Multifunctional PPK electrospun fibrous membrane with active oxygen response and preparation method and application of multifunctional PPK electrospun fibrous membrane

Abstract

The invention discloses a multifunctional PPK electrospinning fiber membrane with active oxygen response as well as a preparation method and application thereof, and the product is prepared by the following method: S1: preparation of an electrospinning liquid: dissolving PLA, PEGDA-EDT and KNN in a hexafluoroisopropanol solution, and stirring until uniform dispersion to obtain a spinning solution; s2, performing electrostatic spinning on the electrospinning liquid to obtain the electrostatic spinning fiber membrane. The electrostatic spinning fiber membrane has good ROS response, generates electrical stimulation based on the piezoelectric material KNN under ultrasonic stimulation, promotes peripheral nerve injury repair, has good biocompatibility and is an ideal medical material capable of being self-degraded.

Description

Technical Field

[0001] The invention relates to a PPK electrospun fiber membrane and a preparation method and application thereof. Background Art

[0002] During chemotherapy, 50% to 90% of patients will experience chemotherapy-induced peripheral neuropathy, a serious adverse reaction, which greatly limits the tolerated dose and may lead to chemotherapy cessation. About 30% of patients will still experience pain symptoms 6 months or even longer after treatment. Among them, the probability of this phenomenon occurring in colorectal cancer patients 2 years after receiving oxaliplatin treatment is as high as 84%. Chemotherapy-induced peripheral neuropathy usually manifests as neuropathic pain, such as tingling or burning. As a third-generation chemotherapy drug, oxaliplatin has a good effect on tumors such as colorectal cancer, liver cancer, and gastric cancer, but it usually causes chemotherapy-induced peripheral neuropathy and is toxic to dorsal root ganglion neurons.

[0003] Reactive oxygen species (ROS) refers to the general term for oxygen-containing free radicals and peroxides that are easy to form free radicals and are related to oxygen metabolism in the body. Under the stimulation of oxaliplatin, the content of ROS increases significantly. Excessive ROS will activate pain neurons, thereby inducing pain response. It can also destroy the mitochondrial electron transport chain and change the production of ATP in sensory neurons.

[0004] At present, the common practice for chemotherapy pain in clinical practice is systemic analgesia, but it may cause serious side effects. In order to better relieve pain, directly connecting electrodes to the nerve foramen for electrical stimulation has also been used in clinical practice as an emerging therapy for treating pain. However, its cost is high and the electrodes cannot be absorbed by the human body. Therefore, the development of absorbable piezoelectric materials to convert ultrasonic energy into electrical energy to relieve pain has attracted much attention.

[0005] Electrospinning technology is an effective method for preparing organic polymer nanofibers. It has a broad prospect in medical research and is mainly used in drug controlled release systems, medical dressings, tissue engineering, etc. It has the advantages of slowly releasing effective growth substances to promote cell adhesion, proliferation and differentiation on the scaffold. However, there is currently no patent for its application in relieving chemotherapy pain. Summary of the invention

[0006] The purpose of the present invention is to provide a multifunctional PPK electrospun fiber membrane with good ROS response, good biocompatibility and self-degradability and active oxygen response, as well as a preparation method and application thereof.

[0007] The technical solution of the present invention is: A multifunctional PPK electrospun fiber membrane with active oxygen response, characterized in that it is prepared by the following method: S1: preparing electrospinning solution: dissolving PLA, PEGDA-EDT and KNN in hexafluoroisopropanol solution and stirring until uniformly dispersed to obtain spinning solution; S2: The electrospinning liquid is subjected to electrospinning to obtain an electrospinning fiber membrane.

[0008] A method for preparing a multifunctional PPK electrospun fiber membrane with active oxygen response, characterized in that it comprises the following steps: S1: preparing electrospinning solution: dissolving PLA, PEGDA-EDT and KNN in hexafluoroisopropanol solution and stirring until uniformly dispersed to obtain spinning solution; S2: The electrospinning liquid is subjected to electrospinning to obtain an electrospinning fiber membrane.

[0009] In the S1, the concentration of PLA dissolved in the hexafluoroisopropanol solution is 10% (w / v), the concentration of PEGDA-EDT dissolved in the hexafluoroisopropanol solution is 20% (w / v), the concentration of KNN is maintained at 50% (w / v), and the particle size of KNN particles is 1 μm.

