A butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and its preparation method and application

Abstract

The present invention provides a butterfly-shaped potassium ion channel molecule and its preparation method and application for mechanical and pH dual regulation, belonging to the field of biomimetic material technology. The butterfly-shaped potassium ion channel molecule of the present invention is respectively based on three-generation and four-generation dendrimer polypropylene imine (POPAM), and POPAM and ion binding site benzo-21-crown ether-7 are connected with urea bonds. Intramolecular hydrogen bond constraint causes GnC7 to present a symmetrical "butterfly-shaped" topological structure, and the channel has unique motion characteristics, and ultimately achieves efficient and so far the highest potassium-sodium selectivity across the membrane transport of potassium ions. Dynamic characteristics, as the core attribute of GnC7, enable the channel to regulate potassium ion transmission rate according to the membrane tension changes caused by mechanical force. Meanwhile, benzylamine hexafluorophosphate Bh, which is affected by pH, acts as a blocker of GnC7 through host-guest specific composite action, which can realize the switch for pH regulation potassium ion transmission.

Description

Technical Field

[0001] The present invention relates to the technical field of bionic materials, and in particular to a butterfly-shaped potassium ion channel molecule capable of dual mechanical and pH regulation, and a preparation method and application thereof. Background Art

[0002] The natural channel proteins on the cell membrane precisely and selectively control the entry and exit of substances that are not permeable to the membrane. + 、Na + 、H + , Ca 2+ ), anions (Cl - ) and water molecules rely on specific membrane channel proteins for transmembrane transport, thereby participating in the regulation of cell metabolism, cell osmotic pressure, pH value and signal transduction. Therefore, disorders of channel proteins usually lead to a series of ion channel diseases including cystic fibrosis and arrhythmias. In order to grasp the potential key factors of biological functions and provide directions for drug development for the treatment of channel diseases, biomimetic chemists have designed and constructed a series of potassium, chloride, hydrogen ion and water molecule transmembrane transport systems through simple chemical synthesis methods to simulate the structure or function of natural channel proteins. And based on the characteristics of natural channel proteins, scientific researchers are committed to achieving breakthroughs in selectivity and stimulus-responsive properties.

[0003] In recent years, artificial ion channels that respond to various stimuli have been reported. It is worth noting that mechanical sensitivity and pH sensitivity each play an irreplaceable role in the five senses of human beings. Mechanically sensitive ion channels (such as PIEZO1) can sense changes in membrane tension caused by mechanical input (pressure, stretching, shear force, etc.), and then regulate ion permeability to transmit signals, thereby promoting physiological processes such as touch, hearing, and proprioception. pH-dependent ion channels, including acid-sensing ion channels (ASICs), change their open state with changes in pH, allowing humans to perceive pathological conditions such as taste, smell, and inflammation. However, there is currently a lack of artificial ion channels that are both mechanically sensitive and pH-responsive.

[0004] Therefore, developing currently lacking artificial ion channels with both mechanical sensitivity and pH response will help elucidate human sensory abilities and potentially contribute to new treatments for pain, tactile disorders, and other diseases. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, as well as its preparation method and application. The butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation provided by the present invention can achieve efficient potassium-sodium selective potassium ion transmembrane transport and can achieve multi-dimensional regulation of channel function.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, having a structure shown in Formula G3C7 or Formula G4C7:

[0008]

[0009] The present invention provides a method for preparing the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0010] isocyanate-modified POPAM by reacting a dendrimer POPAM having a structure represented by formula a or formula b with di-tert-butyl tricarbonate;

[0011]

[0012] The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with an amino-containing benzo-21-crown ether-7 having a structure shown in formula c to obtain a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 with dual mechanical and pH regulation;

[0013]

[0014] Preferably, the temperature of the isocyanate reaction is 20-27° C., and the time is 40-60 min.

[0015] Preferably, the temperature of the nucleophilic addition reaction is 20-27° C., and the time is 16-24 h.

