Mof modified myocardial cell material, preparation method and application thereof, and biomimetic driver

Abstract

The application belongs to the field of biomedical engineering and bionic robot technology, and particularly relates to a MOF modified myocardial cell material, a preparation method and application thereof, and a bionic driver. The MOF modified myocardial cell material provided by the application comprises myocardial cells and MOF material anchored on the cell membrane surface of the myocardial cells through coordination bonds, and the MOF material comprises UiO-66(Zr) and / or functionalized UiO-66(Zr), wherein the functionalized UiO-66(Zr) comprises aminated UiO-66(Zr) and / or nitrated UiO-66(Zr). The MOF modified myocardial cell material provided by the application realizes precise regulation of the calcium ion dynamics and contraction performance of myocardial cells, can significantly accelerate the contraction frequency of myocardial cells while improving the contraction force and mechanical stiffness of myocardial cells, and is crucial for constructing a high-performance and sustainable bionic driver.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and biomimetic robotics technology, specifically relating to MOF-modified cardiomyocyte materials, their preparation methods and applications, and biomimetic actuators. Background Technology

[0002] Cardiac cardiomyocytes possess the property of autonomous contraction, making them an ideal power source for developing bio-hybrid actuators. Their contraction process is regulated by intracellular calcium ions (Ca). 2+ Precise dynamic control of calcium ion concentration is crucial. Currently, the main approach to intervene in cardiomyocyte contraction is through pharmacological means of regulating calcium ion flow. However, these methods lack spatial specificity, are prone to off-target effects, and are difficult to achieve precise and localized control of contractile behavior.

[0003] Therefore, current bio-hybrid actuators based on cardiomyocytes (such as bionic fish) face key challenges such as inconsistent contractile force output and rapid functional decline under physiological stress. The root cause lies in the difficulty of simultaneously enhancing the structural integrity of cells and regulating local calcium ion flow. Summary of the Invention

[0004] The purpose of this invention is to provide MOF-modified cardiomyocyte materials, their preparation methods and applications, and biomimetic actuators. The MOF-modified cardiomyocyte materials provided by this invention achieve precise control over the dynamics of calcium ions and contractile properties of cardiomyocytes, and can significantly accelerate the contraction frequency of cardiomyocytes while improving the contractile force and mechanical stiffness of cardiomyocytes, which is crucial for constructing high-performance, sustainable biomimetic actuators.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a MOF-modified cardiomyocyte material, comprising cardiomyocytes and MOF material anchored to the cell membrane surface of the cardiomyocytes via coordination bonds. The MOF material comprises UiO-66(Zr) and / or functionalized UiO-66(Zr), wherein the functionalized UiO-66(Zr) comprises aminated UiO-66(Zr) and / or nitrated UiO-66(Zr).

[0007] This invention provides a method for preparing MOF-modified cardiomyocyte material as described in the above technical solution, comprising the following steps:

[0008] MOF materials were dispersed in a culture medium solution to obtain MOF suspensions;

[0009] The MOF suspension and cardiomyocytes were mixed and co-cultured to obtain the MOF-modified cardiomyocyte material.

[0010] Preferably, the preparation method of the MOF material includes the following steps:

[0011] The raw materials for preparing MOF materials are mixed to obtain a mixture. The raw materials for preparing MOF materials include inorganic zirconium salt and terephthalic acid, or the raw materials for preparing MOF materials include inorganic zirconium salt and functionalized terephthalic acid, wherein the functionalized terephthalic acid includes 2-aminoterephthalic acid or 2-nitroterephthalic acid.

[0012] The mixture is reacted to obtain the MOF material.

[0013] Preferably, the reaction temperature is 120~140℃ and the time is 12~24h.

[0014] Preferably, the culture medium solution comprises serum-free culture medium or complete culture medium.

[0015] Preferably, the mass concentration of MOF material in the MOF suspension is 0.1~3.0 mg / mL.

[0016] This invention provides the application of the MOF-modified cardiomyocyte material described in the above-mentioned technical solution or the MOF-modified cardiomyocyte material described in the above-mentioned technical solution in a biomimetic actuator.

[0017] Preferably, the biomimetic actuator is a bio-hybrid actuator based on cardiomyocytes.

[0018] Preferably, the biomimetic actuator is a biomimetic fish actuator.

[0019] The present invention provides a biomimetic actuator, the biomimetic actuator including a driving unit, the driving unit being the MOF-modified cardiomyocyte material described in the above technical solution or the MOF-modified cardiomyocyte material described in the above technical solution.

