Piezoelectric nano-composite with anti-oxidation function and preparation method thereof

Abstract

The invention provides a piezoelectric nano composite with an anti-oxidation function and a preparation method thereof, in particular to piezoelectric hydrogel with an anti-oxidation function and a preparation method thereof, and the hydrogel comprises a hydrogel skeleton formed by aminophenylboronic acid grafted sodium alginate and polyvinyl alcohol; the bacterial cellulose nanocrystal is loaded in the hydrogel skeleton and is modified by polydopamine. The invention further provides application of the hydrogel in preparation of a repair material for brain injury repair.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering technology, specifically to a piezoelectric nanocomposite with antioxidant function and its preparation method, especially a hydrogel, particularly a ROS-responsive piezoelectric hydrogel and its preparation method and application. Background Technology

[0002] Ischemic stroke, also known as cerebral infarction, is a common clinical disease. It refers to the necrosis of brain tissue caused by narrowing or occlusion of the blood supply arteries to the brain (carotid and vertebral arteries), resulting in insufficient blood supply. Currently, the best clinical treatment for ischemic stroke is thrombolytic therapy, which involves intravenous injection of antithrombotic drugs to dissolve blood clots and restore cerebral blood flow. However, due to the short optimal treatment window, most patients cannot receive timely and effective treatment, leading to poor prognosis. During ischemic stroke, large amounts of reactive oxygen species (ROS) such as hydroxyl radicals (OH), superoxide anions (O2-), and hydrogen peroxide are generated in the ischemic site, leading to oxidative stress or excessive inflammatory response, which further aggravates ischemic damage.

[0003] Deep brain stimulation (DBS) and vagus nerve stimulation (VNS) are used to treat brain injury, but safe and effective interventions for the resulting cognitive impairment remain lacking. Transcranial electrical stimulation (tDCS), a treatment method that modulates brain function by applying weak currents to target sites, has gradually become a research hotspot in recent years. tDCS has shown great promise in treating neurological and psychiatric diseases such as Parkinson's disease. High-intensity tDCS treatment can significantly improve neuronal pathological damage in the CA1 region and enhance cognitive abilities such as learning and memory, thereby restoring brain function after ischemia. However, due to the limitations of tDCS technology, individual responses to transcranial electrical stimulation may vary. The stimulation effect may be affected by individual differences in the nervous system, variations in brain structure, and the selection of stimulation parameters. Furthermore, the stimulation site of transcranial electrical stimulation is limited to some extent by the scalp and skull, making it impossible to directly and precisely stimulate specific deep brain regions and improve the ischemic microenvironment while providing stimulation. Summary of the Invention

[0004] According to one aspect of this application, a hydrogel is provided comprising: a hydrogel framework formed of sodium alginate grafted with aminophenylboronic acid and polyvinyl alcohol; and polydopamine-modified bacterial cellulose nanocrystals loaded in the hydrogel framework.

[0005] In some embodiments, the mass ratio of the polyvinyl alcohol: the aminophenylboronic acid-grafted sodium alginate: the polydopamine-modified bacterial cellulose nanocrystals is 10:5-20:0.05-0.2.

[0006] In some embodiments, the amount of polydopamine modification in the polydopamine-modified bacterial cellulose nanocrystals is 5%-10%, based on the mass percentage of nitrogen in the polydopamine-modified bacterial cellulose nanocrystals.

[0007] In some embodiments, the mass ratio of sodium alginate to 3-aminophenylboronic acid in the aminophenylboronic acid-grafted sodium alginate is 1:0.2-1.

[0008] In some embodiments, the bacterial cellulose nanocrystals in the polydopamine-modified bacterial cellulose nanocrystals have a particle size of 100-200 nm.

[0009] In some embodiments, the molecular weight of the polyvinyl alcohol is 22-77 kDa.

[0010] According to another aspect of this application, a method for preparing the aforementioned hydrogel is provided, comprising: preparing a mixed solution of aminophenylboronic acid-grafted sodium alginate and polydopamine-modified bacterial cellulose nanocrystals; uniformly mixing the mixed solution with polyvinyl alcohol; adding sodium hydroxide solution to the resulting mixed system; and carrying out a crosslinking reaction to obtain the hydrogel.

[0011] In some embodiments, in the mixed solution of aminophenylboronic acid-grafted sodium alginate and polydopamine-modified bacterial cellulose nanocrystals, the concentration of aminophenylboronic acid-grafted sodium alginate is 5-20 g / ml, and the concentration of polydopamine-modified bacterial cellulose nanocrystals is 50-200 μg / ml.

