All-hydrogel based on ultrasonic response type hydrogel as well as preparation method and application of all-hydrogel

Abstract

The invention discloses a preparation method of full hydrogel based on ultrasonic response type hydrogel, which comprises the following steps: S1, preparing C60 (at) PDA / AP39 nanoparticles; S2, dissolving GelMA in a diluted C60 (at) PDA / AP39 nanoparticle solution to obtain the full hydrogel. The C60 (at) PDA and AP39 in the invention can clear ROS, relieve or alleviate inflammatory response, and provide more effective and safer treatment choices. The all-hydrogel based on the ultrasonic response type hydrogel prepared in the invention is safe and non-toxic, and provides a more effective and safer treatment choice. The prepared full hydrogel based on the ultrasonic response type hydrogel can relieve pain caused by chemotherapy, and a new solution thought is provided for pain caused by chemotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of medical material preparation, and in particular to a preparation method of a full hydrogel with active oxygen scavenging capability and application thereof in the treatment of chemotherapy pain. Background Art

[0002] Chemotherapy-induced peripheral neuropathy (CIPN) is common in patients receiving anticancer treatment and can affect patients' survival and quality of life after treatment. Symptoms of peripheral neuropathy are mainly sensory and can be divided into functional enhancement features, including burning, tingling, and hypersensitivity to cold or touch, and functional loss features, including loss of proprioception, decreased vibration and pinprick sensation, and numbness. CIPN is an adverse reaction to multiple drug treatments, including platinum-based drugs, microtubule-targeted drugs (Tau), protease inhibitors, and angiogenesis inhibitors. There is currently a lack of effective drugs to prevent or control CIPN, and only duloxetine is recommended for the treatment of CINP. Other drugs used for other neuropathic pain may be given due to limited treatment options for CINP, partly due to incomplete understanding of the mechanisms by which chemotherapy alters the sensitivity of sensory neurons. Therefore, research to understand the pathophysiology of CINP and develop new therapeutic drugs for chemotherapy-induced neuropathic pain is crucial.

[0003] At present, the use of opioids in the treatment of chemotherapy pain in clinical practice may lead to drug dependence and side effects, such as drug resistance, drug dependence and the risk of overdose, and the treatment effect is limited. CIPN caused by chemotherapy is often difficult to treat, and no curative treatment for chronic painful post-chemotherapy neuropathy has been found. Current research on biomaterials in the treatment of CIPN includes: nerve conduits are used to repair damaged nerves and promote nerve regeneration; nanomaterials are used for drug delivery to improve efficacy and reduce side effects; biological scaffolds provide structural support; neurotrophic factors such as NGF and BDNF promote nerve repair and regeneration. Biomaterials show great potential in drug delivery, nerve repair, etc., and are expected to provide more effective treatment options for CIPN in the future. A functional material of ultrasound-responsive hydrogel is urgently needed to solve the problem of pain caused by chemotherapy. Summary of the invention

[0004] The purpose of the present invention is to provide a full hydrogel based on ultrasound responsive hydrogel with active oxygen scavenging ability, and a preparation method and use thereof.

[0005] The technical solution of the present invention is: A full hydrogel based on ultrasound responsive hydrogel, characterized in that it is prepared by the following method: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated in Tris-HCl buffer and continuously stirred before ultracentrifugation to obtain C60@PDA / AP39 nanoparticles; S2: Dissolve GelMA in the diluted C60@PDA / AP39 nanoparticle solution to obtain a full hydrogel.

[0006] A method for preparing a full hydrogel based on an ultrasound-responsive hydrogel is characterized by comprising the following steps: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated in Tris-HCl buffer and continuously stirred before ultracentrifugation to obtain C60@PDA / AP39 nanoparticles; S2: Dissolve GelMA in the diluted C60@PDA / AP39 nanoparticle solution to obtain a full hydrogel.

