CN116531352B

CN116531352B - Nanofiber membrane for promoting wound healing

Info

Publication number: CN116531352B
Application number: CN202310798032.XA
Authority: CN
Inventors: 陈祥纪; 黄玲玉; 张庆娇; 梁萍
Original assignee: Beijing Kairui Keqi Medical Technology Co ltd
Current assignee: Beijing Kairui Keqi Medical Technology Co ltd
Priority date: 2023-07-03
Filing date: 2023-07-03
Publication date: 2023-10-27
Anticipated expiration: 2043-07-03
Also published as: CN116531352A

Abstract

A nanofiber membrane for promoting wound healing comprising a drug-loaded layer, wherein the drug-loaded layer comprises an analgesic, at least one high molecular polymer fiber that degrades faster, and at least one high molecular polymer fiber that degrades slower, and the analgesic is mixed in the fiber that degrades faster.

Description

Nanofiber membrane for promoting wound healing

Technical Field

The invention relates to a nanofiber membrane for promoting wound healing, in particular to a nanofiber membrane for promoting wound healing, which contains an analgesic drug and two or more degradable biological materials.

Background

Some body surface wounds, such as burns, scalds, ulcers, deep abrasions, are difficult to heal, and the nerves on the patient's body surface are destroyed, resulting in severe pain. Pain not only brings pain to the burn and scald patient and affects the daily life, social interaction, emotion and sleep of the patient, but also brings a series of psychological and social problems. Meanwhile, pain also affects prognosis of burn and scald patients, and can directly affect healing speed and quality of wound surfaces. At present, the treatment of the wound pain mainly uses an analgesic pump and an oral analgesic drug for systemic administration. However, these systemic administration treatments have significant adverse effects on patients and no effective topical pain treatment is currently available.

Electrospinning is a known method of preparing a spun yarn from a charged polymer solution in a high voltage electric field to form an accelerated jet. The aperture of the nonwoven nanofiber membrane prepared by single spinning is generally 50 nm-1 mu m, the nonwoven nanofiber membrane has good nanofiber structure, the porosity and the void communication rate are very high, and the nonwoven nanofiber membrane can be used for blocking wound infection caused by bacteria. However, as the tissue heals, the newly generated fibroblasts require more space to facilitate their crawling and growth, while fibrous membranes with too dense pore sizes can impede cell ingrowth.

Various functional dressings are used in clinic for various wound healing and pain relieving requirements, such as burns, scalds, ulcers, bruises. The wound medical dressing can play a good role in protecting wounds and promoting healing. However, most of the current dressing materials on the market are made of non-degradable materials and need to be replaced during the healing process. However, during replacement, damage to the new tissue can occur, causing pain and secondary damage to the wound.

For body surface wounds, particularly burns and scalds, it is desirable to provide a dressing product that promotes wound healing and reduces pain.

Disclosure of Invention

According to one aspect of the present invention, a nanofiber membrane for wound healing is provided. In one implementation of the invention, the nanofiber membrane is composed of fibers formed from two degradable polymers. One of the polymer fibers is susceptible to degradation and contains a drug; the other polymer fiber is not easily degraded. In the healing process, the first fiber releases the medicine to realize the pain relieving of the body surface wound, and degrades with time to release the space to provide space for the proliferation of cells. The second fiber continuously provides a scaffold for cell attachment, proliferation provides a favorable environment, and the degradation rate of the two fibers can be adjusted by adopting the design, so that the drug release within 3 days and complete degradation within a certain time period, such as 2-4 weeks, are more favorable, and wound healing is favorable.

According to one aspect of the present invention there is provided a nanofiber membrane for promoting wound healing comprising a drug-loaded layer, wherein the drug-loaded layer comprises an analgesic and at least two high molecular polymer fibers having different degradation rates, and the analgesic is mixed in the fibers having faster degradation rates.

The nanofiber membrane according to wherein the faster degrading fiber degrades at least twice as fast as the slower degrading fiber.

The nanofiber membrane according to which the weight ratio (dry matter) of faster degrading fibers to slower degrading fibers is 0.4: 1-3: 1.

according to one aspect of the present invention there is provided a drug-containing nanofiber membrane for wound repair comprising a drug-loaded layer comprising:

a first fiber comprising a first polymer which is degradable and an analgesic, the first polymer comprising one or more of polylactic acid-glycolic acid copolymer (PLGA), polydioxanone (PDO), polyglycolic acid (PGA), polydioxanone (PPDO), polyethylene glycol-glycolic acid copolymer (PEG-PGA), collagen, hyaluronic acid, gelatin, and

a second fiber comprising a second polymer that is degradable, including one or more of polylactic acid-glycolic acid copolymer (PLGA), polydioxanone, polylactic acid (PLA), polycaprolactone (PCL), polylactic acid-caprolactone copolymer (PLCL), polyglycolic acid-caprolactone copolymer (PGA-PCL), polylactic acid-trimethylene carbonate copolymer (PLA-PTMC), polyglycolic acid-trimethylene carbonate copolymer (PGA-PTMC),

Wherein the first fibers degrade at a higher rate than the second fibers.

The nanofiber membrane according to claim, wherein the first fiber comprises a first PLGA having a molar ratio of lactic acid residues (L) to glycolic acid residues (G) of 1%:99% -65%: 35, the second fiber comprises a second PLGA having a ratio of L to G of 70%:30% -99%: 1%.

The nanofiber membrane according to claim, wherein the second fiber comprises a molar content of caprolactone residues in the PLCL or polyglycolic acid-caprolactone copolymer of 10% -90%.

The nanofiber membrane of claim wherein the first fiber degradation rate is at least twice the second fiber degradation rate.