[0016] Preferably, after the nucleophilic addition reaction, the obtained nucleophilic addition reaction product is subjected to post-treatment, and the post-treatment includes:

[0017] The nucleophilic addition reaction product is precipitated using pre-cooled heptane, and the obtained solid product is purified by Al2O3 chromatography to obtain a pure butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation;

[0018] The mobile phases for the Al2O3 chromatography column purification were EtOAc / MeOH and DCM / MeOH, respectively.

[0019] The present invention provides the application of the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation in potassium ion transmembrane transport.

[0020] The present invention provides a method for regulating the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0021] A mixture of glucose, vesicles, mechanical and pH-dual regulated butterfly potassium channel molecules is provided. By regulating the concentration of glucose in the mixture, the osmotic pressure inside and outside the vesicles is induced to change, thereby causing changes in the vesicle membrane tension, thereby regulating the potassium ion transmission rate of the butterfly potassium channel molecules.

[0022] The present invention provides a method for regulating the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0023] mixing benzylamine hexafluorophosphate, a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and a pH regulator to obtain a premixed solution;

[0024] The premixed solution is mixed with the vesicle solution. When the pH value of the premixed solution is less than 10, the potassium ion channel is closed, and when the pH value is greater than or equal to 10, the potassium ion channel is opened.

[0025] The present invention provides the use of the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation in the preparation of anticancer drugs.

[0026] Preferably, the anticancer drug includes an anti-glioblastoma drug or an anti-melanoma drug.

[0027] The present invention provides a butterfly-shaped potassium ion channel molecule GnC7 (n=3, 4) with dual mechanical and pH regulation. The butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation of the present invention is based on the third-generation and fourth-generation dendrimer polypropylene imine (POPAM) as the core, respectively. POPAM and the ion binding site benzo-21-crown ether-7 are connected by a urea bond. The transmembrane ability of GnC7 is related to the alkyl chains rich in POPAM. Therefore, in order to stabilize the membrane, GnC7 with POPAM as the core structure should adopt a stretched state, thereby exposing the hydrophobic alkyl chains as much as possible to interact with the hydrophobic layer of the phospholipid membrane. The spatial restriction caused by the multi-layer branching points of POPAM causes the crown ether to form a channel around its periphery, and this channel is stabilized by the intramolecular hydrogen bonds formed by the urea bond. Intramolecular hydrogen bond constraints make GnC7 present a symmetrical "butterfly-shaped" topological structure, and the channel has unique motion characteristics, which ultimately achieves efficient and so far the highest potassium-sodium selectivity of potassium ion transmembrane transport. The potassium / sodium selectivity values of G3C7 and G4C7 are as high as 34.38 and 41.31, respectively, which are higher than the highest potassium / sodium selectivity value of 32 in the prior art (see J.Am.Chem.Soc.2021,143,3284-3288). Dynamic properties, as the core attribute of GnC7, enable the channel to regulate the potassium ion transport rate according to changes in membrane tension caused by mechanical force. At the same time, benzylamine hexafluorophosphate Bh, which is affected by pH, acts as a blocker of GnC7 through host-guest specific complexing, which can realize the switch of pH-regulated potassium ion transport. In addition, GnC7 has shown great potential in cancer treatment. The results of the examples show that G4C7 can achieve an IC of 7.6μM by drastically disrupting the potassium ion concentration in cancer cells. 50 Effectively induces apoptosis in U87MG cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A synthetic route for butterfly-shaped potassium ion channel molecules with dual mechanical and pH regulation;

[0029] Figure 2 For G3C7 1 H NMR spectrum;

[0030] Figure 3 For G4C7 1 H NMR spectrum;

[0031] Figure 4 The results of the transport activity test of GnC7 are shown;

[0032] Figure 5 The transport mechanism and potassium / sodium selectivity of GnC7 were tested;

[0033] Figure 6Taking G3C7 as an example, the results of the motility study of GnC7 are shown;

[0034] Figure 7 The results of the mechanical sensitivity study of GnC7 are presented;

