CN113274538A

CN113274538A - bFGF slow-release nano dressing with wound surface active repair function and preparation method and application thereof

Info

Publication number: CN113274538A
Application number: CN202110592637.4A
Authority: CN
Inventors: 张松平; 苏志国; 车诗怡
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-08-20
Anticipated expiration: 2041-05-28
Also published as: CN113274538B

Abstract

The invention relates to a bFGF slow-release nano dressing with a wound surface active repair function, and a preparation method and application thereof. According to the invention, adenosine triphosphate or salt thereof and metal ions are used as bFGF stabilizers, a bFGF-electronegative macromolecule-metal ion ternary composite system is formed through electrostatic interaction and coordination among bFGF, metal ions and electronegative macromolecules, a bFGF stabilization strategy is simply and efficiently established, the activity of bFGF in dressing is synergistically improved, the traditional animal-derived component stabilizer which is easy to generate immunogenicity is replaced, and meanwhile, complex operation of microsphere embedding is avoided. The long-acting slow-release effect of bFGF is also promoted by a core-shell structure and a ternary composite system existing in the nanofiber formed by the dressing through electrostatic spinning.

Description

bFGF slow-release nano dressing with wound surface active repair function and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, relates to a bFGF slow-release nano dressing and a preparation method and application thereof, and particularly relates to a bFGF slow-release nano dressing with a wound surface active repair function and a preparation method and application thereof.

Background

Wounds are quite common in daily life, the treatment period of some wounds with large wound surfaces is long, and the burdens of manpower, medical treatment and cost are easily caused; in the face of small wounds, the self-healing of the small wounds is realized only by the self-repairing mechanism of the body without effective intervention, and scars are easy to leave, so that the visual aesthetic feeling is influenced.

Growth factors are a class of polypeptides that have the activity of stimulating cell growth, and regulate cell growth and other cellular functions by binding to specific, high affinity receptors. The exogenous addition of growth factors can start the self active repair capability of the human body, accelerate the re-epithelialization process of the wound surface while promoting the formation of granulation tissues, and play an important role in the healing process of the wound surface.

Basic fibroblast growth factor (bFGF) is the most early-discovered, most active and most powerful factor in human. In acute wounds, bFGF is capable of promoting fibroblast proliferation, regulating the synthesis and deposition of various extracellular matrix (ECM) components, promoting granulation tissue formation and epithelial regeneration. However, it is extremely low in vivo content, sensitive to heat and acid, and easy to inactivate, which makes clinical application extremely challenging, and therefore, it is very critical to select a proper dosage form for administration.

The traditional bFGF dosage forms mainly comprise spray, freeze-dried powder and gel, the bFGF in the spray exists in a solution form, and the bFGF is very easy to inactivate in links of transportation, storage, use and the like; the freeze-dried powder is also administrated in a solution form, can be quickly combined with the wound surface, but is not beneficial to wound recovery and cannot realize long-term slow release of bFGF due to quick response, quick in vivo metabolism and short half-life; the gel is easy to lose when exposed on the body surface, and needs to be repeatedly administered within one day, thus limiting the clinical application of the gel to a certain extent. Therefore, the controlled release and the sustained release of the medicine are realized by proper medicine carrying materials, and the prolonging of the quality guarantee period is the key point for improving the clinical application of growth factor medicines.

The electrostatic spinning is a technology capable of continuously producing the nano-fiber, and the obtained nano-fiber membrane integrally presents the form of non-woven fabric, and has a compact porous structure and a high specific surface area. Meanwhile, the pore size of the nanofiber membrane can be adjusted by controlling the spinning time, the pore diameter of most of the nanofiber membranes is in a nanometer level and is far smaller than the diameters of various pathogenic bacteria, and the nanofiber membrane has high-efficiency bacteria isolation. Much research is currently devoted to the development of new formulations of bFGF electrospun.

CN107185025A discloses a traditional Chinese medicine nano-spinning composite fiber membrane dressing, a preparation method and an application thereof, the composite fiber membrane dressing takes PLGA as a carrier, and carries Curcumin (CUR) and fibroblast growth factor (bFGF), and a degradable nano-spinning composite material is prepared by an electrostatic spinning method. Wherein the curcumin accounts for 1-3% of the mass fraction of the polylactic acid-glycollic acid copolymer, and the fibroblast growth factor accounts for 8.33-33.33 multiplied by 10 of the mass fraction of the polylactic acid-glycollic acid copolymer^-4% of the total weight of the composition. The composite fiber film dressing has a good nano structure, has the characteristics of maintaining the drug slow release effect and reducing the easy loss of the fibroblast growth factor when being independently used, has good air permeability and waterproof function, and the nano-size aperture is beneficial to the proliferation of cells and the healing of wounds.

CN109602953A discloses a novel long-acting slow-release VEGF and bFGF degradable biological nano-membrane and a preparation method thereof, the method is to dissolve a biodegradable polymer material in a polar and volatile organic solvent, and prepare the biodegradable nano-fiber membrane with a nano-scale structure by an electrostatic spinning method; loading the growth factor onto the biodegradable nanometer fiber membrane, freeze drying, sterilizing and packaging to obtain the required biological nanometer membrane. The biological nanometer membrane can be applied to insufficient regeneration of blood vessels after indirect vascular bypass surgery in neurosurgery, the treatment effect of related ischemic cerebrovascular diseases is improved, and the reoccurrence risk of ischemic stroke is reduced.

