%.
  • the stirrer was stirred at a speed of 800 rpm for 24 hours to obtain a rheologically modified silk protein nanofiber gel dressing.
  • a to c are the pressure spreading effect diagrams of the drug-loaded silk protein nanofiber gel dressing obtained in Example 1. It can be seen that the gel can spread evenly on the glass plate and animal wound surface, and will not shrink after the pressure is removed. ; in Figure 2, d is the effect of pressure spreading of silk protein nanofiber gels with different concentrations of rheological changes. It can be seen that if the concentration is too high, the gel will shrink after the pressure is removed, and if the concentration is too low, the fluidity of the gel will be reduced. Great, easy to drain.
  • Figure 3 is a morphological diagram of the silk protein nanofiber gel with a concentration of 2.5 wt % diluted to 0.8 wt % without shearing. It can be seen from Figure 3 that the unsheared gel does not have reasonable fluidity and does not have reasonable pressure spreading properties.
  • Fig. 4 is the animal experiment result diagram of the drug-loaded silk protein nanofiber gel dressing obtained in Example 1, wherein Control is a control figure, without any treatment, SNF is the silk protein nanofiber whose rheological change is obtained in step (1).
  • Fiber gel, SNF-DFO is the drug-loaded silk protein nanofiber gel dressing obtained in Example 1. It can be seen from Figure 4 that the drug-loaded silk protein nanofiber gel dressing obtained in Example 1 can be evenly distributed between the skin flap and the wound base, and cover the entire skin flap, the survival rate of the skin flap and the vascularization network. were significantly improved.
  • the rats were anesthetized by intraperitoneal injection of 4% chloral hydrate (1ml/100g), the back hair was shaved, the rats were placed on the operating table in a prone position at room temperature, and the bilateral posterior superior iliac spines were marked.
  • the scalpel incised the full thickness of the skin along the design line, leaving the pedicle connection, lift the skin flap from the sarcoma layer, after the full thickness was separated, back to the transplant, and sutured in situ with 5-0 absorbable suture, before the end of the interrupted suture
  • the operation time is controlled within 30 minutes, no antibiotics are used after surgery, and the rats are kept in separate cages.
  • Figure 5 shows the morphology of the silk protein nanofiber gel with a concentration of 1wt% before and after stirring. It can be seen that good fluidity and spreadability are obtained after stirring, and the gel without stirring has no reasonable fluidity. , does not have reasonable pressure spreading properties.
  • Figure 6 shows the rheological properties of the silk protein nanofiber hydrogel with a concentration of 1wt% and the silk protein nanofiber hydrogel loaded with 60uM DFO in step (3).
  • the rheological properties of the fibroin nanofiber hydrogel did not change significantly, and the loading of the drug only caused a slight increase in the viscosity of the silk protein nanofiber hydrogel at low shear rates.
  • Figure 7 shows the Raman spectra of 1 wt% silk protein nanofiber hydrogel before and after loading with deferoxamine.
  • the Raman spectrum after loading with deferoxamine has both the characteristic peaks of silk protein and deferoxamine, confirming that deferoxamine load.
  • Figure 8 is the images of the silk protein nanofiber hydrogel loaded with endothelial cell growth factor and the silk protein nanofiber hydrogel at a concentration of 1.1 wt% before and after removing the bubbles under vacuum.
  • Figure 9 shows the images of silk protein nanofiber hydrogel loaded with endothelial cell growth factor at a concentration of 2 wt % and silk protein nanofiber hydrogel at a concentration of 1.3 wt % before and after removal of air bubbles under vacuum.

