Application of wireless ultrasonic transduction electrical stimulation in treatment of brain injury

Abstract

The invention provides a polydopamine modified anisotropic cellulose piezoelectric hydrogel and a preparation method thereof. The polydopamine modified anisotropic cellulose piezoelectric hydrogel comprises an anisotropic cellulose piezoelectric hydrogel skeleton and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton. The invention also provides an application of wireless ultrasonic transduction electrical stimulation in treatment of brain injury, and particularly provides a wireless stimulation system containing the anisotropic cellulose piezoelectric hydrogel modified by polydopamine and a set product for treatment of brain injury. The invention also provides a brain injury treatment method using the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a polydopamine-modified anisotropic cellulose piezoelectric hydrogel and its application in treating brain injuries. Background Art

[0002] Traumatic brain injury (TBI) is a severe form of head injury. Neural stem cells (NSCs), multipotent cells in the central nervous system (CNS), can secrete neurotrophic factors and differentiate into central and peripheral nervous system cells, playing a crucial role in the treatment of TBI. However, their slow spontaneous differentiation hinders their application in TBI treatment. Therefore, accelerating NSC differentiation into mature neurons and promoting neurological recovery are key to effective TBI treatment. Neural tissue scaffolds that incorporate electrical signals have attracted extensive attention in improving neural repair. For example, in vivo electrical stimulation (ES) delivered via an external power source and percutaneous wires has been shown to significantly promote NSC neural differentiation and neurogenesis. However, this approach still requires wires connected to implanted electrodes to stimulate cells. Furthermore, additional surgery is required to remove the electrodes or wires after stimulation. These procedures can lead to secondary injury and infection. Therefore, scaffolds that enable electrode- and battery-free electrical stimulation hold great promise for clinical applications in tissue regeneration and the treatment of nerve injury.

[0003] With the increasing potential applications of neural devices, new technologies that make neural interventions safer, more durable, and less invasive have sparked interest in small, wireless devices. Remote powering from piezoelectric nanomaterials not only allows for smaller device sizes but also eliminates the need for bulky devices or surgical battery replacement, as they mechanically deform in response to various physical stimuli, generating electrical signals. Traditional inorganic piezoelectric ceramic materials (such as zinc oxide, barium titanate, and lead zirconium titanate) have low biocompatibility and poor structural flexibility, making them a poorly accepted solution for clinical trials. While piezoelectric composite nanogenerators offer excellent flexibility, they suffer from issues such as low density and poorly dispersed piezoelectric phases within the matrix, resulting in a limited piezoelectric filler content and discontinuous, controllable electrical output. These factors result in low overall system output performance, hindering practical application. Polymer piezoelectric materials, such as polyvinylidene fluoride (PVDF), offer superior flexibility and excellent biocompatibility, but are difficult to degrade. Polylactic acid, a reported degradable piezoelectric polymer, produces acidic small molecules upon degradation, leading to an acidic microenvironment and cytotoxicity. To address these challenges, there is an urgent need to find flexible piezoelectric materials with good compatibility, biodegradability, and more controllable and consistent energy conversion output. The natural polymer material nanocellulose is considered a sustainable nanomaterial due to its large production volume, low price, biodegradability, biocompatibility, and piezoelectric properties. However, the low intrinsic piezoelectric properties of biodegradable cellulose piezoelectric polymers significantly limit their applications. A well-known approach to address this limitation is to maximize the proportion of the ferroelectric β phase with a well-defined polarization direction.

[0004] Although piezoelectric nanomaterials have been reported to promote the differentiation of neural stem cells through electrical stimulation, the precise control of electrical stimulation parameters such as current density, pulse width, stimulation frequency and operation timeliness remains a challenge for the application of self-powered piezoelectric nanogenerators in battery-free neural stimulators.

[0005] Considering the specificity of in vivo nerve injury repair applications, the development of high-performance piezoelectric biomaterials modified with enabling materials is an urgent need for regenerative medicine. Summary of the Invention

[0006] According to one aspect of the present application, a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, comprising: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.

[0007] In some embodiments, the modification amount of polydopamine is 0.4%-2% based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel. Optionally, the stretchability of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%.

[0008] According to another aspect of the present application, a method for preparing a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, comprising:

[0009] In an alkaline solution, dopamine is in situ polymerized on the anisotropic cellulose piezoelectric hydrogel to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.

[0010] In some embodiments, the preparation method comprises immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH value of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then washing with water to terminate the reaction to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel. Optionally, the alkaline solution is Tris-HCl buffer.

[0011] In some embodiments, the anisotropic cellulose piezoelectric hydrogel is prepared by the following steps:

[0012] 1) adding a crosslinking agent to the piezoelectric cellulose solution to chemically crosslink and gel to obtain a cellulose piezoelectric hydrogel; and

[0013] 2) Pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to align the cellulose in the cellulose piezoelectric hydrogel, and then immersing it in an aqueous sulfuric acid solution to permanently fix the oriented structure through physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.