[0035] Figure 8 The pH controllability test results of GnC7;

[0036] Figure 9 G4C7 induces the efflux of intracellular potassium ions in U87 MG;

[0037] Figure 10 These are the test results of the anticancer activity of GnC7. DETAILED DESCRIPTION

[0038] The present invention provides a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, having a structure shown in Formula G3C7 or Formula G4C7:

[0039]

[0040] The present invention provides a method for preparing the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0041] isocyanate-modified POPAM by reacting a dendrimer POPAM having a structure represented by formula a or formula b with di-tert-butyl tricarbonate;

[0042]

[0043] The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with an amino-containing benzo-21-crown ether-7 having a structure shown in formula c to obtain a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 with dual mechanical and pH regulation;

[0044]

[0045] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0046] In the present invention, a dendrimer POPAM having a structure represented by Formula a or Formula b is isocyanated with di-tert-butyl tricarbonate to obtain isocyanate-modified POPAM. In the present invention, the dendrimer POPAM is a third-generation POPAM (Formula a) or a fourth-generation POPAM (Formula b). As a specific embodiment of the present invention, the dendrimer POPAM is purchased from Sigma-Aldrich.

[0047] In the present invention, the mass ratio of the dendrimer POPAM having the structure represented by formula a to di-tert-butyl tricarbonate is preferably 1:3 to 5, more preferably 1:4. In the present invention, the isocyanate reaction is preferably carried out in an organic solvent, and the organic solvent is preferably dichloromethane (DCM).

[0048] In the present invention, the isocyanate reaction is preferably carried out under nitrogen protection, the temperature of the isocyanate reaction is preferably 20-27°C, more preferably 25°C, and the time is preferably 40-60 minutes, more preferably 40-50 minutes. After the isocyanate reaction, the present invention preferably does not perform post-treatment and directly proceeds to the next reaction.

[0049] After obtaining the isocyanate-modified POPAM, the isocyanate-modified POPAM undergoes a nucleophilic addition reaction with benzo-21-crown-7 with an amino group having a structure shown in formula c to obtain a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation having a structure shown in formula G3C7 or formula G4C7.

[0050] In the present invention, the mass ratio of the dendrimer POPAM having the structure shown in formula a to the amino-containing benzo-21-crown ether-7 having the structure shown in formula c is preferably 1:19-30, more preferably 1:22-25.

[0051] In the present invention, the temperature of the nucleophilic addition reaction is preferably 20-27° C., more preferably 25° C., and the time is preferably 16-24 h, more preferably 16-20 h.

[0052] After the nucleophilic addition reaction, the present invention further preferably includes post-processing the obtained nucleophilic addition reaction product, and the post-processing preferably includes:

[0053] The nucleophilic addition reaction product is precipitated using pre-cooled heptane, and the obtained solid product is purified by Al2O3 chromatography column to obtain a pure butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation.

[0054] In the present invention, the pre-cooling temperature is preferably 0-4° C. The purpose of the pre-cooling is to reduce the solubility of the product in heptane and to better precipitate the product.

[0055] In the present invention, the mobile phases for the Al2O3 chromatography column purification are preferably EtOAc / MeOH and DCM / MeOH, respectively. In the present invention, the volume ratio of EtOAc / MeOH is preferably 40:1, and the volume ratio of DCM / MeOH is preferably 20:1.

[0056] In the present invention, the synthesis route of the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation is as follows Figure 1 shown.

[0057] The present invention provides the application of the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation in potassium ion transmembrane transport.

[0058] The present invention provides a method for regulating the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0059] A mixture of glucose, vesicles, mechanical and pH-dual regulated butterfly potassium channel molecules is provided. By regulating the concentration of glucose in the mixture, the osmotic pressure inside and outside the vesicles is induced to change, thereby causing changes in the vesicle membrane tension, thereby regulating the potassium ion transmission rate of the butterfly potassium channel molecules.

