Preparation method of mechano-electric hydrogel microspheres for precise cell electrical stimulation

Abstract

The invention discloses a preparation method of force-electricity hydrogel microspheres for precise cell electrical stimulation, which comprises the following steps: dropwise adding methacrylic anhydride into a hyaluronic acid aqueous solution, dropwise adding NaOH, and staying overnight in an ice-water bath; centrifuging to take a supernatant, dialyzing and drying to obtain HAMA; preparing a mixed solution from HAMA and BT, and mixing the mixed solution with a photoinitiator to serve as a dispersion phase; span 80 and paraffin oil are mixed to serve as a continuous phase; preparing microspheres by using a microfluidic technology; freezing the microspheres, and performing ultraviolet radiation; soaking the microspheres in a polydopamine solution, and incubating at room temperature to obtain microspheres Piez (at) pDA; and activating the microspheres, incubating the microspheres with a buffer solution containing an E7 peptide fragment, and collecting the microspheres to obtain the electro-hydrogel microspheres Piezo (at) pDA (at) E7. The microspheres can induce directional aggregation of specific types of stem cells, and can sensitively respond to external mechanical stimulation to generate electric signals, so that accurate and minimally invasive cell electrical stimulation is realized, the tissue regeneration and repair efficiency is improved, and new possibility is provided for application of electrical stimulation therapy in the fields of tissue engineering, regenerative medicine and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing electromechanical hydrogel microspheres for precise cell electrical stimulation. Background Art

[0002] Electrical stimulation therapy is a medical technology that uses electric current to promote tissue regeneration and repair. Due to its simple operation, this therapy has been widely used in actual clinical applications, mainly for large-scale tissue repair and regeneration. However, the efficacy of traditional electrical stimulation therapy is limited by the heterogeneity of current, including spatial heterogeneity, temporal heterogeneity and biological heterogeneity. Due to the low spatial resolution, the current will be transmitted to highly conductive tissues with low resistance, such as blood vessels and nerve tissues. This spatial heterogeneity of uneven electric field distribution can easily cause adverse consequences of electrical overload. During the electrical stimulation process, the temporal heterogeneity of current changes at different time points makes it difficult to form a standardized scheme. Biological heterogeneity includes differences in conductivity of different tissues and differences in current response of different cells, which leads to inconsistent treatment effects. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing electromechanical hydrogel microspheres for precise cell electrical stimulation, which can induce the directional aggregation of specific types of stem cells and can sensitively respond to external mechanical stimulation to generate electrical signals, thereby achieving precise and minimally invasive cell electrical stimulation to improve the efficiency of tissue regeneration and repair.

[0004] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0005] A method for preparing electromechanical hydrogel microspheres for precise cell electrical stimulation comprises the following steps:

[0006] (1) Add methacrylic anhydride to a 2 wt % hyaluronic acid aqueous solution, then add 5 M NaOH solution at a flow rate of 10 ml / h, and place the reaction system in an ice-water bath overnight; centrifuge to obtain the supernatant, dialyze for 3 days, and freeze-dry to obtain methacryloyl hyaluronic acid (HAMA);

[0007] (2) HAMA was mixed with modified barium titanate nanoparticles (BT) to prepare a mixed solution with a total concentration of 4 wt%, and then the mixed solution was mixed with a photoinitiator as a dispersed phase; Span 80 and paraffin oil were mixed as a continuous phase;

[0008] (3) Microspheres were prepared using microfluidic technology, where the flow rate ratio of the dispersed phase to the continuous phase was 1:23;

[0009] (4) After the collected microspheres were frozen at -40°C, they were irradiated with 365 nm ultraviolet light for 10 min to promote cross-linking of the microspheres. The microspheres were then removed of paraffin oil and surfactant using anhydrous ether. The microspheres were then immersed in PBS and rinsed repeatedly.