CN110013567A discloses a degradable drug-loaded controlled-release myocardial repair patch with a multi-stage structure, which is prepared by blending vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF), polypyrrole/metal microparticles and levorotatory polylactic acid/polyethylene lactone (PLLA/PCL) in a mild solvent, and using ultrasonic emulsification and multi-fluid orientation electrospinning techniques at room temperature. The surface of the myocardial repair patch is made into a multi-stage structure by using a laser engraving technology, and has the functions of degrading drug loading and controlling release; the surface layer is coated with polydopamine, which is beneficial to tissue adhesion.

CN110975007A discloses a bFGF-loaded guided tissue regeneration membrane with a core-shell structure and a preparation method thereof, wherein the guided tissue regeneration membrane is constructed by alkaline fibroblast growth factor, a synthetic high molecular compound polylactic acid-glycolic acid copolymer and natural components of wool keratin, the content of the alkaline fibroblast growth factor is 0.03-0.3 g/L, the concentration of the polylactic acid-glycolic acid copolymer is 15-20%, and the concentration of the wool keratin is 1.0-1.5%. The guided tissue regeneration membrane has the advantages of simple preparation method, mild membrane forming conditions, good cell compatibility and capability of actively inducing regeneration of periodontal tissues, meets the requirements of in vivo application, and has good application prospect as a biological membrane with the function of inducing tissue regeneration.

CN105040280B authorizes a polypropylene mesh/electrostatic spinning nanofiber membrane and a preparation method and application thereof, the invention creatively compounds the polypropylene mesh and the electrostatic spinning nanofiber membrane, improves the biocompatibility of the polypropylene mesh by using the nanofiber membrane, improves the tensile strength of the nanofiber membrane by using the polypropylene mesh, supplements the two, and introduces polylactic acid-glycolic acid copolymer PLGA microspheres for preparing embedded basic fibroblast growth factor bFGF to realize drug controlled release, thereby filling a plurality of defects of the clinical existing polypropylene mesh.

In the existing strategy of carrying bFGF by electrostatic spinning, in order to improve the slow release performance of bFGF, emulsion electrospinning is usually adopted to prepare the drug-loaded nanofiber dressing with a core-shell structure, and long-term slow release of bFGF is realized by continuous degradation of shell layer materials, so that the preparation method is simple and convenient to operate and feasible in production amplification. However, if the emulsion electrospinning is applied to the preparation of the bFGF-loaded novel wound dressing, the preparation process of the emulsion and the influence on the activity of the protein drug need to be considered. In order to improve the activity of bFGF, bovine serum albumin is usually used as a stabilizer of a growth factor in the prior art, or the growth factor is prepared into nano microspheres for pre-embedding. But the addition of the bovine serum albumin introduces animal-derived components, which increases the complexity of the components of the growth factor preparation; and the preparation of the nano microspheres increases the complexity of the preparation process of the nano fibers.

Therefore, it is important to develop a nano-dressing which maintains the activity of bFGF for a long time, has a good sustained and controlled release effect of bFGF, and reduces the complexity of the operation.

Disclosure of Invention

Aiming at the defects that the prior art easily causes bFGF inactivation and cannot realize good slow release effect, the invention aims to provide a bFGF slow release nano dressing and a preparation method and application thereof, and in particular relates to a bFGF slow release nano dressing with a wound surface active repair function and a preparation method and application thereof. The nano dressing integrates the promotion effect of bFGF, a stabilizing agent and a nano fiber membrane on wound healing, and provides a research basis for the product development and application of bioactive wound dressings.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a bFGF sustained release nano dressing with a wound surface active repair function, which is an electrostatic spinning nano fibrous membrane prepared from an emulsion containing basic fibroblast growth factor, adenosine triphosphate or salt thereof and metal ions.

The bFGF slow-release nano dressing creatively takes adenosine triphosphate or salt thereof and metal ions as a stabilizer of bFGF, and because the surface of bFGF molecules is rich in positive charges, a ternary complex system of bFGF-negative electricity macromolecules-metal ions is formed through electrostatic interaction and coordination among the bFGF, the metal ions and the negative electricity macromolecules, a stability strategy of the bFGF is simply and efficiently established, the activity of the bFGF in the dressing is synergistically improved, the traditional animal-derived component stabilizer which is easy to generate immunogenicity is replaced, and meanwhile, the complex operation of embedding microspheres is avoided. The long-acting slow-release effect of bFGF is also promoted by a core-shell structure and a ternary composite system existing in the nanofiber formed by the dressing through electrostatic spinning. Meanwhile, the nano dressing integrates the bFGF, metal ions, adenosine triphosphate or salt thereof and the promotion effect of the electrostatic spinning nanofiber membrane on wound healing. The nanometer dressing has good cell proliferation activity and blood compatibility, effectively promotes wound healing, and has wide application prospect.

Preferably, the emulsion comprises an internal aqueous phase comprising basic fibroblast growth factor, adenosine triphosphate or a salt thereof, metal ions and an external oil phase comprising a biocompatible polymeric material.

Preferably, the adenosine triphosphate or its salt includes any one of adenosine triphosphate, adenosine disodium triphosphate, adenosine magnesium triphosphate, adenosine tetralithium triphosphate or a combination of at least two of them; the combination of at least two of the above-mentioned components, such as the combination of adenosine triphosphate and disodium adenosine triphosphate, the combination of disodium adenosine triphosphate and magnesium adenosine triphosphate, etc., may be selected in any combination manner, and will not be described in detail herein.

Preferably, the metal ions include any one or a combination of at least two of zinc ions, magnesium ions, copper ions, ferrous ions, or nickel ions; the combination of at least two of the above-mentioned compounds, such as the combination of zinc ion and magnesium ion, the combination of copper ion and ferrous ion, the combination of ferrous ion and nickel ion, etc., any other combination mode can be selected, and it is not repeated here.