The nanofiber membrane according to claim, wherein the first fiber comprises a first high molecular polymer PLGA having a molar ratio of L to G of 50%:50%; the second fiber comprises a second high molecular polymer PLGA with a molar ratio of L to G of 75%:25%.

The nanofiber membrane according to claim, wherein the first fiber comprises a first high molecular polymer PLGA having a molar ratio of lactic acid residues L and glycolic acid residues G of 50%:50%; the second fibers comprise a second high molecular polymer PLCL or a polyglycolic acid-caprolactone copolymer having a molar content of caprolactone residues of 20% to 50%, preferably 25% to 35%.

The nanofiber membrane according to the present invention, wherein the first fiber comprises a first polymer PLGA having an intrinsic viscosity of 0.2 to 1.8 dL/g; the second fiber comprises a second high molecular polymer having an intrinsic viscosity of 0.35-2.5 dL/g.

According to the nanofiber membrane, the diameter of the first fiber is between 100 and 350 nanometers, and the diameter of the second fiber is between 400 and 3000 nanometers.

According to the nanofiber membrane, the diameter of the first fiber is between 50 and 1000 nanometers, and the diameter of the second fiber is between 400 and 4000 nanometers.

The nanofiber membrane of claim, wherein the first fibers and the second fibers comprise a weight ratio of 2: 1-1: 2.

the nanofiber membrane is characterized in that the drug carrying layer is a membrane formed by mixing and weaving the first fibers and the second fibers.

The nanofiber membrane according to claim, wherein the membrane thickness is 0.01-1 mm.

The nanofiber membrane according to claim, wherein the analgesic is one or more pharmaceutically acceptable salts of bupivacaine, lidocaine, levobupivacaine, ropivacaine, procaine, tetracaine, dyclonine, prilocaine, aspirin, sodium salicylate, bis-salicylates, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, tolmetin, diclofenac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, mefenamic acid, meclofenamic acid, piroxicam, meloxicam, lornoxicam, tenoxicam, nabumetone.

The pharmaceutical dosage form according to wherein the analgesic agents are salts of their hydrophilic form.

The pharmaceutical dosage form according to wherein the analgesic is lidocaine hydrochloride or bupivacaine hydrochloride.

The nanofiber membrane according to claim, wherein the analgesic is present in an amount of 5% to 50% by mass of the entire drug-carrying layer.

According to the nanofiber membrane, the drug carrying layer is manufactured through electrostatic spinning, the voltage of the high-voltage generator is 10-25 KV, and the receiving distance of the receiving device is adjusted to be 10-25 cm.

According to another aspect of the present invention, there is provided an electrospinning preparation method, which can prepare a drug-containing nanofiber membrane for wound repair, comprising a drug-carrying layer comprising:

a second fiber comprising a degradable second high molecular polymer comprising one or more of polylactic acid-glycolic acid copolymer (PLGA), polydioxanone (PDO), polydioxanone (PPDO), polylactic acid (PLA), polycaprolactone (PCL), polylactic acid-caprolactone copolymer (PLCL), polyglycolic acid-caprolactone copolymer (PGA-PCL), polylactic acid-trimethylene carbonate copolymer (PLA-PTMC), polyglycolic acid-trimethylene carbonate copolymer (PGA-PTMC), wherein the first fiber degrades at a higher rate than the second fiber.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

FIG. 1 is a Scanning Electron Microscope (SEM) photograph of the sample prepared in example 1 of the present invention degraded on days 0, 1 and 3.

Fig. 2 is SEM photographs of degradation of the samples prepared in example 1 of the present invention at days 7, 14 and 28.

FIG. 3 is a SEM photograph of the degradation of the sample prepared in example 2 of the present invention at day 0, day 1, and day 3.

Fig. 4 is SEM photographs of degradation of the samples prepared in example 2 of the present invention at days 7, 14 and 28.

Fig. 5 is an SEM photograph of the sample prepared in example 3 of the present invention degraded on day 0, day 1, and day 3.

Fig. 6 is SEM photographs of degradation of the samples prepared in example 3 of the present invention at days 7, 14 and 28.

Fig. 7 is SEM photographs of degradation of the samples prepared in example 4 of the present invention at day 0, day 14 and day 28.

FIG. 8 is a graph of drug dissolution profile in vitro for a portion of the sample.

FIG. 9 is an in vitro drug dissolution profile for a portion of the sample.

FIG. 10 is a photograph of a portion of the results of an animal experiment with a sample.

Fig. 11 is a fiber SEM comparison of analgesic and no analgesic added.

Description of the embodiments

A comfortable composite is provided for promoting wound healing while simultaneously providing analgesia to a patient. More particularly, the present invention provides an effective topical wound analgesic nanofiber membrane for pain relief by releasing analgesic drugs over a period of time, e.g., 3-5 days. Has the advantages of accurate pain relief, less medicine consumption, small side effect and long pain relief time. Still further, the nanofiber membranes of the present invention can be manufactured into casting products.

For various body surface wounds, it is critical to be able to promote rapid healing. The nanofiber membrane of the invention contains a drug-carrying layer which is composed of two or more degradable biological materials. The two materials are each independently present as fibres of the order of nanometers to micrometers in diameter. Wherein the first fiber material degrades faster than the second fiber and the design drug is present in the first fiber material that degrades faster. The two fibers are staggered and overlapped to form a porous membrane. The nanofiber membrane promotes wound healing and effectively relieves pain during the wound healing process.