[0020] This invention provides a MOF-modified cardiomyocyte material, comprising cardiomyocytes and MOF material anchored to the cell membrane surface of the cardiomyocytes via coordination bonds. The MOF material comprises UiO-66(Zr) and / or functionalized UiO-66(Zr), wherein the functionalized UiO-66(Zr) comprises aminated UiO-66(Zr) and / or nitrated UiO-66(Zr). In this invention, the MOF material is adsorbed onto the cell membrane surface of the cardiomyocytes via chemical bonding. This invention utilizes MOF material adsorbed and bonded to the cell membrane surface of cardiomyocytes to form a functional ion-regulating barrier on the cell membrane surface, significantly accelerating the contraction frequency of cardiomyocytes while enhancing cell contractility and mechanical stiffness.

[0021] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0022] This invention successfully utilizes MOF materials (UiO-66(Zr) and / or functionalized UiO-66(Zr)) to achieve a synergistic enhancement of cardiomyocyte contraction frequency and contractile force, with a contraction frequency increase of over 100% and a Young's modulus increase of over 15%.

[0023] The MOF-modified cardiomyocyte material provided by this invention, when applied to a biomimetic actuator, can successfully translate the enhancement effect at the cellular level into the macroscopic biomimetic actuator, thereby comprehensively and significantly improving the swimming performance (frequency, amplitude, thrust, and power) of the biomimetic fish.

[0024] This invention provides a method for preparing MOF-modified cardiomyocyte material as described in the above-mentioned technical solution, comprising the following steps: dispersing MOF material in a culture medium solution to obtain a MOF suspension; mixing the MOF suspension with cardiomyocytes for co-culturing to obtain the MOF-modified cardiomyocyte material. The preparation method provided by this invention is also a method for regulating the contractile properties of cardiomyocytes. The MOF-modified cardiomyocyte material obtained by the preparation method provided by this invention offers an innovative technical path for constructing high-performance bio-hybrid robots. Attached Figure Description

[0025] Figure 1 X-ray diffraction patterns of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments of the present invention;

[0026] Figure 2 The nitrogen adsorption-desorption test results of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared for the embodiments of the present invention;

[0027] Figure 3 Fourier transform infrared spectra of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared for embodiments of the present invention;

[0028] Figure 4 Fluorescence inverted microscope images of cardiomyocytes and MOF-modified cardiomyocyte products prepared for embodiments of the present invention;

[0029] Figure 5 Scanning electron microscope (SEM) images of cardiomyocytes and MOF-modified cardiomyocyte products prepared for embodiments of the present invention;

[0030] Figure 6Atomic force microscopy (AFM) images of cardiomyocytes and MOF-modified cardiomyocyte products prepared for embodiments of the present invention;

[0031] Figure 7 EDS spectra of SDCM and SDCM@U prepared in embodiments of the present invention;

[0032] Figure 8 The results of the CCK-8 cytotoxicity assay for UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared for the embodiments of the present invention are shown in the figure.

[0033] Figure 9 The image shows the AFM assay results of SDCM, SDCM@U, and SDCM@UC prepared according to embodiments of the present invention.

[0034] Figure 10 Characterization results of spontaneous shrinkage recorded using AFM fixed probes for SDCM@U and SDCM@UC prepared in the embodiments of the present invention;

[0035] Figure 11 The SDCM@U and SDCM@UC prepared for embodiments of the present invention are characterized by the results of contraction-calcium transient-action potential under electrical stimulation (1 Hz) synchronously recorded using an IonOptix system;

[0036] Figure 12 Featured swing frames of biomimetic fish, Fish@U and Fish@UC, prepared for embodiments of the present invention;

[0037] Figure 13 The tail wagging states of Fish, Fish@U, and Fish@UC biomimetic fish prepared according to embodiments of the present invention;

[0038] Figure 14 The thrust diagrams of Fish, Fish@U, and Fish@UC prepared for embodiments of the present invention are calculated based on a fluid dynamics model (Morison equations);

[0039] Figure 15 The power diagrams of Fish, Fish@U, and Fish@UC prepared for embodiments of the present invention are calculated based on a fluid dynamics model (Morison equation). Detailed Implementation

[0040] This invention provides a MOF-modified cardiomyocyte material, comprising cardiomyocytes and MOF material anchored to the cell membrane surface of the cardiomyocytes via coordination bonds. The MOF material comprises UiO-66(Zr) and / or functionalized UiO-66(Zr), wherein the functionalized UiO-66(Zr) comprises aminated UiO-66(Zr) and / or nitrated UiO-66(Zr).