Preferably, the biocompatible polymer material comprises any one of or a combination of at least two of polylactic acid, polyglycolic acid copolymer, polycaprolactone, polytetrafluoroethylene and polylactic acid-polyethylene glycol block copolymer. The combination of at least two of the above-mentioned polymers, such as the combination of polylactic acid and polyglycolic acid copolymer, the combination of polycaprolactone and polytetrafluoroethylene, the combination of polytetrafluoroethylene and polylactic acid-polyethylene glycol block copolymer, etc., can be selected in any combination manner, and will not be described in detail herein.

In the invention, the basic fibroblast growth factor, adenosine triphosphate or its salt and metal ions form a ternary complex system through electrostatic interaction and coordination.

Preferably, the ratio of the basic fibroblast growth factor, the metal ion, the adenosine triphosphate or the salt thereof is (1-100) mg, (0.01-10) mmol.

The (1-100) mg means that specific points can be selected from 1mg, 2mg, 5mg, 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 85mg, 90mg, 100mg and the like.

The (0.01-10) mmol means that the specific point value can be selected from 0.01mmol, 0.05mmol, 0.1mmol, 0.5mmol, 1mmol, 1.5mmol, 2mmol, 3mmol, 4mmol, 5mmol, etc.

The specific point values within the above ranges can be selected, and are not described in detail herein.

Preferably, the proportion of the basic fibroblast growth factor, the metal ions, the adenosine triphosphate or the salt thereof is (2-50) mg, (1-5) mmol.

Preferably, the diameter of the nanofibers of the electrospun nanofiber membrane is 250-550nm, such as 250nm, 280nm, 300nm, 325nm, 350nm, 375nm, 400nm, 425nm, 450nm, 500nm, 550nm, and the like, and specific values within the numerical range can be selected, which is not described herein again.

In a second aspect, the present invention provides a preparation method of the bFGF sustained release nano dressing with active wound repair function according to the first aspect, the preparation method includes:

and (2) carrying out electrostatic spinning on the emulsion containing the basic fibroblast growth factor, the adenosine triphosphate or the salt thereof and the metal ions, and drying to prepare the bFGF slow-release nano dressing with the wound active repair function.

Preferably, the parameters of electrospinning include: the flow rate of the spinning solution is 0.2-0.8mL/h, the spinning voltage is 20-30kV, the distance between a needle head and a collecting plate is 15-25cm, and the number of the spinning needle head is 4-7.

The flow rate of the spinning solution may be 0.2mL/h, 0.3mL/h, 0.4mL/h, 0.5mL/h, 0.6mL/h, 0.7mL/h, 0.8mL/h, etc.

The spinning voltage can be 20kV, 22kV, 23kV, 24kV, 25kV, 26kV, 27kV, 28kV, 29kV, 30kV and the like.

The distance between the needle and the collecting plate can be 15cm, 16cm, 17cm, 18cm, 20cm, 21cm, 22cm, 24cm, 25cm and the like.

The spinning needle head can be selected from No. 4, No. 5, No. 6 and No. 7.

Preferably, the drying is natural drying, oven drying, vacuum oven drying or freeze drying.

Preferably, the preparation method of the emulsion comprises the following steps:

(1) mixing basic fibroblast growth factor, buffer solution, metal salt, adenosine triphosphate or its salt to prepare inner water phase solution; mixing a biocompatible high polymer material and an organic solvent to prepare an external oil phase solution;

(2) mixing and emulsifying the inner water phase solution, the outer oil phase solution and the emulsifier to obtain the emulsion.

The preparation process is schematically shown in figure 1.

Preferably, the buffer comprises any one or a combination of two of phosphate buffer, Tris-HCl buffer or HEPES buffer; the combination of at least two of the buffer solutions can be selected from any combination mode, such as the combination of a phosphate buffer solution and a Tris-HCl buffer solution, the combination of a Tris-HCl buffer solution and a HEPES buffer solution, and the like, and the description is omitted.

Preferably, the organic solvent comprises any one or two of dichloromethane, trichloromethane, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide or tetrahydrofuran; the combination of at least two of the above-mentioned compounds, such as the combination of dichloromethane and chloroform, the combination of N, N-dimethylformamide and N, N-dimethylacetamide, the combination of dimethylsulfoxide and tetrahydrofuran, etc., can be selected in any combination manner, and thus, the details are not repeated herein.

Preferably, the concentration of the biocompatible polymer material in the external oil phase solution is 2-20% (w/v), for example, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 20%, etc., and specific values within the numerical range can be selected, which is not described herein again. Preferably 5-12% (w/v).

Preferably, the volume ratio of the solution of the inner water phase to the solution of the outer oil phase in step (2) is 0.1-10% (v/v), such as 0.1%, 0.5%, 1%, 2%, 4%, 5%, 6%, 8%, 10%, etc., and specific values within the value range can be selected, which is not described herein again. Preferably 0.5-6% (v/v).

Preferably, the specific operation method in the step (2) comprises the following steps: and (3) uniformly mixing the emulsifier and the external oil phase solution, dropwise adding the internal water phase solution into the mixture under ice-bath stirring, and emulsifying to obtain the emulsion.

Preferably, the emulsifier comprises any one of a nonionic emulsifier, a zwitterionic emulsifier, an anionic emulsifier or a cationic emulsifier or a combination of at least two thereof.