The drug-carrying layer of the present invention is typically in the form of a polymer film, for example, a film comprising polymer fibers. The polymer film may be made from one or more natural and/or synthetic polymers, homopolymers and/or copolymers. In particular, the natural polymer may be a polysaccharide and/or a sugar polymer, the protein, the synthetic polymer may be a hydrophilic or hydrophobic swellable or erodable polymer, a mucoadhesive polymer, and/or a stimuli-responsive polymer, preferably a degradable polymer. Particularly suitable polymers for forming the film are polylactic acid-glycolic acid copolymer (PLGA), polydioxanone (PDO), polydioxanone (PPDO), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol-lactic acid copolymer (PEG-PLA), polyethylene glycol-glycolic acid copolymer (PEG-PGA), polycaprolactone (PCL), polylactic acid-caprolactone copolymer (PLCL), polyglycolic acid-caprolactone copolymer (PGA-PCL), polylactic acid-trimethylene carbonate copolymer (PLA-PTMC), polyglycolic acid-trimethylene carbonate copolymer (PGA-PTMC). It is contemplated that one or more polymers in the drug-loaded layer may be selected so as to allow the drug dosage form to respond to pH, moisture, tissue fluids, temperature, etc., and thus facilitate the delivery and diffusion of an active substance, such as an analgesic, in the drug-loaded layer to a particular area. Various methods are contemplated to control the release of the active agent from the dosage form. For example, it is contemplated that the polymer may be cross-linked to delay and prolong the release of the active agent(s) from the dosage form, with the introduced cross-links forming an insoluble network, thereby slowing and prolonging the release of the active agent. Modulation of the degree of densification of the fibers is also contemplated to modulate drug release.

The term "PLGA" refers to poly (lactic-co-glycolic acid), namely a polylactic acid-glycolic acid copolymer, also known as a polyglycolide-lactide copolymer, which is formed by random polymerization of two monomers, lactic acid and glycolic acid, is a degradable functional polymer organic compound, has good biocompatibility, is nontoxic, and is widely applied to medical instruments and drug transport. In various countries around the world, a variety of different types of PLGA have been approved by regulatory authorities and formally incorporated into the pharmacopoeia as pharmaceutical excipients. Different monomer ratios can produce different types of PLGA, for example: PLGA75:25 represents that the polymer consists of 75% lactic acid residues (molar ratio, expressed as "L") and 25% glycolic acid residues (molar ratio, expressed as "G"). Depending on the PLGA composition and molecular weight, it can be dissolved in a variety of chlorinated solvents. The rate of degradation of PLGA is affected by a number of factors, such as the ratio of L and G; optical isomerism of L (d or L or dl), L used in the present invention is racemic isomerism (dl); and the initial molecular weight of PLGA. In general, the rate of in vivo degradation of PLGA is generally PLGA50: 50. 1-2 months, PLGA65:35 for 3-4 months, PLGA75:25 for 4-5 months, PLGA85:15 is 5-6 months. Such in vivo absorption cycles are slow with respect to the present invention. After PLGA is processed into a certain form, the degradation rate of PLGA with the form is also affected by the surface area, thickness and volume. The present inventors have found that if other substances, such as drug molecules, are added to polymers, such as PLGA, the hydrophilicity or hydrophobicity of these added substances also affects the degradation rate of PLGA. Hydrophilic substances can increase contact between PLGA and water, accelerate hydrolysis reaction of PLGA, and realize faster degradation. When the added substance leaves the PLGA by diffusion or the like, gaps are formed, the surface area of the PLGA and the entry of moisture are increased, and the degradation rate is increased.

Depending on the degradation rate of the different polymers, a combination of fibers of different degradation rates may be performed. For example, when the slower degrading fiber is polydioxanone or polydioxanone composition, the faster degrading fiber may be selected to be PLGA, preferably PLGA having a G content of at least 50%.

However, the drug-loaded layer design of the present invention allows the overall fiber degradation rate to reach within 2-4 weeks by processing PLGA into fine nanofibers and adding an analgesic to the rapidly degrading fibers, and allows the analgesic drug to be released more rapidly, e.g., within substantially 3 days.

The term "analgesic" refers to a topical analgesic, anesthetic, such as bupivacaine, levobupivacaine, ropivacaine, lidocaine, procaine, tetracaine, prilocaine, dyclonine, or a non-steroidal anti-inflammatory analgesic, such as one or more of aspirin, sodium salicylate, bis-salicylates, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, tolmetin, diclofenac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, mefenamic acid, meclofenamic acid, piroxicam, meloxicam, lornoxicam, tenoxicam, nabumetone, and the like, useful in the treatment of trauma. One drug can be generally selected, or two or more drugs can be selected according to specific needs. The active substance, for example an analgesic, may be mixed with the polymer by mixing with a solvent or by heating to melt, the fibrous structure typically being formed by electrospinning or centrifugal spinning. Hydrophilic forms of these analgesics are preferred, such as bupivacaine hydrochloride, levobupivacaine hydrochloride, ropivacaine hydrochloride, lidocaine hydrochloride, procaine hydrochloride, tetracaine hydrochloride, prilocaine hydrochloride, dyclonine hydrochloride, diclofenac sodium, and the like. The salt form of the analgesic has a higher hydrophilicity than its free base form. For example, bupivacaine hydrochloride has a higher hydrophilicity than bupivacaine free base.

The term "intrinsic viscosity" refers to the most commonly used method of expressing the viscosity of a polymer solution. Defined as the reduced viscosity when the concentration of the polymer solution approaches zero. That is, the contribution of a single molecule to the solution viscosity is a viscosity reflecting the characteristic of a polymer, and the value thereof does not vary with the concentration. Often expressed in [ eta ], a common unit is deciliter per gram (dL/g). Since the intrinsic viscosity has a quantitative relationship with the relative molecular weight of the high molecular weight polymer, the value of [ eta ] is usually used to determine the relative molecular weight, or as a measure of the molecular weight. The value is measured by a capillary viscometer.

