A piezoelectric hydrogel loaded with a response polypeptide and application thereof

Abstract

This invention relates to the field of peptide drugs, specifically to the field of ultrasound physical field-mediated regulation of biological signals and bone tissue repair technology, and more specifically to a piezoelectric hydrogel loaded with responsive peptides and its application. The application is based on a biological signal regulation method of ultrasound-induced responsive peptides LLPS and its application in promoting bone differentiation. It achieves spatiotemporal specific activation of biological signal pathways through precise regulation mediated by physical fields.

Description

Technical Field

[0001] This invention belongs to the field of ultrasound physical field regulation of biological signals and bone tissue repair technology. Specifically, it relates to a piezoelectric hydrogel loaded with responsive peptides and its biological signal regulation method based on ultrasound-induced responsive peptides LLPS and its application in promoting bone differentiation. It achieves spatiotemporal specific activation of biological signal pathways through precise regulation mediated by physical fields. Background Technology

[0002] Systemic bottlenecks in existing biosignal precision regulation technologies

[0003] Tissue regeneration, particularly in osteoporosis-related bone repair, relies heavily on the precise temporal, spatial, and dosage control of key biological signals (such as growth factors and gene expression products). However, existing technologies face significant bottlenecks: In gene delivery, widely used viral vectors (such as adeno-associated virus AAV) typically have high transfection efficiency but pose a potential risk of genome integration, potentially leading to long-term safety concerns. Non-viral vectors offer relatively better safety but often suffer from low transfection efficiency, delayed target gene expression, and unstable duration of expression, making it difficult to meet the need for rapid initiation of repair. In the delivery of bioactive molecules, growth factor-based sustained-release scaffolds (such as hydrogels and microspheres) often experience rapid initial release rates leading to a sudden increase in local concentration, easily inducing side effects such as ectopic ossification in non-target areas, and their release behavior is difficult to dynamically control according to the repair process. Regarding physical control tools, while emerging optogenetic technologies can achieve high-precision temporal and spatial control, the tissue penetration of their excitation light is limited (especially in dense structures such as bone), making it difficult to effectively target deep areas. Furthermore, they typically require invasive implantation of the light source device, increasing the complexity and risk of clinical applications. These bottlenecks collectively limit the safety and effectiveness of biosignal modulation therapy.

[0004] The core contradictions and clinical needs in the field of bone repair

[0005] Focusing on bone repair, the aforementioned technical bottlenecks manifest as core contradictions that urgently need to be addressed: First, during local injection, drugs or growth factors are easily lost from the defect site, making it difficult to maintain effective therapeutic concentrations. This not only reduces efficacy but is also a key factor in inducing heterotopic ossification. Second, while gene therapy strategies have the potential for long-term regulation, their effects are often delayed, making it difficult to meet the urgent needs of scenarios requiring rapid bone formation (such as acute injury repair or bridging of large bone defects). Crucially, existing technology platforms generally struggle to simultaneously achieve three key performance indicators: effective action on deep tissues (such as centimeter-level bone defect centers), rapid response (the speed required to simulate dynamic changes in physiological signals), and completely non-invasive operation. This dilemma of balancing "depth, response speed, and non-invasiveness" is the fundamental obstacle restricting the development of next-generation intelligent bone repair technologies and the core challenge facing the clinical realization of efficient, controllable, and safe bone regeneration.

[0006] The Potential and Transformation Bottlenecks of Emerging Technologies—Phase Separation and Piezoelectric Materials