[0012] In some embodiments, the concentration of the sodium hydroxide solution is 0.1-1 mol / L.

[0013] In some embodiments, the sodium hydroxide solution is added at a volume ratio of 1:10 to the volume of the mixed system.

[0014] In some embodiments, the aminophenylboronic acid-grafted sodium alginate is prepared by the following steps: dissolving sodium alginate and 3-aminophenylboronic acid in deionized water, then adding EDC·HCl, stirring the reaction at room temperature, dialyzing the reaction through a dialysis bag after the reaction is completed, and freeze-drying to obtain the aminophenylboronic acid-grafted sodium alginate.

[0015] In some embodiments, the polydopamine-modified bacterial cellulose nanocrystals are obtained by in-situ polymerization of dopamine on bacterial cellulose nanocrystals in an alkaline solution, optionally, the alkaline solution being a pH 7.5-10tris-HCl buffer.

[0016] In some embodiments, the bacterial cellulose nanocrystals are cellulose nanocrystals with carboxyl functional groups obtained by using bacterial cellulose as raw material and by sulfuric acid hydrolysis and hydrogen peroxide oxidation.

[0017] According to another aspect of this application, a hydrogel injection is provided, comprising the aforementioned hydrogel or a hydrogel prepared according to the aforementioned preparation method.

[0018] According to another aspect of this application, the aforementioned hydrogel or the hydrogel prepared according to the aforementioned preparation method is provided for use in the preparation of a repair material for brain injury repair.

[0019] In some implementations, the repair material is injectable.

[0020] In some implementations, the brain injury is caused by ischemic stroke and degenerative neurological diseases. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Characterization results of bacterial cellulose nanocrystals (BCN) and PDA-modified bacterial cellulose nanocrystals (PDA@BCN) prepared according to exemplary embodiments of this application, wherein a: physical image of BCN; b: atomic force microscopy (AFM) image of BCN; c: particle size distribution map of BCN; d: physical image of PDA@BCN; e: AFM image of PDA@BCN; f: powder X-ray diffraction (XRD) patterns of bacterial cellulose (BC), BCN and PDA@BCN.

[0023] Figure 2 Characterization results of BCN and PDA@BCN prepared according to exemplary embodiments of this application by piezoelectric microscopy (PFM): (a) butterfly amplitude loop of BCN; (b) phase potential hysteresis diagram of BCN; (c) butterfly amplitude loop of PDA@BCN; (d) phase potential hysteresis diagram of PDA@BCN.

[0024] Figure 3 Cumulative release curves of PDA@BCN of ROS-responsive antioxidant piezoelectric hydrogel prepared according to exemplary embodiments of this application in PBS buffer with different concentrations of hydrogen peroxide.

[0025] Figure 4The results of live and dead staining of PDA@BCN at different concentrations prepared according to the exemplary embodiments of this application and co-cultured with SD rat neuronal cells, wherein (a): green represents live cells and red represents dead cells; (b): shows the statistical results of survival rate.

[0026] Figure 5 The total free radical scavenging rate of PDA@BCN at different concentrations was detected using the 2,2'-nitro-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay kit.

[0027] Figure 6 Free radical scavenging experiment: DHE and DCFH-DA staining of neurons differentiated from neural stem cells after 3 days of culture.

[0028] Figure 7 HE and Nissal staining were performed on rats 7 days after ischemia-reperfusion 5 days later by in situ injection of hydrogel prepared according to the exemplary embodiment of this application.

[0029] Figure 8 HE staining of heart, liver, spleen, lung and kidney after 7 days of treatment with hydrogel prepared according to the exemplary embodiment of this application 5 days after ischemia-reperfusion in rats. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] hydrogel

[0032] According to one aspect of this application, a hydrogel, particularly a bacterial cellulose nanocrystal / sodium alginate / PVA composite hydrogel, is provided, comprising: a hydrogel framework formed by aminophenylboronic acid-grafted sodium alginate (A) and polyvinyl alcohol (PVA); and polydopamine (PDA)-modified bacterial cellulose nanocrystals loaded in the hydrogel framework.

[0033] This application provides a hydrogel in which the hydrogel skeleton has ROS-responsive release properties, and the loaded polydopamine-modified bacterial cellulose nanocrystals have ROS scavenging and piezoelectric properties.

