Piezoelectric nanosheet for regulating and controlling distribution and expression of cell membrane protein and preparation method thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a piezoelectric nanosheet for regulating and controlling distribution and expression of cell membrane protein and a preparation method of the piezoelectric nanosheet. The piezoelectric nanosheet is a bismuth oxyiodate layered nanosheet with an Aurivillius structure, and the surface of the piezoelectric nanosheet is modified by polyvinylpyrrolidone (PVP) and is abbreviated as BIOP; the nanosheet is synthesized by bismuth nitrate pentahydrate, potassium iodate and PVP through a hydrothermal method in one step, and the average size is 93 nm. BIOP can be attached to the surface of a cell, responds to ultrasonic irradiation with different intensities, generates a nanoscale periodic oscillation electric field, and performs electrostatic interaction with charged cell membrane protein on a cell membrane, so that the original hydrophobic force-electrostatic force balance steady state of the membrane protein is broken, internalization and degradation of the membrane protein are efficiently mediated, and regulation and control of distribution and expression of the cell membrane protein are realized. Compared with biochemical strategies such as antagonist drugs, gene editing and the like, the strategy has the unique advantages of being accurate and controllable, high in penetration depth and the like, and has important significance for revealing an intercellular communication mechanism and treating cell membrane protein dependent diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ultrasonic-electric conversion piezoelectric nanosheet and its preparation method. Background Technology

[0002] Cell membrane proteins are a special class of proteins located on the surface of cell membranes. They are essential components of the cell membrane, responding to external signaling molecules or physical stimuli and participating in biological processes such as signal transduction, substance transport, cell recognition, and cell-cell interactions. They are closely related to various major health problems, including cancer, neurodegenerative diseases, autoimmune diseases, mental health, and pain management. For example, cancer pain is a significant factor affecting the quality of life of cancer patients, primarily due to the continuous activation of the capsaicin receptor (TRPV1) on the surface of nerve cell membranes by stimulating factors secreted by tumors, leading to persistent and intense pain. Furthermore, overexpression of human epidermal growth factor receptor-2 (HER2) can lead to the proliferation and progression of breast and gastric cancers. Therefore, membrane proteins have become a promising emerging target. However, current methods for regulating membrane protein function mainly include small molecule inhibitors, protein-targeted degradation technologies, and gene editing technologies. These methods are based on the principles of traditional chemical coordination or covalent binding, and suffer from drawbacks such as low protein subtype response rates, easy drug resistance, inability to controllably trigger on demand, and short effective duration, severely weakening their therapeutic effects. In summary, there is an urgent need to develop a novel strategy for regulating membrane protein function that is drug-resistant, long-term effective, safe, controllable, and non-biochemical dependent.

[0003] Studies have shown that the activity of membrane proteins is closely related to their localization on the cell membrane. Regulating the spatial distribution and expression of cell membrane proteins is a crucial method for elucidating their mechanisms of action and intervening in physiological and pathological processes. From a biophysical perspective, the distribution of membrane proteins on the cell membrane lipid layer is influenced by both hydrophobic and electrostatic forces, ultimately achieving a force-balanced homeostasis and stabilizing them on the cell membrane surface. The inventors discovered that membrane proteins can interact electrostatically with external electric fields, disrupting this force-balanced homeostasis and mediating internalization (entering the cell from the cell membrane surface). This provides a novel biophysical mechanism for regulating membrane protein function and may offer new treatment methods for major health threats such as cancer pain. However, achieving efficient, precise, safe, and controllable electrochemical regulation of membrane protein distribution at the single-cell scale remains a technological challenge.