[0007] To seek breakthroughs, cutting-edge research explores combining the LLPS phenomenon with ultrasound, a deep-penetrating physical field. LLPS, formed through biomolecular condensates, can enhance the specificity and efficiency of local signal transduction, offering possibilities for constructing biomimetic regulatory nodes. However, its practical application faces challenges: the spatiotemporal controllability of the spontaneous nucleation process of endogenous phase separation is poor; exogenous chemical inducers carry the risk of cytotoxicity. More importantly, there is currently a lack of physical modulation methods that can efficiently and non-invasively trigger LLPS in deep tissues. Meanwhile, because ultrasound mechanical energy induces piezoelectric materials to generate local electrical signals or chemical microenvironment changes, it is considered a potential pathway to achieve non-invasive deep modulation. However, its biomedical translation faces bottlenecks: on the one hand, existing piezoelectric material-based delivery systems struggle to achieve intelligent and high-precision control of release kinetics (such as on-demand initiation / termination, precise dose adjustment); on the other hand, the local stimuli (such as electric fields) generated by the piezoelectric effect often decay rapidly, making it difficult to maintain a lasting and stable effect. The fundamental challenge lies in the current lack of an integrated platform capable of effectively coupling penetrating physical fields (such as ultrasound) with intelligent LLPS response mechanisms, making it impossible to achieve dynamic and precise control of "on-demand activation and instant termination" in deep tissues. Developing such an intelligent coupling system is of great significance for promoting breakthroughs in tissue regeneration towards non-invasive, precise, and dynamic control. Summary of the Invention

[0008] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solution:

[0009] This invention provides a piezoelectric hydrogel loaded with responsive peptides, the preparation method of which includes the following steps:

[0010] 1) Mix 5-40 wt% methacrylamide gelatin (GelMA) with 5-20 wt% oxidized β-cyclodextrin, and after clarification, cool to room temperature;

[0011] 2) Add the responsive peptide to the clarified solution until its concentration reaches 10-50 μg / mL, and dissolve it completely;

[0012] 3) Add barium titanate micron particles to the above solution until the concentration reaches 5-20 mg / mL, stir evenly, pour into a mold, and after solidification, obtain a piezoelectric hydrogel with loaded responsive peptides.

[0013] The responsive peptide undergoes liquid-liquid phase separation under ultrasonic conditions.

[0014] Preferably, the responsive peptide comprises a hydrophobic nucleating domain, a π-π stacking domain, and a rearrangeable β-sheet domain.

[0015] Preferably, the hydrophobic nucleating domain comprises several hydrophobic amino acids that form a β-sheet structure under ultrasonic conditions, wherein the hydrophobic amino acids are selected from one or more of valine (Val), leucine (Leu), and phenylalanine (Phe).

[0016] Preferably, the π-π stacking domain contains at least two pairs of aromatic amino acid pairs for driving the directional assembly and stabilization of the polypeptide at the phase-separated droplet interface.

[0017] Preferably, a pair of aromatic amino acid pairs in the π-π stacked domain is located in a hydrophobic nucleation domain.

[0018] Preferably, the rearranged β-sheet domain is composed of glycine flexible hinges to achieve dynamic conformational transformation.

[0019] Preferably, the amino acid sequence of the hydrophobic nucleating domain is as shown in SEQ ID NO: 1, the aromatic amino acid pair is selected from Tyr-Phe or Phe-Trp, and the amino acid sequence of the rearranged β-sheet domain is as shown in SEQ ID NO: 2.

[0020] Preferably, the amino acid sequence of the responsive polypeptide is shown in SEQ ID NO: 3.

[0021] Preferably, the barium titanate is modified with APTES ethanol solution.

[0022] Preferably, the mass concentration of the methacrylamide gelatin (GelMA) is 8-12 wt%; the mass concentration of the oxidized β-cyclodextrin is 8-12 wt%; the concentration of the responsive peptide is 25-35 μg / mL; and the concentration of the barium titanate microparticles is 8-12 mg / mL.

[0023] Preferably, the mass concentration of the methacrylamide gelatin (GelMA) is 10 wt%; the mass concentration of the oxidized β-cyclodextrin is 10 wt%; the concentration of the responsive peptide is 30 μg / mL; and the concentration of the barium titanate microparticles is 10 mg / mL.

[0024] In a preferred embodiment of the present invention, the present invention also provides a pharmaceutical composition comprising a piezoelectric hydrogel of the loading responsive peptide described herein and a pharmaceutically acceptable carrier or excipient.

