Butterfly-shaped potassium ion channel molecule with mechanical and pH dual regulation and control functions as well as preparation method and application of butterfly-shaped potassium ion channel molecule

Abstract

The invention provides a mechanical and pH dual-regulated butterfly potassium ion channel molecule as well as a preparation method and application thereof, and belongs to the technical field of biomimetic materials. According to the butterfly-shaped potassium ion channel molecule disclosed by the invention, third-generation and fourth-generation dendrimer polypropyleneimine (POPAM) are respectively taken as a core, and POPAM is connected with an ion binding site benzo-21-crown ether-7 through a urea bond. Intramolecular hydrogen bond constraint enables GnC7 to present a symmetrical butterfly-shaped topology structure, and the channel has unique motion characteristics, and finally, efficient potassium ion transmembrane transmission with highest potassium-sodium selectivity up to now is realized. The dynamic characteristic is used as the core attribute of GnC7, so that the channel can adjust the potassium ion transmission rate according to the membrane tension change caused by mechanical force. Meanwhile, benzylamine hexafluorophosphate Bh influenced by pH is used as a blocking agent of GnC7 through a host-guest specific compounding effect, and switching of pH-regulated potassium ion transmission can be realized.

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, channel protein disorders usually lead to a series of ion channel diseases including cystic fibrosis and arrhythmia. In order to grasp the potential key factors of biological functions and provide directions for drug development for the treatment of channel diseases, bionic 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 response properties.

[0003] In recent years, artificial ion channels that respond to various stimuli have been reported. It is worth noting that mechanosensitivity and pH sensitivity each play an irreplaceable role in the five senses of human beings. Mechanosensitive 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 mechanosensitive and pH-responsive.

[0004] Therefore, developing artificial ion channels with both mechanosensitive and pH-responsive properties, which are currently lacking, 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 potassium ion channel molecule with dual mechanical and pH regulation and its preparation method and application. The butterfly potassium ion channel molecule with dual mechanical and pH regulation provided by the present invention can realize efficient potassium-sodium selective potassium ion transmembrane transport, and can realize 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] The dendrimer POPAM having a structure shown in formula a or formula b is subjected to isocyanate reaction with di-tert-butyl tricarbonate to obtain isocyanate-modified POPAM;

[0011]

[0012] The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with benzo-21-crown-7 having an amino group and having a structure shown in formula c, thereby obtaining a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 and having dual mechanical and pH control.

[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 comprises:

[0017] The nucleophilic addition reaction product is precipitated using precooled heptane, and the obtained solid product is purified by Al2O3 chromatography column to obtain a pure butterfly 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 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, which in turn causes the vesicle membrane tension to change, 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, the 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 respectively based on the third-generation and fourth-generation dendrimer polypropylene imine (POPAM) as the core, and 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 be in a stretched state, so as to expose the hydrophobic alkyl chain 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 bonds. 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 transfer 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 shows great potential in cancer treatment. The results of the examples show that G4C7 can drastically disrupt the potassium ion concentration in cancer cells with an IC of 7.6μM. 50 Effectively induces apoptosis of 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 transmission activity test of GnC7;

[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 test of GnC7 are shown;

[0034] Figure 7 The results of the mechanical sensitivity study of GnC7 were tested;

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

[0036] Fig. 9 G4C7 induces the efflux of potassium ions in U87 MG cells;

[0037] Fig.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] The dendrimer POPAM having a structure shown in formula a or formula b is subjected to isocyanate reaction with di-tert-butyl tricarbonate to obtain isocyanate-modified POPAM;

[0042]

[0043] The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with benzo-21-crown-7 having an amino group and having a structure shown in formula c, thereby obtaining a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 and having dual mechanical and pH control.

[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 shown in formula a or formula b is subjected to an isocyanate reaction 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 shown in formula a to di-tert-butyl tricarbonate is preferably 1:3-5, 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; the time is preferably 40-60 min, more preferably 40-50 min. After the isocyanate reaction, the present invention preferably does not perform post-treatment and directly proceeds to the next step of 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 a structure shown in formula a to the amino-containing benzo-21-crown ether-7 having a 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 to 27° C., more preferably 25° C., and the time is preferably 16 to 24 h, more preferably 16 to 20 h.

[0052] After the nucleophilic addition reaction, the present invention also preferably includes post-treatment of the obtained nucleophilic addition reaction product, and the post-treatment preferably includes:

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

[0054] In the present invention, the precooling temperature is preferably 0-4° C. The purpose of the precooling 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 purification by the Al2O3 chromatography column 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 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, which in turn causes the vesicle membrane tension to change, 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 0.9-1.3 μM. In the present invention, the vesicles are preferably provided in the form of vesicle buffer, which is specifically a buffer containing 10 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic 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 vesicle 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] Mixing benzylamine hexafluorophosphate (Bh), a butterfly-shaped potassium ion channel molecule with dual mechanical and pH regulation, and a pH regulator 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 with 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 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, and more preferably 1:30-35; when the butterfly 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, and more preferably 1:35-40.

[0068] In the present invention, the concentration of the butterfly potassium ion channel molecules regulated by both mechanical and pH 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 a 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, and when the pH value is ≥10, as the pH value increases, the potassium ion channel slowly opens, specifically, when the pH value = 10, the potassium ion channel opens 30%, when the pH value = 11, the potassium ion channel opens 90%, and when the pH value = 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 molecules with dual mechanical and pH regulation provided by the present invention, as well as their preparation methods and applications are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

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

[0073] Under stirring, a DCM solution (5 mL) of the third generation POPAM (30 mg) was added to a DCM solution (15 mL) containing di-tert-butyl tricarbonate (119 mg), and stirred at room temperature for 40 minutes under argon protection. After the formation of isocyanate, benzo-21-crown ether-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. After purification on an Al2O3 chromatography column (mobile phase: EtOAc / MeOH, 40:1 and DCM / MeOH, 20:1), a light yellow oily product was obtained.

