All-hydrogel based on ultrasound-responsive hydrogel and preparation method and use thereof

Abstract

The application discloses a preparation method of a full hydrogel based on an ultrasonic response type hydrogel, and comprises the following steps: S1, preparing C60@PDA / AP39 nanoparticles; S2, dissolving GelMA in a diluted C60@PDA / AP39 nanoparticle solution to obtain the full hydrogel. In the application, C60@PDA and AP39 can remove ROS, relieve or reduce inflammatory reactions, and provide more effective and safe treatment options. The full hydrogel based on the ultrasonic response type hydrogel prepared in the application is safe and non-toxic, and provides more effective and safe treatment options. The full hydrogel based on the ultrasonic response type hydrogel prepared in the application can relieve pain caused by chemotherapy, and provides a new solution for chemotherapy pain.

Description

Technical Field

[0001] This invention relates to the field of medical material preparation technology, and in particular to a method for preparing a fully hydrogel with reactive oxygen species scavenging ability and its application in the treatment of chemotherapy pain. Background Technology

[0002] Chemotherapy-induced peripheral neuropathy (CIPN) is common in patients undergoing anticancer treatment and can impact their survival and post-treatment quality of life. Symptoms of CIPN are primarily sensory, ranging from enhancement symptoms (burning, tingling, and hypersensitivity to cold or touch) to loss-of-function features (loss of proprioception, decreased vibration and pinprick sensation, and numbness). CIPN is an adverse reaction to various medications, including platinum-based drugs, microtubule-targeting drugs (Tau), protease inhibitors, and angiogenesis inhibitors. Currently, there are no effective drugs for the prevention or control of CIPN; only duloxetine is recommended for its treatment. Due to the limited treatment options for CIPN, other medications used for other neuropathic pain may be administered, partly due to an incomplete understanding of the mechanisms by which chemotherapy alters the sensitivity of sensory neurons. Therefore, understanding the pathophysiology of CIPN and developing new treatments for chemotherapy-induced neuropathic pain are crucial.

[0003] Currently, the use of opioids in the clinical treatment of chemotherapy pain may lead to drug dependence and side effects, such as drug resistance, dependence, and the risk of overdose, and the treatment effect is limited. Chemotherapy-induced chronic neuropathic pain (CIPN) is often difficult to treat, and there is currently no curative treatment for chronic chemotherapy-induced neuropathy. Current research on biomaterials in CIPN treatment includes: nerve conduits for repairing damaged nerves and promoting nerve regeneration; nanomaterials for drug delivery to improve efficacy and reduce side effects; biological scaffolds to provide structural support; and neurotrophic factors such as NGF and BDNF to promote nerve repair and regeneration. Biomaterials have shown great potential in drug delivery and nerve repair, and are expected to provide more effective treatment options for CIPN in the future. There is an urgent need for a functional material such as an ultrasound-responsive hydrogel to address the problem of chemotherapy-induced pain. Summary of the Invention

[0004] The purpose of this invention is to provide a fully hydrogel based on an ultrasonically responsive hydrogel with reactive oxygen species scavenging capability, as well as its preparation method and applications.

[0005] The technical solution of this invention is: A fully hydrogel based on an ultrasonically 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 and stirred continuously in Tris-HCl buffer, followed by ultracentrifugation to obtain C60@PDA / AP39 nanoparticles. S2: Dissolve GelMA in a diluted solution of C60@PDA / AP39 nanoparticles to obtain a fully hydrogel.

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

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

[0008] In step S1, 25 mg of C60 was dissolved in 400 ml of pH 8.5 Tris-HCl buffer, and sonicated for 30 min to ensure uniform dispersion in the solution. 100 μl of 10 mM AP39 was added, along with 25 mg of dopamine, and the mixture was stirred continuously at 60 °C for 24 h.

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

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

[0011] Application of an ultrasound-responsive hydrogel-based all-hydrogel in the preparation of materials for treating pain caused by chemotherapy-induced peripheral neuropathy.