[0025] In another preferred embodiment of the present invention, the present invention also provides the use of the piezoelectric hydrogel of the loaded responsive peptide described herein and the pharmaceutical composition thereof in the preparation of a medicament for treating bone injuries.

[0026] Compared with the prior art, the main advantages of this invention are:

[0027] Compared to existing technologies, the core advantage of this invention lies in its innovative construction of a three-tiered cascade regulatory system of "ultrasound-piezoelectricity-responsive peptide," which breaks through the key problem of the lack of coupling between deep-penetrating physical fields and intelligent LLPS interfaces in the background technology. Specifically:

[0028] Non-invasive intervention in deep tissues: Externally applied 1MHz focused ultrasound can penetrate centimeter-level biological tissues (such as bone defect areas) and precisely act on implanted piezoelectric composite carriers (BaTiO3 / GelMA).

[0029] Directed energy-signal conversion: The piezoelectric carrier converts ultrasonic mechanical energy into a localized electrical signal microenvironment (experiments have shown it to be at the millivolt level);

[0030] Molecular-level dynamic switching control: This electrical signal specifically triggers conformational rearrangement of the electroresponsive β-sheet domain (RG3RGR) in the peptide, dynamically regulating its phase separation tendency through the glycine hinge;

[0031] Precise spatiotemporal control on demand: Combining the inherent hydrophobic nucleation domain (SVLYF) of peptides with the π-π stacking interface, it achieves: on-demand activation (ultrasound activation → electric field generation → β-sheet enhancement → phase separation and aggregation); and instant termination (ultrasound deactivation → electric field dissipation → conformational restoration → droplet dissociation).

[0032] Thus, for the first time, in vitro regulation and in vivo biological signal amplification were achieved in deep tissues (such as bone defect areas), providing an innovative solution for clinical challenges such as bone repair.

[0033] This invention creatively constructs a piezoelectric hydrogel-peptide composite system, develops a method for precise ultrasound-induced regulation of LLPS, and innovatively applies it to the field of bone regeneration, forming a complete and unique technical solution from material preparation, mechanism of action to clinical application.

[0034] In this system, the preparation of piezoelectric hydrogels has been scientifically verified, possessing mechanical properties and piezoelectric response characteristics suitable for tissue engineering; the process of ultrasound-induced polypeptide LLPS has been fully demonstrated through multi-dimensional experiments to demonstrate its controllability, reversibility, and molecular-level mechanism of action; in bone regeneration applications, in vitro and in vivo experiments have clearly shown a strong activation of osteogenic signals and a significant promoting effect on bone defect repair.

[0035] This technology breaks through the limitations of traditional bone regeneration strategies, providing a new path for the repair of complex bone defects and the development of intelligent scaffolds for tissue engineering in orthopedic clinical practice, and has the advantages of originality, practicality and clinical translation potential.

[0036] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0037] Figure 1 This is a diagram illustrating the mechanism of ultrasound-driven response peptide formation of LLPS and biosignal amplification.

[0038] Figure 2 The preparation and characterization of piezoelectric hydrogels include: a) Scanning electron microscope (SEM) images of barium titanate microspheres prepared by hydrothermal method; b) Elemental analysis results; c) SEM images of the microstructure of EPH; d) Rheological testing; e) Dynamic strain cyclic rheology; f) Circular dichroism; g) Raman spectroscopy; hi) XPS (C, N spectra); j) Ultrasonic-induced disintegration of the EPH gel network (rheological behavior evidence); k) Under ultrasonic stimulation, the hydrogel-peptide system generates a local electrical signal of approximately 10 mV; and l) Piezoelectric butterfly curve characterizing the linear electromechanical response.

[0039] Figure 3 The figures show the experimental results of ultrasound-induced LLPS formation from EPH in vitro; a) statistical results of LLPS formation at different power levels for different concentrations of EPH; b) the effect of ultrasound treatment of EPH for different durations on LLPS; c) optical microscope images of ultrasound-induced LLPS droplet formation from EPH; d) FRAP experimental results; e) 1 H nuclear magnetic resonance spectroscopy; f) Cell dead / live staining results.