[0004] This invention innovatively constructs a piezoelectric nanosheet, BIOP, capable of adhering to the cell membrane surface. By responding to exogenous ultrasonic irradiation, lattice distortion is induced, generating a locally polarized periodic electric field on the material surface. This, in turn, disrupts the electrostatic equilibrium of the cell membrane protein (TRPV1), causing it to internalize from the cell membrane surface into the cytoplasm and degrade. This achieves drug-resistant, highly efficient, precise, safe, and controllable spatial distribution regulation of membrane proteins, possessing potential for cancer pain treatment. Furthermore, this ultrasound-to-electric conversion platform can precisely control the electric field parameters (intensity and oscillation period) on the nanosheet surface by adjusting ultrasonic parameters (power density and frequency), enabling the regulation of the distribution and activity of a series of membrane proteins with different charge characteristics. This invention provides a novel tool for elucidating the biological mechanisms of membrane proteins and offers a reliable technology for treating cell membrane protein-dependent diseases such as cancer pain. Summary of the Invention

[0005] The purpose of this invention is to provide a piezoelectric nanosheet with ultrasound-to-electric conversion properties, capable of responding to exogenous ultrasound irradiation and dynamically regulating the distribution and expression of the cell membrane protein TRPV1, and its preparation method.

[0006] This invention provides a piezoelectric nanosheet that regulates the distribution and expression of the cell membrane protein TRPV1. It is a layered nanosheet of bismuth oxyiodate (BiOIO3) with an Olivieris structure, the surface of which is modified with polyvinylpyrrolidone (PVP-K30), abbreviated as BIOP. The piezoelectric nanosheet is synthesized in one step by hydrothermal method from bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), potassium iodate (KIO3) and PVP, with an average size of 80-100 nm.

[0007] This invention also provides a method for preparing piezoelectric nanosheets that regulate the distribution and expression of the cell membrane protein TRPV1, the specific steps of which are as follows:

[0008] Step 1: Add appropriate amounts of PVP and Bi(NO3)3·5H2O to ultrapure water, and stir continuously to ensure they are fully dissolved and mixed evenly to form a Bi precursor solution; add appropriate amounts of KIO3 to ultrapure water, and stir continuously to ensure they are fully dissolved to form an IO3 precursor solution.

[0009] Step 2: Add the IO3 precursor solution obtained in Step 1 dropwise to the Bi precursor solution under high-speed stirring, and control the molar concentrations of I and Bi to be consistent and appropriate to form a uniformly dispersed precursor suspension.

[0010] Step 3: Transfer the suspension obtained in Step 2 to a polytetrafluoroethylene liner, seal it, and place it in a high-temperature oven with a gradient heating program to carry out a hydrothermal reaction. After the reaction is completed, centrifuge at high speed and discard the supernatant. Then, wash the product multiple times with ultrapure water and finally freeze-dry to obtain piezoelectric nanosheets, abbreviated as BIOP.

[0011] Furthermore:

[0012] In step one, the mass concentration of the PVP solution is 1-5 mg / mL. -1 The molar concentration of Bi(NO3)3·5H2O is 10-100 mM, and the mass ratio of PVP to Bi(NO3)3·5H2O is 0.1-2; the final molar concentration of IO3 is 10-100 mM. Preferably, the mass concentration of the PVP solution is 4 mg / mL. -1 The molar concentration of Bi(NO3)3·5H2O was 40 mM, the mass ratio of PVP to Bi(NO3)3·5H2O was 0.2, and the final molar concentration of IO3 was 40 mM.

[0013] In step two, the dropping rate of the IO3 precursor solution is 5-20 mL / h. -1 The total concentration of the mixed solution is 10-1000 mg / mL. -1 Preferably, the dropping rate of the IO3 precursor solution is 15 mL / h. -1 The total concentration of the mixed solution is 500 mg / mL. -1 The stirring speed is 300-600 rpm, and the stirring time is 30-120 min.

[0014] In step three, the gradient heating rate is 5-10 °C min. -1 The holding temperature is 110-180 °C, and the total hydrothermal reaction time is 3-24 h; the centrifugation speed is 11000-18000 rpm. Preferably, the gradient heating rate is 5 °C / min. -1 The insulation temperature was 120 °C, and the total hydrothermal reaction time was 6 h; the centrifugal speed was 13000 rpm.