[0060] In the present invention, the concentration of GnC7 in the mixed solution is preferably 0.9 to 1.3 μM. In the present invention, the vesicles are preferably provided in the form of a vesicle buffer, specifically a buffer containing 10 mM HEPES (4-hydroxyethylpiperazine ethylsulfonic acid), 100 mM NaCl and pH = 7.0.

[0061] In the present invention, the concentration of glucose in the mixed solution is preferably 0-350 mM, specifically 0, 200 mM or 350 mM, and the concentration of glucose in the vesicles is preferably 200 mM.

[0062] The present invention provides a method for regulating the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, comprising the following steps:

[0063] benzylamine hexafluorophosphate (Bh), a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and a pH regulator are mixed to obtain a premixed solution;

[0064] The premixed solution is mixed with the vesicle solution. When the pH value of the premixed solution is less than 10, the potassium ion channel is closed, and when the pH value is greater than or equal to 10, the potassium ion channel is opened.

[0065] The present invention mixes benzylamine hexafluorophosphate, a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and a pH regulator to obtain a premixed solution. In the present invention, the structural formula of the benzylamine hexafluorophosphate is shown in formula d:

[0066]

[0067] In the present invention, when the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation is G3C7, the molar ratio of G3C7 to benzylamine hexafluorophosphate is preferably 1:26-40, more preferably 1:30-35; when the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation is G4C7, the molar ratio of G4C7 to benzylamine hexafluorophosphate is preferably 1:32-45, more preferably 1:35-40.

[0068] In the present invention, the concentration of the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation in the mixed solution is preferably 0.5 to 5 μM. In the present invention, the pH regulator is preferably NaOH and / or HCl.

[0069] The present invention mixes the premix with the vesicle solution. In the present invention, the volume ratio of the premix to the vesicle solution is preferably 1:5 to 50. As a specific embodiment of the present invention, the volume ratio of the premix to the vesicle solution is 1:5, 1:10, 1:20, 1:30, 1:40 or 1:50. In the present invention, when the pH value of the premix is less than 10, preferably, when the pH value is ≤9, the potassium ion channel is completely closed. When the pH value is ≥10, as the pH value increases, the potassium ion channel slowly opens. Specifically, when the pH value is 10, the potassium ion channel opens 30%, when the pH value is 11, the potassium ion channel opens 90%, and when the pH value is 12, the potassium ion channel is completely open.

[0070] The present invention provides the use of the above-mentioned butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation in the preparation of anticancer drugs. Preferably, the anticancer drug includes an anti-human brain glioblastoma drug or an anti-melanoma drug.

[0071] The butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation provided by the present invention, as well as its preparation method and application, are described in detail below with reference to the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0072] Example 1 Preparation of butterfly-shaped potassium ion channel molecule G3C7:

[0073] To a stirred solution of di-tert-butyl tricarbonate (119 mg) in DCM (15 mL) was added a solution of trigeneration POPAM (30 mg) in DCM (5 mL), and the mixture was stirred at room temperature under argon for 40 minutes. After isocyanate formation, benzo-21-crown-7 (578 mg) with an amino group was added to the reaction mixture. After 16 hours, the crude product was precipitated with heptane (250 mL) precooled at 0-4°C. Purification on an Al2O3 column (mobile phase: EtOAc / MeOH, 40:1 and DCM / MeOH, 20:1) afforded the product as a pale yellow oil.

[0074] The obtained product was characterized by H NMR spectroscopy. 1 H NMR spectrum Figure 2 The characterization data are as follows:

[0075] G3C7. 1 H NMR (500MHz, CDCl3) δ6.74 (d, J = 27.2Hz, 48H), 6.37 (s, 24H),

[0076] 4.10(dq,J=20.8,6.7,6.3Hz,130H),3.84(s,64H),3.77-3.72(m,64H) ,3.71-3.67(m,64H),3.63(s,128H),3.13(s,32H),2.73-2.35(m,84H).