[0014] In some embodiments, step 1) comprises dissolving the piezoelectric cellulose in a lithium hydroxide and urea solution to obtain the piezoelectric cellulose solution. Optionally, in the lithium hydroxide and urea solution, the concentration of lithium hydroxide is 3.6-5.4 wt %, and the concentration of urea is 12-18%. Preferably, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1 wt %-5 wt %. Optionally, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled at a temperature of -20°C to 0°C.

[0015] Optionally, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose and cotton.

[0016] Optionally, the cross-linking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin and polyethylene glycol diglycidyl ether,

[0017] Optionally, in step 1), the molar ratio of the cross-linking agent to the deoxyglucose unit in the piezoelectric cellulose solution is 1:1-3:1,

[0018] Optionally, step 1) includes dropwise adding the crosslinking agent to the cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring into a template, and gelling at 0-10° C. for 10 h to 100 h to obtain the cellulose piezoelectric hydrogel;

[0019] Optionally, the stretching rate of the pre-stretching in step 2) is 80%-215%;

[0020] Optionally, step 2) comprises soaking the pre-stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution for physical crosslinking and terminating chemical crosslinking, and then washing with water to obtain the anisotropic cellulose piezoelectric hydrogel. Preferably, the concentration of the dilute sulfuric acid solution is 1 wt%-5 wt%.

[0021] According to another aspect of the present application, there is provided a polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to any of the aforementioned embodiments or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments for use as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.

[0022] According to another aspect of the present application, a wireless electric stimulation system is provided, comprising: a polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to any of the aforementioned embodiments, or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the action of ultrasound of the ultrasonic device.

[0023] Optionally, the ultrasonic frequency, ultrasonic intensity and ultrasonic time of the ultrasonic device can be programmably controlled.

[0024] Optionally, the ultrasound intensity of the ultrasound action is 0-5W / cm 2 .

[0025] According to another aspect of the present application, there is provided the use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of any of the aforementioned embodiments or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments in the preparation of a repair material for treating brain injury, especially traumatic brain injury.

[0026] According to another aspect of the present application, a product package for treating brain injury is provided, comprising: neural stem cells; and the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of any of the aforementioned embodiments or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments.

[0027] Beneficial effects

[0028] The technical solution of this application can achieve at least one of the following effects:

[0029] (1) The polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in this application has the advantages of high piezoelectric output, antioxidant properties, flexibility, good biocompatibility, and degradability. The polydopamine-modified anisotropic cellulose hydrogel gives the cellulose hydrogel antioxidant ability and increases the piezoelectric output performance.

[0030] (2) The polydopamine-modified anisotropic cellulose piezoelectric hydrogel structure provided in this application can simulate the directional structure of regenerated natural tissues (such as nerves) and support and guide axonal projection.

[0031] (3) Polydopamine-modified anisotropic cellulose piezoelectric hydrogels give the cellulose hydrogels the ability to actively regulate the microenvironment and controllable continuous electrical output.

[0032] (4) By using programmed ultrasound as a remote mechanical stimulus, the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of the present application can provide on-demand wireless electrical stimulation (ES) with adjustable timing, duration, and intensity.

[0033] (5) Programmable ultrasound combined with polydopamine-modified anisotropic cellulose piezoelectric hydrogel forms a radio stimulation system or radio neural regulation system, which provides on-demand radio stimulation, accelerates the process of neural stem cell differentiation and promotes the development of neurons.

[0034] (6) Programmable ultrasound combined with polydopamine-modified anisotropic cellulose piezoelectric hydrogel to form a radio stimulation system or radio neural regulation system can improve the injured immune microenvironment, promote the in situ differentiation of neural stem cells at the injured site, and promote nerve regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. The drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 A schematic diagram of the process for preparing polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH) according to an embodiment of the present application is shown.

[0037] Figure 2 The stretchability of the chemically cross-linked cellulose hydrogel prepared in the examples of the present application is shown.

[0038] Figure 3A physical picture of PDA / ACH-4 prepared in an example of the present application is shown.

[0039] Figure 4 Scanning electron microscope images of chemically cross-linked cellulose hydrogels ACH-1 (before stretching) and ACH-4 (after stretching, stretching rate 120%) prepared in the examples of the present application are shown, where the left image is ACH-1 and the right image is ACH-4.

[0040] Figure 5 The scanning electron microscopy observation and elemental analysis results of the polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH-4) prepared in the examples of the present application are shown, wherein the left figure is a scanning electron microscope image of PDA / ACH-4, and the right figure is an elemental analysis diagram of PDA / ACH-4.

[0041] Figure 6 The two-dimensional small-angle X-ray scattering (SAXS) (inset) and the azimuthal integrated intensity distribution curve of the SAXS pattern of different cellulose hydrogels prepared according to the examples of the present application are shown, where 0° represents the vertical direction.