[0010] After the solution in S1 is added, ultrasonic treatment is performed for 15 minutes to fully stir the solution until it is evenly dispersed, thereby obtaining a spinning solution.

[0011] The electrospinning parameters in S2 include: during the spinning process, the voltage parameter is 14.5-15 V, the distance between the spinneret and the aluminum foil is 10-15 cm, and the injection speed of the injection pump is 1 mL / h.

[0012] After the electrospinning in S2 is completed, the film is dried in an oven for 48 h to evaporate the residual hexafluoroisopropanol, and then sealed and stored away from light.

[0013] Application of a multifunctional PPK electrospun fiber membrane with active oxygen response in the preparation of materials for relieving pain from oxaliplatin chemotherapy.

[0014] The electrospun fiber membrane PPK in the present invention is prepared by electrospinning from a system of PLA, PEGDA-EDT and KNN. PLA is a biocompatible, biodegradable and non-toxic polymer, which has a wide range of applications in biomedical fields such as tissue engineering and medical dressings. It can be used as a substrate support material to spin electrospun nanofibers as regenerative scaffold materials or drug delivery systems. PEGDA (polyethylene glycol diacrylate) is a non-toxic and harmless organic biomaterial, and EDT (1,2-ethanedithiol) is a safe complexing agent. PEGDA and EDT react with thiol-ene under the action of a catalyst to generate a polymer that can react with ROS, PEGDA-EDT, and this compound containing a thioether group is easily oxidized into a hydrophilic substance and soluble in water. Therefore, PEGDA-EDT is a potential ROS-responsive material; KNN, as a piezoelectric material, can convert various mechanical stimuli into electrical signals based on the piezoelectric effect, and generate electrical stimulation under the action of ultrasound to promote nerve repair.

[0015] The electrospun fiber membrane of the present invention has a good ROS response, generates electrical stimulation based on the piezoelectric material KNN under ultrasonic stimulation, promotes the repair of peripheral nerve damage, and has good biocompatibility, and is an ideal self-degradable medical material. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0017] Figure 1-Figure 5 This is a material characterization diagram of the electrospun PKK fiber membrane in one embodiment of the present invention; in, Figure 1 : NMR images of electrospun PKK fiber membrane; Figure 2 : Infrared spectrum of electrospun PKK fiber membrane; Figure 3 : SEM image of electrospun PKK fiber membrane (scale bar: 100 μm); Figure 4 : SEM image of electrospun PKK fiber membrane (scale bar: 50 μm); Figure 5 : Scanning electron microscopy image of electrospun PKK fiber membrane (scale bar: 2 μm); Figure 6-10 This is a performance verification and biosafety verification diagram of the electrospun PKK fiber membrane in one embodiment of the present invention; wherein, Figure 6 :Cyclic voltammetry verifies the current change before and after ultrasound; Figure 7 : Conductivity diagram of PLA / PEGDA-EDT fiber membrane and PLA / PEGDA-EDT / KNN fiber membrane; Figure 8 :The turbidity change of PEGDA-EDT under the stimulation of different concentrations of H2O2; Fig. 9 , 10: Scanning electron microscopy images of electrospun PKK fiber membranes after being immersed in H2O2 simulated ROS environment for 3 days and 7 days.

[0018] Fig.11 This is a schematic diagram reflecting that the electrospun PKK fiber membrane has good biocompatibility and can maintain high cell activity when co-cultured with cells under ultrasonic stimulation.

[0019] Fig.12 Compared with the normal group, the body weight of rats in the pathological group continued to decrease from day 0 to day 7, and began to recover after the cessation of oxaliplatin injection on the eighth day. The body weight of rats in the experimental group gradually increased from the fourth day.

[0020] Fig.13 Compared with the normal group, the heat pain threshold of rats in the pathological group continued to decrease from 0 to 7 days, and began to recover after the cessation of oxaliplatin injection on the 8th day. The heat pain threshold of rats in the experimental group gradually recovered from the 4th day and was close to the level of the normal group from the 8th day.