[0007] In the step S1, C60 is first dissolved in Tris-HCl buffer and then ultrasonicated for 30 minutes to be evenly dispersed in the solution, and then AP39 is added; and then dopamine powder is added to the above solution.

[0008] In step S1, 25 mg of C60 was dissolved in 400 ml of Tris-HCl buffer at pH 8.5, and ultrasonicated for 30 min to make it evenly dispersed in the solution, 100 μl of 10 mM AP39 and 25 mg of dopamine were added, and the mixture was stirred continuously at 60° C. for 24 h.

[0009] After continuous heating and stirring, the solution in S1 was ultracentrifuged at 16000 g for 2 h, and washed three times with Milli-Q water to obtain C60@PDA / AP39 nanoparticles, which were finally resuspended with Milli-Q water for further use.

[0010] The concentration of GelMA dissolved in the nanoparticle solution in S2 is 30%.

[0011] The invention discloses an application of a full hydrogel based on ultrasound responsive hydrogel in preparing a material for treating pain caused by peripheral neuropathy induced by chemotherapy.

[0012] The all-hydrogel of the present invention has the ability to scavenge reactive oxygen species, and the all-hydrogel of the present invention contains C60 and AP39. Among them, C60 has been studied for use as an antioxidant, drug delivery, and photodynamic therapy. C60 and its derivatives have a unique ability to scavenge ROS. Dopamine is used for surface modification and coating due to its adhesiveness and self-polymerization properties. PDA is a bioactive molecule that simulates dopamine and has excellent antioxidant and free radical scavenging abilities. PDA-modified nanoparticles have stronger hydrophilicity, good biocompatibility, appropriate biodegradability, strong photothermal conversion ability, reactive oxygen species (ROS) scavenging ability, and ultrasonic responsiveness. AP39, a synthetic H2S donor, is capable of transporting H2S to mitochondria. H2S is a gas signaling molecule with antioxidant, anti-inflammatory, and cytoprotective effects. AP39 regulates cellular energy metabolism and reduces oxidative stress by targeting mitochondria. Photocurable gelatin-methacryloyl (GelMA) is a safe and effective biological carrier that can provide a good transplantation environment for cells and biological factors. It has good biocompatibility, degradability and mechanical properties and is considered to be the most promising biomaterial in the biomedical field. In this study, AP39-modified C60@PDA nanoparticles were dissolved in GelMA to construct a full hydrogel based on ultrasound-responsive hydrogel to remove excess ROS in the body to reduce inflammation and relieve pain. The ultrasound-responsive full hydrogel of the present invention has good antioxidant function, can reduce inflammation and relieve pain, and at the same time make drug release smoother. It is an ideal self-degradable medical material.

[0013] The whole hydrogel based on ultrasound-responsive hydrogel prepared in the present invention, wherein C60@PDA and AP39 can remove ROS, relieve or reduce inflammatory response, and provide a more effective and safe treatment option. The whole hydrogel based on ultrasound-responsive hydrogel prepared in the present invention is safe and non-toxic, providing a more effective and safe treatment option. The whole hydrogel based on ultrasound-responsive hydrogel prepared in the present invention can relieve pain caused by chemotherapy, providing a new solution to chemotherapy pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0015] Figure 1 is a material characterization diagram of C60@PDA and GelMA in one embodiment of the present invention; Among them, A: Potential diagram of C60@PDA and C60; B: Particle size diagram of C60@PDA and C60; C: Infrared spectra of C60@PDA and C60; D: Scanning electron microscopy of Gelma (scale bar: 50 μm); E: Transmission electron microscopy of C60@PDA and C60; F: Scanning electron microscopy of C60@PDA and C60 (scale bar: 200 nm); Figure 2 It is the performance verification and biosafety verification of the nanoparticles in one embodiment of the present invention; Among them, G: ROS fluorescence graph under the intervention of different component culture media; H: ROS quantitative statistical graph under the intervention of different component culture media; IK: Expression of inflammatory factors (IL-1β, IL-6, TNF-α) in cell supernatant under the intervention of different component culture media.