[0077] Example 2 Preparation of butterfly-shaped potassium channel molecule G4C7:

[0078] The difference from Example 1 is that the third-generation POPAM is replaced by the fourth-generation POPAM.

[0079] The obtained product was characterized by H NMR spectroscopy. 1 H NMR spectrum Figure 3 The characterization data are as follows:

[0080] G4C7. 1 H NMR (500MHz, CDCl3) δ6.69 (s, 96H), 6.39 (d, J = 55.9Hz, 48H),

[0081] 4.04(s,137H),3.84(s,128H),3.74(s,128H),3.69(s,125H),3.63(s,256H),3.14(s,64H),2.41(s,181H),1.65(d,J=45.9Hz,383H).

[0082] Test Example 1 Transport Activity, Channel Activity and Potassium / Sodium Selectivity Test

[0083] Ion transport activity was measured by the phosphatidylcholine (EYPC)-based 8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium (HPTS) assay. The pH-sensitive fluorophore 8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium (HPTS) and 100 mM sodium chloride were entrapped into EYPC-based large unilamellar vesicles (LUVs) at pH 7.0, and the extravesicular buffer was pH = 7.6 and contained 100 mM metal chloride MCl (M = Na, Li, K, Rb, Cs), thereby generating a pH and ion gradient inside and outside the LUV. + Transport from the outer coat to the LUV can be detected by the change in the fluorescence intensity of HPTS. The EC 50 The concentration required to reach 50% activity within 300 seconds is used to quantitatively represent the transport activity of the measured molecule for the corresponding ion. The faster and higher the fluorescence rise, the higher the transport activity of the molecule for the ion. The final data were normalized and presented as relative fluorescence values of 0 to 1.

[0084] Testing process: After adding 1 mL of extravesicle buffer + 25 μL of vesicle solution into the cuvette, start the fluorescence test. After running the baseline for 10 seconds, add the GnC7 sample to start transmission.

[0085] The results of the GnC7 transport activity test are as follows Figure 4 shown. Figure 4 The upper left figure shows the transport activity test of G3C7 (2 μM) to different alkali metal cations (K + ,Cs + ,Rb + ,Na + ,Li + ) test results (DMSO with the same volume as that of G3C7 solution was used as negative control group). The results showed that G3C7 had an effect on K + , Cs + ,Rb + The transport activity of ions is very good, and the Na + ,Li + The transmission performance is poor.

[0086] Figure 4 The upper right figure shows the transport activity test of G4C7 (2 μM) to different alkali metal cations (K + ,Cs + ,Rb + ,Na + ,Li + ) test results (DMSO with the same volume as that of G4C7 solution was used as negative control group). The results showed that G4C7 had an effect on K + , Cs + ,Rb +The transport activity of ions is very good, and the Na + ,Li + The transmission performance is poor.

[0087] The present invention focuses on biological applications, so only the physiological ion K is specifically studied. + ,Na + In order to quantitatively verify the superior K + The transport activity of G3C7 (0-2 μM) and G4C7 (0-3.1 μM) at different concentrations was tested. The concentration-activity data (activity value is the relative fluorescence value of transport 300 seconds) were used to calculate the transport activity of G3C7 (0-2 μM) and G4C7 (0-3.1 μM). 50 / [C]) n ) for analysis (where n is the Hill coefficient, and a value close to 1 indicates that a single molecule plays a role).

[0088] The test results showed that both G3C7 and G4C7 exhibited high transport activity, EC 50K+ 0.72μM ( Figure 4 in the lower left panel) and 0.9 μM ( Figure 4 (lower right image in ).

[0089] Using a lipid bilayer workstation, single-channel current signals are recorded on a planar lipid bilayer membrane (BLM) to determine the mechanism of ion transport (channel or carrier) of the measured molecule. In this experiment, only continuous transport caused by a channel will show a regular, square-shaped current signal. This experiment also allows the conductivity to reflect the transport rate and can be used to test transport selectivity.