[0015] The piezoelectric nanosheets prepared in this invention, which regulate the spatial distribution and expression of the cell membrane protein TRPV1, are composite nanomaterials. Specifically, they are layered nanosheets of bismuth iodate oxyiodide with an orthorhombic crystal system and a surface modified with PVP, with an average size of 80-100 nm. The IO3 structural units within the crystals exhibit excellent piezoelectric response characteristics, enabling them to respond to exogenous ultrasonic irradiation in cells and organisms, controllably generating a localized periodic oscillating electric field, thereby effectively regulating the force balance state of the membrane protein TRPV1.

[0016] This invention utilizes biomedical technology to construct novel ultrasound-to-electric conversion piezoelectric nanobiomaterials, addressing the bottlenecks of existing membrane protein regulation technologies, such as low protein subtype response rates, susceptibility to drug resistance, inability to controllably trigger on demand, and short effective duration, from a biophysical perspective. The high anisotropy and large local curvature of the layered piezoelectric nanosheets, along with surface-modified PVP, not only enhance the stabilizing effect between the nanosheets and the cell membrane surface but also significantly improve the aqueous dispersion performance of the nanosheets. Under ultrasound irradiation, the piezoelectric nanosheets efficiently convert exogenous ultrasound waves into a localized, periodic oscillating electric field on the surface, which then interacts electrostatically with the cell membrane protein TRPV1. This disrupts the steady-state equilibrium between hydrophobic and electrostatic forces, thereby mediating the internalization and degradation of TRPV1, achieving precise regulation of the spatial distribution and expression of the membrane protein TRPV1.

[0017] This invention also provides an application of the above-mentioned piezoelectric nanosheets in regulating the internalization of the SH-SY5Y cell line membrane protein TRPV1. Specifically, the piezoelectric nanosheets are prepared as adhesive sheets and attached to the surface of the SH-SY5Y cell membrane; the IO3 structural units within the crystal can respond to exogenous ultrasonic irradiation, causing lattice distortion and generating a locally polarized periodic electric field on the material surface; this electric field can interact electrostatically with TRPV1, disrupting its hydrophobic-electrostatic equilibrium on the membrane surface, mediating the internalization and degradation of TRPV1, thereby blocking a series of TRPV1-related cell signaling pathways and inhibiting its related functions (such as the generation of cancer pain).

[0018] Furthermore, this invention utilizes ultrasound with high tissue penetration depth as a mechanical wave excitation source. By mediating the polarization of IO3 structural units and BIOP lattice distortion, a nanoscale polarized electric field is formed on the material surface. The intensity and oscillation period of this electric field are precisely controlled by the power density and frequency of the exogenous ultrasound. Therefore, when BIOP adheres to the cell membrane surface, the surface generates a responsive localized polarized electric field through ultrasound-electrolysis, which interacts electrostatically with the charged cell membrane protein TRPV1. This disrupts the hydrophobic-electrostatic equilibrium of TRPV1 on the cell membrane surface, efficiently mediating TRPV1 internalization and promoting its degradation in lysosomes, thus achieving precise regulation of the spatial distribution and expression of TRPV1 membrane proteins. This invention proposes a novel technology based on ultrasound-electrolysis to dynamically regulate the spatial distribution and expression of cell membrane proteins, which can be used for the treatment of cell membrane protein-dependent diseases.

[0019] The main features and advantages of this invention are:

[0020] (1) Develop a process for preparing piezoelectric nanosheets BIOP that regulates the spatial distribution and expression of cell membrane protein TRPV1. The process is stable and controllable and can be prepared on a gram-scale under laboratory conditions.

[0021] (2) Develop a technology to regulate the spatial distribution and expression of cell membrane protein TRPV1 by using a local polarized periodic electric field on the surface of a nanopiezoelectric sheet; this technology can respond to high tissue-penetrating ultrasonic irradiation and generate a local polarized periodic electric field, and has the unique advantage of being able to control the spatial distribution and expression of charged cell membrane proteins in situ in vivo.