[0034] In some embodiments, the mass ratio of polyvinyl alcohol: aminophenylboronic acid-grafted sodium alginate: polydopamine-modified bacterial cellulose nanocrystals in the hydrogel of this application is 10:5-20:0.05-0.2, for example, 10:(any value in the range of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20 or any value in the range of two of these) :(any value in the range of 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2 or any value in the range of two of these).

[0035] In some embodiments, in the aminophenylboronic acid-grafted sodium alginate, the mass ratio between sodium alginate and 3-aminophenylboronic acid is 1:0.2-1, for example, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, or any value within a range of two of these.

[0036] In some embodiments, the amount of polydopamine modification in the polydopamine-modified bacterial cellulose nanocrystals is 5%-10%, for example, any value within the range of 5%, 6%, 7%, 8%, 9%, 10%, or a combination thereof, based on the mass percentage of nitrogen in the polydopamine-modified bacterial cellulose nanocrystals.

[0037] In some embodiments, the bacterial cellulose nanocrystals in the polydopamine-modified bacterial cellulose nanocrystals have a particle size of 100-200 nm, such as any range of 100 nm, 120 nm, 130 nm, 148 nm, 160 nm, 170 nm, 192 nm, 200 nm, or a combination thereof. The PDA surface is intermolecularly coupled to the bacterial cellulose nanocrystals via hydroxyl groups.

[0038] In some embodiments, the powder X-ray diffraction pattern of the bacterial cellulose nanocrystals provided in this application has characteristic diffraction peaks at the following 2θ (°) angles: 14°±2°, 16°±2° and 21°±2°, which represent the three crystal planes (1-10), (110) and (020) of the bacterial cellulose (BC) structure, respectively. 21° represents β-crystalline cellulose. The sharper the peak, the higher the crystal content.

[0039] In some embodiments, the polydopamine-modified bacterial cellulose nanocrystals include bacterial cellulose nanocrystals as a core and PDA coated on the surface of the bacterial cellulose nanocrystals.

[0040] In some implementations, the molecular weight of polyvinyl alcohol is 22-77 kDa, for example 77 kDa.

[0041] Hydrogel preparation method

[0042] According to another aspect of this application, a method for preparing hydrogels, particularly bacterial cellulose nanocrystal / sodium alginate / PVA composite hydrogels, is provided, comprising: preparing a mixed solution of aminophenylboronic acid-grafted sodium alginate and PDA-modified bacterial cellulose nanocrystals; uniformly mixing the mixed solution with PVA; adding sodium hydroxide solution to the resulting mixed system; and carrying out a cross-linking reaction to obtain the hydrogel.

[0043] In some embodiments, in a mixed solution of aminophenylboronic acid-grafted sodium alginate and PDA-modified bacterial cellulose nanocrystals, the concentration of aminophenylboronic acid-grafted sodium alginate is 5-20 mg / ml (e.g., any value within the range of 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, or a combination thereof), and the concentration of PDA-modified bacterial cellulose nanocrystals is 50-200 μg / ml (e.g., any value within the range of 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 120 μg / ml, 150 μg / ml, 180 μg / ml, 200 μg / ml, or a combination thereof). Within this concentration range, the resulting hydrogel exhibits better injectability and biocompatibility.

[0044] Aminophenylboronic acid-grafted sodium alginate and PDA-modified bacterial cellulose nanocrystals are first mixed to form a homogeneous solution, and then PVA is added. This avoids the uneven dispersion of PDA-modified bacterial cellulose nanocrystals caused by hydrogen bonds and entanglement between aminophenylboronic acid-grafted sodium alginate and PVA.

[0045] In some embodiments, the hydrogel preparation method of this application uses a sodium hydroxide solution with a concentration of 0.1-1 mol / L, for example, any value within the range of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, or a combination thereof. In some embodiments, the sodium hydroxide solution is added at a volume ratio of 1:10 to the mixed system. The OH- in the sodium hydroxide solution... - It combines with borate groups to form hydroxyborate ions, which then react with the 1,2 and 1,3 diol structures in the PVA molecule to form borate esters. Within this concentration and ratio range, a balance between hydrogel formation and biocompatibility can be maintained.

[0046] In some embodiments, the PVA used in the hydrogel preparation method of this application has a molecular weight of 22-77 kDa, for example 77 kDa.