[0090] The transport mechanism and potassium / sodium selectivity test results of GnC7 are as follows Figure 5 As shown. In a symmetrical chamber (forward chamber = reverse chamber = 1M KCl), the channel currents of G3C7 (1.6μM) and G4C7 (1.6μM) were recorded. The voltage was varied between 100mV and 150mV and the current signal and current value were recorded. The γ of G3C7 and G4C7 was determined by linear fitting using the IV curve. K+ (K + The results are as follows. Figure 5 The left four figures in the figure are shown. In an asymmetric tank (1M KCl in the anti-chamber was replaced with 1M NaCl), the channel currents of G3C7 (1.6μM) and G4C7 (1.6μM) were recorded. The IV curves of G3C7 and G4C7 were linearly fitted to obtain the reversal potential (ε rev ) are -90.84mV and -95.55mV respectively. Then use the GHK equation (ε rev =-(RT / F)*ln(PK+ / P Na+ )) Determine the ion permeability ratio (P K+ / P Na+ ) to reflect K + / Na + Selectivity. F = 96485C mol -1 ,R=8.314JK -1 mol -1 ,T=298K. The results are as follows Figure 5 As shown in the four pictures on the right.

[0091] The results showed that after GnC7 was added to the cis chamber containing 1M KCl solution (transverse chamber = 1M KCl), a regular square current signal appeared when voltage was applied to the membrane, confirming that GnC7 mediates transmembrane ion transport through a channel mechanism.

[0092] Furthermore, during the test, a voltage between -150mV and 100mV was applied to the membrane, and the conductivity values of the IV curves were 66.2±1.2pS and 37.4±1.6pS, respectively. The high conductivity indicated the high efficiency of ion transport. Then, an asymmetric planar lipid bilayer experiment (forward chamber = 1M KCl, reverse chamber = 1M NaCl) was conducted to evaluate the ion permeability (P K+ / P Na+ The reversal potential (εrev) of G3C7 and G4C7 was calculated by using the Goldman-Hodgkin-Katz (GHK) equation. K+ / P Na+ The values are as high as 34.38 and 41.31, respectively, indicating that the potassium / sodium selectivity has reached an unprecedented level.

[0093] Test Case 2: Study on Dynamic Characteristics

[0094] The dynamic nature of GnC7 was verified by a dipalmitoylphosphatidylcholine (DPPC)-based HPTS assay. When the temperature is below the glass transition temperature of DPPC (45°C), the phospholipid membrane of DPPC-LUVs freezes, causing ion transport dependent on carrier movement to cease, while ion transport in stationary channels across the phospholipid membrane remains unaffected.

[0095] Testing process: After adding 1 mL of extravesicle buffer + 25 μL of vesicle solution into the cuvette, start the fluorescence test. After running the baseline for 10 seconds, add the GnC7 sample to start transmission.

[0096] Taking G3C7 as an example (the final concentration of G3C7 is 7 μM), the results of the motility study of GnC7 are as follows: Figure 6 shown. Figure 6The left figure shows that, compared to the control group with an equal volume of DMSO added, which was unaffected by temperature, the relative fluorescence change induced by G3C7 in DPPC-LUVs containing HPTS at 25°C and 45°C showed a significant 30% difference within just 100 seconds. First-order kinetic studies revealed that the initial transport rate of the test sample within 15 seconds at 25°C was an order of magnitude lower than that at 45°C. This is consistent with the channel mechanism demonstrated by GnC7, a representative example of G3C7, in lipid bilayer experiments and suggests that GnC7 is a mobile channel with unique motility properties, which are crucial for its significantly enhanced ion transport activity.

[0097] Test Example 3: Study of Mechanical Sensitivity

[0098] The mechanosensitivity of GnC7 was demonstrated using EYPC-based HPTS assays under varying membrane tensions. By manipulating the glucose concentration in the extravesicular fluid (constantly maintained at 200 mM within the vesicle), the osmotic pressure shift, which in turn altered membrane tension, was induced. The vesicles used were EYPC-based vesicles containing HPTS.