[0040] Figure 4 The images show the experimental results of ultrasound-induced EPH to induce LLPS in vivo; ab) in vivo imaging results; cd) two-photon microscopy images.

[0041] Figure 5 Figure 1 shows the results of an in vitro biosignal amplification experiment on ultrasound-induced EPH to produce LLPS; ac) Effect of ultrasound-induced EPH to produce LLPS on the expression levels of BMPR2 and Smad1 / 5 / 9 in BMSCs; df) Level of LLPS-state peptides recruiting the cell membrane surface receptor BMPR2; gi) LLPS-state peptides increasing the phosphorylation level of Smad1 / 5 / 9 in the cell nucleus; jl) Content of free EAP in the culture medium after ultrasound-induced EPH.

[0042] Figure 6 The figure shows the experimental results of ultrasound-induced EAP-LLPS promoting bone formation; ad) ultrasound-induced EPH-LLPS affecting ALP and alizarin red expression levels in BMSCs.

[0043] Figure 7 The image shows the results of an experiment on ultrasound-induced EPH and LLPS promoting bone defect regeneration in rats; ac) Bone volume fraction (BV / TV) of ultrasound-induced EPH and LLPS in a transverse femoral defect model in SD rats. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1: Construction of a piezoelectric hydrogel-responsive peptide composite system (EPH)

[0046] (1) Steps for preparing GelMA hydrogel by hydrothermal method:

[0047] Preparation of GelMA hydrogel: Dissolve 10g of gelatin in 100mL of PBS, stir at 60℃ for 1 hour until dissolved, cool to 50℃, and add 8mL of methacrylic anhydride (MA) dropwise at a rate of 0.5mL / min. Continue stirring for 2 hours, maintaining the pH at 9 with carbonate buffer. After the reaction, add 200mL of PBS to terminate the reaction, transfer to an 8-14kDa dialysis bag, dialyze with deionized water for 7 days (changing the water 3 times a day), and freeze-dry to obtain GelMA powder.

[0048] (2) Steps for preparing barium titanate micron-sized particles (BaTiO3) by hydrothermal method:

[0049] 99.9% pure BaCO3 and TiO2 were accurately weighed at a molar ratio of 1:1. The raw materials were added to a planetary ball mill, using agate balls as the grinding medium (ball-to-material ratio 5:1), with anhydrous ethanol as a dispersant. The mixture was ground at 350 rpm for 3 hours, followed by vacuum drying to remove the ethanol. The dried powder was placed in a corundum crucible and placed in a muffle furnace. The temperature was increased to 1200℃ at a rate of 5℃ / min and held for 3 hours to complete the crystal phase synthesis. The mixture was then cooled to room temperature with the furnace. The calcined product was ground a second time in a planetary ball mill (200 rpm, 1 hour), passed through a 20μm standard sieve to control the particle size, and then vacuum dried after sieving. SEM analysis showed that it consisted of spherical particles with a diameter of 20μm. Figure 2 (a).

[0050] (3) Preparation of response peptide (EAP):

[0051] A responsive peptide with a molecular weight of approximately 4.4 kDa was designed, and its amino acid sequence is shown in SEQ ID NO: 3: ISVLYFDDSSNVILKKYEHDKEFWGRGRGRGWY. This responsive peptide was synthesized via organic solid-phase synthesis.

[0052] (4) Preparation steps of BaTiO3-GelMA (EPH) piezoelectric hydrogel:

[0053] 20 μm BaTiO3 powder was ultrasonically modified with 2% APTES ethanol solution for 1 hour, followed by centrifugation and drying to enhance compatibility. A 10 wt% solution of methacrylamide gelatin (GelMA) was mixed with 8 wt% oxidized β-cyclodextrin to form a clear solution, which was then cooled to room temperature. 30 μg / mL of the responsive peptide EAP was added to the clear solution and dissolved completely. Then, 10 mg / mL of barium titanate microparticles were added, and the mixture was stirred at 100 rpm for 5 min. The mixture was then poured into a mold and allowed to stand for 30 min to obtain EPH gel.