[0010] (5) Soak the microspheres in a polydopamine solution, incubate at room temperature for 1 day, and repeatedly rinse with sterile deionized water to obtain microspheres Piezo@pDA;

[0011] (6) Activate the microspheres and incubate them with Tris buffer solution containing E7 peptide at 4°C for 24 h. Collect the modified microspheres to obtain Piezo@pDA@E7 hydrogel microspheres.

[0012] Preferably, in the above technical solution, in step (2), the barium titanate nanoparticle modification treatment is performed by polarization at a voltage of 17 kV for 10 minutes, and the distance between electrodes is 1.8 cm.

[0013] Preferably, in the above technical solution, in step (2), in a mixed solution with a total concentration of 4wt%, the mass volume ratio of the barium titanate nanoparticles to the HAMA is 0.0wv%-4.0wv%; and the concentration of the photoinitiator is 0.5wt% of the total mass of the dispersed phase.

[0014] Preferably, in the above technical solution, in step (2), the mass volume ratio of the barium titanate nanoparticles to the HAMA is 0.0wv%, 0.5wv%, 1.0wv%, 2.0wv%, 4.0wv%.

[0015] Preferably, in the above technical solution, in step (2), the continuous phase is a mixture of Span 80 and paraffin oil in a ratio of 5 wt%.

[0016] Preferably, in the above technical solution, in step (5), the polydopamine solution is a 2 mg / ml polydopamine solution prepared by dissolving dopamine hydrochloride in Tris-HCL.

[0017] Preferably, in the above technical solution, in step (6), the microspheres are activated with PBS containing 0.15 M NaCl.

[0018] Preferably, in the above technical solution, in step (6), the concentration of E7 is 100 ug / mL.

[0019] A kind of electromechanical hydrogel microsphere is prepared according to the above preparation method.

[0020] A kind of electromechanical hydrogel microspheres for applications in cell culture, tissue regeneration, drug delivery and biosensors.

[0021] The above technical solution of the present invention has the following beneficial effects:

[0022] This application achieves minimally invasive and efficient cell electrical stimulation by precisely inducing specific types of stem cells to aggregate and generate electrical signals in response to external mechanical stimulation. This innovative method not only improves the efficiency of tissue regeneration and repair, but also reduces treatment time and cost. It has significant advantages such as minimal invasiveness, strong adaptability, and a wide range of applications. It provides new possibilities for the application of electrical stimulation therapy in the fields of tissue engineering, regenerative medicine, etc., and is expected to promote the innovation of related medical technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0024] Figure 1 This is a flow chart for the preparation of electromechanical hydrogel microspheres of this application.

[0025] Figure 2 The figure is a flow chart of the preparation and characterization of injectable piezoelectric hydrogel microspheres. A is a transmission electron microscope image of barium titanate nanoparticles and a representative image of the corresponding selected area electron diffraction (inset image). B is a representative image of a high-resolution transmission electron microscope image of barium titanate nanoparticles. C is a piezoelectric response phase curve of barium titanate nanoparticles. D is a piezoelectric response amplitude curve of barium titanate nanoparticles. E is a representative scanning electron microscope image of piezoelectric hydrogel microspheres modified with polydopamine. F is the loading efficiency of stem cell recruitment peptides in hydrogel microspheres. G is a representative image of the scanning electron microscope and corresponding element mapping of stem cell recruitment functionalized piezoelectric hydrogel microspheres. H is a schematic diagram of the detection device for ultrasonically triggered hydrogel microsphere electrical output. I is an oscilloscope output open-circuit voltage waveform of ultrasonically stimulated piezoelectric hydrogel microspheres in deionized water. J is an oscilloscope output short-circuit current waveform of ultrasonically stimulated piezoelectric hydrogel microspheres in deionized water. K is the stem cell recruitment peptide release curve of HM@CR (non-piezoelectric sample) and 2% piezo@CR (piezoelectric sample) under ultrasonic stimulation. The red area represents the instant when the ultrasound stimulation was applied.