[0042] Figure 7 The piezoelectric characterization results of different cellulose hydrogels prepared in the examples of the present application are shown, where a: piezoelectric output of ACH-1, ACH-4, and PDA / ACH-4 under ultrasound, b: schematic diagram of ultrasonic piezoelectricity, c: butterfly amplitude ring of PDA / ACH-4, and d: phase potential hysteresis.

[0043] Figure 8 The antioxidant characterization results of different cellulose hydrogels prepared in the examples of this application are shown.

[0044] Figure 9 A schematic diagram of treating traumatic brain injury (TBI) in rats using the wireless neuromodulation system according to an embodiment of the present application is shown.

[0045] Figure 10 The free radical scavenging characterization results of the radio-neuromodulatory system of an embodiment of the present application are shown, which were observed 3 days after TBI using superoxide anion fluorescent probe (DHE) staining to visualize the damaged area. From the left, Figure 1: TBI group; Figure 2: TBI+ACH-4+ultrasound (TAU) group; Figure 3: TBI+PDA / ACH-4+ultrasound (TPAU) group, where red represents DHE staining, blue represents DAPI staining, *T represents the damaged area, and the white dotted line separates the damaged area and normal tissue.

[0046] Figure 11The results of evaluating the radio-neuromodulatory system of an embodiment of the present application in promoting the transformation of microglia to anti-inflammatory M2 by immunofluorescence staining of the anti-inflammatory marker CD206 and the pro-inflammatory marker CD16 / 32 are shown. From the left, the first column: TBI group; the second column: TAU group; the third column: TPAU group, wherein green represents anti-inflammatory marker CD206 or pro-inflammatory marker CD16 / 32 staining, blue represents DAPI staining, *T represents the damaged area, and the white dotted line separates the damaged area and normal tissue.

[0047] Figure 12 The figure shows the evaluation of the differentiation of exogenous neural stem cells at the injury site by immunofluorescence staining of astrocyte markers (GFAP) and mature neuron markers (MAP2). From the left, Figure 1: TBI group; Figure 2: TBI+NSC (TC) group; Figure 3: PDA / ACH-4+NSC+ultrasound (TPACU) group; Figure 4: Local enlargement of the TPACU group, where red indicates CM-Dil staining, green indicates MAP2 staining, yellow indicates GFAP staining, and blue indicates DAPI staining.

[0048] Figure 13 The results of the radio-neuromodulatory system of an embodiment of the present application promoting neurological function recovery after TBI observed by fluorescence imaging are shown, with Figure 1 from the left: TBI group; Figure 2: TC group; Figure 3: TPACU group, wherein green represents DCX staining, red represents CM-Dil staining, blue represents DAPI staining, *T represents the injured area, and the white dotted line separates the injured area and normal tissue.

[0049] Figure 14 The results of detecting the axon area of the injured area using axon markers (neurofilament, NF) 7 days after TBI injury are shown. The first to third figures from the left are fluorescence imaging of the TBI group, TC group, and TPACU group, respectively (wherein green represents NF staining, blue represents DAPI staining, *T represents the injured area, and the white dotted line separates the injured area and normal tissue). Figure 4 shows the percentage of the area occupied by NF in each group.

[0050] Figure 15 The figure shows the recovery of learning and memory ability of rats after brain trauma evaluated by the Morris water maze task, where a: swimming paths of the sham group, TBI group, TC group and TPACU group; b: time to reach the platform, number of times crossing the platform, and regional time statistics of each group. DETAILED DESCRIPTION

[0051] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions of this application will be described in detail below. Obviously, the embodiments described are only some of the embodiments of this application, rather than all of them. Based on the embodiments in this application, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of this application.

[0052] Following primary external injury, secondary blood-brain barrier damage, such as cerebral edema and neuroinflammation, can cause long-term brain pathology and lead to severe neurodegenerative diseases. This is caused by the excessive production of reactive oxygen species (ROS), such as superoxide radicals (O2), hydroxyl radicals (OH), and hydrogen peroxide (H2O2). However, endogenous antioxidant enzymes are insufficient to remove excess ROS. The introduction of exogenous ROS scavengers is a key strategy to mitigate ROS-mediated secondary damage and improve the efficacy of stem cell therapy for TBI. The acute oxidative stress that occurs rapidly after nerve injury leads to excessive production of reactive oxygen species (ROS). ROS recruits microglia to polarize and secrete a large number of inflammatory factors to form an immune microenvironment, which damages the cellular microenvironment, exacerbates nerve damage, and greatly affects the effect of nerve regeneration and repair. Immune microenvironment regulation by scavenging oxygen free radicals and controlling macrophage transformation has become an essential prerequisite for the survival of NSCs and nerve repair. The inventors of the present application have studied PDA-modified anisotropic acoustic-electric transducer devices as an excellent strategy for treating traumatic brain injury (TBI) and regulating the brain injury microenvironment to promote neural regeneration.

[0053] According to one aspect of the present application, a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, which comprises: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.