[0022] (3) A new type of nanotechnology for regulating the spatial distribution of membrane proteins based on biophysical effects is proposed; the BIOP-mediated TRPV1 internalization and related pathway inhibition by piezoelectric nanosheets have unique advantages of being non-drug resistant, highly efficient, precise, safe and controllable; a novel strategy for regulating the distribution of membrane proteins by local polarization periodic electric fields is proposed, which can achieve precise regulation of a series of membrane proteins with different charge characteristics by continuously and finely changing ultrasound parameters, and has distinctive features in the development of applied research tools and clinical treatment technologies. Compared with biochemical strategies such as antagonist drugs and gene editing, this strategy has unique advantages such as precision and controllability and high penetration depth, which is of great significance for revealing the intercellular communication mechanism and treating cell membrane protein-dependent diseases. Attached Figure Description

[0023] Figure 1 Transmission electron microscope (TEM) image of the piezoelectric nanosheet BIOP prepared in Example 1 of this invention.

[0024] Figure 2 The X-ray diffraction (XRD) patterns of the piezoelectric nanosheets BIOP and bismuth oxyiodate (BiOIO3) standard cards prepared in Example 1 of this invention.

[0025] Figure 3 The piezoelectric response force microscopy (PFM) pattern of the piezoelectric nanosheet BIOP prepared in Example 1 of this invention.

[0026] Figure 4 These are confocal micrographs of the cell membrane protein TRPV1 stained with fluorescence in (A) human neuroblastoma cells (SH-SY5Y) and (B) human embryonic kidney cells (HEK293).

[0027] Figure 5 The bar chart shows the mechanical pain sensitivity in the C57BL / 6 mouse sciatic nerve injury (SNI) model. Detailed Implementation

[0028] The following detailed description, in conjunction with the accompanying drawings, provides further illustrations. Except as specifically mentioned below, the processes, conditions, and experimental methods described in these embodiments are all common knowledge and general information in the field, and this invention does not impose any particular limitations.

[0029] Example 1: Preparation of PVP-modified piezoelectric nanosheets BIOP

[0030] First, 200 mg of polyvinylpyrrolidone (PVP) was thoroughly dissolved in 35 mL of ultrapure water. Then, 970 mg of bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) and 428 mg of potassium iodate (KIO3) were added to the PVP solution and thoroughly mixed under ultrasonication to form a uniformly dispersed precursor suspension. The suspension was then transferred to a 50 mL polytetrafluoroethylene liner, sealed, and placed in a high-temperature oven with a heating rate of 5 °C / min. -1 The mixture was heated to 120 °C and subjected to a hydrothermal reaction for 6 h. After the reaction was completed and cooled to room temperature, the product mixture was centrifuged at high speed and the supernatant was discarded. The product was then washed multiple times with ultrapure water to thoroughly remove residual surfactants and unreacted solvents. The centrifugation speed and time were 13000 rpm and 6 min, respectively. Finally, the product was freeze-dried overnight to obtain PVP-modified piezoelectric nanosheets BIOP.

[0031] All chemical reagents used in step 1 of this invention were purchased directly from the reagent company. Unless otherwise specified, none of the chemical reagents underwent any purification.

[0032] Figure 1 This is a transmission electron microscope (TEM) image of the piezoelectric nanosheets BIOP prepared in Example 1 of this invention. The nanosheets shown in the image are in the form of thin sheets with an average size of 93 nm.

[0033] Figure 2 The X-ray diffraction (XRD) patterns of the piezoelectric nanosheet BIOP prepared in Example 1 of this invention and the standard card (ICSD-262019) are shown in the figure. As shown in the figure, the characteristic peak positions of the piezoelectric nanosheet BIOP and the standard card are consistent, confirming that Example 1 of this invention successfully synthesized orthorhombic piezoelectric nanosheet BIOP with good crystallinity.