[0026] Figure 3Recruiting cells with functionalized piezoelectric hydrogel microspheres for injectable stem cell recruitment. A is a schematic diagram of ultrasound-stimulated stem cell recruitment peptide release to promote cell migration. Ultrasound stimulates the redistribution of charges within barium titanate nanoparticles, thereby destroying the electrostatic adsorption force between the peptide and the microsphere. At the same time, the vibration generated by ultrasound also promotes the release of the peptide. B is a representative image of live / dead staining of bone marrow mesenchymal stem cells after 2D co-culture with stem cell recruitment functionalized piezoelectric hydrogel microspheres. C is a representative image of scanning electron microscopy (i) and live / dead staining (ii) of bone marrow mesenchymal stem cells after 3D co-culture with stem cell recruitment functionalized piezoelectric hydrogel microspheres in a low-adhesion well plate. The green area represents mesenchymal stem cells. D is a representative image of crystal violet staining of bone marrow mesenchymal stem cells migrating to the upper chamber after 48 hours of co-culture with microspheres. E is a representative image of live / dead staining of bone marrow mesenchymal stem cells in the scratch area after 5 days of co-culture with microspheres. F is the number of bone marrow mesenchymal stem cells that migrated after 2D co-culture with microspheres. G is the coverage area of ​​bone marrow mesenchymal stem cells adhered to microspheres after 3D co-culture with microspheres in low-adhesion well plates. H is the count of BMSCs migrated to the upper chamber after 48 hours of co-culture with microspheres. I is the wound healing rate after 5 days of scratching. In all data, n = 3 independent samples, data are expressed as mean ± SD. ns = no significance, *P < 0.05, **P < 0.01, and ***P < 0.001.

[0027] Figure 4 Functionalized piezoelectric hydrogel microspheres for injectable stem cell recruitment promote chondrogenic differentiation of bone marrow mesenchymal stem cells. A is a schematic diagram of the electrical stimulation of functionalized piezoelectric hydrogel microspheres for stem cell recruitment under ultrasound activation to promote chondrogenic differentiation of stem cells. Ultrasound stimulates the redistribution of charge within the barium titanate nanoparticles inside the microspheres, and the integrated electrical stimulation enhances the influx of calcium ions into the cells, promoting the differentiation of stem cells into cartilage. B is the relative expression of cartilage genes ACAN, COL2A1 and SOX-9 detected by quantitative real-time polymerase chain reaction (RT-qPCR). C is the protein level of ACAN, COL2A1 and SOX-9 detected by Western blot WB. D is the visualization of ACAN, COL2A1 and SOX-9 proteins (green), cytoskeleton (red) and nucleus (blue) of bone marrow mesenchymal stem cells on different hydrogel microspheres by immunofluorescence. E is the detection of glycosaminoglycans (GAGs) in bone marrow mesenchymal stem cells after 14 days of co-culture with different hydrogel microspheres by Alcian blue staining. F is the detection of glycosaminoglycans (GAG) in bone marrow mesenchymal stem cell particles after 14 days of co-culture with different hydrogel microspheres by Alcian blue staining. G is the visualization of COL2A1 protein (green) in bone marrow mesenchymal stem cell particles after 14 days of co-culture with different hydrogel microspheres by immunofluorescence. In all data, n = 3 independent samples, data are expressed as mean ± SD. ns = not significant, ***P < 0.001.