Electrospinning is known as an efficient spinning and film-forming process that can be used to produce fibrous polymer films comprising fiber diameters in the nanometer to micrometer range. The polymer for electrospinning as described above, together with one or more active substances, such as drugs, solvents and excipients, may be mixed in the form of a solution, suspension, emulsion or melt, and a voltage of from about 5 to 30kV, preferably 10 to 25kV, more preferably 11 to 20kV may be applied to the polymer solution, suspension, emulsion or melt. The active substances can be incorporated by direct dissolution, suspension or in the form of an emulsion. Suitable solvents for preparing the electrospinning solution include water and organic solvents. The auxiliary materials can be plasticizer, solubilizer, surfactant and/or defoamer, etc.

The active substances in the dosage form of the invention, such as analgesics, can exert the analgesic effect, and the polymer fibers in the dosage form are synthetic or natural polymer materials with good biocompatibility for human bodies, such as PLGA, collagen, and the like, can exert the functions of providing a tissue regeneration bracket or a three-dimensional structure, regulating the physiological functions of cells, protecting immunity, and the like, and can degrade into nontoxic small molecules to be absorbed by the body along with the time and tissue healing.

The invention utilizes the biodegradability of biomedical polymer materials and the biodegradation speed difference between different materials of fibers prepared by electrostatic spinning, considers and utilizes the influence of drugs mixed into the fibers on the diameter and degradation of the fibers, and realizes the control of the degradation speed of the nanofiber membrane material and the release of analgesic drugs in a certain time. Sustained release products and techniques are known in the art and generally have a major purpose focused on sustained release or the need to maintain physical properties of the product (e.g. as a regenerative stent), and drug release times as long as 7-10 days have to be achieved and are not suitable for the purposes of the present invention. The invention utilizes the fibers with different degradation speeds and discovers and utilizes the influence of the analgesic on the fibers, such as the diameter and the degradation speed, so that the analgesic drug is released in a large amount within about 3 days according to the actual requirement of the invention while the mechanical strength of the nanofiber membrane is maintained. When more than two fibers are present, for example, one fiber degrades faster than the other, it is preferred that the analgesic is mixed in the fiber that degrades faster for the purposes of the present invention.

In one embodiment of the present invention, PLGA in contact with wound tissue is found and utilized, because of the different degradation rates of L and G at different ratios, the addition of an analgesic in combination with PLGA can further accelerate the degradation of such fibers to adjust the degradation rate of the material. The findings can also be applied to other degradable polymers. The pharmaceutical dosage form of the present invention may comprise an electrospun film comprising at least two different fibers. In the case of PLGA materials, one of the fibers has a L content close to or less than the other, for example, the molar ratio of L to G in the lower L content fibers is 1% 99% to 65% 35%, for example, the L content may be 5%,10%,15%, 20%,25%,30%,35%,40%,45%,50%,52%,54%,55%,60%,62%,65%, preferably 55% or less, more preferably 52% or less; whereas in fibers with higher L content the L to G ratio was 99%:1% -70%: 30%, for example, the L content may be 95%,90%,88%,86%,84%,82%,80%,78%,76%,74%,72%,70%,68%, preferably 70% or more, more preferably 73% or more. Whereas the weight ratio (dry matter) of fibers that degrade faster, such as low L content fibers, to fibers that degrade slower, such as high L content fibers, is 0.4: 1-3: 1, for example 0.5:1,0.85:1,0.9:1,0.95:1,1:1,1.2:1,1.4:1,1.6:1,1.8:1,2:1,2.2:1,2.4:1,2.6:1,2.8:1, preferably 1:1.

The fibers that degrade rapidly have all or a majority of their diameters, e.g., at least 60%, at least 70%, at least 80% of their fibers have diameters between 50 and 1000 nanometers, preferably between 80 and 800 nanometers, more preferably between 100 and 600 nanometers, and even more preferably between 120 and 350 nanometers. All or a substantial portion of the slow degrading fibers, e.g., at least 60%, at least 70%, at least 80% of the fibers have a fiber diameter in the range of 400 to 5000 nanometers, preferably 450 to 4500 nanometers, more preferably 500 to 4000 nanometers, and even more preferably 550 to 3500 nanometers.

The present invention also finds that the amount of analgesic in the fiber affects the drug release and the amount released, as well as the mechanical properties and degradation rate of the fiber. Too high an amount of analgesic will result in reduced strength of the fiber and a large release in a short period of time (e.g., 24 hours), but too low a drug loading will affect the analgesic effect. The mass content of the analgesic in the fiber mixture which is degraded more rapidly can be 2% -70%; preferably 5% to 50%, or more preferably 10% to 40%. The mass content of the analgesic in the whole drug-carrying layer may be 5% to 50%, preferably 8% to 30%.

The invention designs a fiber with slower degradation speed and larger diameter, which provides main mechanical strength support, and the formed matrix with larger aperture is beneficial to cell attachment and proliferation and can also prevent external bacteria from invading. And another fiber with higher degradation speed is designed to be mixed with the analgesic drug, and holes are formed by the release of the drug and the degradation of the fiber, so that the degradation of the fiber and the release of the drug are accelerated. While the preferred analgesic increases the conductivity of the spinning liquid, the resulting fibers spun have finer diameters, further promoting fiber degradation. The diameter of the fiber affects the absorption rate and fine spinning is more easily degraded and absorbed.