[0016] Figure 3 The effect of the ultrasound-responsive all-hydrogel in one embodiment of the present invention on oxaliplatin-induced chemotherapy pain in rats; wherein L: HE staining of different organs of rats after intrathecal injection of the all-hydrogel; M: changes in body weight of rats with chemotherapy pain; N: changes in the threshold of thermal pain in rats with chemotherapy pain; O: changes in the threshold of mechanical pain in rats with chemotherapy pain. DETAILED DESCRIPTION

[0017] A method for preparing a full hydrogel based on an ultrasound-responsive hydrogel comprises the following steps: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated in Tris-HCl buffer and continuously stirred before ultracentrifugation to obtain C60@PDA / AP39 nanoparticles; S2: Dissolve GelMA in the diluted C60@PDA / AP39 nanoparticle solution to obtain a full hydrogel.

[0018] In step S1, 25 mg of C60 was dissolved in 400 ml of Tris-HCl buffer at pH 8.5, 100 μl of 10 mM AP39 was added, and ultrasonication was performed for 30 min to uniformly disperse it in the solution, 25 mg of dopamine was added, and stirring was continued at 60° C. for 24 h, followed by ultracentrifugation at 16000 g for 2 h, and washed three times with Milli-Q water to obtain C60@PDA / AP39 nanoparticles, which were finally resuspended with Milli-Q water for further use.

[0019] The concentration of GelMA dissolved in the nanoparticle solution in S2 is 30%.

[0020] The following is an explanation of the above content in combination with specific verification experiments: 1: Synthesis and characterization of nanoparticle materials 1.1 Experimental methods: 1.1.1 Malvern particle size analyzer to measure the potential and particle size of nanoparticles Take 1 μl of the sample and add it to 99 μl of Milli-Q water. Mix well and add it to the cuvette. Use Malvern particle size analyzer to detect the potential and particle size.

[0021] 1.1.2 Characteristics of Nanoparticles and Gelma Detected by FT-IR, TEM and SEM About 90 mg of C60@PDA nanoparticles were obtained by freeze drying, and the freeze-dried samples were characterized by Fourier transform infrared spectroscopy (FTIR). The morphology of C60 and C60@PDA was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Gelma was freeze-dried and its surface morphology was observed by scanning electron microscopy (SEM).

[0022] 1.2 Experimental results: (1) The nanoparticles C60@PDA obtained after C60 modification are negatively charged, as shown in Figure A.

[0023] (2) The particle size of C60@PDA after C60 modification becomes larger, as shown in Figure B.

[0024] (3) Intramolecular vibration of the C60 skeleton structure, as shown in Figure C.

[0025] (4) Gelma exhibits a porous and sponge-like microstructure.

[0026] (5) Scanning electron microscopy and transmission electron microscopy showed that C60@PDAC60 nanoparticles exhibited a uniform spherical structure and a rough surface, and PDA was successfully modified onto fullerene (C60) through self-polymerization.

[0027] 2: Ultrasound-responsive all-hydrogel has ROS scavenging and anti-inflammatory effects 2.1 Experimental methods: 2.1.1 Quantitative detection of intracellular reactive oxygen species levels under different treatments using reactive oxygen species detection kit Prepare cell suspension and count cells; inoculate cell suspension (1 mL / well) in a six-well plate at a density of approximately (1×104); stimulate the positive control well with hydrogen peroxide; add culture medium and hydrogen peroxide of different components to the intervention wells in the culture plate, and incubate the culture plate in an incubator for 6 h; dilute DCFH-DA with serum-free culture medium at 1:1000; remove the cell culture medium, add 1 mL of diluted DCFH-DA to each well to fully cover the cells; incubate in a 37 ℃ cell culture incubator for 20 min; wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. Take photos under a fluorescence microscope.