[0028] Figure 5 Therapeutic effect of functionalized piezoelectric hydrogels for stem cell recruitment to enhance cartilage regeneration in a rabbit osteochondral defect model. A is the study design of microspheres for osteochondral defect rabbits. B is the representative gross images and micro-CT reconstruction images of defect-only, non-piezoelectric, and piezoelectric hydrogel microsphere groups with ultrasound stimulation (1 month). The red circle indicates the defect area. C is the representative gross images and micro-CT reconstruction images of defect-only, non-piezoelectric, and piezoelectric hydrogel microsphere groups with ultrasound stimulation (2 months). D is the representative image of H&E, Safranin O / Fast Green, and IHC staining of COL 2A1 on rabbit femoral OC defect sections with ultrasound stimulation (1 month) and defect-only, non-piezoelectric, and piezoelectric hydrogel microsphere groups. E is the representative image of H&E, Safranin O / Fast Green, and IHC staining of COL 2A1 on rabbit femoral OC defect sections with ultrasound stimulation (2 months) and defect-only, non-piezoelectric, and piezoelectric hydrogel microsphere groups. F is the International Cartilage Repair Society (ICRS) gross cartilage assessment score of rabbit femurs without (or with) ultrasound stimulation 1, 2 months later. G is the subchondral bone volume of rabbit femurs reconstructed by micro-CT without (or with) ultrasound stimulation and with ultrasound stimulation 1, 2 months later. In all data, n = 4 independent samples, data are expressed as mean ± SD. ns = no significance, *P < 0.05, **P < 0.01.

[0029] Figure 6 In vitro compatibility of functionalized piezoelectric hydrogels for stem cell recruitment. A is a representative image of live / dead staining of bone marrow mesenchymal stem cells after 1, 3, and 5 days of co-culture with hydrogel microspheres. Green represents live cells and red represents dead cells. B is the viable cell count of bone marrow mesenchymal stem cells after 1, 3, and 5 days of co-culture with hydrogel microspheres. C is the CCK8 result of bone marrow mesenchymal stem cells after 1, 3, and 5 days of co-culture with hydrogel microspheres. D is a representative image of live / dead staining of chondrocytes after 1, 3, and 5 days of co-culture with hydrogel microspheres. Green represents live cells and red represents dead cells. E is the viable cell count of chondrocytes after 1, 3, and 5 days of co-culture with hydrogel microspheres. F is the CCK8 result of chondrocytes after 1, 3, and 5 days of co-culture with hydrogel microspheres. In all data, n = 3 independent samples, and data are expressed as mean ± SD. ns = not significant.

[0030] Figure 7 In vivo biocompatibility of functionalized piezoelectric hydrogel microspheres for injectable stem cell recruitment, representative HE-stained images of heart, liver, spleen, lung, and kidney, indicating that the microspheres were non-toxic to major internal organs after in vivo injection. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0032] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.

[0033] The present application uses microfluidic technology to prepare methacryloyl hyaluronic acid hydrogel microspheres loaded with 2%wt barium titanate piezoelectric nanoparticles, and crosslinks and solidifies the microspheres under ultraviolet light. The surface coating of the microspheres is then modified with dopamine to enhance the cell adhesion of the microspheres and provide the binding force of the polypeptide. Furthermore, the modified microspheres are mixed and stirred overnight with the stem cell recruitment polypeptide E7 to achieve effective binding under the action of π-π conjugation and electrostatic adsorption.

[0034] Example 1 Preparation of electrodynamic hydrogel microspheres

[0035] This embodiment describes in detail a method for preparing electromechanical hydrogel microspheres for precise cell electrical stimulation, which comprises the following steps (e.g. Figure 1 shown):

[0036] (1) Preparation of methacryloyl hyaluronic acid (HAMA):

[0037] 20 mL of methacrylic anhydride was added dropwise to a 2 wt % aqueous solution of hyaluronic acid (molecular weight 46 kDa).

[0038] Subsequently, 20 mL of NaOH (5 M) was added dropwise to the solution at a flow rate of 10 ml / h using a microsyringe.

[0039] The entire reaction system was reacted overnight in an ice-water bath, and the solution after the reaction was centrifuged at 7000 rpm for 15 min. The supernatant was transferred to a dialysis bag (molecular weight cutoff 8000 Da) and dialyzed for 3 days to obtain a purified product.

[0040] Finally, the obtained HAMA solution was freeze-dried and stored at -80°C for future use.

[0041] (2) Modification and characterization of nanoparticles:

[0042] In order to obtain barium titanate nanoparticles (BT) with surface potential, polarization was performed at a voltage of 17 kV for 10 min, and the distance between the electrodes was 1.8 cm.