[0021] Fig.14 Compared with the normal group, the mechanical pain threshold of rats in the pathological group showed a downward trend from 0 to 7 days, and began to recover after the injection of oxaliplatin was stopped on the 8th day. The mechanical pain threshold of rats in the experimental group gradually recovered from the 8th day and was close to the level of the normal group from the 8th day. DETAILED DESCRIPTION

[0022] A method for preparing a multifunctional PPK electrospun fiber membrane with active oxygen response includes the preparation of a spinning solution and the preparation of electrospinning. The specific steps of the preparation method are as follows: S1: Preparation of spinning solution: PLA, PEGDA-EDT and KNN were dissolved in hexafluoroisopropanol solution and stirred until uniformly dispersed to obtain a spinning solution; Specifically, in one embodiment, the preparation of the spinning solution comprises the following steps: A1: PLA was dissolved in hexafluoroisopropanol solution to a concentration of 10% (w / v) in hexafluoroisopropanol.

[0023] A2: PEGDA-EDT and KNN were added to the above solution respectively, wherein the concentration of PEGDA-EDT was maintained at 20% (w / v) and the concentration of KNN was maintained at 50% (w / v).

[0024] A3: Ultrasonicate for 15 minutes to fully stir until it is evenly dispersed to obtain the spinning solution.

[0025] S2: Electrospinning: The spinning solution in S1 is subjected to electrospinning to obtain the electrospun PKK fiber membrane.

[0026] Specifically, in one embodiment, the parameters of the electrospinning include a spinning voltage of 15 V, a distance between the spinneret and the aluminum foil of 5-10 cm, and an injection speed of the injection pump of 1 mL / h.

[0027] The electrospinning is carried out in a fume hood, and an electrospun PKK fiber membrane is obtained after the electrospinning is completed.

[0028] The obtained electrospun PKK fiber membrane was placed in an oven and dried for 48 h to volatilize the organic solvent in the fiber membrane. After drying, it was sealed and stored away from light.

[0029] The present application also provides a multifunctional PPK electrospun fiber membrane with active oxygen response, which is obtained using the above preparation method, and its material characterization diagram is as follows Figure 1 , Figure 2 shown.

[0030] The present application provides the use of the electrospun PKK fiber membrane described above in the preparation of an oxaliplatin chemotherapy pain product. The electrospun PKK fiber membrane described above has good biocompatibility, ROS scavenging effects and the like.

[0031] Specifically, the electrospun PKK fiber membrane is used in the preparation of materials for alleviating pain from oxaliplatin chemotherapy.

[0032] The following is an explanation of the above content in combination with specific verification experiments: 1: Verification of wireless discharge capability of electrospun PKK fiber membrane 1.1 Experimental methods: 1.1.1 Electrochemical cyclic voltammetry to detect the wireless discharge capability of electrospun PKK fibers Ultrasonic stimulation was applied under the same potential conditions, and the corresponding current changes were recorded. By comparing the current response before and after ultrasonic stimulation, the ability of the electrospun PKK fiber membrane to convert ultrasonic energy into electrical energy after stimulation can be quantitatively analyzed.

[0033] 1.1.2 Measuring the resistance of electrospun PKK fiber membrane with high resistance meter Under the condition that the surface of the electrospun fiber membrane sample is clean and free of dust or pollutants, a high resistance meter is used to detect the PLA / PEGDA-EDT electrospun fiber membrane and the PLA / PEGDA-EDT / KNN electrospun fiber membrane. The resistance of the electrospun fiber membrane containing KNN is significantly higher than that of the control group, and the discharge rate is slower, which can achieve the effect of slowly releasing current.

[0034] 1.2 Experimental results: (1) The electrospun PKK fiber membrane has wireless discharge capability when stimulated by ultrasound, such as Figure 6 shown.

[0035] (2) The electrospun PKK fiber membrane has a greater resistance than the control group, such as Figure 7 shown.

[0036] 2: Electrospun PKK fiber membrane has ROS response and good biosafety performance verification 2.1.1 Observation of electrospun fiber morphology after co-culture with H2O2 under SEM 0%, 2.5%, 5%, and 7.5% H2O2 were prepared respectively, and 4 portions of PEGDA-EDT of the same mass were weighed and immersed in them. The turbidity of the solution was analyzed to verify that PEGDA-EDT is responsive to active oxygen. Due to the increase of hydrophobic thioether, the PEGDA-EDT solid dissolved with the increase of H2O2 concentration, and the turbidity continued to rise.