Preferably, the non-ionic emulsifier comprises any one of Span20, Span40, Span60, Span80, Span85, Tween20, Tween40, Tween60, Tween80 or Tween85 or a combination of at least two thereof.

Preferably, the zwitterionic emulsifier comprises any one of lecithin, amino acid type emulsifier, betaine type emulsifier or triethanolamine soap of stearic acid or a combination of at least two of the above.

Preferably, the anionic emulsifier comprises any one of sodium dodecylbenzene sulfonate, oleic acid, lauric acid, sulfated castor oil or calcium stearate or a combination of at least two of them.

Preferably, the cationic emulsifier comprises any one of or a combination of at least two of benzyltriethylammonium chloride, quaternary ammonium salt compounds or alkyl ammonium salt compounds.

Preferably, the amount of the emulsifier added to the solution of the external oil phase is 0.1-5% (v/v), such as 0.1%, 0.2%, 1%, 2%, 3%, 4%, 5%, etc., and specific values within this range can be selected, which is not described herein again. Further preferably 0.2 to 2% (v/v).

Preferably, the emulsifying method comprises any one of a magnetic stirring method, a homogeneous dispersion method, an ultrasonic emulsification method, a syringe mutual pushing method or a membrane emulsification method or a combination of at least two of the methods.

Preferably, the emulsifying time is 1-30min, such as 1min, 2min, 3min, 5min, 10min, 12min, 15min, 20min, 22min, 25min, 30min, etc., and specific point values within the numerical range can be selected, which is not described in detail herein. Preferably 2-12 min.

In a third aspect, the invention provides an application of the bFGF sustained-release nano dressing with active wound healing function in preparing a material for promoting wound healing.

Compared with the prior art, the invention has the following beneficial effects:

the bFGF slow-release nano dressing creatively takes adenosine triphosphate or salt thereof and metal ions as a stabilizer of bFGF, and because the surface of bFGF molecules is rich in positive charges, a ternary complex system of bFGF-negative electricity macromolecules-metal ions is formed through electrostatic interaction and coordination among the bFGF, the metal ions and the negative electricity macromolecules, a stability strategy of the bFGF is simply and efficiently established, the activity of the bFGF in the dressing is synergistically improved, the traditional animal-derived component stabilizer which is easy to generate immunogenicity is replaced, and meanwhile, the complex operation of embedding microspheres is avoided.

The long-acting slow-release effect of bFGF is also promoted by a core-shell structure and a ternary composite system existing in the nanofiber formed by the dressing through electrostatic spinning.

Meanwhile, the nano dressing integrates the bFGF, metal ions, adenosine triphosphate or salt thereof and the promotion effect of the electrostatic spinning nanofiber membrane on wound healing. The nanometer dressing has good cell proliferation activity and blood compatibility, effectively promotes wound healing, and has wide application prospect. Compared with the bFGF nano dressing without a stabilizer, the bFGF slow-release nano dressing has the advantages that the activity of promoting the proliferation of human dermal fibroblasts is improved by 1.39 times, the volume of type I collagen is improved by 3.45 times, the hemolysis rate is obviously reduced, and the biocompatibility is good.

The bFGF slow-release nano dressing has a good treatment effect on a mouse skin defect model. Compared with a blank group and the bFGF nano dressing without the stabilizer, the bFGF slow-release nano dressing has the advantages that the speed of promoting wound healing is higher in the same time, the epidermal thickness and the collagen bulk density of the wound of a mouse are not significantly different from those of normal skin in 14 days, and the bFGF slow-release nano dressing has a wide application prospect.

Drawings

FIG. 1 is a schematic diagram of the preparation of a bFGF sustained-release nano-dressing according to the invention;

FIG. 2 is an SEM image of each group of bFGF sustained-release nano-dressings;

FIG. 3 is TEM image of each group of bFGF sustained-release nano-dressings;

FIG. 4 is a bFGF in vitro release curve chart of each group of bFGF sustained-release nano-dressings;

FIG. 5 is a graph of sirius red staining results of various groups of bFGF sustained-release nano-dressings;

FIG. 6 is a graph showing the results of wound healing in mice of each group.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The embodiment provides a bFGF sustained release nano dressing (abbreviated as bFGF/PCL), which is prepared by the following steps:

(1) a bFGF stock solution was prepared by redissolving 100 μ g of lyophilized powder of recombinant human basic fibroblast growth factor (bFGF) in 100 μ L of Tris (5mM, pH 7.6) and diluting to 100 μ g/mL with 5% trehalose in PBS stock solution (pH 7.2);

(2) 2.383g of HEPES was weighed and dissolved in 700mL of ultrapure water, the pH was adjusted to 7.4 with a sodium hydroxide solution, and the solution was diluted to 1000mL, and sterilized by filtration to prepare a HEPES buffer (10mM, pH 7.4);

(3) mix 50 μ L of stock bFGF with 50 μ L of HEPES buffer to prepare 100 μ L of an aqueous inner phase solution;

(4) dissolving 200mg of polycaprolactone in 2mL of chloroform to prepare a polycaprolactone solution with the concentration of 10% (w/v) as an external oil phase solution;

(5) adding 1.5% (v/v) (30 mu L) of Span80 into 2mL of polycaprolactone solution obtained in the step (4), uniformly mixing, dropwise adding 100 mu L of internal water phase solution obtained in the step (3) into the solution under ice bath stirring, and emulsifying for 5min by adopting an ultrasonic method to obtain emulsion;

(6) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. After completion of electrospinning, the fibrous membrane was placed in a fume hood overnight to remove residual organic solvent, yielding bFGF/PCL.