[0043] (3) Preparation of piezoelectric HAMA / BT microspheres:

[0044] Hydrogel microspheres were prepared using microfluidic technology, and the flow rate ratio of the dispersed phase to the continuous phase was 1:23.

[0045] BT and HAMA solutions (4 wt%) were prepared into mixed solutions in different mass volume ratios (0.0 wv%, 0.5 wv%, 1.0 wv%, 2.0 wv% and 4.0 wv%), and then the solutions were mixed with 0.5 wt% photoinitiator as dispersed phases. The above solutions were used as dispersed phases.

[0046] Span 80 and paraffin oil were mixed at a ratio of 5 wt% as the continuous phase.

[0047] The dispersed phase and the continuous phase are pushed into the channels of the microfluidic device by syringe pumps respectively.

[0048] After the collected microspheres were frozen at -40°C, they were irradiated with ultraviolet light at a wavelength of 365 nm for 10 min to promote cross-linking of the microspheres.

[0049] Subsequently, the microspheres were washed repeatedly in PBS after removing paraffin oil and surfactant with anhydrous ether.

[0050] (4) Preparation of piezoelectric microspheres with stem cell recruitment function:

[0051] Dissolve dopamine hydrochloride in Tris-HCl to prepare a 2 mg / ml polydopamine solution.

[0052] The prepared microspheres were completely immersed in the above solution and incubated at room temperature for 1 day.

[0053] Subsequently, the microspheres were rinsed repeatedly with sterile deionized water to obtain Piezo@pDA microspheres for subsequent experiments.

[0054] (5) Activation and functionalization of microspheres:

[0055] Piezo@pDA microspheres were first activated with PBS containing 0.15 M NaCl.

[0056] Subsequently, the Piezo@pDA microspheres were incubated with Tris buffer solution containing or not containing 100 ug / mL E7 (EPLQLKM, purity 95%, purchased from Beijing Kexing Yaguang Biotechnology Co., Ltd.) at 4°C for 24 h.

[0057] The modified microspheres were collected to obtain piezoelectric microspheres Piezo@pDA@E7 with stem cell recruitment function.

[0058] The mixture was washed repeatedly with sterile deionized water for 5 times. According to the previous experimental results, the BT ratio of Piezo@pDA@E7 used was 2.0 wv%.

[0059] The electromechanical hydrogel microspheres prepared in the present application can, on the one hand, induce the directional aggregation of specific types of stem cells, and on the other hand, can sensitively respond to external mechanical stimulation to generate weak electrical stimulation of up to 451mV to promote stem cell differentiation.

[0060] Figure 2 The flow chart of the preparation and characterization of injectable piezoelectric hydrogel microspheres. The AK diagram illustrates the preparation process, structural characteristics, piezoelectric properties, electrical signal response and peptide release behavior of Piezo@pDA@E7 microspheres under ultrasound stimulation, thus verifying the potential application of this microsphere in precise cell electrical stimulation therapy. Figure 3 Recruiting cells with functionalized piezoelectric hydrogel microspheres for injectable stem cell recruitment. This figure verifies the effect of injectable stem cell recruitment functionalized piezoelectric hydrogel microspheres (Piezo@pDA@E7) in promoting the migration, adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs), and demonstrates the mechanism of peptide release under ultrasound stimulation. Together, these images and data support the potential application of microspheres in cell recruitment and tissue repair, and provide experimental evidence for the effectiveness of microspheres as a new precision cell electrical stimulation therapy tool. Figure 4 Functionalized piezoelectric hydrogel microspheres for injectable stem cell recruitment promote chondrogenic differentiation of bone marrow mesenchymal stem cells. This figure verifies that Piezo@pDA@E7 microspheres provide comprehensive experimental data and image evidence in promoting chondrogenic differentiation of BMSCs. Figure 7 This indicates that the microspheres have no toxicity to major internal organs after in vivo injection.