[0047] In some embodiments, in the aminophenylboronic acid-grafted sodium alginate used in the hydrogel preparation method of this application, the mass ratio between sodium alginate and 3-aminophenylboronic acid is 1:0.2-1, for example, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1 or any value within the range of two of these.

[0048] In some embodiments, in the polydopamine-modified bacterial cellulose nanocrystals used in the hydrogel preparation method of this application, the amount of polydopamine modification is 5%-10%, for example, any value within the range of 5%, 6%, 7%, 8%, 9%, 10%, or a combination thereof, based on the mass percentage of nitrogen element in the polydopamine-modified bacterial cellulose nanocrystals. PDA modification increases the piezoelectric properties of the bacterial cellulose nanocrystals and imparts them with antioxidant properties.

[0049] In some embodiments, the bacterial cellulose nanocrystals in the polydopamine-modified bacterial cellulose nanocrystals used in the hydrogel preparation method of this application have a particle size of 100-200 nm, such as any range of 100 nm, 120 nm, 130 nm, 148 nm, 160 nm, 170 nm, 192 nm, 200 nm, or a combination thereof.

[0050] In some embodiments, in the hydrogel preparation method of this application, the mass ratio of polyvinyl alcohol, aminophenylboronic acid-grafted sodium alginate, and polydopamine-modified bacterial cellulose nanocrystals used is 10:5-20:0.05-0.2, for example, 10:(any value within the range of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or any value within the range of two of these) :(any value within the range of 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, or any value within the range of two of these). In some embodiments, during the formation of the hydrogel, all of the polyvinyl alcohol, aminophenylboronic acid-grafted sodium alginate, and polydopamine-modified bacterial cellulose nanocrystals used in the preparation process participate in the synthesis of the hydrogel.

[0051] Preparation of aminophenylboronic acid-grafted sodium alginate

[0052] In some embodiments, the aminophenylboronic acid-grafted sodium alginate of this application is prepared by the following steps: dissolving sodium alginate and 3-aminophenylboronic acid in deionized water, then adding EDC·HCl, stirring the reaction at room temperature, dialyzing through a dialysis bag after the reaction is complete, and freeze-drying to obtain aminophenylboronic acid-grafted sodium alginate (Alg-PBA). The synthesis of Alg-PBA uses EDC·HCl as a coupling agent to graft 3-aminophenylboronic acid onto the sodium alginate backbone via amide bonds.

[0053] In some implementations, the mass ratio of sodium alginate: 3-aminophenylboronic acid: EDC·HCl is 1:0.2-1:1-2, for example, 1:0.4:1.

[0054] In some implementations, the stirring reaction time is 6-24 hours, for example, 24 hours.

[0055] In some implementation schemes, the molecular weight cutoff of the dialysis bag is 8-14 kDa, and the dialysis time is 3-6 days.

[0056] Preparation of PDA-modified bacterial cellulose nanocrystals

[0057] In some embodiments, the PDA-modified bacterial cellulose nanocrystals of this application are obtained by in-situ polymerization of dopamine (DA) on bacterial cellulose nanocrystals in an alkaline solution.

[0058] In some implementations, the alkaline solution used in the preparation of PDA-modified bacterial cellulose nanocrystals is a pH 7.5-10 tris-HCl buffer.

[0059] In some embodiments, the ratio of dopamine to bacterial cellulose nanocrystals is 0.3-1:6. PDA modification increases the piezoelectric properties of the bacterial cellulose nanocrystals and imparts them with antioxidant properties.

[0060] In some implementations, in-situ polymerization is carried out at room temperature for 6-12 hours.

[0061] In some embodiments, the bacterial cellulose nanocrystals in this application are cellulose nanocrystals with carboxyl functional groups obtained by using bacterial cellulose as raw material and by sulfuric acid hydrolysis and hydrogen peroxide oxidation.

[0062] In some implementations, bacterial cellulose is derived from commercially available sources.

[0063] Application of bacterial cellulose nanocrystals / sodium alginate / PVA composite hydrogel

[0064] According to another aspect of this application, a hydrogel injection, particularly a hydrogel injection for brain injury repair, is provided, comprising the hydrogel provided in this application. The hydrogel injection may also contain other excipients, such as some nutritional factors for treating nerve damage, such as drugs like BDNF.

[0065] According to another aspect of this application, the use of the hydrogel of this application, particularly the bacterial cellulose nanocrystal / sodium alginate / PVA composite hydrogel, in the preparation of a repair material for brain injury repair is provided.