[0054] (5) Characterization of EPH hydrogel:

[0055] Figure 2 The a-type particle size is ~20 μm (spherical). Elemental analysis shows that the main constituent elements of EPH are: Ba, Ti and C ( Figure 2 (b) Figure 2 Figure c shows the microstructure of EPH. Rheological results show that G' is always much larger than G'', indicating that EPH is more like an "elastic solid" than a viscous fluid, and that EPH crosslinks uniformly. Figure 2 (d). Dynamic strain cyclic rheological testing shows that EPH is a physically cross-linked self-healing gel that can be used in fatigue-resistant scenarios. Figure 2(e). Circular dichroism chromatogram results showed that ultrasonic treatment altered the chiral conformation of the EPH system: the signals of the original ordered structures (such as helices and folds) were weakened, presumably due to ultrasonic-induced conformational rearrangement. Figure 2 Raman spectroscopy characterization shows that ultrasound-induced EPH conformational rearrangement and orderly stacking reflect its structural dynamic response characteristics under force field. Figure 2 X-ray photoelectron spectroscopy (XPS) showed that ultrasound induced spatial conformational rearrangement of the EPH amide bonds, with a significant decrease in the C=O peak intensity and a broadening of the CN peak, suggesting a reshaping of the chemical environment of the carbon-based functional groups. Figure 2 Furthermore, the NH peak binding energy decreases and the peak shape becomes more diffuse, leading to a reshaping of the chemical state of the nitrogen functional group (h). Figure 2 (i). Ultrasonic-induced disintegration of the gel network directly manifests as a change in the mobility of solvent molecules from "restricted" to "free". Figure 2 The hydrogel-peptide composite system can generate a local electrical signal of 10 mV under ultrasonic stimulation. Figure 2 K). In the piezoelectric response butterfly curve experiment, the amplitude curve exhibits the linear response characteristics of the inverse piezoelectric effect, and the phase curve shows the hysteresis effect of deformation and electric field (K). Figure 2 The above results directly reflect the structural dynamic response of EPH under ultrasonic action, and the deformation and electric field show a symmetrical response.

[0056] Example 2: Ultrasound-induced in vitro LLPS generation of EPH

[0057] Piezoelectric hydrogels loaded with different concentrations of peptides were subjected to ultrasonic intervention of different intensities for 1 minute. It was found that the peptides at a concentration of 30 μg / mL readily underwent LLPS (low-density polymorphism). Figure 3 (a) Based on a peptide concentration of 30 μg / mL, ultrasound interventions of varying intensities were applied for a series of times, and it was found that at 1 W / cm... 2 Ultrasound intensity intervention for 3 minutes most readily induces the formation of LLPS in peptides ( Figure 3 (b). Optical microscopy shows a peptide concentration of 30 μg / mL at 1 W / cm². 2 Ultrasonic intensity intervention for 3 minutes resulted in the formation of 5µm LLPS droplets. Figure 3 c); Fluorescence recovery after bleaching (FRAP) assays observed the fluorescence recovery rate, indicating that the molecules exhibit dynamic exchange rather than a completely rigid phase structure, consistent with the dynamic reversible characteristics of LLPS. Figure 3 d). 1H NMR spectroscopy confirmed that the polypeptide EPH underwent LLPS and generated hydrogen bonds under ultrasonic irradiation. Figure 3 (e); In a BMSCs culture system, LLPS was induced in EPH. After 3 days of culture, cell dead / live staining was performed. The results showed that LLPS induced by EPH did not affect cell viability. Figure 3 f).