[0099] The conversion between the osmotic pressure difference inside and outside the vesicle and the membrane tension is shown in Equation 1:

[0100] Δσ=(ΔCRTr) / 2, Equation 1;

[0101] In formula 1, ΔC is the difference in glucose concentration inside and outside the vesicle; T is the temperature; r is the vesicle radius; R = 8.314 J·mol -1 ·K -1 .

[0102] like Figure 7 The figure clearly shows that the membrane relaxation tension (Δσ=25mN m -1 ) vesicles, G3C7 (final concentration 0.94 μM) caused a 44% relative fluorescence change within 100 s, which was 16% higher than that of the tension-free group (28%). -1 The significant difference in transport activity under different membrane tensions strongly supports the responsiveness of the channel molecule to mechanical stimulation.

[0103] Test Example 4 pH Controllability Study

[0104] The inhibitor Bh inhibits GnC7 channel activity through a reliable host-guest complex between protonated benzylamine and 21-crown-7 ether, thereby blocking the channel. The protonation state of Bh is pH-dependent, so pH regulation of GnC7 channel activity can be achieved by adjusting the pH value between 6 and 12 by adding NaOH and HCl. The results of the pH controllability test of GnC7 are shown in Figure 2. Figure 8 shown. Figure 8 The upper left panel shows a schematic diagram of the host-guest interaction between Bh and dibenzo-21-crown-7-ether. The upper right panel shows the normalized potassium transport activity of G3C7 (2 μM) in the presence of different ratios of Bh blocker. The lower left panel shows the normalized potassium transport activity of G4C7 (3 μM) in the presence of different ratios of Bh blocker. The lower right panel shows that in the presence of Bh (52 μM), alternating pH values between 6 and 12 results in multiple ON / OFF cycles in the potassium transport activity of G3C7 (2 μM).

[0105] Optimizing the G3C7 / G4C7 and Bh ratios for optimal shut-off results revealed that transport was completely blocked at molar ratios of 1:26 and 1:32, respectively. By adjusting the pH value, the transport activity of G3C7 could be precisely controlled over three consecutive cycles.

[0106] Test Example 5: Study on Mediating Intracellular Potassium Ion Efflux

[0107] Using confocal laser scanning microscopy (CLSM) combined with the potassium ion probe EPG-4AM, dynamic changes in potassium ion concentration in human astrocytic glioblastoma cells (U87 MG) after two hours of G4C7 treatment were monitored in real time. The method involved staining adherent cells cultured in eight-well chamber slides with the probe and imaging them under CLSM, with one image taken every 45 seconds. After 5-6 images were taken, a buffer containing the molecule was added to the chamber containing the cells and imaging continued. Monitoring began for two hours from the time the molecule was added. The final G4C7 concentration was 64 μM.

[0108] The effect of G4C7 on the intracellular potassium ion in U87 MG is as follows Figure 9 As shown, Figure 9 The upper figure shows representative CLSM images of U87MG cells at different time points after the addition of G4C7 (64 μM). Scale bar: 20 μm. The lower figure shows the effect of G4C7 (64 μM) on K + Quantitative analysis of concentration effects.

[0109] The results showed that the addition of 64 μM G4C7 caused a 70% drop in fluorescence intensity within only 9 minutes, rapidly disrupting potassium ion homeostasis.

[0110] Test Example 6 Cytotoxicity Study

[0111] The viability of U87 MG or melanoma B16F10 cells exposed to G3C7 or G4C7 was assessed using the CellCounting Kit-8 (CCK-8) assay. Briefly, 10,000 cells were plated in 100 μL of DMEM (10% FBS) in a 96-well plate (culture area, 0.32 cm²). After 12 hours of incubation, the medium was removed and 100 μL of serum-free DMEM containing various concentrations of G3C7 or G4C7 (0.25 to 64 μM, containing 1 wt% DMSO) was added. Cells not exposed to the channel molecules served as a control group. After 24 hours of culture, the medium was removed and the cells were incubated with 100 μL of DMEM (10% FBS) containing 10% v / v CCK-8 for 2-3 hours. The absorbance of each well was measured at 450 nm using a Tecan Spark multi-function microplate reader. The cell viability of the control group was 100%, and the absorbance of each well was normalized to the absorbance value of the control group.