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Abstract

本发明提供了一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料的制备方法，包括以下步骤：S1)将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合，得到载药丝蛋白纳米纤维凝胶；S2)将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。与现有技术相比，本发明通过调控丝蛋白纳米纤维凝胶的流变特性，实现凝胶温和压力下铺展定型形成超薄涂层的特殊功能，同时以丝蛋白纳米纤维为载体加载促血管再生药物，从而满足皮瓣均匀血管化的特殊需求。

Description

具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用
本申请要求于2021年03月08日提交中国专利局、申请号为202110251223.5、发明名称为“具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于创面修复技术领域，尤其涉及一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用。
背景技术
皮瓣移植是整形、烧伤、创伤等常用的治疗方式，能对创面提供有效保护，恢复皮肤功能。皮瓣移植成功的关键在于皮瓣和创基血管化网络的快速重构，从而能为皮瓣提供营养，避免皮瓣坏死。由于血供不足导致的皮瓣坏死是皮瓣移植手术失败的重要因素，也是制约皮瓣使用尺寸、形状和应用领域的核心问题。因此，如何提高皮瓣的促血管化能力，促进皮瓣和创基血管化网络的快速有效重构是皮瓣移植的关键问题，相关技术和产品在创面修复领域具有重要意义和广泛应用前景。
目前已有多种能够促进血管化的药物，并成功应用于临床，为实现皮瓣快速均匀血管化的要求，需要合适的载体对促血管化药物进行加载控释。具有优异生物相容性的凝胶体系不仅能够控制药物的控释，同时可为皮瓣再生提供基质，被认为是合适的载体材料，包含促血管化药物的凝胶敷料在促进真皮再生、骨组织修复等方面也表现出良好效果。然而，尽管多种生物材料载药凝胶也被尝试用于促进皮瓣的再生和血管化，由于皮瓣对血管化速率和均匀性的特殊要求，常规的载药凝胶难以获得良好预期效果。主要原因包括：(1)传统凝胶难以形成均一的涂层为皮瓣提供均匀的血管化刺激，而这种不均匀性自然带来皮瓣部分坏死；(2)尽管所载药物能够促进血管化，但凝胶本身会在皮瓣和创基之间形成物理障碍，限制两者之间血管化网络的快速形成。因此，理想的载药物凝胶敷料应该具有良好的可铺展性，能够均匀布满皮瓣和创基，同时所形成涂层应该足够薄，以避免凝胶本身的物理阻碍，根据其他研究结果，最好涂层的厚度能在200微米以下。同时，考虑到临床实际应用，具有压力可铺展性的 凝胶，使用过程不会改变现有临床医生常规术式，是载药促血管化凝胶敷料的理想选择。然而，具有压力可铺展性，且所形成涂层满足低于200微米厚度要求的生物材料凝胶未见任何报道。
丝蛋白已被广泛尝试应用于创面修复领域，其具有优异的生物相容性，低炎症反应以及可吸收性。近年来，通过不同方式，研究者制备出不同构象和尺寸的丝蛋白纳米纤维凝胶和海绵，以进一步改善其对组织的修复能力。并且纳米纤维同时还可以加载不同药物，通过药物的控释实现不同功能。因此，考虑到丝蛋白在不同组织修复、凝胶制备以及不同药物加载控释等多方面的显著优势，以丝蛋白纳米纤维为基质，开发制备能满足皮瓣血管化特殊要求的凝胶敷料具有一定可行性，然而同其它生物材料相似，具有如上压力可铺展性的丝蛋白纳米纤维凝胶未见报道。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用，该丝蛋白纳米纤维凝胶敷料可在温和压力下铺展定型形成超薄涂层。
本发明提供了一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料的制备方法，包括以下步骤：
S1)将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；