[0058] Example 3: Ultrasound-induced LLPS in vivo during EPH treatment

[0059] C57BL / 6 mice were implanted subcutaneously with EPH labeled with FITC peptide in their backs. After percutaneous ultrasound induction, local enhancement of fluorescence signal was observed using in vivo imaging technology, suggesting that ultrasound may percutaneously induce EPH aggregation in vivo. Figure 4 (a, b). EPH labeled with FITC peptide was subcutaneously implanted in the thigh of nude mice. After percutaneous ultrasound induction, two-photon microscopy revealed the formation of LLPS (LLPS) in vivo by ultrasound-induced EPH. Figure 4 (c, d).

[0060] Example 4: Ultrasound-induced EPH generates LLPS, which amplifies biological signals in vitro.

[0061] Western blot experiments showed that ultrasound-induced EPH and LLPS promoted the expression of BMPR2 protein in BMSCs and significantly enhanced the phosphorylation level of Smad1 / 5 / 9. Figure 5 ac). Immunofluorescence staining showed that peptides in the LLPS state recruited the cell membrane surface receptor BMPR2 and increased the phosphorylation level of Smad1 / 5 / 9 in the cell nucleus ( ). Figure 5 After sonication induction, very little EAP diffused into the culture medium, suggesting that most of the free EAP formed LLPS (di). Figure 5 (jl). The above results suggest that ultrasound-induced polypeptide LLPS can amplify biological signals with only a low concentration.

[0062] Example 5: Ultrasound-induced EPH to promote LLPS and bone formation

[0063] Ultrasound-induced EPH and the formation of LLPS can upregulate alkaline phosphatase (ALP) activity in BMSCs and enhance the formation of alizarin red-stained mineralized nodules, thereby promoting their osteogenic differentiation. Figure 6 (ad).

[0064] Example 6: Ultrasound-induced EPH and LLPS promote bone defect regeneration in rats

[0065] A 3 mm diameter transverse bone defect was created in the distal femur of SD rats, and EPH was implanted into the defect site. Percutaneous ultrasound induction was performed for 3 minutes. Micro-CT showed that after 8 weeks, the bone volume fraction (BV / TV) increased by approximately 300%. Figure 7 (ac).

[0066] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A piezoelectric hydrogel loaded with a responsive polypeptide, the preparation method of which includes the following steps: 1) Mix 5-40 wt% methacrylamide gelatin (GelMA) solution with 5-20 wt% oxidized β-cyclodextrin solution to form a clear solution, and cool to room temperature; 2) Add the responsive peptide to the clarified solution until its concentration reaches 25-50 μg / mL, and dissolve it completely; 3) Add barium titanate micron particles to the above solution until the concentration reaches 5-20 mg / mL, stir evenly, pour into a mold, and after solidification, obtain a piezoelectric hydrogel with loaded responsive peptides. The responsive peptide undergoes liquid-liquid phase separation under ultrasonic conditions; The amino acid sequence of the responsive polypeptide is shown in SEQ ID NO:

3.

2. The piezoelectric hydrogel of the loaded responsive peptide according to claim 1, characterized in that, The barium titanate was modified with APTES ethanol solution.

3. The piezoelectric hydrogel of the loaded responsive peptide according to claim 1, characterized in that, The concentration of the methacrylamide gelatin (GelMA) is 8-12 wt%; the concentration of the oxidized β-cyclodextrin is 8-12 wt%; the concentration of the responsive peptide is 25-35 μg / mL; and the concentration of the barium titanate microparticles is 8-12 mg / mL.

4. The piezoelectric hydrogel with a loading response peptide according to claim 3, characterized in that, The concentration of the methacrylamide gelatin (GelMA) is 10 wt%; the concentration of the oxidized β-cyclodextrin is 10 wt%; the concentration of the responsive peptide is 30 μg / mL; and the concentration of the barium titanate microparticles is 10 mg / mL.

5. A pharmaceutical composition comprising a piezoelectric hydrogel of a loaded responsive polypeptide as described in any one of claims 1-4 and a pharmaceutically acceptable excipient.

6. The use of the piezoelectric hydrogel of the loaded responsive polypeptide according to any one of claims 1-4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating bone injuries.

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