[0054] The anisotropic cellulose piezoelectric hydrogel skeleton can also be called a multi-dimensionally oriented cellulose piezoelectric hydrogel skeleton. The anisotropy is generated from the directional distribution of cellulose chains in the hydrogel network.

[0055] Herein, "piezoelectric hydrogel" refers to a hydrogel having a piezoelectric effect, including but not limited to anisotropic cellulose hydrogel and bacterial cellulose hydrogel.

[0056] In some embodiments, the modification amount of polydopamine is 0.4%-2%, for example, 0.4%, 0.5%, 1%, 1.5%, or 2%, based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.

[0057] In some embodiments, the stretchability of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%, for example, 80%, 100%, 120%, 180%, 200%, 215%, or any value within a range thereof. As used herein, "stretchability" refers to the rate of change in the length of the stretched hydrogel (hydrogel skeleton) relative to the length of the hydrogel (hydrogel skeleton) before stretching in the stretching direction. This can improve the piezoelectric properties and oriented structure of the cellulose piezoelectric hydrogel.

[0058] According to another aspect of the present application, a method for preparing anisotropic cellulose piezoelectric hydrogel modified with polydopamine is provided, comprising: in an alkaline solution, in situ polymerizing dopamine on the anisotropic cellulose piezoelectric hydrogel to obtain the anisotropic cellulose piezoelectric hydrogel modified with polydopamine.

[0059] In some embodiments, a method for preparing an anisotropic cellulose piezoelectric hydrogel modified with polydopamine is provided, comprising immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then terminating the reaction by washing with water to obtain the anisotropic cellulose piezoelectric hydrogel modified with polydopamine. In one embodiment, the alkaline solution is a Tris-HCl buffer. This can improve the piezoelectric properties and biocompatibility of the cellulose piezoelectric hydrogel and impart antioxidant properties to the cellulose piezoelectric hydrogel.

[0060] In some embodiments, a method for preparing anisotropic cellulose piezoelectric hydrogel is provided, comprising:

[0061] 1) adding a crosslinking agent to the piezoelectric cellulose solution to chemically crosslink and gel to obtain a cellulose piezoelectric hydrogel; and

[0062] 2) pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to align the cellulose in the cellulose piezoelectric hydrogel, and then immersing it in an aqueous sulfuric acid solution to permanently fix the oriented structure through physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.

[0063] In some embodiments, physical crosslinking is achieved through hydrogen bonding.

[0064] In some embodiments, step 1) includes dissolving a piezoelectric cellulose source in a lithium hydroxide and urea solution to obtain a piezoelectric cellulose solution. According to one embodiment, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1 wt%-5 wt%. In some embodiments, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 2 wt%. In some embodiments, the concentration of lithium hydroxide in the lithium hydroxide and urea solution is 3.6-5.4 wt%, and the concentration of urea is 12-18 wt%, for example, the concentration of lithium hydroxide is 4.5 wt%, and the concentration of urea is 15 wt%. This can produce a cellulose hydrogel with a low elastic modulus and a longer tensile length, providing the prerequisite for pre-stretching.

[0065] In some embodiments, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled to a temperature between -20°C and 0°C, for example, at -18°C, thereby obtaining a cellulose solution with higher solubility.

[0066] In some embodiments, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose, and cotton (e.g., cotton lint). In some embodiments, the piezoelectric cellulose is derived from filter paper, such as Advantec Toyo quantitative filter paper.

[0067] In some embodiments, the crosslinking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin, and polyethylene glycol diglycidyl ether. In some embodiments, the crosslinking agent is epichlorohydrin. Thus, a cellulose piezoelectric hydrogel with chemical crosslinking of hydroxyl groups is obtained.

[0068] In some embodiments, in step 1), the molar ratio of the crosslinking agent to the deoxyglucose units in the piezoelectric cellulose solution is 1:1-3:1, for example, 1.5:1. This can produce a cellulose hydrogel with a low elastic modulus and a longer tensile length, providing a prerequisite for pre-stretching.

[0069] In some embodiments, step 1) includes dropwise adding a crosslinking agent to the piezoelectric cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring the product into a template, and gelling at 0-10° C. for 10-100 hours to obtain the cellulose piezoelectric hydrogel. In some embodiments, gelling is performed at 4° C. for 30 hours.

[0070] In some embodiments, the pre-stretching rate in step 2) is 80%-215%, thereby improving the piezoelectric performance and oriented structure of the cellulose piezoelectric hydrogel.

[0071] In some embodiments, step 2) includes immersing the stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution to physically crosslink and terminate chemical crosslinking, followed by washing with water to obtain an anisotropic cellulose piezoelectric hydrogel. According to one embodiment, the concentration of the dilute sulfuric acid solution is 1 wt% to 5 wt%, for example, 3 wt%. This results in an anisotropic cellulose piezoelectric hydrogel with a lower elastic modulus.

[0072] According to another aspect of the present application, there is provided the use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in any of the above embodiments or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the above embodiments as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.