The diameter of the prepared nanofiber is 271 +/-66 nm by SEM measurement.

Example 2

The embodiment provides a bFGF sustained release nano dressing (bFGF-Zn/PCL for short), which is prepared by the following steps:

(3) weighing 27.26g of zinc chloride powder, dissolving in 100mL of deionized water, adding hydrochloric acid for assisting dissolution to obtain Zn with the concentration of 2M²⁺Mother liquor, which was diluted to 40mM to prepare Zn²⁺Storing liquid;

(4) 50 μ L of stock bFGF, 10 μ L of Zn²⁺Stock solution and 40. mu.L HEPES buffer were mixed to prepare 100. mu.L of an inner aqueous phase solution;

(5) dissolving 200mg of polycaprolactone in 2mL of chloroform to prepare a polycaprolactone solution with the concentration of 10% (w/v) as an external oil phase solution;

(6) adding 1.5% (v/v) (30 mu L) of Span80 into 2mL of polycaprolactone solution obtained in the step (5), uniformly mixing, dropwise adding 100 mu L of internal water phase solution obtained in the step (4) into the solution under ice bath stirring, and emulsifying for 5min by adopting an ultrasonic method to obtain emulsion;

(7) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. After completion of electrospinning, the fibrous membrane was placed in a fume hood overnight to remove the residual organic solvent, yielding bFGF-Zn/PCL.

The diameter of the prepared nano-fiber is 324 +/-127 nm determined by SEM.

Example 3

The embodiment provides a bFGF sustained release nano dressing (bFGF-ATP/PCL for short), which is prepared by the following steps:

(3) weighing 605.2mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing a 40mM ATP solution;

(4) mixing 50. mu.L of stock bFGF, 10. mu.L of ATP solution and 40. mu.L of HEPES buffer solution to prepare 100. mu.L of an aqueous inner phase solution;

(7) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. After completion of electrospinning, the fibrous membrane was placed in a fume hood overnight to remove the residual organic solvent and to obtain bFGF-ATP/PCL.

The diameter of the prepared nanofiber is 324 +/-146 nm by SEM measurement.

Example 4

The embodiment provides a bFGF sustained release nano dressing (bFGF-ATP-Zn/PCL for short), which is prepared by the following steps:

(3) weighing 605.2mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing a 40mM ATP solution; weighing 27.26g of zinc chloride powder, dissolving in 100mL of deionized water, adding hydrochloric acid for assisting dissolution to obtain Zn with the concentration of 2M²⁺Mother liquor, which was diluted to 40mM to prepare Zn²⁺Storing liquid;

(4) 50 μ L of stock bFGF, 5 μ L of ATP solution, 5 μ L of Zn²⁺Stock solution and 40. mu.L HEPES buffer were mixed to prepare 100. mu.L of an inner aqueous phase solution;

(7) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. And after the electrospinning is finished, the fiber membrane is placed in a fume hood overnight to remove residual organic solvent, so that the bFGF-ATP-Zn/PCL is obtained.

The diameter of the prepared nanofiber is 364 +/-138 nm determined by SEM.

Example 5

The embodiment provides a bFGF sustained release nano dressing (bFGF-ATP-Mg/PCL for short), which is prepared by the following steps:

(3) weighing 605.2mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing a 40mM ATP solution; 38.08Mg of magnesium chloride was weighed out and dissolved in 100mL of deionized water to obtain 40mM Mg²⁺Storing liquid;

(4) 50 μ L of stock bFGF, 5 μ L of ATP solution, 5 μ L of Mg²⁺Stock solution and 40. mu.L HEPES buffer were mixed to prepare 100. mu.L of an inner aqueous phase solution;

(7) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. And after the electrospinning is finished, placing the fiber membrane in a fume hood overnight to remove residual organic solvent to obtain the bFGF-ATP-Mg/PCL.

The diameter of the prepared nanofiber is 550 +/-113 nm as determined by SEM.

Example 6

(3) weighing 605.2mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing a 40mM ATP solution; weighing 27.26g of zinc chloride powder, dissolving in 100mL of deionized water, adding hydrochloric acid for assisting dissolution to obtain Zn with the concentration of 2M²⁺Mother liquor, which was diluted to 20mM to prepare Zn²⁺Storing liquid;

The diameter of the prepared nano-fiber is 350 +/-104 nm by SEM measurement.

Example 7

(3) weighing 302.6mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing 20mM ATP solution; weighing 27.26g of zinc chloride powder, dissolving in 100mL of deionized water, adding hydrochloric acid for assisting dissolution to obtain Zn with the concentration of 2M²⁺Mother liquor, which was diluted to 40mM to prepare Zn²⁺Storing liquid;

The diameter of the prepared nano-fiber is 300 +/-78 nm by SEM measurement.

Example 8

(3) weighing 302.6mg of ATP disodium salt, dissolving in 50mL of sterile deionized water, and preparing 20mM ATP solution; weighing 27.26g of zinc chloride powder, dissolving in 100mL of deionized water, adding hydrochloric acid for assisting dissolution to obtain Zn with the concentration of 2M²⁺Mother liquor, which was diluted to 20mM to prepare Zn²⁺Storing liquid;

The diameter of the prepared nanofiber is 250 +/-96 nm through SEM measurement.