将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合，得到载药丝蛋白纳米纤维凝胶；
S2)将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。
优选的，所述第一丝蛋白纳米纤维凝胶的浓度为2～5wt％；所述第二丝蛋白纳米纤维凝胶的浓度为0.3～2wt％；所述流变性改变的丝蛋白纳米纤维凝胶的浓度为0.6～1.5wt％。
优选的，所述第一丝蛋白纳米纤维凝胶中的丝蛋白纳米纤维为高结晶丝蛋白纳米纤维；所述高结晶丝蛋白纳米纤维的结晶度大于等于40％；所述高结晶丝蛋白纳米纤维的直径为10～30nm；长度为0.1～3μm。
优选的，所述步骤S1)中混合搅拌的速率为200～5000rpm；混合搅拌的时间为1～48h；
混合的速率为10～800rpm；混合的时间为10min～12h；
混合后，离心，得到载药丝蛋白纳米纤维凝胶；
所述离心的速率为2000～15000rpm；离心的时间为5min～2h。
优选的，所述步骤S2)中流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶的体积比为(8～24)：1。
优选的，所述S2)中混合搅拌的速率为200～5000rpm；混合搅拌的时间为1～48h。
优选的，所述步骤S2)中混合搅拌后，还进行抽真空处理，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。
优选的，所述抽真空处理的真空度为0.01～100Pa；抽真空处理的时间为10～60min。
本发明还提供了一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料，所述丝蛋白纳米纤维凝胶敷料在压力作用下可铺展成厚度小于等于200微米的薄膜，且压力取消后仍维持铺展状态。
本发明还提供了一种上述丝蛋白纳米纤维凝胶敷料作为皮瓣移植用凝胶敷料的应用。
本发明提供了一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料的制备方法，包括以下步骤：S1)将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合，得到载药丝蛋白纳米纤维凝胶；S2)将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。与现有技术相比，本发明通过调控丝蛋白纳米纤维凝胶的流变特性，实现凝胶温和压力下铺展定型形成超薄涂层的特殊功能，同时以丝蛋白纳米纤维为载体加载促血管再生药物，从而满足皮瓣均匀血管化的特殊需求，且制备得到的丝蛋白纳米纤维凝胶敷料在温和压力下可均匀铺展，在压力除去后仍可保持铺展状态，得到的涂层厚度低至200微米以下，从而消除凝胶对皮瓣和创基血管网络重建的阻碍作用；再者敷料加载的药物可缓慢释 放，为皮瓣的血管化提供均匀稳定的刺激性环境，结合丝蛋白纳米纤维本身的生物相容性和促进组织修复的能力，有效改善皮瓣血管化水平，提高皮瓣的存活效率；并且该制备方法简单、得到的敷料性能稳定，易于规模化制备，促进血管化能力显著，不仅满足皮瓣血管化的需求，同时对其他创面的血管化具有良好的应用价值，市场前景广阔。
附图说明
图1为本发明提供的具备压力可铺展性的丝蛋白纳米纤维凝胶敷料作为创面修复凝胶敷料使用方法示意图；
图2为本发明实施例1中得到的载药丝蛋白纳米纤维凝胶敷料及不同浓度的流变性改变的丝蛋白纳米纤维凝胶的压力铺展效果测试图；
图3为本发明实施例1中浓度为2.5wt％的丝蛋白纳米纤维凝胶稀释至0.8wt％未经剪切后的形态图；
图4为本发明实施例1中得到的载药丝蛋白纳米纤维凝胶敷料的动物实验结果图；
图5为本发明实施例2中1wt％的丝蛋白纳米纤维凝胶在搅拌前后的形态图；
图6为本发明实施例2步骤(3)中浓度为1wt％的丝蛋白纳米纤维凝胶及加载60uM DFO的丝蛋白纳米纤维水凝胶的流变性能图；
图7为本发明实施例2中1wt％的丝蛋白纳米纤维水凝胶加载去铁胺前后的拉曼光谱图；
图8为本发明实施例3中加载内皮细胞生长因子后的丝蛋白纳米纤维水凝胶与1.1wt％浓度的丝蛋白纳米纤维水凝胶混合后在真空下去除气泡前后的图像；
图9为本发明实施例4中加载内皮细胞生长因子后的浓度为2wt％的丝蛋白纳米纤维水凝胶与1.3wt％浓度的丝蛋白纳米纤维水凝胶混合后在真空下去除气泡前后的图像；