[0012] This invention relates to a fully hydrogel with reactive oxygen species (ROS) scavenging capabilities. The fully hydrogel contains C60 and AP39. C60 has been studied for its potential as an antioxidant, drug delivery, and photodynamic therapy. C60 and its derivatives possess unique ROS scavenging abilities. Dopamine, due to its adhesive and self-polymerizing properties, is used for surface modification and coatings. PDA is a dopamine-mimicking bioactive molecule with excellent antioxidant and free radical scavenging capabilities. PDA-modified nanoparticles exhibit enhanced hydrophilicity, good biocompatibility, appropriate biodegradability, strong photothermal conversion capacity, and ROS scavenging ability, and also possess ultrasonic responsiveness. AP39, a donor for synthesizing H2S, can deliver H2S to mitochondria. H2S is a gaseous signaling molecule with antioxidant, anti-inflammatory, and cytoprotective effects. AP39 regulates cellular energy metabolism and reduces oxidative stress by targeting mitochondria. Photocurable methacryloyl gelatin (GelMA) is a safe and effective biocarrier that provides a favorable transplantation environment for cells and biological factors. It possesses excellent biocompatibility, biodegradability, and mechanical properties, and is considered one of the most promising biomaterials for clinical applications in the biomedical field. In this study, AP39-modified C60@PDA nanoparticles were dissolved in GelMA to construct a fully hydrogel based on an ultrasound-responsive hydrogel. This hydrogel effectively reduced inflammation and relieved pain by scavenging excess ROS in vivo. The ultrasound-responsive fully hydrogel of this invention exhibits excellent antioxidant properties, reduces inflammation and relieves pain, and simultaneously promotes more stable drug release, making it an ideal self-degradable medical material.

[0013] This invention provides a fully hydrogel based on an ultrasound-responsive hydrogel, in which C60@PDA and AP39 can scavenge ROS, alleviate or reduce inflammatory responses, and provide a more effective and safer treatment option. This fully hydrogel based on an ultrasound-responsive hydrogel is safe and non-toxic, offering a more effective and safer treatment option. Furthermore, this fully hydrogel based on an ultrasound-responsive hydrogel can alleviate chemotherapy-induced pain, providing a new solution for chemotherapy-related pain. Attached Figure Description

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

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

[0016] Figure 3 This invention describes the effect of an ultrasound-responsive hydrogel on oxaliplatin-induced chemotherapy pain in rats, as described in one embodiment of the invention. L represents HE staining images of different organs in rats after intrathecal injection of the hydrogel; M represents changes in body weight in the chemotherapy-induced pain rats; N represents changes in the threshold of thermal pain in the chemotherapy-induced pain rats; and O represents changes in the threshold of mechanical pain in the chemotherapy-induced pain rats. Detailed Implementation

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

[0018] In step S1, 25 mg of C60 was dissolved in 400 ml of pH 8.5 Tris-HCl buffer, 100 μl of 10 mM AP39 was added, and the mixture was sonicated for 30 min to ensure uniform dispersion in the solution. 25 mg of dopamine was added, and the mixture was stirred continuously at 60 °C for 24 h. Then, the mixture was centrifuged at 16000 g for 2 h and washed three times with Milli-Q water to obtain C60@PDA / AP39 nanoparticles. Finally, the nanoparticles were resuspended in Milli-Q water for further use.

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

[0020] The above content will be explained in conjunction with specific verification experiments: 1: Synthesis and Characterization of Nanoparticle Materials 1.1 Experimental Methods: 1.1.1 Malvern particle size analyzer for detecting the potential and particle size of nanoparticles Take 1 μl of sample and add it to 99 μl of Milli-Q water. Mix well and then add it to a cuvette. Use Malvern particle size analyzer to determine the potential and particle size.

[0021] 1.1.2 Infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy were used to detect the characteristics of nanoparticles and gelma. Approximately 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 is larger, as shown in Figure B.

[0024] (3) Intramolecular vibrations of the C60 framework 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 show that C60@PDAC60 nanoparticles exhibit a uniform spherical structure and a rough surface. PDA was successfully modified onto fullerene (C60) through self-polymerization.

[0027] 2: Ultrasound-responsive hydrogels have 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 a reactive oxygen species detection kit Cell suspensions were prepared and cells were counted. Cell suspensions were seeded into six-well plates (1 mL / well) at a density of approximately 1 × 10⁴. Positive control wells were stimulated with hydrogen peroxide. Intervention wells were treated with different compositions of culture medium and hydrogen peroxide. The plates were incubated for 6 h. DCFH-DA was diluted 1:1000 with serum-free culture medium. The cell culture medium was removed, and 1 mL of diluted DCFH-DA was added to each well to fully cover the cells. Cells were incubated at 37 °C for 20 min. Cells were washed three times with serum-free cell culture medium to remove any uninfiltrated DCFH-DA. Images were taken under a fluorescence microscope.