[0041] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0042] The MOF-modified cardiomyocyte material provided by this invention includes cardiomyocytes.

[0043] The MOF-modified cardiomyocyte material provided by this invention comprises MOF material anchored to the cell membrane surface of cardiomyocytes via coordination bonds. In this invention, the MOF material comprises UiO-66(Zr) and / or functionalized UiO-66(Zr), wherein the functionalized UiO-66(Zr) comprises aminated UiO-66(Zr) and / or nitrated UiO-66(Zr).

[0044] In this invention, metal-organic framework (MOF) materials have the advantages of adjustable porosity, large specific surface area, and convenient surface functionalization. The UiO-66(Zr) and / or functionalized UiO-66(Zr) in this invention exhibit excellent water stability and chemical stability.

[0045] This invention uses UiO-66(Zr) and / or functionalized UiO-66(Zr) as a "cardiomyocyte membrane barrier" to directly regulate ion transport at the cardiomyocyte membrane interface, while also enhancing the mechanical properties of cardiomyocytes, thus facilitating the construction of a high-performance, sustainable biohybrid drive system.

[0046] This invention provides a method for preparing MOF-modified cardiomyocyte material as described in the above technical solution, comprising the following steps:

[0047] MOF materials were dispersed in a culture medium solution to obtain MOF suspensions;

[0048] The MOF suspension and cardiomyocytes were mixed and co-cultured to obtain the MOF-modified cardiomyocyte material.

[0049] This invention disperses MOF materials in a culture medium solution to obtain a MOF suspension.

[0050] In this invention, the method for preparing the MOF material preferably includes the following steps:

[0051] The raw materials for preparing MOF materials are mixed to obtain a mixture. The raw materials for preparing MOF materials include inorganic zirconium salt and terephthalic acid, or the raw materials for preparing MOF materials include inorganic zirconium salt and functionalized terephthalic acid, wherein the functionalized terephthalic acid includes 2-aminoterephthalic acid or 2-nitroterephthalic acid.

[0052] The mixture is reacted to obtain the MOF material.

[0053] This invention involves mixing the raw materials for preparing MOF materials to obtain a mixture. In this invention, the inorganic zirconium salt can be zirconium oxychloride, and in the examples, it can be zirconium oxychloride octahydrate (ZrOCl2·8H2O). The molar ratio of the inorganic zirconium salt to the terephthalic acid is preferably 1:1. The molar ratio of the inorganic zirconium salt to the functionalized terephthalic acid is preferably 1:1. The mixing is preferably performed by grinding. The grinding time is preferably 5-20 minutes, ensuring that the raw materials for preparing the MOF material are thoroughly ground and mixed uniformly.

[0054] After obtaining the mixture, the present invention reacts the mixture to obtain the MOF material. In the present invention, the reaction is preferably carried out in a high-pressure reactor lined with polytetrafluoroethylene. The reaction is preferably carried out under closed conditions. The reaction temperature is preferably 120-140°C, and in the examples, it can be 130°C. The reaction time is preferably 12-24 hours, and in the examples, it can be 12 hours. After the reaction is completed, a solid product is obtained; the solid product is then washed and dried sequentially to obtain the MOF material powder. The washing reagent is preferably anhydrous ethanol. The washing temperature is preferably 60-65°C. The washing is preferably centrifugal washing. The drying is preferably vacuum drying, and the vacuum drying temperature is preferably 130-150°C.

[0055] In this invention, the culture medium solution preferably comprises serum-free culture medium or complete culture medium. The serum-free culture medium can be fresh serum-free culture medium. The complete culture medium can be fresh complete culture medium. Preferably, the MOF material is pre-sterilized, and then the pre-sterilized MOF material is dispersed in the culture medium solution to obtain a MOF suspension. In this invention, the dispersion is preferably carried out under ultrasonic conditions. This invention does not have special requirements for the specific parameters of the ultrasonic treatment, as long as the MOF material is uniformly dispersed.

[0056] In this invention, the mass concentration of MOF material in the MOF suspension is preferably 0.1~3.0 mg / mL, and in the examples it can be 1 mg / mL.

[0057] The MOFs suspension was obtained, and the present invention mixed the MOFs suspension with cardiomyocytes for co-culture to obtain the MOF-modified cardiomyocyte material.