[0028] 2.1.1 ELISA detection of intracellular inflammatory factor levels stimulated by LPS in different culture media The cells were seeded on a 6-well plate at a density of 24×104 per well. After incubation overnight to allow the cells to adhere to the wall, the cells were co-cultured with culture media of different components for 6 hours, the supernatant was aspirated, and the levels of inflammatory factors (IL-1β, IL-6, TNF-α) in the cell supernatant were detected by ELISA kit. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20 minutes, and the remaining strips were sealed with a ziplock bag and returned to 4°C. Standard wells and sample wells were set up, and 50μL of standards of different concentrations were added to each standard well; 10μL of the sample to be tested was first added to the sample well, and then 40μL of the sample diluent was added; no blank wells were added. Except for the blank wells, 100μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and sample well, and the reaction wells were sealed with a sealing film, and incubated in a 37°C water bath or incubator for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well, incubate at 37℃ in the dark for 15 minutes. Add 50μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450nm within 15 minutes.

[0029] 2.1 Experimental results: (1) The ultrasound-responsive all-hydrogel has good reactive oxygen species scavenging ability, as shown in Figures GH.

[0030] (2) Ultrasound-responsive all-hydrogel has good anti-inflammatory effect, and the level of inflammatory factors in the supernatant of the intervened cells is reduced, such as Figure 2 As shown in IK.

[0031] 3: Effects of ultrasound-responsive all-hydrogel on chemotherapy pain in rats 3.1 Experimental methods: 3.1.1 Establishment of a pain model of chemotherapy pain in rats and the effect of ultrasound-responsive all-hydrogel 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, surgery was performed on the pathological group and the experimental group. After the rats were anesthetized, the hair on the back was removed, and the skin layer and muscle layer were cut layer by layer from the outside to the inside. 40ul of the full hydrogel (i.e., the full hydrogel based on the ultrasound-responsive hydrogel of the present invention) at a dose of 10 mg / ml was injected into the rat body with an insulin needle sheath, and the wound was sutured. The rat DRG was then ultrasounded every 2 days, with an ultrasound frequency of 1 MHz and a sound pressure of 100 kPa, and each time lasted for 10 minutes.

[0032] 3.1.2 Effects of ultrasound-responsive all-hydrogel on mechanical pain in a rat model of chemotherapy pain The mechanical pain threshold of rats was evaluated by the Von Frey test. The rats with confirmed groups and numbers were placed in a box on a metal mesh and waited for 15-30 minutes until the rats remained quiet in the unfamiliar environment (not walking around in the cage or licking themselves). The Von Frey hair was applied to the hairless area on the left palm and sole of the rat. The Von Frey hair was "S" shaped. The action time was 6-8 seconds, and the interval between adjacent stimulations was at least 7 seconds. Positive reaction: During the action time, the rat lifted its foot or licked the stimulated side. During the stimulation process, if the rat walked or lifted its foot as soon as it was stimulated, it was suspected to be positive. It was necessary to wait until the rat was quiet before measuring. Positive results were recorded as X and negative results as O. When measuring, the middle value of 10.0g was selected as the starting point, and the measurement was performed in ascending order. When the first crossover occurred, the following four measurements were made. The corresponding log unit value of the Von Frey hair measured in the last measurement was substituted into the formula for calculation.

[0033] 3.1.3 Effect of ultrasound-responsive all-hydrogel on thermal pain in a rat model of chemotherapy pain The thermal pain threshold of rats was tested using a plantar heat pain meter. The rats were placed on the hot plate instrument. After the rats became quiet and stopped licking their hair, the test head was slid under the soles of the rats' feet and the start button was pressed. The off button was immediately pressed after the rats showed any pain reactions such as lifting their feet or licking their hind paws. The time was recorded with an upper limit of 20 seconds and repeated 3 times for each rat.

[0034] 3.1.4 HE staining of various organs in rats treated with all-hydrogel for chemotherapy pain The rat tissue sections to be stained were dewaxed with xylene for 10 minutes each time, twice. 100%, 95%, 85% and 75% ethanol were used for 5 minutes each, and then washed with distilled water twice, 2 minutes each time. Hematoxylin stain was used for 3 minutes, and then the floating color was washed away with distilled water.