[0061] In in vitro experiments, electromechanical hydrogel microspheres can induce directional migration of stem cells, and on the 7th day of co-culture, they are almost 100% covered on the surface of the microspheres. Experiments such as PCR and WB have shown that the content of cartilage-specific markers, such as type 2 collagen and glycosaminoglycans, has been significantly increased. In in vivo experiments, in the rabbit osteochondral defect model, electromechanical hydrogel microspheres enhanced cartilage regeneration at the lesion site, close to natural cartilage, and this conclusion was confirmed by both Micro-CT and histological evaluations. In the rat OA model, cartilage destruction was alleviated and osteophyte proliferation was reduced. Gait analysis, pain threshold test and motion trajectory monitoring showed that the behavior of rats was significantly improved. In addition, the results of histological sections showed that the structure of cartilage was significantly improved.

[0062] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for preparing electromechanical hydrogel microspheres for precise cell electrical stimulation, characterized in that: The following steps are involved: (1) Add methacrylic anhydride to a 2 wt % hyaluronic acid aqueous solution, then add 5 M NaOH solution at a flow rate of 10 ml / h, and place the reaction system in an ice-water bath overnight; centrifuge to obtain the supernatant, dialyze for 3 days, and freeze-dry to obtain methacryloyl hyaluronic acid (HAMA); (2) HAMA was mixed with modified barium titanate nanoparticles (BT) to prepare a mixed solution with a total concentration of 4 wt%, and then the mixed solution was mixed with a photoinitiator as a dispersed phase; Span 80 and paraffin oil were mixed as a continuous phase; (3) Microspheres were prepared using microfluidic technology, where the flow rate ratio of the dispersed phase to the continuous phase was 1:23; (4) After the collected microspheres were frozen at -40°C, they were irradiated with 365 nm ultraviolet light for 10 min to promote cross-linking of the microspheres. The microspheres were then removed of paraffin oil and surfactant using anhydrous ether. The microspheres were then immersed in PBS and rinsed repeatedly. (5) Soak the microspheres in a polydopamine solution, incubate at room temperature for 1 day, and repeatedly rinse with sterile deionized water to obtain microspheres Piezo@pDA; (6) Activate the microspheres and incubate them with Tris buffer solution containing E7 peptide at 4°C for 24 h. Collect the modified microspheres to obtain Piezo@pDA@E7 hydrogel microspheres.

2. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (2), the barium titanate nanoparticle modification treatment is performed by polarization at a voltage of 17 kV for 10 minutes, with an inter-electrode distance of 1.8 cm.

3. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (2), in a mixed solution with a total concentration of 4 wt %, the mass volume ratio of the barium titanate nanoparticles to the HAMA is 0.0 wv %-4.0 wv %; and the concentration of the photoinitiator is 0.5 wt % of the total mass of the dispersed phase.

4. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (2), the mass volume ratio of the barium titanate nanoparticles to the HAMA is 0.0 wv%, 0.5 wv%, 1.0 wv%, 2.0 wv%, and 4.0 wv%.

5. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (2), the continuous phase is a mixture of Span 80 and paraffin oil at a ratio of 5 wt%.

6. The method for preparing electromechanical hydrogel microspheres according to claim 1, characterized in that: In step (5), the polydopamine solution is prepared by dissolving dopamine hydrochloride in Tris-HCL to prepare a 2 mg / ml polydopamine solution.

7. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (6), the microspheres are activated with PBS containing 0.15 M NaCl.

8. The method for preparing electrodynamic hydrogel microspheres according to claim 1, characterized in that: In step (6), the concentration of E7 is 100 ug / mL.

9. Electromechanical hydrogel microspheres prepared according to the preparation method described in any one of claims 1 to 8.

10. Application of the electromechanical hydrogel microspheres prepared by the preparation method according to any one of claims 1 to 8 in cell culture, tissue regeneration, drug delivery and biosensors.

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