图10为本发明实施例5中得到的载药丝蛋白纳米纤维凝胶的膨胀性能测试图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料的制备方法，包括以下步骤：S1)将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合，得到载药丝蛋白纳米纤维凝胶；S2)将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。
本发明首先通过调整丝蛋白纳米纤维的浓度，调控丝蛋白纳米纤维凝胶的流变性到合适范围，随后通过合适的剪切作用，对流变性进行进一步调控，最终获得理想的流变性能，实现凝胶的温和压力可铺展。随后，将负载药物的丝蛋白纳米纤维同上述体系复合，通过两者比例的调整降低加入载药纳米纤维对体系流变性的影响，并通过再次搅拌的剪切处理，在实现药物均匀分散的同时，获得理想的性能。上述技术的关键在于对丝蛋白纳米纤维深度理解的基础上，优化丝蛋白纳米纤维浓度和剪切作用的协同，最终实现材料性能的突破，获得具有压力可铺展性能，且铺展后涂层厚度低于200微米的丝蛋白纳米纤维载药凝胶敷料。
并且，本发明所采用的制备方法，全部采用易于控制的物理方式，在水相中实现，方法简单可控，重复性好，有利于保持活性物质的活性，易于规模化生产，临床应用前景广阔。
其中，本发明对所有原料的来源并没有特殊的限制，为市售即可。
将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；所述第一丝蛋白纳米纤维凝胶的浓度优选为2～5wt％，更优选为2～4wt％，再优选为2～3wt％；在本发明提供的实施例中，所述第一丝蛋白纳米纤维凝胶的浓度具体为2wt％、2.5wt％或3wt％；所述第一丝蛋白纳米纤维凝胶按照本领域技术人员熟知的制备方法制备即可，并无特殊的限制，在本发明中优选为：将丝蛋白纳米纤维水溶液40℃～60℃浓缩至浓度为6～12wt％的 第一丝蛋白纳米纤维水溶液；将第一丝蛋白纳米纤维水溶液在20℃～35℃浓缩至浓度为18～24wt％的第二丝蛋白纳米纤维水溶液；将所述第二丝蛋白纳米纤维水溶液加水稀释至第三丝蛋白纳米纤维水溶液；将所述第三丝蛋白纳米纤维水溶液在50℃～70℃密封孵育，得到第一丝蛋白纳米纤维凝胶。所述第一丝蛋白纳米纤维凝胶中的丝蛋白纳米纤维优选为高结晶丝蛋白纳米纤维；高结晶丝蛋白纳米纤维具有高结晶度和高电荷密度，从而使得丝蛋白纳米纤维之间存在疏水作用和电荷作用的动态平衡，因此基于浓度的变化，丝蛋白纳米纤维在水中会存在由溶液态向凝胶态的逐渐转变，从而存在凝胶与溶液的中间态，能够在压力下铺展形成涂层，又能够铺展后固定在创面，为皮瓣提供持续支持；同时，丝蛋白纳米纤维高的负电荷密度赋予凝胶剪切变稀的性能，且由于负电荷导致丝蛋白纳米纤维之间的斥力，可以维持剪切处理后的流变性能，使得剪切后的凝胶在保持凝胶固态的前提下，铺展性能得到明显改善，因此，将浓度调控和剪切处理结合，在深入理解高晶丝蛋白纳米纤维内在结构和性能关系的基础上，通过浓度和剪切处理的协同作用，获得丝蛋白纳米纤维凝胶的理想状态，再结合丝蛋白纳米纤维载药特性，有望突破现有技术屏障，获得适用于皮瓣血管化的特殊丝蛋白纳米纤维凝胶敷料；所述高结晶丝蛋白纳米纤维的结晶度优选大于等于40％，更优选为40％～80％，再优选为40％～60％，最优选为40％～50％；所述高结晶丝蛋白纳米纤维的直径优选为10～30nm，更优选为15～25nm，再优选为20nm；长度优选为0.1～3μm，更优选为0.5～2.5μm，再优选为0.5～1.5μm，最优选为1μm；将第一丝蛋白纳米纤维凝胶与水混合搅拌；所述水优选为去离子水；所述混合搅拌的速率优选为200～5000rpm，更优选为500～4000rpm，再优选为600～3000rpm；在本发明提供的实施例中，所述混合搅拌的速率具体为800rpm、900rpm、600rpm、3000rpm或1600rpm；所述混合搅拌的时间优选为1～48h，更优选为2～46h；在本发明提供的实施例中，所述混合搅拌的时间具体为24h、46h、2h或3h；混合搅拌后得到的流变性改变的丝蛋白纳米纤维凝胶的浓度优选为0.6～1.5wt％，更优选为0.8～1.3wt％；在本发明提供的实施例中，所述流变性改变的丝蛋白纳米纤维凝胶的浓度具体为0.8wt％、1wt％、1.1wt％、1.3wt％、1.3wt％或1.2wt％。本发明通过特定构象和纳米结构的丝蛋白纳米纤维的选择，充分利用其疏水性和高负电 性，通过纳米纤维浓度变化和剪切的协同作用调控疏水性和高负电性对凝胶理化性能的影响，最终获得能够在压力下铺展，形成厚度低于200微米涂层的丝蛋白纳米纤维凝胶，满足皮瓣再生的特殊要求。