Example 1

Preparation of sample #1

Solution a: PLGA (Shandong national academy of sciences of pharmacy, L: G=50:50) and bupivacaine hydrochloride are dissolved in a certain amount of hexafluoroisopropanol according to a mass ratio of 70:30 to form a solution with a total solid mass ratio of 14%.

Solution B: PLGA (DLG 75-12) (L: G=75:25) as a B component was dissolved in hexafluoroisopropanol to form a 5% mass concentration solution.

And then the solution A is filled into an electrostatic spinning injector A, the solution B is filled into an electrostatic spinning injector B, the speed of a microinjection pump of the injector A is adjusted to 5 milliliters/hour, the speed of the microinjection pump of the injector B is adjusted to 13.7 milliliters/hour according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B of 1:1, the voltage of a high-voltage generator is adjusted to 17KV, and the receiving distance of a receiving device is adjusted to 15 cm. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed. Sample #1 was prepared.

FIG. 1 is a photograph of a Scanning Electron Microscope (SEM) of sample #1 prepared in example 1 (0 d, i.e., 0 day) and a photograph of degradation over time, 1 day (1 d), 3 days (3 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification. FIG. 2 is a photograph of sample #1 prepared in example 1 degraded at time 7 days (7 d), 14 days (14 d) and 28 days (28 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification.

Both the thickness and the shape of the fiber can be clearly seen. The fine fibers are formed as the A component and the coarse fibers are formed as the B component. Most of the fine fibers have a diameter of 300nm or less, and most of the coarse fibers have a diameter of 400nm or more. Over time, the fine A-component fibers degrade rapidly exposing more B-component fibers and forming cavities that can facilitate cell entry and tissue healing. It can be seen that on day 3, the a-component fibres have not yet broken but a large number of holes have been made, which marks the release of the drug and the degradation of the fibres; the B component fiber has no holes. On day 7, the A-component fibers had a large number of breaks and disappeared, and the B-component fibers, similar to the A-component fibers on day 3, also began to appear a large number of holes. On day 14, the fibers of the A component were found to have been substantially invisible and degraded, while the B fibers had a number of breaks and deletions, while retaining the basic morphology, despite some interference in the field of view. At 28 days, the B fibers were also substantially degraded. From the degradation of both fibers, it can be seen that the B fibers take approximately twice as long to reach a degradation state similar to that of the a fibers, with the a component fibers degrading at substantially twice the rate of the B component fibers.

Example 2

Preparation of sample #2

Solution a: PLGA (L: G=50:50) and bupivacaine hydrochloride are dissolved in a certain amount of hexafluoroisopropanol according to a mass ratio of 70:30 as a component A to form a solution with a solid mass ratio of 14%.

Solution B: PLGA (L: G=75:25) was dissolved as the B component in hexafluoroisopropanol to form a 5% strength by mass solution.

And then the solution A is filled into an electrostatic spinning injector A, the solution B is filled into an electrostatic spinning injector B, the speed of an injector micro-A injection pump is adjusted to 4 milliliters/hour according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B of 1:2, the speed of the injector micro-A injection pump is adjusted to 27.7 milliliters/hour, the voltage of a high-voltage generator is adjusted to 18KV, and the receiving distance of a receiving device is adjusted to 15 cm. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed. Sample #2 was prepared.

Fig. 3 is a SEM photograph (0 d, i.e., 0 day) and a photograph of sample #2 prepared in example 2 degraded over time, 1 day (1 d), 3 days (3 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification. FIG. 4 is a photograph of sample #2 prepared in example 2 degraded over time, 7 days (7 d), 14 days (14 d) and 28 days (28 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification.

Both the thickness and the shape of the fiber can be clearly seen. Fine a fibers are formed as a component a and coarse B fibers are formed as B component B. Over time, the fine a fibers degrade faster, exposing more B fibers and forming cavities that facilitate cell entry and tissue healing. It can be seen that on day 3, the a fibers began to develop holes and breaks, which marked drug release and fiber degradation. On day 7, more holes are commonly present in the a fibers; most of the B fibers have not yet appeared holes. On day 14, the a fibers were severely absent, only some residue was visible, and the a fibers were essentially complete in degradation; the B fibers begin to exhibit small holes. On day 28, the B fibers had some fiber morphology but had substantially lost support morphology. From the degradation of both fibers, it can be seen that the B fibers take more than twice as long to reach a degradation state similar to that of the a fibers, with the a component fibers degrading more than twice as fast as the B component fibers.

Example 3

Preparation of sample #3

Then filling the solution A into an electrostatic spinning injector A, filling the solution B into an electrostatic spinning injector B, and regulating the speed of a microinjection pump of the injector A to 5 ml/h according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B of 2:1 to obtain A fibers; the rate of the syringe B microinjection pump was 6.67 ml/hr, the voltage of the high voltage generator was adjusted to 18 KV, and the receiving distance of the receiving device was adjusted to 15 cm, thereby obtaining the B fiber. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed. Sample #3 was prepared.

Fig. 5 is SEM photograph (0 d) and photograph of sample #3 prepared in example 3 degraded over time, 1 day (1 d), 3 days (3 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification. FIG. 6 is a photograph of sample #3 prepared in example 3 degraded over time, 7 days (7 d), 14 days (14 d) and 28 days (28 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification.