[0028] 2.1.1 ELISA detection of intracellular inflammatory factor levels stimulated by LPS under different culture medium treatments Cells were seeded at a density of 24 × 10⁴ cells per well in 6-well plates. After incubation overnight until cell attachment, the cells were co-cultured with different culture media for 6 hours. The supernatant was collected, and the levels of inflammatory factors (IL-1β, IL-6, TNF-α) in the cell supernatant were detected using an ELISA kit. The required strips were removed from the aluminum foil bag after equilibration at room temperature for 20 minutes, and the remaining strips were sealed in a resealable bag and returned to 4°C. Standard wells and sample wells were prepared. 50 μL of different concentrations of standard were added to each standard well. 10 μL of the sample to be tested was added to each sample well, followed by 40 μL of sample diluent. No reagents were added to the blank wells. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each standard and sample well. The reaction wells were sealed with sealing film and incubated in a water bath or incubator at 37°C for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 1 minute, shake off the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and within 15 minutes, measure the OD value of each well at a wavelength of 450 nm.

[0029] 2.1 Experimental Results: (1) Ultrasonic-responsive hydrogels have good reactive oxygen species scavenging ability, as shown in Figure GH.

[0030] (2) Ultrasound-responsive hydrogels have good anti-inflammatory effects, reducing the level of inflammatory factors in the supernatant of the intervened cells, such as... Figure 2 As shown in IK.

[0031] 3: Effects of ultrasound-responsive hydrogels on rats experiencing chemotherapy pain 3.1 Experimental Methods: 3.1.1 Establishment of a rat model of chemotherapy-induced pain and the effect of ultrasound-responsive hydrogel on rat body weight Six-week-old rats were divided into a normal group, a pathological group, and an experimental group. Rats in the pathological and experimental groups were injected intraperitoneally with 4 mg / kg oxaliplatin for 7 consecutive days, while the normal group was injected with physiological saline. Their weight was measured and recorded daily. On day 0, the pathological and experimental groups underwent surgery. After anesthesia, the hair on the back of the rats was removed, and the skin and muscle layers were cut layer by layer from the outside to the inside. 40 μL of a 10 mg / ml all-hydrogel (i.e., the all-hydrogel based on ultrasound-responsive hydrogel of this invention) was injected into the rats through an insulin needle sheath. The wound was sutured. Subsequently, the rats' DRGs were subjected to ultrasound every 2 days at a frequency of 1 MHz, a sound pressure level of 100 kPa, and a duration of 10 minutes each time.

[0032] 3.1.2 Effects of ultrasound-responsive hydrogels on mechanical pain in a rat model of chemotherapy-induced pain The mechanical pain threshold of rats was assessed using the Von Frey test. Rats of the confirmed group and number were placed in a box on a metal mesh and allowed to rest for 15-30 minutes until they remained calm in the unfamiliar environment (no longer walking around the cage or licking themselves). Von Frey hair was applied to the hairless area of ​​the left palmar-plantar region of the rat, arranged in an "S" shape, for 6-8 seconds, with at least a 7-second interval between adjacent stimuli. A positive reaction was indicated by the rat raising its paw or licking the stimulated side during the stimulation period. If the rat moved during stimulation or raised its paw immediately upon stimulation, it was considered a suspicious positive, and the measurement should only be performed after the rat calmed down. A positive result was recorded as X, and a negative result as O. For measurement, the median value of 10.0g was used as the starting point, and measurements were taken in ascending order. If the first crossover occurred, four more measurements were taken. The log unit value of the Von Frey hair from the last measurement was substituted into the formula for calculation.

[0033] 3.1.3 Effects of ultrasound-responsive hydrogels on thermal pain in a rat model of chemotherapy pain The heat pain threshold of rats was detected using a plantar heat stimulation device. The rat was placed on a hot plate and waited until it was quiet and stopped licking its fur. The test head was slid under the rat's foot and the start button was pressed. The rat showed pain response such as lifting its foot or licking its hind foot. The stop button was pressed immediately afterward and the time was recorded. The upper limit of the recording time was 20 seconds. Each rat was repeated 3 times.