将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合；所述促血管再生的药物为本领域技术人员熟知的促血管化的药物即可，并无特殊的限制，其可为小分子药物、大分子药物或其他具有促血管化能力的功能分子，在本发明中优选为去铁胺或生长因子VEGF；所述第二丝蛋白纳米纤维凝胶的浓度优选为0.3～2wt％，更优选为0.6～2wt％；在本发明提供的实施例中，所述第二丝蛋白纳米纤维凝胶的浓度具体为1.2wt％、1wt％、2wt％或0.6wt％；所述第二丝蛋白纳米纤维凝胶可按照上述第一丝蛋白纳米纤维凝胶的方法进行制备即可；所述第二丝蛋白纳米纤维凝胶中丝蛋白纳米纤维也优选为高结晶丝蛋白纳米纤维；所述高结晶丝蛋白纳米纤维的结晶度优选大于等于40％，更优选为40％～80％，再优选为40％～60％，最优选为40％～50％；所述高结晶丝蛋白纳米纤维的直径优选为10～30nm，更优选为15～25nm，再优选为20nm；长度优选为0.1～3μm，更优选为0.5～2.5μm，再优选为0.5～1.5μm，最优选为1μm；所述混合的速率优选为10～800rpm，更优选为50～700rpm，再优选为100～600rpm；在本发明提供的实施例中，所述混合的速率具体为200rpm、400rpm、600rpm或100rpm；混合的时间优选为10min～12h，更优选为0.5～10h，再优选为2～10h；混合后体系中促血管再生的药物的浓度优选为0.01～5mg/ml，更优选为0.05～3mg/ml，再优选为0.06～2mg/ml；混合后，优选离心，得到载药丝蛋白纳米纤维凝胶；所述离心的速率优选为2000～15000rpm，更优选为5000～15000rpm，更优选为5000～14000rpm；在本发明提供的实施例中，所述离心的速率具体为12000rpm、14000rpm、9000rpm或5000rpm；所述离心的时间优选为5min～2h，更优选为10～100min，再优选为20～80min，最优选为25～60min。通过离心可除去未加载的药物。
在本发明中上述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶的制备并无先后顺序之分。
将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌；所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶体 积比优选为(8～24)：1；在本发明提供的实施例中，所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶体积比具体为8：1、15：1、24：1或12：1；所述混合搅拌的速率优选为200～5000rpm，更优选为500～4000rpm，再优选为500～2000rpm；在本发明提供的实施例中，所述混合搅拌的速率具体为1000rpm、600rpm、2000rpm或500rpm；混合搅拌的时间优选为1～48h，更优选为2～40h，再优选为2～30h，最优选为2～24h；在本发明提供的实施例中，所述混合搅拌的时间具体为6h、14h、4h、2h或24h。通过丝蛋白纳米纤维加载促血管化药物，利用相同丝蛋白纳米纤维易于相互分散的优势，实现药物在凝胶中的均匀分散，结合超薄涂层实现对皮瓣血管化的均匀作用，解决现有技术体系难以均匀作用的问题以及凝胶物理阻碍作用的问题，性能显著提高；同时药物负载在纳米纤维上，再同处理后的纳米纤维凝胶复合，并通过两者比例的调整，降低载药纳米纤维对凝胶体系本体流变性的影响，使得体系具有更高灵活性，可以根据不同要求，选择合适药物，以满足不同应用需求。