Both the thickness and the shape of the fiber can be clearly seen. Fine a fibers are formed as a component a and coarse B fibers are formed as B component B. Over time, the fine a fibers degrade faster, exposing more B fibers and forming cavities that facilitate cell entry and tissue healing. On day 3, fiber a breaks, which marks the release of drug and degradation of fiber; the B fibers remain intact. On day 7, a fiber appeared a large number of holes, and the supporting form could not be maintained; the fiber B has complete shape and is rarely perforated. On day 14, only a portion of the a fibers remained, essentially completing degradation; b fibers exhibit a large number of holes but remain substantially intact. At 28 days, only a portion of the fiber morphology was visible for the B fibers, with most of the substantial degradation completed. From the degradation of both fibers, it can be seen that the B fibers take more than twice as long to reach a degradation state similar to that of the a fibers, with the a fibers degrading more than twice as fast as the B fibers.

Example 4

Preparation of sample #4

PLGA (L: g=75:25) was dissolved in hexafluoroisopropanol to form a 5% mass concentration solution. Then the solution is put into an electrostatic spinning injector, the speed of a micro injection pump of the injector is regulated to 20 milliliters/hour, the voltage of a high voltage generator is regulated to 12KV, and the receiving distance of a receiving device is regulated to 15 cm. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed. Sample #4 was prepared. Sample #4 was made from B fibers alone.

Fig. 7 is SEM photograph (0 d) and photograph (14 d) of sample #4 prepared in example 4 and degraded over time for 14 days (28 d). The upper photo is at 1000 times magnification, and the lower photo is at 5000 times magnification. At 28 days, the support morphology remained despite the small holes on the surface of the individual fibers, and degradation was slow.

Example 5

Preparation of sample #5

PDLLA was dissolved in hexafluoroisopropanol to form a 5.5% strength by mass solution. Then the solution is put into an electrostatic spinning injector, the speed of a micro injection pump of the injector is regulated to be 14 milliliters/hour, the voltage of a high voltage generator is regulated to be 13KV, and the receiving distance of a receiving device is regulated to be 15 cm. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed. Sample #5 was prepared.

Example 6

Preparation of sample #6 and determination of in vitro drug dissolution

Solution a: PLGA (L: G=50:50) and bupivacaine hydrochloride were dissolved in hexafluoroisopropanol at a mass ratio of 70:30 to form a solution with a total solid concentration of 14%.

Solution B: PLCL (lactic acid residue L: caprolactone residue cl=70:30) was dissolved in hexafluoroisopropanol to form a solution having a total concentration of 10% by mass.

Then the solution A is filled into an electrostatic spinning injector A, the solution B is filled into an electrostatic spinning injector B, and the speed of an injector micro-A injection pump is regulated to 6 milliliters/hour according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B of 1:1, so as to obtain A fibers; the syringe B microinjection pump was set at a rate of 8.62 ml/hr to obtain B fiber, the voltage of the high voltage generator was adjusted to 16KV, and the receiving distance of the receiving device was adjusted to 15 cm. The receiving apparatus used a rotating roll, and after spinning was completed, the film was removed from the receiving roll to prepare sample #6.

Example 7

Preparation of sample #7 and determination of in vitro drug dissolution

Solution a: PLGA (L: g=50:50) and lidocaine hydrochloride were dissolved in hexafluoroisopropanol at a mass ratio of 70:30 to form a solution with a total solid concentration of 14%.

Solution B: PLCL (L: cl=70:30) was dissolved in hexafluoroisopropanol to form a solution with a total concentration of 10% by mass.

Then the solution A is filled into an electrostatic spinning injector A, the solution B is filled into an electrostatic spinning injector B, and the speed of an injector micro-A injection pump is regulated to 6 milliliters/hour according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B of 1:1, so as to obtain A fibers; the syringe B microinjection pump was set at a rate of 8.62 ml/hr to obtain B fiber, the voltage of the high voltage generator was adjusted to 16KV, and the receiving distance of the receiving device was adjusted to 15 cm. The receiving device used a rotating roller, and after spinning was completed, the film was removed from the receiving roller to prepare sample #7.

Example 8

Solution a: collagen and lidocaine hydrochloride are dissolved in hexafluoroisopropanol according to a mass ratio of 70:30 to form a solution with a total solid concentration of 8%.

And then the solution A is filled into an electrostatic spinning injector A, the solution B is filled into an electrostatic spinning injector B, the speed of an injector micro-A injection pump is regulated to be 6 milliliters/hour according to the solid mass ratio of a liquid storage pipe A in the injector A to a liquid storage pipe B in the injector B to obtain A fibers, the speed of the injector micro-B injection pump is regulated to be 4.8 milliliters/hour to obtain B fibers, the voltage of a high-voltage generator is regulated to be 16KV, and the receiving distance of a receiving device is regulated to be 15 cm. The receiving device used a rotating roller, and after spinning was completed, the film was removed from the receiving roller to prepare sample #8.

Example 9

Preparation #9 of sample containing bupivacaine base

First, bupivacaine hydrochloride powder was dissolved in distilled water at a concentration of 10%. 1M NaOH was added dropwise to the solution, stirring was continued until no more precipitate formed. Washing with fresh distilled water, and filtering to obtain solid oxybuprocaine. The hydroxybutanil was moved to a clean beaker with water and stirred for 5 hours or more. Then carefully washed with distilled water to remove additional NaOH. After washing, air dried overnight at less than 80 degrees vacuum. Drying to obtain bupivacaine free alkali as white soft powder. Bupivacaine base and PLGA (L: g=50:50) were weighed in mass ratios of 30:70 each and completely dissolved in an appropriate amount of hexafluoroisopropanol solution to form a solution with a solid mass concentration of 17%. After magnetic stirring, the resulting PLGA/BUP solution was collected in a syringe. The syringe was fixed to a bolus pump and the bolus was made at a rate (about 4 mL/h). While the receiver rotates at a certain speed (about 200 r/min). The voltage of the high voltage generator is regulated to 16KV, and the receiving distance of the receiving device is regulated to 15 cm. The receiving device used a rotating roller, and after spinning was completed, the film was removed from the receiving roller to prepare sample #9 containing bupivacaine base.