[0037] 2.1.2 Turbidimeter to detect ROS response The electrospun PKK fiber membrane with an area of ​​1*1cm was immersed in 7.5% H2O2 for 3 days and 7 days, and the appearance of the electrospun PKK fiber membrane after ROS response was observed by SEM. The electrospun PKK fiber membrane expanded to a certain extent after being immersed in H2O2, and a large area of ​​dissolution and fracture occurred. The electrospun PKK fiber had a large number of jagged or worm-eaten defects, and the pores were enlarged. The characteristics of electrospun PKK fiber dissolving and breaking in H2O2 environment indicate that the electrospun fiber membrane has ROS responsiveness.

[0038] 2.1.3 Testing the biosafety of electrospun fiber membranes by cell live-dead staining The electrospun PKK fiber membranes with different compositions were immersed in complete culture medium, placed in a cell culture incubator at 37°C for 24 hours, and the supernatant was taken to obtain culture medium with different compositions.

[0039] PC12 cells were co-cultured with the above-mentioned culture medium with different components for 24 h, the culture medium was discarded, and the cells were washed once with PBS.

[0040] Calcein / PI fluorescence staining was used to detect cell activity and cytotoxicity. Calcein AM stained live cells with green fluorescence, while PI stained dead cells with red fluorescence. The two probes can detect intracellular esterase activity and cell membrane integrity, respectively, thus reflecting cell activity and cytotoxicity.

[0041] After adding the fluorescent probe, incubate at 37 °C in the dark for 30 min, and observe the staining effect under a fluorescence microscope.

[0042] 2.2 Experimental results: (1) As the H2O2 concentration increases, the turbidity of the PEGDA-EDT solution gradually decreases. Figure 8 shown.

[0043] (2) The electrospun PKK fiber membrane dissolved and fractured when immersed in H2O2, and was ROS responsive, such as Fig. 9 , 10 shown.

[0044] (3) The electrospun PKK fiber membrane has good biocompatibility and can maintain high cell activity when co-cultured with cells under ultrasonic stimulation. Fig.11 shown.

[0045] Effects of electrospun PKK fiber membrane on CIPN rats 3.1 Experimental methods: 3.1.1 Establishment of the pain model of oxaliplatin chemotherapy rats by electrospun PKK fiber membrane and its effect on rat body weight Six-week-old rats were divided into a normal group, a pathological group, and an experimental group. The rats in the pathological group and the experimental group were intraperitoneally injected with 4 mg / kg oxaliplatin for 7 consecutive days, and the rats in the normal group were injected with normal saline. Their body weights were weighed and recorded daily. On day 0, the pathological group and the experimental group underwent surgery. After the rats were anesthetized, the back hair was removed, and the skin layer and muscle layer were cut layer by layer from the outside to the inside to expose the right transverse process of the 4th-5th lumbar vertebrae. The electrospun fiber membrane with a dose of 30 mg / kg KNN was placed as close to the rat DRG as possible, and the wound was sutured. The rat DRG was then ultrasonicated every 2 days, with an ultrasonic frequency of 1 MHz and a sound pressure of 100 kPa, and each time lasted for 10 minutes.

[0046] 3.1.2 Effect of electrospun PKK fiber membrane on mechanical pain in rat model of oxaliplatin chemotherapy pain The mechanical pain threshold of rats was assessed by the Von Frey test. The rats were placed on the experimental platform and the experimental environment was quiet. The rats were allowed to adapt for about 10-15 minutes to reduce disturbance to the rats. Starting with a 15g Von Frey ciliary mechanical stimulation needle, gently press until it bends, and then hold for a few seconds. Observe the rat's reaction, such as withdrawal, shaking, or licking the test site. If the rat does not react, gradually increase the hardness of the stimulation needle until the minimum force that causes a reaction is found. Each mechanical ciliary stimulation needle was tested at least 3 times, and the mechanical pain threshold of the rat was recorded and calculated.