[0073] According to another aspect of the present application, a radio stimulation system or a radio neural regulation system is provided, comprising: a polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in any of the above embodiments or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared by the preparation method according to any of the above embodiments; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the ultrasonic action of the ultrasonic device.

[0074] In some embodiments, the ultrasonic frequency, ultrasonic intensity, and ultrasonic time of the ultrasonic device in the wireless stimulation system or wireless neural regulation system provided by the present application can be programmably controlled. In some embodiments, the ultrasonic intensity of the ultrasonic action in the wireless stimulation system or wireless neural regulation system provided by the present application is 0-5W / cm 2 .

[0075] According to another aspect of the present application, there is provided use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of the present application in preparing a repair material for treating brain injury, especially traumatic brain injury (TBI).

[0076] According to another aspect of the present application, a product package for treating brain injury is provided, comprising: neural stem cells; and the polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided by the present application.

[0077] According to another aspect of the present application, a method for treating brain injury is provided, comprising: covering the site of brain injury with the polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided by the present application; and applying ultrasound to cause the polydopamine-modified anisotropic cellulose piezoelectric hydrogel to generate radio signals to stimulate nerve regeneration and repair at the site of brain injury.

[0078] In some embodiments, in the methods of treating brain injury of the present application, the brain injury is traumatic brain injury (TBI).

[0079] In some embodiments, the brain injury treatment method of the present application further comprises injecting exogenous neural stem cells into the brain injury site to promote the differentiation of the exogenous neural stem cells into neurons through electrical signal stimulation generated by ultrasound.

[0080] In some embodiments, in the method for treating brain injury of the present invention, the ultrasound intensity of the ultrasound effect is 0-5 W / cm 2 .

[0081] In some embodiments, in the method for treating brain injury of the present application, the ultrasound frequency, ultrasound intensity, and ultrasound time of the ultrasound action are adjustable or programmable.

[0082] This application prepares polydopamine-modified anisotropic cellulose piezoelectric hydrogels by chemical crosslinking, pre-stretching, physical crosslinking and in situ polymerization. By increasing the structural order and polydopamine modification, the piezoelectric properties of the hydrogel are improved. It is used as an acoustic energy generator to convert ultrasonic energy into electrical energy, especially radio signals. The radio stimulation system or radio neuromodulation system based on the polydopamine-modified anisotropic cellulose piezoelectric hydrogel and ultrasound can provide an antioxidant immune microenvironment and on-demand radio stimulation for accelerating neural stem cell-based neural regeneration and repair. It has been confirmed that anisotropic acoustic-electric transducer (nano) devices have been successfully applied to the in vivo rat TBI model. In the early stage of TBI, by scavenging free radicals, microglia are promoted to transform into anti-inflammatory M2 cells, inflammation is downregulated, and the post-injury immune microenvironment is improved. In addition, by using programmed ultrasound as a remote mechanical stimulation, anisotropic cellulose piezoelectric hydrogels can provide on-demand electrical stimulation with adjustable time, duration and intensity, promote the differentiation of endogenous neural stem cells at the site of injury, and thus improve the recovery of learning and memory ability after TBI.

[0083] Example

[0084] The test materials used in the examples of this application are all conventional test materials in the field and can be purchased through commercial channels.

[0085] Example 1

[0086] Preparation of polydopamine (PDA) modified anisotropic cellulose hydrogel (PDA / ACH), such as Figure 1 As shown:

[0087] (1) Preparation of chemically cross-linked cellulose hydrogel: Advantec Toyo quantitative filter paper was completely dried at 60°C and dissolved in a 4.5wt% lithium hydroxide / 15wt% urea solution pre-cooled at -18°C to obtain a transparent cellulose solution with a concentration of 2wt%. Epichlorohydrin was added dropwise to the dissolved cellulose solution, and the crude cellulose gel containing an epichlorohydrin (ECH) / anhydroglucose unit (AGI) molar ratio of 1.5 was centrifuged to remove bubbles, poured into the template, and reacted in a refrigerator at 4°C for 30 hours to obtain a chemically cross-linked cellulose hydrogel.

[0088] (2) Preparation of anisotropic cellulose hydrogel (ACH): Applying external force to the chemically cross-linked cellulose hydrogel obtained in step (1) to stretch the cellulose molecular chains inside the gel to cause the cellulose hydrogel to be oriented, such as Figure 2 As shown in Figure 2, the stretchability of the chemically cross-linked hydrogel reached 215%. Using a hydrogel stretched to a specific length ratio of 120%, and soaked in a 3% H2SO4 solution, the oriented structure was permanently fixed by physical cross-linking (hydrogen bonding) between adjacent cellulose chains, and thoroughly washed with water, anisotropic cellulose hydrogel (ACH) was obtained.