[0066] In some embodiments, the brain injury repair material in this application is injectable.

[0067] This application also provides methods for repairing or treating brain injuries, including applying the hydrogel provided in this application to the brain injury area of ​​a subject in need.

[0068] In some embodiments, the brain injury in this application is caused by ischemic stroke and degenerative neurological disease.

[0069] The hydrogel provided in this application is a ROS-responsive antioxidant piezoelectric hydrogel. It can be injected into the cerebral infarction site via in-situ injection, generating electrical signals in response to applied external field stimulation. Simultaneously, the ROS-responsiveness of the hydrogel allows the release of PDA-modified bacterial cellulose nanocrystals. Polydopamine (PDA) is a melanin-like polymer produced from the neurotransmitter dopamine, exhibiting excellent biocompatibility and biodegradability. Due to its abundant phenolic groups, PDA possesses ROS-scavenging properties. PDA can be used to remove ROS from the damaged microenvironment, while bacterial cellulose can generate localized electrical stimulation under external ultrasonic stimulation, repairing damaged neurons.

[0070] PDA-modified bacterial cellulose nanocrystals, a type of ROS-responsive hydrogel with acoustic-electric transduction, offer new insights for the treatment of brain injuries (such as those caused by ischemic stroke and degenerative neurological diseases), providing novel therapeutic methods for treating inflammation, reducing nerve damage, and repairing damaged nerves.

[0071] Example

[0072] Example 1

[0073] 1. Preparation of ROS-responsive antioxidant piezoelectric hydrogels:

[0074] Synthesis of bacterial cellulose nanocrystals: 5g of bacterial cellulose (BC) was dissolved in 80ml of 50wt% sulfuric acid solution. The mixture was stirred rapidly at 45℃ for 3 hours, then 320ml of deionized water was added to quench the reaction, and simultaneously 8ml of 30wt% hydrogen peroxide solution was added for oxidation. The mixture was then centrifuged twice at 9000rpm for 15min. Afterward, the solution was dialyzed for 8 days using a dialysis bag with a molecular weight cutoff of 3500 to obtain a bacterial cellulose nanocrystal (BCN) solution.

[0075] 2. Preparation of PDA-modified bacterial cellulose nanocrystals (PDA@BCN): 100 ml of bacterial cellulose nanocrystal solution (6 mg / ml) was mixed thoroughly with 1 ml of pH 8.8 tris-HCl buffer. 40 mg of dopamine (DA) was then added to the mixture, and the mixture was reacted at room temperature for 12 h to obtain a solution of PDA-modified bacterial cellulose nanocrystals (PDA@BCN). The PDA was then freeze-dried to obtain lyophilized PDA@BCN. The modification amount of PDA was 8% based on the mass percentage of nitrogen in the PDA@BCN.

[0076] The structural characterization of the prepared BCN and PDA@BCN is as follows: Figure 1 As shown.

[0077] Figure 1 Images a, b, and c show the physical image, atomic force microscopy (AFM) image, and particle size distribution diagram of the prepared BCN solution, respectively. It can be seen that the prepared BCN has excellent dispersibility in water and does not precipitate even after prolonged standing. The AFM images show a uniform particle size distribution of the BCN. Figure 1 The particle size distribution of c shows that the particle size of BCN is about 148 nm, indicating that BCN was successfully prepared.

[0078] Figure 1 Images d and e show the physical image and atomic force microscopy (AFM) observation of the prepared PDA@BCN solution, respectively. Figure 1 f shows the powder X-ray diffraction (XRD) patterns of BC, BCN, and PDA@BCN. From Figure 1 As can be seen from d and e, PDA successfully encapsulated BCN, and the successful preparation of PDA@BCN was further verified by XRD.

[0079] 3. Preparation of aminophenylboronic acid-grafted sodium alginate (Alg-PBA): Weigh 1.0 g of sodium alginate and 0.4 g of 3-aminophenylboronic acid and dissolve them in 100 mL of deionized water, then add 1.0 g of EDC·HCl. Stir the mixture at room temperature for 24 h. After the reaction is complete, dialyze the solution using a dialysis bag with a molecular weight cutoff of 8-14 kDa for 3 days, and freeze-dry to obtain aminophenylboronic acid-grafted sodium alginate (Alg-PBA).