混合搅拌后，优选还进行抽真空处理，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料；所述抽真空处理的真空度优选为0.01～100Pa，更优选为1～100Pa，再优选为5～100Pa，最优选为10～100Pa；抽真空处理的时间优选为10～60min，更优选为20～60min。通过抽真空处理可除去其中的气泡。
本发明通过调控丝蛋白纳米纤维凝胶的流变特性，实现凝胶温和压力下铺展定型形成超波涂层的特殊功能，同时以丝蛋白纳米纤维为载体加载促血管再生药物，从而满足皮瓣均匀血管化的特殊需求，且制备得到的丝蛋白纳米纤维凝胶敷料在温和压力下可均匀铺展，在压力除去后仍可保持铺展状态，得到的涂层厚度低至200微米以下，从而消除凝胶对皮瓣和创基血管网络重建的阻碍作用；再者敷料加载的药物可缓慢释放，为皮瓣的血管化提供均匀稳定的刺激性环境，结合丝蛋白纳米纤维本身的生物相容性和促进组织修复的能力，有效改善皮瓣血管化水平，提高皮瓣的存活效率；并且该制备方法简单、得到的敷料性能稳定，易于规模化制备，促进血管化能力显著，不仅满足皮瓣血管化的需求，同时对其他创面的血管化具有良好的应用价值，市场前景广阔。
本发明还提供了一种上述方法制备的具备压力可铺展性的丝蛋白纳米纤维凝胶敷料，所述丝蛋白纳米纤维凝胶敷料在压力作用下可铺展成厚度小于等 于200微米的薄膜，且压力取消后仍维持铺展状态。
其中，所述压力优选为500g。
本发明还提供了上述方法制备的具备压力可铺展性的丝蛋白纳米纤维凝胶敷料作为创面修复凝胶敷料的应用，尤其是皮瓣移植用凝胶敷料的应用。参见图1，图1为本发明提供的具备压力可铺展性的丝蛋白纳米纤维凝胶敷料作为创面修复凝胶敷料使用方法示意图。
为了进一步说明本发明，以下结合实施例对本发明提供的一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用进行详细描述。
以下实施例中所用的试剂均为市售。
实施例1
(1)将浓度为2.5wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到0.8wt％，利用磁力搅拌器在800rpm的速率下，搅拌24小时；
(2)将去铁胺粉末同浓度为1.2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)混合，使得去铁胺浓度为1mg/ml，在200rpm的速率下搅拌4小时，随后在12000rpm的转速下离心30分钟，底部为载药的丝蛋白纳米纤维凝胶；
(3)将上述载药纳米纤维凝胶和(1)中搅拌处理的凝胶按照1:15的体积比混合，然后在1000rpm的转速下搅拌6小时，使得两种凝胶充分混合，且混合凝胶的流变性调整到合适程度；
(4)将上述丝蛋白载药纳米纤维凝胶在60Pa的真空度下抽真空50分钟，去除气泡，获得最终的载药丝蛋白纳米纤维凝胶敷料。
同时将浓度为2.5wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到0.3～2wt％，利用磁力搅拌器在800rpm的速率下，搅拌24小时，得到流变性改变的丝蛋白纳米纤维凝胶敷料。
对实施例1中得到的载药丝蛋白纳米纤维凝胶敷料及不同浓度的流变性改变的丝蛋白纳米纤维凝胶的压力铺展效果进行测试，压力为500g，得到其结果如图2所示，其中a～c为实施例1中得到的载药丝蛋白纳米纤维凝胶敷料 的压力铺展效果图，可以看到凝胶在玻璃板和动物创面上均可以均匀铺展，且压力去除后不会收缩；图2中d为不同浓度的流变性改变的丝蛋白纳米纤维凝胶的压力铺展效果图，可以看到，浓度过高，凝胶在压力取消后会收缩，浓度过低，凝胶流动性太好，容易流失。
图3为浓度为2.5wt％的丝蛋白纳米纤维凝胶稀释至0.8wt％未经剪切后的形态图。从图3中可以看出未经剪切处理的凝胶没有合理的流动性，不具备合理的压力铺展性能。