Comparative example 1

The comparative example provides a bFGF-free nanocoating (PBS/PCL for short) as a blank control, and the preparation method thereof is:

(1) dissolving 200mg of polycaprolactone in 2mL of chloroform to prepare a polycaprolactone solution with the concentration of 10% (w/v) as an external oil phase solution;

(2) adding 1.5% (v/v) (30 muL) of Span80 into 2mL of polycaprolactone solution obtained in the step (1), uniformly mixing, dropwise adding 100 muL of PBS solution (pH 7.2) into the solution under ice-bath stirring, and emulsifying for 5min by adopting an ultrasonic method to obtain emulsion;

(3) the emulsion was electrospun using a 5-gauge needle at a flow rate of 0.5mL/h with an injection pump set to 25kV and a collector plate distance set to 20 cm. After completion of electrospinning, the fibrous membrane was placed in a fume hood overnight to remove residual organic solvent, yielding PBS/PCL.

Comparative example 2

The comparative example provides a bFGF gel as a control for bFGF nanopaste, which was prepared as follows:

(1) taking 9400.85 g of carbomer, uniformly dispersing with 15g of glycerol and 5g of 1, 2-propylene glycol, adding 57g of 0.1M NaOH, adding 10mM PBS to 100g, stirring uniformly, heating in water bath at 80 ℃ for 30min, and preparing a gel matrix;

(2) sterilizing the gel matrix obtained in the step (1) for 30min by adopting a high-pressure steam sterilization pot (105Kpa, 121 ℃), and cooling;

(3) and (3) uniformly mixing 1mL of the gel matrix obtained in the step (2) with 100 mu L of bFGF solution (4 mu g/mL) to obtain the bFGF gel.

Test example 1

The test example is the characterization of the fiber morphology structure and the contact angle of the bFGF sustained-release nano dressing, and the specifically used characterization method comprises the following steps: the surface morphology of the bFGF sustained-release nano dressing is characterized by SEM and a contact angle, the contact angle test data is shown in Table 1, and the SEM schematic diagram is shown in FIG. 2; the core-shell structure was characterized by TEM and the results are shown in figure 3.

TABLE 1

As can be seen from fig. 2: the bFGF/PCL nano-dressing of example 1 has more spike-shaped filaments, the surface of the bFGF-Zn/PCL of example 2 has a plurality of spikes, and the surface morphologies of the bFGF-ATP/PCL of example 3 and the bFGF-ATP-Zn/PCL of example 4 are better. As can be seen from Table 1, the bFGF/PCL of example 1 has certain hydrophilicity, while the bFGF nano-dressings of examples 2-4 have significantly increased water contact angles, indicating that the hydrophobicity is increased.

Test example 2

The test example is the mechanical property characterization of the bFGF sustained-release nano dressing, and the specific method comprises the following steps: the nano dressing was fixed on a jig, and a tensile external force was applied to the nano dressing by a Dynamic Mechanical Analyzer (DMA) until the film broke, and the test results were obtained as shown in table 2.

TABLE 2

	Nanometer dressing type	Tensile Strength (MPa)	Elongation at Break (%)
				Example 1	bFGF/PCL	3.12	73.43
Example 2	bFGF-Zn/PCL	9.79	111.81
				Example 3	bFGF-ATP/PCL	9.93	88.13
Example 4	bFGF-ATP-Zn/PCL	10.46	111.92

As can be seen from Table 2, the bFGF/PCL nano-dressing of example 1 has lower tensile strength and elongation at break; in examples 2-4, ATP and Zn²⁺The tensile strength and the elongation at break of the bFGF-loaded nano dressing are obviously improved by adding the (B) and the (B) modified nano dressing, and the effect of example 4 is the best. In particular, in examples 2 and 4, Zn²⁺The maximum breaking elongation of the nanofiber membrane can reach more than 100 percent by adding the nano-fiber membrane, and the nano-fiber membrane has very important application significance for wounds on frequently moving parts such as finger joints, ankle joints and the like.

Test example 3

The test example is an in vitro release performance evaluation test of the bFGF sustained-release nano dressing, and the specific method comprises the following steps:

(1) cutting each group of bFGF slow-release nano dressing into 3mg, accurately weighing, immersing into 2mL sterile PBS solution, and oscillating in a shaker at 37 ℃ at a constant speed of 100 rpm;

(2) taking out 2mL of release solution from the centrifuge tube in a preset time, simultaneously supplementing 2mL of fresh PBS solution, and continuously putting the solution into a shaking table;

(3) determining the concentration of the released solution taken out in the step (2) by enzyme-linked immunosorbent assay (ELISA) according to the instruction of the kit; the test results are shown in fig. 4 and table 3.

TABLE 3

	Nanometer dressing type	14 days Release (%)
			Example 1	bFGF/PCL	33.54
Example 2	bFGF-Zn/PCL	27.36
			Example 3	bFGF-ATP/PCL	28.12
Example 4	bFGF-ATP-Zn/PCL	22.76

From the test data in table 3, it can be seen that, compared with example 1, the formation of bFGF-ATP-Zn ternary complex and bFGF-ATP, bFGF-Zn binary complex provides significant slow release effect of bFGF in the nano-dressing, wherein the slow release effect of bFGF-ATP-Zn/PCL nano-dressing is optimal.