[0080] 4. Preparation of ROS-responsive antioxidant piezoelectric hydrogel: Weigh the lyophilized PDA@BCN obtained in step 2 and the lyophilized Alg-PBA obtained in step 3, add water and mix evenly to obtain a mixed solution containing 200 μg / mL PDA@BCN and 10 mg / mL Alg-PBA. Then, mix the two solutions directly with polyvinyl alcohol (PVA, molecular weight 77 kDa) at a mass ratio of Alg-PBA:polyvinyl alcohol of 2:1. Add a 0.3 mol / L NaOH solution (NaOH solution to mixed solution volume ratio of 1:10) to the mixed solution. After mixing evenly, let stand for half a minute to form a ROS-responsive antioxidant piezoelectric hydrogel.

[0081] Test Example 1: Piezoelectric detection of PDA@BCN:

[0082] The local piezoelectric activity of the BCN and PDA@BCN prepared in Example 1 was studied using atomic force microscopy, and their piezoelectric response amplitude curves and phase images were obtained. The piezoelectric response amplitude curve, i.e., the butterfly curve, directly reflects the strain response of the region below the conductive probe under the action of an electric field.

[0083] like Figure 2 As shown, the amplitude curve of PDA@BCN is as follows when the bias voltage changes from -10V to 10V. Figure 2 (c) exhibits a significant hysteresis, sufficient to achieve polarity reversal. Looking at the phase curve of PDA@BCN, PDA@BCN ( Figure 2 (d) and BCN ( Figure 2 Compared to (b), the tip offset shows a wide hysteresis loop, indicating the existence of polarity in PDA@BCN, which can be reversed under an applied electric field. This polarity reversal is mainly due to the presence of the polar β phase. Experimental results also demonstrate that PDA@BCN possesses excellent piezoelectric properties, and the modification of PDA enhances the piezoelectric properties of BCN.

[0084] Test Example 2: Detection of the responsiveness of ROS-responsive piezoelectric hydrogels

[0085] Detection of PDA@BCN release from hydrogel: Two mL of the hydrogel prepared in Example 1 was immersed in two mL of PBS buffer and PBS buffer containing 20 μM, 100 μM, 200 μM, and 500 μM hydrogen peroxide, respectively. 200 μL samples were taken at intervals of 0, 2, 4, 12, 24, 72, and 120 h. Finally, the UV absorbance of the PDA@BCN sample solutions was measured at 490 nm using a UV-Vis spectrophotometer. The cumulative release curve of PDA@BCN from the hydrogel was obtained. Figure 3 The study found that the release of PDA@BCN from the ROS-responsive piezoelectric hydrogel increased with increasing H2O2 concentration, while the release was minimal in the PBS group. These results indicate that the ROS-responsive piezoelectric hydrogel exhibits excellent responsiveness to H2O2, and its ROS-responsive release capability lays the foundation for stroke repair after injection at the infarct site.

[0086] Test Example 3: In vitro biocompatibility of PDA@BCN

[0087] The biocompatibility of PDA@BCN prepared in Example 1 with neuronal cells was tested.

[0088] Neuronal cell culture medium: Neurobasel + 1% L-glutamine + 2% B-27 additive.

[0089] First, contain 4×10 5 A suspension of SD rat neuronal cells was seeded onto a glass slide covered with poly-L-lysine. Different amounts of lyophilized PDA@BCN (0-300 μg / ml) prepared according to the method in Example 1 were co-cultured with the neuronal cells to obtain the culture system. Cell viability and mortality staining were performed after 3 days of culture, and the results are as follows: Figure 4 As shown. Figure 4 As shown in (b), when the concentration of PDA@BCN was 0-200 μg / mL, the cell survival rate reached more than 90%, indicating that PDA@BCN has good biocompatibility. The following experiments were conducted using 200 μg / mL PDA@BCN.

[0090] Test Example 4: Detection of the Antioxidant Performance of PDA@BCN

[0091] The lyophilized PDA@BCN prepared according to Example 1 was diluted with water to prepare solutions of different concentrations (200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1000 μg / mL). The antioxidant potential of different concentrations of PDA@BCN was investigated using a 2,2'-nitro-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) kit. The free radical scavenging effect of 300 μL of different concentrations of PDA@BCN or water (control group) was measured. After incubation at room temperature for 10 minutes in 700 μL of ABTS solution, the OD (optical density) value of the mixture at 734 nm was measured using a microplate reader (BioTek Cytation 3). The free radical scavenging effect was obtained from the following equation.