[0047] 3.1.3 Effect of electrospun PKK fiber membrane on thermal pain in rat model of oxaliplatin chemotherapy pain The thermal pain threshold of rats was tested using a plantar thermal allergy meter. The test head was slid under the sole of the rat's foot and the start button was pressed. The off button was pressed the instant the rat lifted its foot and the time was recorded. The upper limit of the recording was 20 seconds and repeated 3 times for each rat.

[0048] 3.2 Experimental results: (1) Compared with the normal group, the body weight of the rats in the pathological group continued to decrease from day 0 to day 7, and began to recover after the cessation of oxaliplatin injection on day 8. The body weight of the rats in the experimental group gradually increased from day 4. Fig.12 shown.

[0049] (2) Compared with the normal group, the heat pain threshold of the rats in the pathological group continued to decrease from day 0 to day 7, and began to recover after the injection of oxaliplatin was stopped on day 8. The heat pain threshold of the rats in the experimental group gradually recovered from day 4 and was close to the level of the normal group from day 8. Fig.13 shown.

[0050] (3) Compared with the normal group, the mechanical pain threshold of the rats in the pathological group showed a downward trend from day 0 to day 7, and began to recover after the injection of oxaliplatin was stopped on day 8. The mechanical pain threshold of the rats in the experimental group gradually recovered from day 8 and was close to the level of the normal group from day 8. Fig.14 shown.

[0051] This proves that the electrospun fiber membrane provided in this application can effectively remove ROS, and by stably converting ultrasonic stimulation into an electrical signal mode, achieve continuous and effective management of chemotherapy pain. In this study, we constructed a multifunctional electrospun fiber membrane with simple process according to the characteristics of biomaterials. We implanted the electrospun PKK fiber membrane into the sheath, which can not only release electrical stimulation through ultrasonic stimulation to promote nerve repair, but also consume ROS accumulation in DRG caused by chemotherapy, which is an ideal medical material.

Claims

1. A multifunctional PPK electrospun fiber membrane with active oxygen response, characterized by: Prepared by the following method: S1: preparing electrospinning solution: dissolving PLA, PEGDA-EDT and KNN in hexafluoroisopropanol solution and stirring until uniformly dispersed to obtain spinning solution; S2: The electrospinning liquid is subjected to electrospinning to obtain an electrospinning fiber membrane.

2. A method for preparing a multifunctional PPK electrospun fiber membrane with active oxygen response, characterized in that: The following steps are involved: S1: preparing electrospinning solution: dissolving PLA, PEGDA-EDT and KNN in hexafluoroisopropanol solution and stirring until uniformly dispersed to obtain spinning solution; S2: The electrospinning liquid is subjected to electrospinning to obtain an electrospinning fiber membrane.

3. The method for preparing the multifunctional PPK electrospun fiber membrane with active oxygen response according to claim 2, characterized in that: In the S1, the concentration of PLA dissolved in the hexafluoroisopropanol solution is 10% (w / v), the concentration of PEGDA-EDT dissolved in the hexafluoroisopropanol solution is 20% (w / v), the concentration of KNN is maintained at 50% (w / v), and the particle size of KNN particles is 1 μm.

4. The method for preparing the multifunctional PPK electrospun fiber membrane with active oxygen response according to claim 2 or 3, characterized in that: After the solution in S1 is added, ultrasonic treatment is performed for 15 minutes to fully stir the solution until it is evenly dispersed, thereby obtaining a spinning solution.

5. The method for preparing the multifunctional PPK electrospun fiber membrane with active oxygen response according to claim 2 or 3, characterized in that: The electrospinning parameters in S2 include: during the spinning process, the voltage parameter is 14.5-15 V, the distance between the spinneret and the aluminum foil is 10-15 cm, and the injection speed of the injection pump is 1 mL / h.

6. The method for preparing the multifunctional PPK electrospun fiber membrane with active oxygen response according to claim 2 or 3, characterized in that: After the electrospinning in S2 is completed, the film is dried in an oven for 48 h to evaporate the residual hexafluoroisopropanol, and then sealed and stored away from light.

7. Application of a multifunctional PPK electrospun fiber membrane with active oxygen response in the preparation of materials for relieving pain from oxaliplatin chemotherapy.

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