Test example 4

The test example is a blood compatibility evaluation test of the bFGF sustained-release nano dressing, and specifically is a method for detecting the hemolysis rate of the bFGF nano dressing by using erythrocytes, which comprises the following steps:

(1) cutting each group of bFGF slow-release nano dressing into round pieces with the diameter of 1.5cm, irradiating each surface for 15min by ultraviolet, and placing the round pieces at the bottom of a 24-hole culture plate;

(2) blood is taken from Balb/c mouse eyeballs, 1mL of blood is centrifuged to remove upper serum so as to separate red blood cells, the red blood cells are washed twice, 30mL of PBS (10mM, pH 7.2) is used for heavy suspension and dilution, 1mL of diluted red blood cells are added to a nano dressing of a 24-hole culture plate, and the incubation is carried out for 1h at 37 ℃;

(3) collecting the leaching liquor of each well, centrifuging at 1000rpm for 10min, separating the supernatant, measuring the absorbance of the supernatant at 540nm by using a microplate reader, adding 0.1% Triton X-100 into red blood cells as a positive control (n is 4), and adding 0.1% PBS into the red blood cells as a blank control;

(4) the hemolysis rate was calculated as: (absorbance of sample group-blank absorbance)/(absorbance of positive control group-blank absorbance) × 100%; the test results are shown in table 4.

TABLE 4

	Nanometer dressing type	Hemolysis ratio (%)
			Example 1	bFGF/PCL	14.72
Example 2	bFGF-Zn/PCL	3.36
			Example 3	bFGF-ATP/PCL	7.29
Example 4	bFGF-ATP-Zn/PCL	3.13
			Comparative example 1	PBS/PCL	15.02

As can be seen from Table 4, the PBS/PCL nanofiber membrane in comparative example 1 has a relatively high hemolysis rate; the hemolysis rate (14.72%) of bFGF/PCL in example 1 was comparable to that of comparative example 1. Adding ATP or Zn²⁺Thereafter, the hemolysis rate of examples 2-4 decreased dramatically, indicating ATP or Zn²⁺The addition of (2) effectively improves the blood compatibility of the bFGF-loaded nano dressing, wherein the blood compatibility of the example 4 is the best.

Test example 5

The test example is a cell compatibility evaluation test of the bFGF sustained release nano dressing, specifically, a cell proliferation experiment of the nano dressing using human dermal fibroblasts, the method is as follows:

(1) cutting each group of bFGF slow-release nano dressing into round pieces with the diameter of 1.5cm, irradiating each side for 15min by ultraviolet, placing at the bottom of a 24-hole culture plate, after human dermal fibroblasts grow and are fully fused, digesting by trypsin, counting, and counting by 5.0 multiplied by 10 per hole⁴The number of cells is planted on the nanometer dressing in each pore plate, and the culture medium is replaced every two days; and (3) detecting the adhesion and proliferation of cells on the nano dressing by using a CCK-8 kit. After inoculating cells for 2d, 4d and 6d, washing with PBS, adding 400 mu L of DMEM medium containing 10% CCK8 into each well, incubating for 2h at 37 ℃, then sucking 150 mu L of the DMEM medium out of each well, adding the DMEM medium into a 96-well plate, and reading the plate at the wavelength of 450nm by using an enzyme-labeling instrument; the test results are shown in table 5;

TABLE 5

As is apparent from Table 5, the cell proliferation activities of examples 1 to 4 and comparative examples 1 to 2 were increased over a period of 6 days. Compared with the comparative example 1, the OD value of the bFGF nano dressings prepared in the examples 1-2 and 4 is higher, which indicates that the cell activity is higher; whereas the bFGF gel of comparative example 2 has the highest OD value due to the rapid release of bFGF from the gel matrix by dissolution of the gel in the liquid medium; the OD value of the bFGF-ATP/PCL nano-dressing prepared by the embodiment 3 is the lowest. Example 4 OD value at day 6 was close to that of the coupleThe bFGF gel in ratio 2, which had the optimum cell proliferation activity in the four examples, was increased 1.39-fold as compared with example 1, thereby indicating that bFGF, ATP and Zn²⁺Has synergistic effect on cell proliferation.

(2) The fibroblast collagen secretion condition is characterized by adopting sirius red staining, after the 6 th day of cell inoculation, a stainless steel ring is taken down, a fiber membrane is cleaned by PBS, the fiber membrane is fixed by 4% paraformaldehyde for 30min at 25 ℃, the fiber membrane is soaked and dyed by sirius red staining solution for 1h after being cleaned by deionized water, redundant dye is removed by washing, a light microscope is used for staining observation and analysis, and the test result is shown in figure 5; semi-quantitative analysis was performed using image J software, and the analysis results are shown in Table 6.

TABLE 6

	Nanometer dressing type	Collagen volume (%)
			Example 1	bFGF/PCL	6.02
Example 2	bFGF-Zn/PCL	6.97
			Example 3	bFGF-ATP/PCL	2.13
Example 4	bFGF-ATP-Zn/PCL	20.78
			Comparative example 1	PBS/PCL	4.05
Comparative example 2	bFGF gel	29.33

As can be seen from Table 6, in examples 1-4, the collagen volume of bFGF-ATP-Zn/PCL in example 4 was the highest (20.78%), but there was still a gap (29.33%) compared to comparative example 2; the amount of bFGF-ATP/PCL collagen produced in example 3 was the lowest, only 2.13%. The results prove that the bFGF-ATP-Zn/PCL prepared in example 4 has good biocompatibility, and can effectively promote cell proliferation and collagen secretion when applied to in vitro cell culture.

Test example 6

The test example is a wound healing effect test of the bFGF sustained-release nano dressing, and the specific method comprises the following steps:

BALB/c mice were weighed and randomly divided into 5 groups of 4 mice each, and after anaesthetizing the mice, the back hair was shaved with an electric razor and the local depilatory cream treatment was performed to prevent the interference of the new hair growth too fast. The method comprises the steps of manufacturing a full-skin wound with the diameter of about 6mm on the back of a mouse by using an ophthalmologic scissors after iodophor disinfection, photographing after an operation to record the initial wound area, performing iodophor disinfection on the wound, covering corresponding sterilized dressings according to the requirements of each experimental group, fixing the dressings by using a circular band-aid and sterile gauze, observing the healing condition of the wound part and photographing to record the

area

3, 7, 10 and 14 days after the wound occurs, wherein the test result is shown in figure 6, the healing rate of the wound is calculated by using image J software, and the calculation result is shown in table 7.