[0058] In this invention, the cardiomyocytes are preferably obtained by seeding enriched primary cardiomyocytes (SDCM) into cell culture plates or dishes and culturing them using complete culture medium. The enriched primary cardiomyocytes (SDCM) are derived from Sprague-Dawley (SD) rat sucklings. This invention does not have special requirements for the enrichment process. The preferred seeding density of the enriched primary cardiomyocytes is 5 × 10⁻⁶. 4 The cells / mL. The complete culture medium is preferably DMEM, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The culture temperature is preferably 37°C. The culture is preferably carried out in a 5% CO2 saturated humidity incubator. After the cells have adhered and grown (24 hours later), the old culture medium is discarded to obtain cardiomyocytes.

[0059] In this invention, the MOF suspension is mixed with cardiomyocytes to ensure that the MOF suspension covers the surface of the cardiomyocytes.

[0060] In this invention, the co-culturing temperature is preferably 37°C. The co-culturing is preferably carried out in a saturated humidity incubator with 5% CO2.

[0061] In this invention, after the co-culture is completed, the cell products are preferably washed gently with PBS buffer 2-3 times to remove unbound MOF particles and obtain MOF-modified cardiomyocyte material.

[0062] This invention provides the application of the MOF-modified cardiomyocyte material described in the above-mentioned technical solution or the MOF-modified cardiomyocyte material described in the above-mentioned technical solution in a biomimetic actuator.

[0063] In this invention, the biomimetic actuator is preferably a bio-hybrid actuator based on cardiomyocytes, which can be a biomimetic fish actuator. The biomimetic actuator provided by this invention is a biomimetic fish, and the tail drive unit of the biomimetic fish is composed of MOF-modified cardiomyocyte material as described in the above technical solution, thereby obtaining higher tail fin oscillation frequency, oscillation amplitude, thrust, and power output.

[0064] This invention provides a biomimetic actuator, which includes a driving unit. The driving unit is either the MOF-modified cardiomyocyte material described in the above-described technical solution or the MOF-modified cardiomyocyte material described in the above-described technical solution. When the biomimetic actuator is a biomimetic fish, the driving unit is located at the tail of the biomimetic fish.

[0065] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] (1) This embodiment provides a method for preparing UiO-66(Zr), using zirconium oxychloride octahydrate (ZrOCl2·8H2O) as the metal source and terephthalic acid as the organic ligand, specifically including the following steps:

[0068] 483 mg of zirconium oxychloride octahydrate (ZrOCl2·8H2O) and 249 mg of terephthalic acid were placed in an agate mortar at a molar ratio of 1:1 and ground thoroughly for 10 minutes to achieve a homogeneous mixture. The ground mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in an oven at 130 °C for 12 hours. After the reaction was completed, the reactor was allowed to cool to room temperature, and the product was removed. The product was washed repeatedly by centrifugation with anhydrous ethanol at 60 °C. Finally, the product was vacuum-dried overnight at 150 °C to obtain pure UiO-66(Zr) powder.

[0069] (2) Co-culture of cardiomyocytes and MOFs

[0070] Primary cardiomyocyte (SDCM) isolation: Hearts were aseptically removed from neonatal (1-3 day old) Sprague-Dawley (SD) rats. The hearts were washed with pre-cooled PBS buffer to remove blood. Cardiomyocytes were dissociated using a mixed enzyme solution of trypsin and collagenase II through multiple stepwise digestion. The digestion fluid containing cells was collected, and non-cardiomyocytes such as fibroblasts were removed using differential adhesion (cell suspension was incubated in an incubator for 1-2 hours) to obtain enriched primary cardiomyocytes (SDCM).

[0071] Cell culture and MOF co-culture: The isolated SDCM cells were cultured at an appropriate density (5 × 10⁶ cells / year). 4MOFs (microcardiomyocytes / mL) were seeded in cell culture plates or dishes and cultured in complete medium (DMEM, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2 in a saturated humidity incubator. After the cells adhered and grew (24 hours later), the old medium was discarded. Pre-sterilized UiO-66(Zr) powder was dispersed in fresh serum-free or complete medium and sonicated to ensure uniform dispersion, preparing a MOF suspension with a concentration of 1.0 mg / mL. This MOF suspension was added to the cultured cells, ensuring that the cell surface was covered. Co-culture was continued for 48 hours. After co-culture, the cells were gently washed 2-3 times with PBS buffer to remove loosely bound MOF particles, yielding MOF-modified cardiomyocyte material, i.e., UiO-66(Zr)-modified cardiomyocyte material, denoted as SDCM@U.