[0089] (3) Preparation of polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH): The ACH obtained in step (2) was immersed in a 10 μM Tris-HCl buffer solution with a pH value of 8.8, and then dopamine at a concentration of 2 mg / mL was added for 2 hours. The reaction was terminated by repeated washing with water. Figure 3 As shown, a polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH) was obtained, wherein the modification amount of polydopamine was 0.96% based on the mass percentage of the N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.

[0090] Example 2 Investigation of the piezoelectric and antioxidant properties of anisotropic cellulose hydrogels

[0091] To facilitate subsequent stem cell differentiation into neurons, an anisotropic cellulose piezoelectric hydrogel with a smaller modulus was selected. The chemically cross-linked cellulose hydrogel was treated with a pre-stretch ratio of 120%. Other steps were followed as in Example 1 to prepare anisotropic cellulose hydrogel (ACH) and polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH), yielding samples ACH-4 and PDA / ACH-4, respectively. The resulting anisotropic cellulose hydrogels were characterized by scanning electron microscopy.

[0092] like Figure 4As shown, the left figure ACH-1 represents the chemically cross-linked cellulose hydrogel before stretching, which shows no directionality, while the right figure ACH-4 represents the chemically cross-linked cellulose hydrogel after stretching (stretching rate 120%), which has obvious directionality. Figure 5 The scanning electron microscopy observation and elemental analysis results of PDA / ACH-4 show that PDA / ACH-4 also has order, indicating that PDA modification does not affect the order of ACH. The nitrogen content in the elemental analysis is 0.96%, indicating the successful modification of PDA.

[0093] Two-dimensional small-angle X-ray scattering (SAXS) was used to characterize the effects of stretching and modification with different PDA contents on the anisotropy of the hydrogels. Figure 6 , inset). For the ACH-1 hydrogel without pre-stretching treatment, a uniform intensity ring appears in the SAXS pattern, reflecting an isotropic structure. When the hydrogel is stretched to 120%, ACH-4 forms a sharp, elongated equatorial stripe, and the ring intensity is anisotropic. The results show that stress-induced submicrobeam orientation occurs along the stress direction, forming an anisotropic structure. At the same time, the azimuth-integrated intensity distribution curve of the pre-stretched hydrogel produces a sharp peak at 90° ( Figure 6 ), further demonstrating the anisotropy of the hydrogel. Furthermore, PDA modification did not significantly change the azimuthal integrated intensity distribution curve, indicating that PDA modification had little effect on the orientation of the cellulose hydrogel.

[0094] The piezoelectric properties of PDA / ACH-4 were tested by ultrasound and oscilloscope. The results are as follows: Figure 7 shown.

[0095] like Figure 7 As shown in Figure a, the voltage of ordered ACH-4 is approximately 1 V, significantly higher than that of disordered ACH-1 (approximately 0.5 V), indicating that the ordered structure improves the piezoelectric properties of the cellulose hydrogel. Furthermore, it was found that PDA modification significantly improved the piezoelectric properties of ACH, with the PDA / ACH-4 voltage reaching 1.5 V. This indicates that the ordered structure and the addition of PDA enhance the piezoelectric properties of the cellulose hydrogel.

[0096] The local piezoelectric activity of the hydrogel was studied using atomic force microscopy, and its amplitude image ( Figure 7 c) and phase image ( Figure 7 d). The hydrogel's piezoelectric response amplitude curve, also known as the butterfly curve, directly reflects the strain response of the area below the conductive probe under the action of the electric field.

[0097] like Figure 7As shown in Figures 7c and 7d, when the bias voltage increases from -10V to +10V, the amplitude curve of PDA / ACH-4 exhibits significant hysteresis, sufficient to achieve polarity reversal. Regarding the piezoelectric properties of PDA / ACH-4, the phase curve of PDA / ACH-4 shows a wide hysteresis loop for the ±10V tip excursion, indicating that the polarity of PDA / ACH-4 exists and can be reversed in the presence of an applied electric field. This polarity reversal is primarily due to the presence of the polar β phase. These experimental results also demonstrate the excellent piezoelectric properties of PDA / ACH-4.

[0098] The antioxidant potential of various cellulose hydrogels was investigated using a 1-diphenyl-2-pyridylhydrazide (DPPH) free radical (DPPH) assay. DPPH free radical scavenging activity was determined by immersing 300 μL of different hydrogels (10 mg / mL hydrogel aqueous solution) or water (control) in 700 μL of a 0.1 mM DPPH solution (95 wt% ethanol) in the dark for 6 hours. The optical density (OD) of the mixture was measured at 517 nm using a microplate reader (BioTekCytation 3). The DPPH free radical scavenging activity was calculated using the following equation.

[0099]

[0100] like Figure 8 As shown, the scavenging capacity of the PDA-modified cellulose hydrogel PDA / ACH-4 was significantly higher than that of ACH-1 and ACH-4, reaching over 60%. The abundant antioxidant groups in PDA (such as phenolic hydroxyl groups) are responsible for scavenging ROS. Therefore, the PDA-modified hydrogel has excellent antioxidant activity.