TABLE 7

As can be seen from Table 7, the bFGF-ATP-Zn/PCL nano dressing prepared in example 4 has the fastest wound healing speed and the smallest wound area at 14 days. Therefore, the bFGF-ATP-Zn ternary composite system and the nano fibers are cooperatively matched in the bFGF sustained-release nano dressing provided by the invention, so that the healing of the wound surface is accelerated together.

The applicant states that the present invention is described by the above embodiments, but the present invention is not limited to the above embodiments, that is, the present invention does not mean that the present invention must be implemented by the above embodiments. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. The bFGF slow-release nano dressing with the wound surface active repair function is characterized in that the bFGF slow-release nano dressing is an electrostatic spinning nano fibrous membrane prepared from an emulsion containing alkaline fibroblast growth factors, adenosine triphosphate or salt thereof and metal ions.

2. The bFGF sustained-release nano dressing having a wound active repair function of claim 1, wherein the emulsion comprises an internal aqueous phase containing basic fibroblast growth factor, adenosine triphosphate or salt thereof, metal ions and an external oil phase containing a biocompatible polymer material;

preferably, the adenosine triphosphate or its salt includes any one of adenosine triphosphate, adenosine disodium triphosphate, adenosine magnesium triphosphate, adenosine tetralithium triphosphate or a combination of at least two of them;

preferably, the metal ions include any one or a combination of at least two of zinc ions, magnesium ions, copper ions, ferrous ions, or nickel ions;

preferably, the biocompatible polymer material comprises any one of or a combination of at least two of polylactic acid, polyglycolic acid copolymer, polycaprolactone, polytetrafluoroethylene and polylactic acid-polyethylene glycol block copolymer.

3. The bFGF slow-release nano dressing with an active wound repair function of claim 1 or 2, wherein the basic fibroblast growth factor, the adenosine triphosphate or the salt thereof and the metal ions form a ternary complex system through electrostatic interaction and coordination;

preferably, the proportion of the basic fibroblast growth factor, the metal ions, the adenosine triphosphate or the salt thereof is (1-100) mg, (0.01-10) mmol;

4. The bFGF slow-release nano dressing with an active wound repair function as claimed in any one of claims 1 to 3, wherein the nanofiber diameter of the electrospun nanofiber membrane is 250-550 nm.

5. The preparation method of the bFGF slow-release nano dressing with an active wound repair function according to any one of claims 1 to 4, wherein the preparation method comprises the following steps:

6. The method of claim 5, wherein the electrospinning parameters comprise: the flow rate of the spinning solution is 0.2-0.8mL/h, the spinning voltage is 20-30kV, the distance between a needle head and a collecting plate is 15-25cm, and the number of the spinning needle head is 4-7;

7. The method of claim 5 or 6, wherein the emulsion is prepared by a method comprising the steps of:

8. The method of claim 7, wherein the buffer comprises any one or a combination of two of phosphate buffer, Tris-HCl buffer, or HEPES buffer;

preferably, the organic solvent comprises any one or two of dichloromethane, trichloromethane, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide or tetrahydrofuran;

preferably, the concentration of the biocompatible polymeric material in the solution of the external oil phase is 2-20% (w/v), preferably 5-12% (w/v).

9. The method according to claim 7 or 8, wherein the volume ratio of the solution of the inner aqueous phase to the solution of the outer oil phase in step (2) is 0.1 to 10% (v/v), preferably 0.5 to 6% (v/v);

preferably, the specific operation method in the step (2) comprises the following steps: uniformly mixing an emulsifier and the external oil phase solution, dropwise adding the internal water phase solution into the mixture under ice-bath stirring, and emulsifying to obtain the emulsion;

preferably, the emulsifier comprises any one of or a combination of at least two of a nonionic emulsifier, a zwitterionic emulsifier, an anionic emulsifier or a cationic emulsifier;

preferably, the non-ionic emulsifier comprises any one of Span20, Span40, Span60, Span80, Span85, Tween20, Tween40, Tween60, Tween80 or Tween85 or a combination of at least two of the same;

preferably, the zwitterionic emulsifier comprises any one or a combination of at least two of lecithin, amino acid type emulsifier, betaine type emulsifier or stearic acid triethanolamine soap;

preferably, the anionic emulsifier comprises any one or a combination of at least two of sodium dodecylbenzene sulfonate, oleic acid, lauric acid, sulfated castor oil or calcium stearate;

preferably, the cationic emulsifier comprises any one or a combination of at least two of benzyltriethylammonium chloride, quaternary ammonium salt compounds or alkyl ammonium salt compounds;

preferably, the addition amount of the emulsifier in the external oil phase solution is 0.1-5% (v/v), more preferably 0.2-2% (v/v);

preferably, the emulsifying method comprises any one or combination of at least two of a magnetic stirring method, a homogeneous dispersion method, an ultrasonic emulsification method, an injector mutual pushing method or a membrane emulsification method;

preferably, the time of emulsification is 1-30min, preferably 2-12 min.

10. Use of the bFGF slow-release nano dressing with active wound healing function of any one of claims 1-4 in preparation of a material for promoting wound healing.