Example 10

Tensile test and experimental results of samples

The samples were subjected to a tensile test after sterilization by irradiation with 25 kGy of radiation, and 5 samples were tested for each of samples #1, #2, #3, #6, # 9. During testing, each sample is left for 2cm along two ends, and the two ends are respectively placed into a die of a tensile machine and tested by the tensile machine. And testing until the breaking is stopped.

The test results were averaged:

sample of	#1	#2	#3	#6	#9
						Maximum tensile force (N)	26	48	22	17	5
Maximum fracture deformation (mm)	10.2	9.9	23.2	51.0	2.7

The mechanical strength (maximum tensile force and maximum breaking deformation) of the film is very important for the use of the product. The tensile force can provide enough strength, so that the strength of the material is ensured to be enough and the material cannot be broken in the use process. The maximum fracture deformation can lead the product to have certain deformation along with the tissues of the human body. Adjusting the proportion of the B component and changing the polymer material of the B component can improve the mechanical strength of the sample. All samples of the present invention had higher mechanical strength than sample #9, which was a single fiber of bupivacaine free base.

Example 11

In vitro dissolution assay of drugs

According to the "adult burn pain management guidelines" published by the journal of Chinese burns, 2013, 6, 29, acute pain of burns and scalds occurs within 2-3 days. Pain from other acute wounds also occurs within 2-3 days after the wound. The analgesic drug is released rapidly and not too slowly. The prepared sample was placed in 1000ml of a phosphate buffer (pH 7.2) at 100 rpm, 10ml of the dissolution solution was taken out at 2.5, 5, 15, 30, 60, 120, 240, and 360 minutes, respectively, and the dissolution medium of the same temperature and the same volume was immediately replenished. The content of the eluted drug was analyzed by high performance liquid chromatography as follows: octadecylsilane chemically bonded silica as packing material (4.6 mm. Times.100 mm,5 μm or column temperature equivalent to the efficacy): 30 ℃, detection wavelength: 240nm, flow rate: 1.5ml/min, sample injection amount: 20 μl, mobile phase: 0.02mol/L phosphate buffer (pH 8.0): acetonitrile=35:65. The cumulative dissolution of the drug at each time point was calculated. In vitro dissolution experiments use a large amount of liquid and intense stirring, and the dissolution rate of the drug measured in vitro is not representative of the dissolution rate of the sample in vivo and on the body surface. In general, the dissolution results of in vitro assays are much faster than those of in vivo, and the dissolution rates of body surfaces are much faster.

FIG. 8 is a measurement of in vitro dissolution profile for sample #1 (triangle), sample #2 (flower star), sample #3 (diamond), sample #6 (dot) according to the in vitro dissolution method described in example 11. The dissolution rate of the drug can be adjusted by the ratio of the a-fiber and the B-fiber. Increasing the proportion of B fibers (sample # 2) slows the dissolution rate of the drug.

Figure 9 shows in vitro release profiles for sample #9 (bupivacaine base, square) and sample #6 of the present invention (dot). The sample #6 of the invention uses bupivacaine hydrochloride, has a faster release speed, and meets the requirement of quick effect of the medicine in auxiliary materials.

Example 12

In-vitro degradation experiment of spinning membrane

Samples #1, #2, #3, #4 prepared in the examples were subjected to hydrolytic degradation in phosphate buffer at 37 ℃. A portion of the sample was removed at 1, 3, 7, 14, 28 days and dried. Degradation of the spun fibers was observed using a scanning electron microscope SEM. The results are set forth and described in the preceding preparation examples.

Example 13

Animal experiment

Experimental animals were purchased from SD rats, male, 2-3 months old, 200 grams, 3 total, SPF grade of s Bei Fu. Feeding into SPF-class animal house, maintaining feeding temperature at 18-26 deg.C and humidity at 40-70%, and feeding 3 experimental animals in 1 cage with photoperiod of 12 h:12 h, and feeding with breeding feed (S Bei Fu). The feeding environment was followed 7 days after entering the animal house, and the adaptation period of the experimenter, veterinarian and breeder was established and the experiment was started after elimination of the environment and personnel stress. The experimental animal feeding welfare and the use regulations specified by the experimental animal management committee are strictly followed. The experimental technique operation is carried out by following the internal animal experiment of the company by using license management SOP, and the animal experiment scheme is checked and passed by the animal management committee of the company.

Samples #1, #4, #5 prepared in the examples were implanted into a scalding model of one rat, and the effects of various samples on wound recovery were compared. Specifically, the rat coat is first removed. Then the skin of the back of the experimental animal is cut to expose subcutaneous tissues, two injuries are caused by immersing the skin of the back of the experimental animal in a part region (3 cm x 2 cm) along two sides of the spine for a certain time at a high temperature of 95 ℃, a single-layer test film is coated on the left side injury of the back of the rat, and the test film is not coated on the right side injury. At the 4 week post-operative time point, 2 exposed skins were taken for each group of animals to compare wound healing and membrane absorption.

Fig. 10 is a 28 day recovery after application of samples #1, #4, #5 (left to right) at the burn site. It can be seen that after 4 weeks post-surgery, the wound (left panel) with sample #1 applied had substantially recovered, the tissue structure was intact, and the patch was fully absorbed. However, the wounds with samples #4 (middle) and #5 (right) had not recovered, granulation tissue had just formed (extensive punctate bulge), bleeding was significant, and the patches had significant residues, with arrows pointing to the residual patches.