[0034] Figure 3 The image shows the piezoelectric response force microscopy (PFM) pattern of the piezoelectric nanosheet BIOP prepared in Example 1 of this invention. As shown in the figure, under the condition of applying a continuous DC bias voltage, the piezoelectric nanosheet BIOP exhibits continuous surface deformation, confirming that the piezoelectric nanosheet BIOP prepared in Example 1 of this invention has a clear and good piezoelectric response characteristic.

[0035] Example 2: BIOP-responsive exogenous ultrasound-mediated internalization of cell membrane protein TRPV1 in piezoelectric nanosheets

[0036] Using TRPV1 expressed on the cell membrane surface as the detection target, confocal laser scanning microscopy was used for fluorescence imaging to evaluate the regulation of TRPV1 spatial distribution mediated by the piezoelectric nanosheets BIOP prepared in Example 1 in human neuroblastoma cells (SH-SY5Y) and transfected human embryonic kidney cells (HEK293). SH-SY5Y and HEK293 cells were respectively subjected to 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density per well in confocal culture dishes and pre-cultured under normoxic conditions for 12 h. Cells were randomly divided into five groups: control group, material group (BIOPs), sonication group (US), short treatment group (BIOPs + US for 5 min), and long treatment group (BIOPs + US for 30 min). The material concentration and sonication power were both 200 μg / mL. -1 and 0.08 W cm -2 After treatment, cells were washed repeatedly with pre-cooled PBS buffer; then fixed with a universal tissue fixative; and blocked with 5% bovine serum albumin (BSA) solution. Next, cells were incubated overnight at 4°C with anti-TRPV1 antibody (mouse-derived) and then incubated for 2 h at room temperature with A488-labeled secondary antibody. Before imaging, the cell nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI). Finally, green fluorescence signals (A488-labeled membrane protein TRPV1) were captured using a confocal laser scanning microscope. All image acquisition parameters were kept consistent to ensure data comparability, and each experiment was independently repeated three times.

[0037] Figure 4 These are confocal micrographs of the cell membrane protein TRPV1 stained with fluorescence in (A) human neuroblastoma cells (SH-SY5Y) and (B) human embryonic kidney cells (HEK293). As shown in the figures, the control group, the material group (BIOPs), and the ultrasound group (US) exhibit strong fluorescence signals, indicating high expression levels of TRPV1 on the cell membrane. The short-treatment group (BIOPs + US for 5 min) and the long-treatment group (BIOPs + US for 30 min) show significantly weakened fluorescence signals, with the long-treatment group (BIOPs + US for 30 min) showing the weakest fluorescence signal. This confirms that the piezoelectric nanosheets BIOP can mediate the internalization and degradation of TRPV1 in lysosomes under ultrasound irradiation stimulation, and the effect becomes more significant with prolonged ultrasound irradiation stimulation time.

[0038] Example 3: BIOP-mediated internalization of cell membrane protein TRPV1 by piezoelectric nanosheets for highly effective pain suppression

[0039] To verify the pain-inhibiting effect of piezoelectric nanosheets BIOP, C57BL / 6 mice were randomly divided into two groups of six each. Both groups underwent sciatic nerve ligation surgery to establish a mouse model of sciatic nerve injury (SNI). After 7 days of stabilization, the control group (5 μL Saline) and the treatment group (5 μL 500 μg mL) were administered intrathecally, respectively. -1 BIOPs; 0.4 W cm -2 The mice were treated with 1 MHz and 4 minUS. At 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatment, the skin on the lateral plantar surface of the hind paw of the mice was vertically stimulated with von Frey fibers, and the stimulation intensity (i.e., mechanical pain threshold) when the mice showed a positive withdrawal reflex was recorded. Then, the area under the curve (i.e., AUC value) of the mechanical pain threshold over time was quantitatively analyzed to reflect the sensitivity of the mice to pain.

[0040] Figure 5 The figure shows a bar chart of mechanical pain sensitivity in a C57BL / 6 mouse sciatic nerve injury (SNI) model. As shown in the figure, the AUC value of the treatment group was significantly higher than that of the control group, confirming that the piezoelectric nanosheet BIOP can mediate TRPV1 internalization and achieve efficient inhibition of nerve pain under ultrasonic irradiation stimulation.