[0034] 3.1.4 HE staining of various organs in rats treated with all-hydrogel for chemotherapy pain Prepare rat tissue sections for staining. Dewax with xylene for 10 minutes each time, twice in total. Then, use 100%, 95%, 85%, and 75% ethanol for 5 minutes each, followed by washing twice with distilled water for 2 minutes each time. Stain with hematoxylin for 3 minutes, then wash away any excess stain with distilled water.

[0035] Treat with 1% hydrochloric acid-alcohol differentiation solution for 30 seconds, then wash with tap water for 3 minutes, repeating twice. Stain with eosin solution for 1-3 minutes, then rinse briefly with distilled water for 30 seconds. Then, sequentially use 80%, 95%, and anhydrous ethanol for 1 minute each, followed by clearing with xylene for 3 minutes each time, repeating twice.

[0036] Use neutral resin 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 pathological group rats showed a decreasing trend from 0 to 7 days and began to recover after the injection of oxaliplatin was stopped on the 8th day. The thermal pain threshold of the experimental group rats gradually recovered from the 4th day. By the 8th day, compared with the case group, the drug-treated 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 pathological group rats showed a decreasing trend from 0 to 7 days. It began to recover after the injection of oxaliplatin was stopped on the 8th day. The mechanical pain threshold of the drug-treated group and the case group rats 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-treated group was closer to the level of the normal group, as shown in Figure O.

[0040] The above experiments demonstrate that the ultrasound-responsive hydrogel provided in this application can effectively remove ROS, reduce inflammatory responses, and alleviate chemotherapy pain. In this study, we constructed a simple ultrasound-responsive hydrogel based on the characteristics of biomaterials. We implanted the ultrasound-responsive hydrogel into the DRG of rats, promoting nerve repair through ultrasound stimulation and consuming ROS accumulation in the DRG caused by chemotherapy, making it an ideal medical material.

Claims

1. An intrathecal injection all-hydrogel based on an 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 and stirred continuously in Tris-HCl buffer, followed by ultracentrifugation to obtain C60@PDA / AP39 nanoparticles. S2: Dissolve GelMA in a diluted solution of C60@PDA / AP39 nanoparticles to obtain a fully hydrogel; In step S1, C60 is first dissolved in Tris-HCl buffer and then sonicated for 30 minutes to disperse it evenly in the solution, and then AP39 is added; then dopamine powder is added to the above solution.

2. A method for preparing an intrathecal injection all-hydrogel based on an ultrasound-responsive hydrogel, characterized in that: Includes the following steps: S1: Preparation of C60@PDA / AP39 nanoparticles: Dopamine, C60, and AP39 were heated and stirred continuously in Tris-HCl buffer, followed by ultracentrifugation to obtain C60@PDA / AP39 nanoparticles. S2: Dissolve GelMA in a diluted solution of C60@PDA / AP39 nanoparticles to obtain a fully hydrogel; In step S1, C60 is first dissolved in Tris-HCl buffer and then sonicated for 30 minutes to disperse it evenly in the solution, and then AP39 is added; then dopamine powder is added to the above solution.

3. The method for preparing an intrathecal injection all-hydrogel based on an ultrasound-responsive hydrogel according to claim 2, characterized in that: In step S1, 25 mg of C60 was dissolved in 400 ml of pH 8.5 Tris-HCl buffer, and sonicated for 30 min to ensure uniform dispersion 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.

4. A method for preparing an intrathecal injection all-hydrogel based on an ultrasound-responsive hydrogel according to claim 2 or 3, characterized in that: After continuous heating and stirring, the solution in S1 was centrifuged at 16000g for 2 hours and washed three times with Milli-Q water to obtain C60@PDA / AP39 nanoparticles. Finally, the nanoparticles were resuspended in Milli-Q water for further use.

5. A method for preparing an intrathecal injection all-hydrogel based on an ultrasound-responsive hydrogel according to claim 2 or 3, characterized in that: The concentration of GelMA dissolved in the nanoparticle solution in S2 is 30%.

6. The application of an intrathecal injection all-hydrogel based on ultrasound-responsive hydrogel in the preparation of materials for treating pain caused by chemotherapy-induced peripheral neuropathy, characterized in that: The ultrasound-responsive hydrogel is an ultrasound-responsive intrathecal injection formulation that promotes nerve repair and can also consume ROS accumulation in the DRG caused by chemotherapy.

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