[0101] Example 3

[0102] This embodiment provides a wireless neuromodulation system, which includes the PDA / ACH-4 and ultrasound equipment provided in the above embodiment, and studies and analyzes the effect of the wireless neuromodulation system on promoting brain injury repair in TBI rats. Figure 9 As shown:

[0103] (1) Male SD rats aged 8 to 10 weeks and weighing 180 to 200 g were selected to establish a fluid percussion injury (TBI) model. Each rat was anesthetized by intraperitoneal injection. After successful anesthesia, the rat was fixed on a stereotactic instrument. The scalp was incised slightly to the right of the midline of the head, and the skull was drilled with a dental drill at the midpoint of the line connecting the right anterior and posterior fontanelles. With this as the center, the bone window was enlarged with a mosquito-type vascular clamp to form a bone window with a diameter of 5 mm. The dura mater was kept intact. A free-fall impact device was used to drop a 60 g weight from a height of 25 cm to hit the striker to act on the brain tissue. The right parietal lobe contusion was caused by the iron block falling less than 1 mm. The impact rod was immediately removed after the impact, and a sterile cotton ball was used to dry the epidural bleeding and expose the dura mater. After the model was successfully established, the rat was placed in a clean, quiet environment at 20°C and away from strong light for feeding.

[0104] (2) Subsequently, rat neural stem cells (rNSC) labeled with CM-Dil (cell density of 1*10 7 / mL, 5 μL) was injected into the brain injury site, and then polydopamine-modified anisotropic cellulose piezoelectric hydrogel (5 mm×5 mm) was adhered to the injury site of the rat.

[0105] (3) The above culture system was placed under ultrasound equipment and ultrasound was applied at 0 and 2 W / cm 2 Treatment, treatment time is 7 days, 5 minutes each time, 2 times a day.

[0106] (4) First, in terms of regulating the immune microenvironment in the early stage of TBI, the TBI group (without ACH gel), the TBI+ACH-4+ultrasound (TAU) group, and the TBI+PDA / ACH-4+ultrasound (TPAU) group were compared to observe the reactive oxygen species levels in the injured area using superoxide anion fluorescent probe (DHE) staining 3 days after TBI. Figure 10 As shown, a significant DHE signal was detected 3 days after TBI, indicating the presence of a large amount of accumulated reactive oxygen species (ROS) in the injured area. While the level of ROS did not decrease in the TAU group, the lowest DHE fluorescence intensity was observed in the injured area after TPAU treatment, demonstrating its strongest ability to scavenge reactive oxygen species in the injured area.

[0107] (5) Subsequently, the distribution of pro-inflammatory M1 microglia and anti-inflammatory M2 microglia in the injury area was detected 3 days after TBI. Figure 11As shown, three days after TBI, immunofluorescence staining for the anti-inflammatory marker CD206 and the pro-inflammatory marker CD16 / 32 was used to evaluate the transformation of microglia in the injured area after different treatments. High green fluorescence intensity of the CD16 / 32 marker and low green fluorescence intensity of the CD206 marker were observed in the TBI and TAU groups, indicating pro-inflammatory polarization of microglia. In the TPAU group, microglia polarized to a neuroprotective phenotype, as demonstrated by a significant increase in CD206 green fluorescence intensity and a significant decrease in CD16 / 32 green fluorescence intensity. This indicates that the wireless neuromodulatory system has significant microglial transformation-promoting and anti-inflammatory functions, and provides a suitable environment for stem cell engraftment.

[0108] (6) To evaluate the differentiation of exogenous neural stem cells after implantation into the TBI injury site, immunofluorescence staining of astrocyte markers (GFAP) and mature neuron markers (MAP2) was performed 7 days after TBI. Figure 12 ), CM-Dil and MAP2, CM-Dil and GFAP overlapping cells are cells differentiated from exogenous neural stem cells. Figure 12 As shown, compared with the TBI+NSC (TC) group, the MAP2 expression level of rats treated with the PDA / ACH-4+NSC+ultrasound (TPACU) group was significantly increased, indicating that the antioxidant wireless neural regulation system provided by the present application significantly promoted the differentiation of exogenous neural stem cells into neurons, which provided favorable conditions for brain injury repair.

[0109] (7)Neurogenesis is considered another key indicator of neural function recovery after brain injury. Histological evaluation of newborn neurons 7 days after surgery showed that ( Figure 13 ), due to the endogenous regeneration mechanism, some new neurons appeared at the injury site. Even in the TBI group that did not implant the anti-inflammatory nanogenerator (PAD-modified cellulose piezoelectric hydrogel provided by this application), a small number of DCX-positive cells appeared at the injury site. Fluorescence imaging showed that the number of newborn neurons in the TPACU group increased after 7 days of treatment. The above results emphasize that the antioxidant wireless neuromodulatory system can promote the migration of endogenous neural stem cells and tissue regeneration, and has a therapeutic effect on TBI repair.