[0041] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A piezoelectric nanosheet for regulating the distribution and expression of cell membrane proteins, characterized in that, It is a layered nanosheet of bismuth iodate with an olivarian structure, whose surface is modified with polyvinylpyrrolidone (PVP), abbreviated as BIOP; the nanosheet is synthesized in one step by hydrothermal method from bismuth nitrate pentahydrate, potassium iodate and PVP, with an average size of 80-100 nm.

2. The method for preparing piezoelectric nanosheets as described in claim 1, characterized in that, The specific steps are as follows: Step 1: Add PVP and Bi(NO3)3·5H2O to ultrapure water and stir continuously until they are fully dissolved and mixed evenly to form a Bi precursor solution; add KIO3 to ultrapure water and stir continuously until they are fully dissolved to form an IO3 precursor solution. Step 2: Add the IO3 precursor solution obtained in Step 1 dropwise to the Bi precursor solution under high-speed stirring, and control the molar concentrations of I and Bi to be consistent and appropriate to form a uniformly dispersed precursor suspension. Step 3: Transfer the suspension obtained in Step 2 to a polytetrafluoroethylene liner, seal it, and place it in a high-temperature oven with a gradient heating program to carry out a hydrothermal reaction. After the reaction is completed, centrifuge at high speed and discard the supernatant. Then, wash the product multiple times with ultrapure water and finally freeze-dry to obtain piezoelectric nanosheets, abbreviated as BIOP.

3. The preparation method according to claim 2, characterized in that, In step one, the mass concentration of the PVP solution is 1-5 mg / mL. -1 The molar concentration of Bi(NO3)3·5H2O is 10-100 mM, the mass ratio of PVP to Bi(NO3)3·5H2O is 0.1-2, and the final molar concentration of IO3 is 10-100 mM.

4. The preparation method according to claim 2, characterized in that, In step two, the dropping rate of the IO3 precursor solution is 5-20 mL / h. -1 The total concentration of the mixed solution is 10-1000 mg / mL. -1 The stirring speed is 300-600 rpm, and the stirring time is 30-120 min.

5. The preparation method according to claim 2, characterized in that, In step three, the gradient heating rate is 5-10°C / min. -1 The heat preservation temperature is 110-180 °C, and the total hydrothermal reaction time is 3-24 h; the centrifugal speed is 11000-18000 rpm.

6. The application of the piezoelectric nanosheets as described in claim 1 in the adhesive sheet for regulating the distribution and expression of the SH-SY5Y cell line membrane protein TRPV1; specifically, the adhesive sheet is attached to the surface of the SH-SY5Y cell membrane; the IO3 structural units within the crystal can respond to exogenous ultrasonic irradiation and generate a locally polarized periodic electric field on the material surface through lattice distortion; this electric field interacts electrostatically with TRPV1, disrupting its hydrophobic-electrostatic equilibrium on the membrane surface, mediating the internalization and degradation of TRPV1, thereby blocking a series of TRPV1-related cell signaling pathways and inhibiting its related functions.

7. The application according to claim 6, characterized in that, Using ultrasound with high tissue penetration depth as a mechanical wave excitation source, a nanoscale polarized periodic electric field is formed on the material surface by mediating the polarization of IO3 structural units and BIOP lattice distortion. The intensity and oscillation period of this electric field are precisely controlled by the power density and frequency of the ultrasound. Therefore, when BIOP is attached to the cell membrane surface, the surface generates a responsive localized polarized electric field through ultrasound-electric conversion, and interacts electrostatically with the charged cell membrane protein TRPV1. This disrupts the hydrophobic-electrostatic equilibrium of TRPV1 on the cell membrane surface, efficiently mediates TRPV1 internalization, and promotes TRPV1 degradation in lysosomes, thereby achieving precise regulation of the spatial distribution and expression of TRPV1 membrane protein.

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