[0092]

[0093] like Figure 5 It can be seen that, at different concentrations, the bacterial cellulose nanocrystal complex has a free radical scavenging rate of over 60%.

[0094] Simultaneously, 200 μg / mL PDA@BCN and 200 μg / mL BCN obtained by recombining lyophilized BCN prepared in Example 1 with water were added to normal or 200 μM hydrogen peroxide-containing neuronal culture media, respectively, and subjected to ultrasonic stimulation with neurons (density: 5*10). 5 Cells were co-cultured at a density of 1 / ml. The neuron culture medium was Neurobasal medium + 1% N2 + 1% B27. On day 3, intracellular superoxide anion levels and the amounts of various ROS were determined by staining with dihydroethidium (DHE, 5 mM) and 2,7-dichlorofluorescein (DCFH-DA, 10 μM), respectively. Figure 6 As can be seen, when PDA@BCN is present, the fluorescence of DHE and DCFH-DA is weakened, while that of the BCN group is not weakened, indicating that the PDA coating plays a crucial role in scavenging free radicals.

[0095] Test Example 5: Application of ROS-responsive piezoelectric hydrogels in ischemic stroke

[0096] To investigate the therapeutic effect of the ROS-responsive piezoelectric hydrogel prepared in Example 1 on ischemic stroke, a MCAO / R model was established using male SD rats weighing 180-250g to simulate clinical ischemia-reperfusion injury. After separating the vagus nerve, the proximal end of the CCA, the distal end of the external carotid artery (ECA), and the internal carotid artery (ICA) were ligated. A small incision was made on the ECA, and a nylon suture plug was inserted into the ECA. The ICA ligation was then lifted, and the suture plug was slowly advanced towards the ICA until the black mark on the tail disappeared into the vessel. The proximal end of the ECA was then ligated, and the proximal end of the CCA ligation was lifted. After 90 minutes, the suture plug was removed, the skin was sutured, and iodine was applied for disinfection. The effectiveness of the ischemia-reperfusion injury model was evaluated using the Longa and Bederson 5-point scale (1-2 points were considered effective) or the modified Garcia JH score. Five days after successful modeling, the brain was fixed to a stereotactic injection device, which was then adjusted to ensure the anterior and posterior fontanelles were on the same horizontal plane. After disinfection, the anterior skin between the ears was incised to expose the anterior and posterior fontanelles. Injection sites were determined based on brain mapping: AP +2 mm, +3 mm, DV -2.5 mm; AP +2 mm, +3 mm, DV -1.5 mm; AP +0 mm, +3 mm, DV -2.5 mm; AP +0 mm, +3 mm, DV -1.5 mm. The skull was drilled open, and 5 μL of ROS-responsive piezoelectric hydrogel was injected at each of the four sites at a rate of 5 μL / min. Healthy SD rats were involved in the intervention in the following groups: Sham group (20 μL PBS solution); cerebral infarction group; and cerebral infarction + hydrogel + ultrasound (US) group (20 μL of ROS-responsive piezoelectric hydrogel prepared in Example 1). Rats in the cerebral infarction + hydrogel + ultrasound (US) group were placed under an ultrasound device and administered 2 W / cm² of ultrasound. 2 The treatment will last for 7 days, with each treatment lasting 5 minutes and twice a day.

[0097] After 7 days of treatment, the above rats were frozen sections. The frozen sections were fixed with acetone and then stained with hematoxylin and eosin (HE) and Nissl. The specific steps for HE staining are as follows: the sections were washed three times with PBS, then immersed in hematoxylin and eosin for 2-3 minutes, washed with water, and then redeiled with 1% ammonia for 2-5 seconds. The sections were then washed with running tap water. After satisfactory microscopic examination, the sections were washed with distilled water, then immersed in 0.5% eosin solution for 3 minutes, washed with water, and dehydrated stepwise in 75%-100% graded alcohol solutions for 2 minutes each. The sections were then cleared in xylene I-II for 5 minutes each, mounted with neutral resin, and photographed using a slide scanner.

[0098] The specific steps for Nissl staining are as follows: Wash the slides three times with PBS, add two drops of Nissl dye, incubate at 37°C for 3-7 minutes, soak in 95% and 100% ethanol for 10 seconds each, then immerse the slides in xylene I for 2 minutes, mount with neutral resin, and photograph using a slide scanner. Results are as follows: Figure 7 As shown.