[0112] The anticancer activity test results of GnC7 are as follows Figure 10 shown. Figure 10 The left figure shows the dose-dependent curve of cell viability assessed by CCK-8 assay after incubation of U87 MG cells with G3C7 (0.025-64 μM) for 24 hours. The half-inhibitory concentration (IC50) of G3C7 on U87 MG cells was obtained by curve analysis. 50 ) was 25.3 μM. Figure 10 The right figure shows the dose-dependent curve of cell viability assessed by CCK-8 assay after incubation of B16F10 and U87 MG cells with G4C7 (0.025-64 μM) for 24 hours. The results show that G4C7 has an IC 50 The values were 7.6 μM and 17.7 μM, respectively, indicating high toxicity.

[0113] The results of CCK-8 experiments showed that both G3C7 and G4C7 showed good anti-cancer potential. G4C7 could significantly disrupt the potassium ion concentration in cancer cells with an IC of 7.6 μM. 50 Effectively induces apoptosis in U87MG cells.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, characterized in that: It has the structure shown in formula G3C7 or G4C7:

2. The method for preparing the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation according to claim 1, comprising the following steps: isocyanate-modified POPAM by reacting a dendrimer POPAM having a structure represented by formula a or formula b with di-tert-butyl tricarbonate; The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with an amino-containing benzo-21-crown ether-7 having a structure shown in formula c to obtain a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 with dual mechanical and pH regulation; 3. The preparation method according to claim 2, characterized in that The temperature of the isocyanate reaction is 20-27° C., and the time is 40-60 minutes.

4. The preparation method according to claim 2, characterized in that The temperature of the nucleophilic addition reaction is 20-27° C., and the time is 16-24 hours.

5. The preparation method according to claim 2, characterized in that After the nucleophilic addition reaction, the obtained nucleophilic addition reaction product is subjected to post-treatment, and the post-treatment includes: The nucleophilic addition reaction product is precipitated using pre-cooled heptane, and the obtained solid product is purified by Al2O3 chromatography to obtain a pure butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation; The mobile phases for the Al2O3 chromatography column purification were EtOAc / MeOH and DCM / MeOH, respectively.

6. Use of the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation according to claim 1 or the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation prepared by the preparation method according to any one of claims 2 to 5 in potassium ion transmembrane transport.

7. A method for regulating the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation according to claim 1 or the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation prepared by the preparation method according to any one of claims 2 to 5, comprising the following steps: A mixture of glucose, vesicles, mechanical and pH-dual regulated butterfly potassium channel molecules is provided. By regulating the concentration of glucose in the mixture, the osmotic pressure inside and outside the vesicles is induced to change, thereby causing changes in the vesicle membrane tension, thereby regulating the potassium ion transmission rate of the butterfly potassium channel molecules.

8. A method for regulating the butterfly-shaped potassium channel molecule with dual mechanical and pH regulation according to claim 1 or the butterfly-shaped potassium channel molecule with dual mechanical and pH regulation prepared by the preparation method according to any one of claims 2 to 5, comprising the following steps: mixing benzylamine hexafluorophosphate, a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and a pH regulator to obtain a premixed solution; The premixed solution is mixed with the vesicle solution. When the pH value of the premixed solution is less than 10, the potassium ion channel is closed, and when the pH value is greater than or equal to 10, the potassium ion channel is opened.

9. Use of the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation according to claim 1 or the butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation prepared by the preparation method according to any one of claims 2 to 5 in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that The anticancer drug includes an anti-human brain glioblastoma drug or an anti-melanoma drug.

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