Example 14

Effects of analgesic on fiber diameter

PLGA (Shandong national academy of sciences of pharmacy, L: G=50:50) and bupivacaine hydrochloride were dissolved in hexafluoroisopropanol at a mass ratio of 80:20 to form a solution with a total mass concentration of 20%. Then the solution is put into an electrostatic spinning injector, the speed of a micro injection pump of the injector is regulated to 6 milliliters/hour, the voltage of a high voltage generator is regulated to 19KV, and the receiving distance of a receiving device is regulated to 15 cm. The receiving device uses a rotating roller, the film is taken off the receiving roller after electrostatic spinning for a period of time, and then the residual solvent on the film sheet is removed by heating. Samples were prepared.

In the SEM photograph of fig. 11, the left hand graph is of the spun fibers without bupivacaine hydrochloride, only PLGA (L: g=50:50), and it is seen that the majority of the fibers are 1-5 microns in diameter. The right panel shows the spinning of PLGA (L: G=50:50) with bupivacaine hydrochloride (20%) added, the majority of fibers having a diameter between 0.1 and 0.8 microns. At the same magnification (1000 times), it can be seen that the fibers are significantly attenuated after the addition of bupivacaine hydrochloride.

Those skilled in the art will appreciate that various modifications (additions and/or deletions) of the various components/parts of the products, formulations, devices, methods, systems and embodiments described herein may be made without departing from the full scope and spirit of the application, which is intended to be covered by such modifications.

Claims

1. A nanofiber membrane for promoting wound healing comprises a drug-loaded layer comprising an analgesic, at least one high molecular polymer fiber with a relatively high degradation rate, and at least one high molecular polymer fiber with a relatively low degradation rate, wherein

The analgesic is mixed in the fibers with higher degradation rate,

the fiber with higher degradation speed comprises a first fiber, the first fiber comprises a first degradable high molecular polymer, the first high molecular polymer comprises one or more of polylactic acid-glycolic acid copolymer, polydioxanone, polyglycolic acid, polydioxanone and polyethylene glycol-glycolic acid copolymer,

The fibers with slower degradation speed comprise second fibers, the second fibers comprise degradable second high molecular polymers, the second high molecular polymers comprise one or more of polylactic acid-glycolic acid copolymer, polydioxanone, polylactic acid, polycaprolactone, polylactic acid-caprolactone copolymer, polyglycolic acid-caprolactone copolymer, polylactic acid-trimethylene carbonate copolymer, polyglycolic acid-trimethylene carbonate copolymer, and

the dry matter weight ratio of the faster degrading fiber to the slower degrading fiber was 0.4: 1-3: 1, wherein the drug-loaded layer is a porous membrane formed by the staggered superposition of the two fibers, wherein the degradation speed of the fiber with higher degradation speed is at least twice that of the fiber with lower degradation speed, and the mass content of the analgesic in the whole drug-loaded layer is 5-50%.

2. The nanofiber membrane according to claim 1, wherein the first fiber comprises a first PLGA having a molar ratio of lactic acid residues L and glycolic acid residues G of 1%:99% -65%: 35, the second fiber comprises a second PLGA having a ratio of L to G of 70%:30% -99%: 1%.

3. The nanofiber membrane of claim 1, wherein the second fiber comprises a molar content of caprolactone residues in the polylactic acid-caprolactone copolymer or the polyglycolic acid-caprolactone copolymer of 10% -90%.

4. The nanofiber membrane according to claim 1, wherein the first fiber comprises a first high molecular polymer PLGA comprising a molar ratio of lactic acid residues L and glycolic acid residues G of 50%:50%; the second fiber comprises a second high molecular polymer PLGA with a molar ratio of L to G of 75%:25%.

5. The nanofiber membrane according to claim 1, wherein the first fiber comprises a first high molecular polymer PLGA comprising a molar ratio of lactic acid residues L and glycolic acid residues G of 50%:50%; the second fiber comprises a second high molecular polymer polylactic acid-caprolactone copolymer or polyglycolic acid-caprolactone copolymer, wherein the molar content of caprolactone residues in the second high molecular polymer polylactic acid-caprolactone copolymer is 20% -50%.

6. The nanofiber membrane according to claim 1, wherein the first polymer PLGA comprised by the first fiber has an intrinsic viscosity of 0.2-1.8 dL/g; the second fiber comprises a second high molecular polymer having an intrinsic viscosity of 0.35-2.5 dL/g.

7. The nanofiber membrane according to claim 1, wherein the diameter of the first fiber is between 50 and 1000 nanometers and the diameter of the second fiber is between 400 and 4000 nanometers.

8. The nanofiber membrane according to claim 1, wherein the weight ratio of the first fibers to the second fibers comprised is 2: 1-1: 2.

9. the nanofiber membrane according to claim 1, wherein the membrane thickness is 0.01-1 mm.

10. The nanofiber membrane according to claim 1, wherein the analgesic is one or several pharmaceutically acceptable salts of bupivacaine, lidocaine, levobupivacaine, ropivacaine, procaine, tetracaine, dyclonine, prilocaine, aspirin, sodium salicylate, salsalate, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, tolmetin, diclofenac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, mefenamic acid, meclofenamic acid, piroxicam, meloxicam, lornoxicam, tenoxicam, nabumetone.

11. The nanofiber membrane according to claim 10, wherein the analgesic agents are salts of their hydrophilic form.

12. The nanofiber membrane according to claim 1, wherein the analgesic is lidocaine hydrochloride or bupivacaine hydrochloride.