[0110] (8) Then, the axonal area of the injured area was detected by axon marker (neurofilament, NF). Figure 14). Seven days after injury, the percentage of NF-positive areas in the injured area was less than 5%, indicating that most neurofilaments (intermediate filaments in the neuronal cytoplasm) were lost. At this time, there were no neurofilaments in the brain injury cavity. It was found that after the implantation of the antioxidant wireless neuromodulation system, the axon area in the area around the injury increased. Seven days after surgery, the increased axon area was about 4.6 times higher than that of the TBI group. These results indicate that the antioxidant wireless neuromodulation system promotes further axonal sprouting.

[0111] (9) Subsequently, the Morris water maze task was used to clarify the degree of macro-neurocognition and evaluate the recovery of learning and memory abilities in rats after brain trauma ( Figure 15 During the 5-day training, the time to find the platform (escape latency), which is the main parameter reflecting the spatial learning ability of TBI rats, gradually decreased. Compared with the TBI group, the rats treated with TPACU group took a shorter time and path length to find the hidden platform ( Figure 15 a). During the 5-day training, the time to reach the plateau decreased more in the TPACU group than in the TBI group ( Figure 15 b), indicating that the antioxidant wireless neural regulation system can effectively enhance the learning ability of TBI rats. On the 6th day, a probe test was conducted without a platform to evaluate memory consolidation. The results of the probe test to evaluate the memory retrieval ability of rats showed that the rats in the TPACU group were able to remember the platform position shown in the swimming path ( Figure 15 a). In contrast, the TBI group exhibited more chaotic swimming paths during the test. Rats in the TPACU group spent significantly more time in the target quadrant and crossed the platform more frequently than rats in the TBI group ( Figure 15 c) These results demonstrate that the antioxidant wireless neuromodulation system can improve memory and learning abilities after brain injury, repair the damaged nervous system, and is a potential auxiliary treatment method for promoting functional recovery after brain injury.

[0112] The above description is merely a specific embodiment of the invention covered by this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. A polydopamine-modified anisotropic cellulose piezoelectric hydrogel, comprising: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.

2. The polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1, wherein the modification amount of polydopamine is 0.4%-2% based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel, and optionally, the elongation rate of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%.

3. A method for preparing anisotropic cellulose piezoelectric hydrogel modified with polydopamine, comprising: In an alkaline solution, dopamine is in situ polymerized on the anisotropic cellulose piezoelectric hydrogel to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.

4. The preparation method according to claim 3, comprising immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH value of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then washing with water to terminate the reaction to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel, optionally, the alkaline solution is Tris-HCl buffer.

5. The preparation method according to claim 3, wherein The anisotropic cellulose piezoelectric hydrogel is prepared by the following steps: 1) adding a cross-linking agent to the piezoelectric cellulose solution to chemically cross-link and gelate to obtain a cellulose piezoelectric hydrogel; and 2) Pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to align the cellulose in the cellulose piezoelectric hydrogel, and then immersing it in an aqueous sulfuric acid solution to permanently fix the oriented structure through physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.

6. The preparation method according to claim 5, wherein step 1) comprises dissolving the piezoelectric cellulose in a lithium hydroxide and urea solution to obtain the piezoelectric cellulose solution, optionally, in the lithium hydroxide and urea solution, the concentration of lithium hydroxide is 3.6-5.4wt%, and the concentration of urea is 12-18%. Preferably, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1wt%-5wt%. Optionally, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled at a temperature of -20°C to 0°C. Optionally, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose and cotton. Optionally, the cross-linking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin and polyethylene glycol diglycidyl ether, Optionally, in step 1), the molar ratio of the cross-linking agent to the deoxyglucose unit in the piezoelectric cellulose solution is 1:1-3:1, Optionally, step 1) includes dropwise adding the crosslinking agent to the cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring into a template, and gelling at 0-10° C. for 10 h to 100 h to obtain the cellulose piezoelectric hydrogel; Optionally, the stretching rate of the pre-stretching in step 2) is 80%-215%; Optionally, step 2) comprises soaking the pre-stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution for physical crosslinking and terminating chemical crosslinking, and then washing with water to obtain the anisotropic cellulose piezoelectric hydrogel. Preferably, the concentration of the dilute sulfuric acid solution is 1 wt%-5 wt%.

7. Use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2 or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any one of claims 3 to 6 as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.

8. Radiofrequency stimulation system, including: The polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2, or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any one of claims 3 to 6; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the ultrasonic action of the ultrasonic device, Optionally, the ultrasonic frequency, ultrasonic intensity and ultrasonic time of the ultrasonic device can be programmably controlled. Optionally, the ultrasound intensity of the ultrasound action is 0-5W / cm 2 .

9. Use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2 or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any one of claims 3 to 6 in the preparation of a repair material for treating brain injury, especially traumatic brain injury.

10. A product package for treating brain injury, including: neural stem cells; and The polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2, or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared by the preparation method according to any one of claims 3 to 6.

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