[0072] Comparative Example 1

[0073] (1) This comparative example provides a method for preparing carboxylated UiO-66(Zr), using zirconium oxychloride octahydrate (ZrOCl2·8H2O) as the metal source and 1,2,4-benzenetricarboxylic acid as the functionalizing ligand, specifically including the following steps:

[0074] 483 mg of zirconium oxychloride octahydrate (ZrOCl2·8H2O) and 315 mg of 1,2,4-benzenetricarboxylic acid were placed in an agate mortar at a molar ratio of 1:1 and ground thoroughly for 10 minutes to achieve a homogeneous mixture. The ground mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in an oven at 130 °C for 12 hours. After the reaction was completed, the reactor was allowed to cool to room temperature, and the product was removed. The product was washed repeatedly by centrifugation with anhydrous ethanol at 60 °C. Finally, the product was vacuum-dried overnight at 150 °C to obtain pure nitrated UiO-66(Zr) powder, i.e., UiO-66(Zr)-COOH.

[0075] (2) Co-culture of cardiomyocytes and MOFs

[0076] The method for obtaining enriched primary cardiomyocytes (SDCM) is the same as in Example 1.

[0077] Cell culture and MOF co-culture: The isolated SDCM cells were cultured at an appropriate density (5 × 10⁶ cells / year). 4MOFs (cells / mL) were seeded in cell culture plates or dishes and cultured in complete medium (DMEM, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2 in a saturated humidity incubator. After the cells adhered and grew (24 hours later), the old medium was discarded. Pre-sterilized UiO-66(Zr)-COOH powder was dispersed in fresh serum-free or complete medium and sonicated to ensure uniform dispersion, preparing a MOF suspension with a concentration of 1.0 mg / mL. This MOF suspension was added to the cultured cells, ensuring that the cell surface was covered. Co-culture was continued for 48 hours. After co-culture, the cells were gently washed 2-3 times with PBS buffer to remove loosely bound MOF particles, yielding MOF-modified cardiomyocyte material, i.e., UiO-66(Zr)-COOH-modified cardiomyocyte material, denoted as SDCM@UC.

[0078] Example 2

[0079] (1) This embodiment provides a method for preparing aminated UiO-66(Zr), using zirconium oxychloride octahydrate (ZrOCl2·8H2O) as the metal source and 2-aminoterephthalic acid as the functionalized ligand, specifically including the following steps:

[0080] 483 mg of zirconium oxychloride octahydrate (ZrOCl2·8H2O) and 272 mg of 2-aminoterephthalic acid were placed in an agate mortar at a molar ratio of 1:1 and ground thoroughly for 10 minutes to achieve a homogeneous mixture. The ground mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in an oven at 130 °C for 12 hours. After the reaction was completed, the reactor was allowed to cool to room temperature, and the product was removed. The product was washed repeatedly by centrifugation with anhydrous ethanol at 60 °C. Finally, the product was vacuum-dried overnight at 150 °C to obtain pure aminated UiO-66(Zr) powder, i.e., UiO-66(Zr)-NH2.

[0081] The remaining preparation of UiO-66(Zr)-NH2 modified cardiomyocyte material is the same as in Example 1, except that the UiO-66(Zr) used in Example 1 is replaced with UiO-66(Zr)-NH2 in this example.

[0082] Example 3

[0083] (1) This embodiment provides a method for preparing nitrated UiO-66(Zr), using zirconium oxychloride octahydrate (ZrOCl2·8H2O) as the metal source and 2-nitroterephthalic acid as the functionalized ligand, specifically including the following steps:

[0084] 483 mg of zirconium oxychloride octahydrate (ZrOCl2·8H2O) and 317 mg of 2-nitroterephthalic acid were placed in an agate mortar at a molar ratio of 1:1 and ground thoroughly for 10 minutes to achieve a homogeneous mixture. The ground mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor, sealed, and placed in an oven at 130 °C for 12 hours. After the reaction was completed, the reactor was allowed to cool to room temperature, and the product was removed. The product was washed repeatedly by centrifugation with anhydrous ethanol at 60 °C. Finally, the product was vacuum-dried overnight at 150 °C to obtain pure nitrated UiO-66(Zr) powder, i.e., UiO-66(Zr)-NO2.

[0085] The remaining preparation of UiO-66(Zr)-NO2 modified cardiomyocyte material is the same as in Example 1, except that the UiO-66(Zr) used in Example 1 is replaced with UiO-66(Zr)-NO2 in this example.