图4为实施例1中得到的载药丝蛋白纳米纤维凝胶敷料的动物实验结果图，其中Control为对照图，不经任何处理，SNF为步骤(1)中得到流变性改变的丝蛋白纳米纤维凝胶，SNF-DFO为实施例1中得到的载药丝蛋白纳米纤维凝胶敷料。由图4可以看出，实施例1中得到的载药丝蛋白纳米纤维凝胶敷料可均匀分布在皮瓣和创基之间，并铺满整个皮瓣，皮瓣的存活率和血管化网络程度均显著改善。动物实验过程中，将大鼠以4％浓度的水合氯醛腹腔注射麻醉(1ml/100g)后，剃除背部毛发，室温下大鼠以俯卧位置于手术台上，标记双侧髂后上棘位置，设计大鼠背部“乒乓拍”形狭长窄蒂皮瓣，蒂部起点为两侧髂棘连线水平，蒂宽1cm，蒂长3cm，圆形瓣部直径3cm；用75％乙醇进行皮肤消毒；手术刀沿设计线切开皮肤全层，保留蒂部连接，将皮瓣自肉膜层掀起，全层分离后，回移植，用5-0可吸收线原位缝合，间断缝合结束前自创口边缘将0.5ml材料(空白组、SNF、SNF-DFO)推注至皮瓣与基底之间，完成缝合，以指腹约500g压力于皮瓣上方轻柔按摩，使材料铺展于皮瓣下方，清洁纱布包扎，手术时长控制在30分钟内，术后不使用抗生素，大鼠分笼饲养。
实施例2
(1)将浓度为2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到1wt％，利用磁力搅拌器在900rpm的速率下，搅拌24小时；
(2)将去铁胺粉末同浓度为1wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)混合，使得去铁胺浓度为2mg/ml，在200rpm的速率下搅拌6小时，随后在14000rpm的转速下离 心25分钟，底部为载药的丝蛋白纳米纤维凝胶；
(3)将上述载药纳米纤维凝胶和(1)中搅拌处理的凝胶按照1:24的体积比混合，然后在600rpm的转速下搅拌14小时，使得两种凝胶充分混合，且混合凝胶的流变性调整到合适程度；
(4)将上述丝蛋白载药纳米纤维凝胶在100Pa的真空度下抽真空60分钟，去除气泡，获得最终的载药丝蛋白纳米纤维凝胶敷料。
图5为浓度为1wt％的丝蛋白纳米纤维凝胶在搅拌前后的形态图，可以看出，经过搅拌获得了良好的流动性和可铺展性，未经搅拌处理的凝胶没有合理的流动性，不具备合理的压力铺展性能。
图6为步骤(3)中浓度为1wt％的丝蛋白纳米纤维凝胶及加载60uM DFO的丝蛋白纳米纤维水凝胶的流变性能图，从中可以看出，加入小分子药物对凝胶材料的流变性能没有明显改变，药物的加载仅使得丝蛋白纳米纤维水凝胶在低剪切速率时有少许黏度增加的现象。
图7为1wt％的丝蛋白纳米纤维水凝胶加载去铁胺前后的拉曼光谱图，加载去铁胺后的拉曼光谱上同时具有丝蛋白和去铁胺的特征峰，证实去铁胺的加载。
实施例3
(1)将浓度为2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到1.1wt％，利用磁力搅拌器在600rpm的速率下，搅拌46小时；
(2)将内皮细胞生长因子粉末同浓度为1.2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)混合，使得内皮生长因子浓度为60μg/ml，在400rpm的速率下搅拌4小时，随后在9000rpm的转速下离心60分钟，底部为载药的丝蛋白纳米纤维凝胶；
(3)将上述载药纳米纤维凝胶和(1)中搅拌处理的凝胶按照1:8的体积比混合，然后在2000rpm的转速下搅拌4小时，使得两种凝胶充分混合，且混合凝胶的流变性调整到合适程度；
(4)将上述丝蛋白载药纳米纤维凝胶在40Pa的真空度下抽真空30分钟，去除气泡，获得最终的载药丝蛋白纳米纤维凝胶敷料。
图8为加载内皮细胞生长因子后的丝蛋白纳米纤维水凝胶与1.1wt％浓度的丝蛋白纳米纤维水凝胶混合后在真空下去除气泡前后的图像。
实施例4
(1)将浓度为3wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到1.3wt％，利用磁力搅拌器在3000rpm的速率下，搅拌2小时；
(2)将内皮细胞生长因子粉末同浓度为2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)混合，使得内皮生长因子浓度为100μg/ml，在600rpm的速率下搅拌2小时，随后在5000rpm的转速下离心60分钟，底部为载药的丝蛋白纳米纤维凝胶；
(3)将上述载药纳米纤维凝胶和(1)中搅拌处理的凝胶按照1:12的体积比混合，然后在2000rpm的转速下搅拌2小时，使得两种凝胶充分混合，且混合凝胶的流变性调整到合适程度；
(4)将上述丝蛋白载药纳米纤维凝胶在10Pa的真空度下抽真空30分钟，去除气泡，获得最终的载药丝蛋白纳米纤维凝胶敷料。
图9为加载内皮细胞生长因子后的浓度为2wt％的丝蛋白纳米纤维水凝胶与1.3wt％浓度的丝蛋白纳米纤维水凝胶混合后在真空下去除气泡前后的图像。