[0035] Treat with 1% hydrochloric acid alcohol differentiation solution for 30 seconds, then wash with tap water for 3 minutes, twice. Stain with eosin solution for 1-3 minutes, then wash with distilled water for 30 seconds. Use 80%, 95% and absolute ethanol for 1 minute each, then use xylene for clearing, 3 minutes each, twice.

[0036] Use neutral gum for mounting.

[0037] 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 injection of oxaliplatin was stopped on day 8. The body weight of the rats in the experimental group gradually increased from day 4, as shown in Figure M.

[0038] (2) Compared with the normal group, the thermal 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 thermal pain threshold of the rats in the experimental group gradually recovered from day 4, and on day 8, compared with the case group, the drug-added group was close to the level of the normal group, as shown in Figure N.

[0039] (3) Compared with the normal group, the mechanical pain threshold of the 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 the rats in the drug-added group and the case group gradually recovered from the 8th day and gradually approached the level of the normal group after 8 days. Compared with the case group, the drug-added group was closer to the level of the normal group, as shown in Figure O.

[0040] Through the above experiments, it can be proved that the ultrasound-responsive all-hydrogel provided in this application can effectively remove ROS, reduce inflammatory response, and achieve the effect of relieving chemotherapy pain. In this study, we constructed an ultrasound-responsive all-hydrogel with a simple process based on the characteristics of biomaterials. We implanted the ultrasound-responsive all-hydrogel into rat DRG, which promoted nerve repair through ultrasonic stimulation and consumed ROS aggregation in DRG caused by chemotherapy. It is an ideal medical material.

Claims

1. A full hydrogel based on ultrasound responsive hydrogel, characterized by: Prepared by the following method: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated in Tris-HCl buffer and continuously stirred before ultracentrifugation to obtain C60@PDA / AP39 nanoparticles; S2: Dissolve GelMA in the diluted C60@PDA / AP39 nanoparticle solution to obtain a full hydrogel.

2. A method for preparing a full hydrogel based on an ultrasound-responsive hydrogel, characterized in that: The following steps are involved: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated in Tris-HCl buffer and continuously stirred before ultracentrifugation to obtain C60@PDA / AP39 nanoparticles; S2: Dissolve GelMA in the diluted C60@PDA / AP39 nanoparticle solution to obtain a full hydrogel.

3. The method for preparing a full hydrogel based on ultrasound responsive hydrogel according to claim 2, characterized in that: In the step S1, C60 is first dissolved in Tris-HCl buffer and then ultrasonicated for 30 minutes to be evenly dispersed in the solution, and then AP39 is added; and then dopamine powder is added to the above solution.

4. The method for preparing a full hydrogel based on ultrasound responsive hydrogel according to claim 3, characterized in that: In step S1, 25 mg of C60 was dissolved in 400 ml of Tris-HCl buffer at pH 8.5, and ultrasonicated for 30 min to make it uniformly dispersed in the solution. Then, 100 μl of 10 mM AP39 and 25 mg of dopamine were added, and the mixture was stirred continuously at 60° C. for 24 h.

5. A method for preparing a full hydrogel based on ultrasound responsive hydrogel according to claim 2, 3 or 4, characterized in that: After continuous heating and stirring, the solution in S1 was ultracentrifuged at 16000 g for 2 h, and washed three times with Milli-Q water to obtain C60@PDA / AP39 nanoparticles, which were finally resuspended with Milli-Q water for further use.

6. A method for preparing a full hydrogel based on ultrasound responsive hydrogel according to claim 2, 3 or 4, characterized in that: The concentration of GelMA dissolved in the nanoparticle solution in S2 is 30%.

7. Application of a full hydrogel based on ultrasound-responsive hydrogel in the preparation of materials for treating pain caused by chemotherapy-induced peripheral neuropathy.

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