[0086] Example 4

[0087] Fabrication of micropatterned gelatin films: A silicon master mold with a micron-scale linear groove structure (feature dimensions: ridge width 25 μm, groove width 4 μm, groove depth 5 μm) was prepared using soft photolithography. Polydimethylsiloxane (PDMS) prepolymer was cast onto the silicon master mold, cured, and then demolded to obtain a PDMS stamp with complementary microstructures.

[0088] 0.2 g of gelatin was dissolved in 1 mL of PBS solution and heated in a water bath at 65°C for 30 minutes to obtain a 20% w / v gelatin PBS solution. 0.08 g of microbial transglutaminase crosslinking agent was dissolved in 1 mL of PBS solution and heated in a water bath at 37°C for 30 minutes to obtain an 8% w / v microbial transglutaminase PBS solution. The 20% w / v gelatin PBS solution was poured into the 8% w / v microbial transglutaminase PBS solution and stirred for 10 minutes to prepare a mixed solution. 0.5 mL of the mixed solution was dropped onto the patterned surface of a PDMS stamp, and then another PDMS stamp was placed with its patterned surface facing each other, applying slight pressure to uniformly fill the microcavities with the gelatin solution. Crosslinking and curing were performed at 25°C and 65% humidity. Finally, the stamps were carefully separated to obtain a micropatterned gelatin film with a bilaterally symmetrical microgroove structure and a thickness of approximately 0.38 mm.

[0089] Bionic Fish Assembly: Following the fish-like structure design proposed by Parker et al. (Lee KY, Park SJ, Matthews DG, et al. An autonomously swimming biohybrid fish designed with human cardiac biophysics[J]. Science, 2022, 375(6581): 639-647.), a passive support system for the bionic fish was constructed. This system includes a rigid paper layer at the front (for providing anchoring support), a flexible gelatin film at the rear (as a deformable matrix for the tail), and lightweight plastic fins (for adjusting buoyancy and motion stability).

[0090] Construction and Integration of the Driving Unit: The cell suspensions of SDCM, SDCM@U, and SDCM@UC prepared in step (2) of Example 1 were seeded onto both sides of a micropatterned gelatin film and cultured statically in an incubator to promote cell adhesion. Under the contact guidance of the microgroove structure, the cardiomyocytes oriented along a preset direction. After several days of culture, the cells self-organized on both sides of the film to form anisotropic double-layered cardiomyocyte tissue, simulating the natural cardiomyocyte structure. The driving unit was integrated with the passive support system and assembled into biomimetic fish samples. The biomimetic fish samples prepared by SDCM, SDCM@U, and SDCM@UC were designated as Fish, Fish@U, and Fish@UC, respectively. During spontaneous contraction, the synchronous beating of the cardiomyocytes on both sides can drive the tail to produce periodic bending-extension movements, thereby realizing fish-like swimming behavior.

[0091] Test case

[0092] Figure 1 The X-ray diffraction patterns of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments and comparative examples of the present invention are shown. Figure 1 X-ray diffraction (XRD) confirmed that the crystal structures of the synthesized materials in the examples and comparative examples conformed to the standard UiO-66 (Zr).

[0093] Figure 2 The nitrogen adsorption-desorption test results are shown for UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments and comparative examples of this invention. Figure 2The specific surface area and pore size distribution of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH were determined by nitrogen adsorption-desorption test, confirming that the UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments and comparative examples of the present invention are microporous materials.

[0094] Figure 3 Fourier transform infrared spectra of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments and comparative examples of the present invention. Figure 3 The successful introduction of the functional groups -NH2, -NO2, and -COOH into UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH was confirmed by Fourier transform infrared spectroscopy (FT-IR).

[0095] Figure 4 Fluorescence inverted microscope image of MOF-modified cardiomyocyte products prepared for embodiments of the present invention. Figure 5 Scanning electron microscope (SEM) image of the MOF-modified cardiomyocyte product prepared for an embodiment of the present invention. Figure 6 Atomic force microscopy (AFM) images of MOF-modified cardiomyocyte products prepared for embodiments of the present invention. Figure 4 (a) Figure 5 (a) and Figure 6 (a) shows the results of fluorescence inverted microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) of the SDCM, respectively. Figure 4 (b) Figure 5 (b) and Figure 6 (b) shows the fluorescence inverted microscope, scanning electron microscope (SEM), and atomic force microscope (AFM) results for SDCM@U, respectively. Figure 4 (c) Figure 5 (c) and Figure 6 (c) shows the results of fluorescence inverted microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) of SDCM@UC, respectively. Figure 4 , Figure 5 and Figure 6 Observations using inverted fluorescence microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) revealed that MOF particles were stably attached to the cell membrane surface, and the cell surface roughness was significantly increased.