实施例5
(1)将浓度为2wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)添加去离子水，将浓度调控到1.2wt％，利用磁力搅拌器在1600rpm的速率下，搅拌3小时；
(2)将去铁胺粉末同浓度为0.6wt％的丝蛋白纳米纤维凝胶(丝蛋白纳米纤维的结晶度为40％，直径为20nm，长度为1μm)混合，使得去铁胺浓度为1.2mg/ml，在100rpm的速率下搅拌10小时，随后在12000rpm的转速下离心60分钟，底部为载药的丝蛋白纳米纤维凝胶；
(3)将上述载药纳米纤维凝胶和(1)中搅拌处理的凝胶按照1:12的体积比混合，然后在500rpm的转速下搅拌24小时，使得两种凝胶充分混合，且混合凝胶的流变性调整到合适程度；
(4)将上述丝蛋白载药纳米纤维凝胶在10Pa的真空度下抽真空20分钟，去除气泡，获得最终的载药丝蛋白纳米纤维凝胶敷料。
图10为制备所得丝蛋白纳米纤维凝胶的膨胀性能测试，经过测试，丝蛋白纳米纤维水凝胶及加载药物的丝蛋白纳米纤维水凝胶在37℃，与模拟人体内环境组织液的pH＝7.4的盐溶液接触时，在长时间(超过24h)下体积未发生明显变化(体积变化＜10％)。

Claims (10)

  1. 一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料的制备方法，其特征在于，包括以下步骤：
    S1)将第一丝蛋白纳米纤维凝胶与水混合搅拌，得到流变性改变的丝蛋白纳米纤维凝胶；
    将促血管再生的药物与第二丝蛋白纳米纤维凝胶混合，得到载药丝蛋白纳米纤维凝胶；
    S2)将所述流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶混合搅拌，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷料。
  2. 根据权利要求1所述的制备方法，其特征在于，所述第一丝蛋白纳米纤维凝胶的浓度为2～5wt％；所述第二丝蛋白纳米纤维凝胶的浓度为0.3～2wt％；所述流变性改变的丝蛋白纳米纤维凝胶的浓度为0.6～1.5wt％。
  3. 根据权利要求1所述的制备方法，其特征在于，所述第一丝蛋白纳米纤维凝胶中的丝蛋白纳米纤维为高结晶丝蛋白纳米纤维；所述高结晶丝蛋白纳米纤维的结晶度大于等于40％；所述高结晶丝蛋白纳米纤维的直径为10～30nm；长度为0.1～3μm。
  4. 根据权利要求1所述的制备方法，其特征在于，所述步骤S1)中混合搅拌的速率为200～5000rpm；混合搅拌的时间为1～48h；
    混合的速率为10～800rpm；混合的时间为10min～12h；
    混合后，离心，得到载药丝蛋白纳米纤维凝胶；
    所述离心的速率为2000～15000rpm；离心的时间为5min～2h。
  5. 根据权利要求1所述的制备方法，其特征在于，所述步骤S2)中流变性改变的丝蛋白纳米纤维凝胶与载药丝蛋白纳米纤维凝胶的体积比为(8～24)：1。
  6. 根据权利要求1所述的制备方法，其特征在于，所述S2)中混合搅拌的速率为200～5000rpm；混合搅拌的时间为1～48h。
  7. 根据权利要求1所述的制备方法，其特征在于，所述步骤S2)中混合搅拌后，还进行抽真空处理，得到具备压力可铺展性的丝蛋白纳米纤维凝胶敷 料。
  8. 根据权利要求7所述的制备方法，其特征在于，所述抽真空处理的真空度为0.01～100Pa；抽真空处理的时间为10～60min。
  9. 一种具备压力可铺展性的丝蛋白纳米纤维凝胶敷料，其特征在于，所述丝蛋白纳米纤维凝胶敷料在压力作用下可铺展成厚度小于等于200微米的薄膜，且压力取消后仍维持铺展状态。
  10. 权利要求1～8任意一项制备方法所述制备的丝蛋白纳米纤维凝胶敷料或权利要求9所述的丝蛋白纳米纤维凝胶敷料作为皮瓣移植用凝胶敷料的应用。
PCT/CN2021/122111 2021-03-08 2021-09-30 具备压力可铺展性的丝蛋白纳米纤维凝胶敷料、其制备方法及应用 WO2022188399A1 (zh)

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