[0099] from Figure 7 In HE staining, we observed neurons near the infarct injury area and found that 7 days after the infarction, the number of intact neurons was significantly reduced, nuclear dissolution and vacuolation were observed, and intercellular spaces were widened. ROS-responsive antioxidant piezoelectric hydrogel combined with ultrasound treatment significantly alleviated the damage, including reduced neuronal deformation, less dark nucleus staining, fewer atrophied cells, and no obvious intercellular space formation, indicating that PDA@BCN plays a crucial role in protecting neurons. In Nissl staining, the reduction in Nissl bodies indicated a decline in neural tissue function. The Nissl body content in the hydrogel + ultrasound group was higher than that in the simple infarction group, indicating that the hydrogel has a certain repair effect on infarction.

[0100] The rats in each group were perfused, and the heart, liver, spleen, lungs, and kidneys were dissected and sectioned for HE staining. Figure 8 HE staining revealed no histopathological abnormalities or biotoxicity in the hydrogel + ultrasound group, indicating that the material has good biocompatibility.

[0101] 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 variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A hydrogel comprising: a hydrogel framework formed of sodium alginate grafted with aminophenylboronic acid and polyvinyl alcohol; and polydopamine-modified bacterial cellulose nanocrystals loaded in the hydrogel framework.

2. The hydrogel according to claim 1, wherein... The mass ratio of the polyvinyl alcohol, the aminophenylboronic acid-grafted sodium alginate, and the polydopamine-modified bacterial cellulose nanocrystals is 10:5-20:0.05-0.2; and / or In the polydopamine-modified bacterial cellulose nanocrystals, the amount of polydopamine modification is 5%-10% based on the mass percentage of nitrogen element in the polydopamine-modified bacterial cellulose nanocrystals; and / or In the aminophenylboronic acid-grafted sodium alginate, the mass ratio of sodium alginate to 3-aminophenylboronic acid is 1:0.2-1; and / or The bacterial cellulose nanocrystals in the polydopamine-modified bacterial cellulose nanocrystals have a particle size of 100-200 nm; and / or The molecular weight of the polyvinyl alcohol is 22-77 kDa.

3. The method for preparing the hydrogel according to claim 1 or 2, comprising: A mixed solution of aminophenylboronic acid-grafted sodium alginate and polydopamine-modified bacterial cellulose nanocrystals was prepared. The mixed solution was then uniformly mixed with polyvinyl alcohol. Sodium hydroxide solution was added to the resulting mixture to carry out a cross-linking reaction, thereby obtaining the hydrogel.

4. The preparation method according to claim 3, wherein... In the mixed solution of aminophenylboronic acid-grafted sodium alginate and polydopamine-modified bacterial cellulose nanocrystals, the concentration of aminophenylboronic acid-grafted sodium alginate is 5-20 g / ml, and the concentration of polydopamine-modified bacterial cellulose nanocrystals is 50-200 μg / ml; and / or The concentration of the sodium hydroxide solution is 0.1-1 mol / L. Preferably, the sodium hydroxide solution is added at a volume ratio of 1:10 to the volume of the mixed system.

5. The preparation method according to claim 3 or 4, wherein the aminophenylboronic acid-grafted sodium alginate is prepared by the following steps: dissolving sodium alginate and 3-aminophenylboronic acid in deionized water, then adding EDC·HCl, stirring and reacting at room temperature, dialyzing with a dialysis bag after the reaction is completed, and freeze-drying to obtain the aminophenylboronic acid-grafted sodium alginate.

6. The preparation method according to any one of claims 3-5, wherein the polydopamine-modified bacterial cellulose nanocrystals are obtained by in-situ polymerization of dopamine on bacterial cellulose nanocrystals in an alkaline solution, optionally, the alkaline solution is a pH 7.5-10tris-HCl buffer solution.

7. The preparation method according to claim 6, wherein the bacterial cellulose nanocrystals are cellulose nanocrystals with carboxyl functional groups obtained by using bacterial cellulose as raw material and by sulfuric acid hydrolysis and hydrogen peroxide oxidation.

8. A hydrogel injection comprising the hydrogel according to claim 1 or 2 or the hydrogel prepared by any one of claims 3-7.

9. Use of the hydrogel according to claim 1 or 2, or the hydrogel prepared by any one of claims 3-7, in the preparation of a repair material for brain injury repair.

10. The use according to claim 9, wherein The repair material is injectable; and / or The brain injury was caused by ischemic stroke and degenerative neurological disease.

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