[0096] Figure 7 (a) in the figure is the EDS energy spectrum of the SDCM prepared in the embodiment of the present invention. Figure 7 (b) is the EDS energy spectrum of SDCM@U prepared in the embodiment of the present invention. Figure 7 The successful binding of MOFs was confirmed by detecting zirconium (Zr) signals on the surface of SDCM@U cells using energy-dispersive X-ray spectroscopy (EDS).

[0097] Figure 8 The figure shows the CCK-8 cytotoxicity test results of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, and UiO-66(Zr)-COOH prepared in the embodiments and comparative examples of the present invention. Figure 8 The CCK-8 cytotoxicity assay showed that at a concentration of 1.2 mg / mL, the material had no significant effect on cell viability. Figure 8 The experimental method is as follows: SDCM is prepared at 5×10⁻⁶ ppm per well. 3 Cells were seeded at a density of 100 μL of complete culture medium in 96-well plates. After 24 hours of culture to allow cell adhesion, the cells were exposed to a series of concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, and 3.0 mg / mL) of UiO-66(Zr), UiO-66(Zr)-NH2, UiO-66(Zr)-NO2, or UiO-66(Zr)-COOH) for 24 hours. Control groups included untreated cells (negative control) and a blank control containing only culture medium (blank control). After treatment, 10 μL of LCK-8 reagent was added to each well, gently vortexed, and then incubated at 37°C in the dark for 1 hour. Absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader (USA), with a reference wavelength of 650 nm. Cell viability was calculated using the following formula: Cell viability (%) = [(A_sample - A_blank) / (A_control - A_blank)] × 100%. Where A_sample, A_control, and A_blank represent the absorbance values ​​of the treated well, negative control well, and blank control well, respectively. All data are expressed as the mean ± standard deviation (SD) of three independent experiments.

[0098] Figure 9 The AFM assay results of SDCM, SDCM@U, and SDCM@UC prepared for embodiments of the present invention are shown in the figure. Figure 9 Figures (a) and (d) in the diagram show the results of AFM assay for cell mechanical properties using SDCM. Figure 9 Figures (b) and (e) in the figure show the AFM results of SDCM@U for detecting cell mechanical properties. Figure 9Figures (c) and (f) in the figure show the results of AFM detection of cell mechanical properties of SDCM@UC. Figure 9 Cell mechanical properties were assessed using AFM: Compared with the control group (SDCM), the Young's modulus of SDCM@U was significantly increased from 3007 Pa to 3469 Pa (an increase of 15.4%), while the increase in modulus of SDCM@UC was smaller.

[0099] Figure 10 Characterization results of spontaneous shrinkage recorded using AFM fixed probes for SDCM, SDCM@U, and SDCM@UC prepared for embodiments of the present invention. Figure 10 Spontaneous contractions were recorded using an AFM-fixed probe: the contraction frequency of SDCM@U increased from 0.77 Hz to 1.61 Hz (an increase of 109%), and the frequency of SDCM@UC increased to 1.44 Hz. However, the diastolic time of SDCM@UC was prolonged by 14.7%.

[0100] Figure 11 The SDCM, SDCM@U, and SDCM@UC prepared for embodiments of the present invention are characterized by the results of contraction-calcium transient-action potential under electrical stimulation (1 Hz) synchronously recorded using the IonOptix system. Figure 11 In this context, (a) represents the sarcomere contraction rate. Figure 11 (b) represents the time required for the sarcomere to contract to 90% of its peak value. Figure 11 (c) represents the change in calcium ion concentration. Figure 11 Simultaneous recording of systolic-calcium transient-action potentials under electrical stimulation (1 Hz) using the IonOptix system revealed significantly increased sarcomere shortening and calcium transient amplitudes in SDCM@U, with faster contraction. SDCM@UC exhibited the largest calcium transient amplitude, but no increase in sarcomere shortening amplitude and prolonged contraction time, indicating calcium overload and decreased systolic-diastolic coupling efficiency.

[0101] Figure 12 Feature swing frames of biomimetic fish, Fish@U and Fish@UC, prepared for embodiments of the present invention. Figure 12 (a) in the text is Fish. Figure 12 (b) in the text refers to Fish@U. Figure 12 (c) in the text refers to Fish@UC.

[0102] Figure 13 Analysis of the images of Fish, Fish@U, and Fish@UC biomimetic fish tail wagging patterns prepared for embodiments of the present invention shows that... Figure 13Analysis of biomimetic fish tail wagging images showed that the tail fin wagging frequency (0.36 Hz vs 0.29 Hz) and wagging amplitude (20.30° vs 18.90°) of Fish@U were higher than those of the control group Fish.

[0103] Figure 14 and Figure 15 The thrust and power diagrams of Fish, Fish@U, and Fish@UC prepared for embodiments of the present invention are calculated based on a fluid dynamics model (Morison equation). Figure 14 and Figure 15 Thrust and power were calculated based on the fluid dynamics model (Morison equation): Fish@U generated significantly higher average oscillating force (6.97 μN vs 4.64 μN) and average power output (21.8 nW vs 13.0 nW) than the control group, with a 50% increase in thrust and a 68% increase in power. In contrast, Fish@UC had lower power output than the control group due to impaired contraction force.

[0104] The above test results show that this invention successfully utilized UiO-66(Zr)MOFs to synergistically enhance the contraction frequency and force of cardiomyocytes, increasing the contraction frequency by over 100% and Young's modulus by over 15%. In contrast, while the carboxyl-functionalized UiO-66(Zr)-COOH prepared in Comparative Example 1 also accelerated the contraction frequency, it induced intracellular calcium ion overload, disrupting calcium homeostasis and consequently leading to decreased cardiomyocyte contractility and impaired diastolic function.

[0105] As demonstrated by the above embodiments, the MOF material in the MOF-modified cardiomyocyte material provided by this invention can adsorb onto the surface of the cardiomyocyte membrane, forming a functional ion regulation barrier. This significantly accelerates the contraction frequency of cardiomyocytes while enhancing the cell's contractile force and mechanical stiffness. This invention utilizes the designability of MOF materials, achieving precise control over the dynamics of calcium ions and contractile performance of cardiomyocytes through the selection of functional groups. The MOF material in the MOF-modified cardiomyocyte material of this invention has a dual function: it acts as both a "mechanical enhancer" and an "ion regulator," enhancing cell membrane rigidity and optimizing calcium transients. Applying the MOF-modified cardiomyocyte material provided by this invention to a biomimetic fish actuator effectively translates the performance improvements at the cellular level into a leap in the motion performance of macroscopic devices, significantly improving the actuator's frequency, force, and efficiency.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of MOF-modified cardiomyocyte material in the fabrication of biomimetic actuators, characterized in that, The MOF-modified cardiomyocyte material includes cardiomyocytes and MOF material anchored to the cell membrane surface of the cardiomyocytes via coordination bonds, wherein the MOF material is UiO-66(Zr).

2. The application according to claim 1, characterized in that, The preparation method of the MOF-modified cardiomyocyte material includes the following steps: MOF materials were dispersed in a culture medium solution to obtain MOF suspensions; The MOF suspension and cardiomyocytes were mixed and co-cultured to obtain the MOF-modified cardiomyocyte material.

3. The application according to claim 2, characterized in that, The preparation method of the MOF material Includes the following steps: The raw materials for preparing MOF materials are mixed to obtain a mixture, wherein the raw materials for preparing MOF materials include inorganic zirconium salt and terephthalic acid; The mixture is reacted to obtain the MOF material.

4. The application according to claim 3, characterized in that, The reaction is carried out at a temperature of 120-140°C for 12-24 hours.

5. The application according to claim 2, characterized in that, The culture medium solution includes serum-free culture medium or complete culture medium.

6. The application according to claim 2 or 5, characterized in that, The mass concentration of MOF material in the MOF suspension is 0.1~3.0 mg / mL.

7. The application according to claim 1, characterized in that, The biomimetic actuator is a bio-hybrid actuator based on cardiomyocytes.

8. The application according to claim 1 or 7, characterized in that, The biomimetic actuator is a biomimetic fish actuator.

9. A bionic actuator, characterized in that, The biomimetic actuator includes a driving unit, which is a MOF-modified cardiomyocyte material. The MOF-modified cardiomyocyte material includes cardiomyocytes and MOF material anchored to the cell membrane surface of the cardiomyocytes via coordination bonds. The MOF material is UiO-66 (Zr).

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