[0074] The obtained product was characterized by hydrogen nuclear magnetic resonance 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 hydrogen nuclear magnetic resonance 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 trisulfonate trisodium (HPTS) assay. The pH-sensitive fluorophore 8-hydroxy-1,3,6-pyrene trisulfonate trisodium (HPTS) and 100 mM sodium chloride were entrapped into EYPC-based large unilamellar vesicles (LUVs) at pH 7.0, and the extravesicular buffer pH = 7.6 contained 100 mM metal chloride MCl (M = Na, Li, K, Rb, Cs), thereby generating a pH and ion gradient inside and outside the LUVs. + Transport from the exocoel to the LUV can be detected by the change in 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 rises, the higher the transport activity of the molecule for the ion. The final data was normalized and presented as a relative fluorescence value of 0 to 1.

[0084] Test 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 the transmission.

[0085] The results of the transport activity test of GnC7 are as follows Figure 4 shown. Figure 4 The upper left figure shows the transport activity test of G3C7 (2 μM) for 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 + , Cs + ,Rb + The ion transport activity is very good, and for 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 the negative control group). The results showed that G4C7 had an + , Cs + ,Rb +The ion transport activity is very good, and for Na + ,Li + The transmission performance is poor.

[0087] The present invention focuses on biological applications, and therefore only specifically studies the physiological ion K + ,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 obtained series of concentration-activity data (activity value is the relative fluorescence value of the transport 300 s) 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 figure in the figure).

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

[0090] The transport mechanism and potassium / sodium selectivity of GnC7 were tested as follows Figure 5 As shown. In a symmetrical tank (forward chamber = reverse chamber = 1M KCl), the channel currents of G3C7 (1.6μM) and G4C7 (1.6μM) were recorded. The voltage value was changed between 100mV--150mV and the current signal and current value were recorded. The IV curve was linearly fitted to determine the γ K+ (K + The results are as follows: Figure 5 The left four figures in the figure are shown. In the asymmetric tank (1M KCl in the counter 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 (transverse chamber = 1M KCl) containing 1M KCl solution, a regular square current signal appeared when voltage was applied to the membrane, confirming that GnC7 mediates the transmembrane transport of ions by a channel mechanism.

[0092] Moreover, during the test, the 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 (cis chamber = 1M KCl, anti chamber = 1M NaCl) was performed to evaluate the ion permeability (P K+ / P Na+ The P values ​​of G3C7 and G4C7 were calculated by analyzing the reversal potential (εrev) 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 dynamics of GnC7 were verified by the HPTS assay based on dipalmitoylphosphatidylcholine (DPPC). When the temperature is lower than the glass transition temperature of DPPC (45°C), the phospholipid membrane of DPPC-LUVs is in a frozen state, resulting in the cessation of ion transport dependent on carrier movement, while ion transport in stationary channels across the phospholipid membrane is not affected.

[0095] Test 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 the 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 in the figure shows that, compared with the control group with an equal volume of DMSO added, which was not affected by temperature, the relative fluorescence change caused by G3C7 in DPPC-LUVs containing HPTS at 25°C and 45°C showed a significant difference of 30% in just 100 seconds. Studies on first-order kinetics showed 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 of GnC7 represented by G3C7 in lipid bilayer experiments, and indicates that GnC7 is a moving channel with unique motion characteristics, and this is extremely critical to the substantial improvement of its ion transport activity.

[0097] Test Example 3 Study of Mechanical Sensitivity

[0098] The mechanical sensitivity of GnC7 was demonstrated by the HPTS test based on EYPC under different membrane tensions. The glucose concentration in the extravesicular fluid was regulated to set the glucose concentration difference inside and outside the vesicle (constant 200mM inside the vesicle) to induce osmotic pressure changes, thereby changing membrane tension. 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 formula 1:

[0100] Δσ=(ΔCRTr) / 2, Formula 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 Control Study

[0104] The inhibitor Bh inhibits the activity of GnC7 channel through reliable host-guest complexation 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 adding NaOH and HCl to adjust the pH value between 6 and 12. The test results of pH controllability of GnC7 are shown in Figure 2. Figure 8 shown. Figure 8 The upper left figure in the figure is a schematic diagram of the host-guest interaction between Bh and dibenzo-21-crown-7-ether. The upper right figure shows the normalized potassium ion transport activity of G3C7 (2μM) under the action of different ratios of Bh blocker. The lower left figure shows the normalized potassium ion transport activity of G4C7 (3μM) under the action of different ratios of Bh blocker. The lower right figure shows that in the presence of Bh (52μM), the alternating changes in pH between 6 and 12 lead to multiple ON / OFF cycles of potassium ion transport activity of G3C7 (2μM).

[0105] The ratio of G3C7 / G4C7 and Bh was optimized to obtain the best shutdown effect, and it was found that the transmission was completely blocked when the molar ratio was 1:26 and 1:32, respectively. By adjusting the pH value, the transmission activity switching state of G3C7 can be precisely controlled for three consecutive cycles.

[0106] Test Example 5 Study on mediating intracellular potassium ion efflux

[0107] Using confocal laser scanning microscopy (CLSM) combined with potassium ion probe EPG-4AM, the dynamic changes of potassium ion concentration in human brain astrocytoblastoma cells (U87 MG) after two hours of G4C7 were monitored in real time. The specific method is: after the cells attached to the wall are cultured in an eight-well chamber slide and stained with the probe, they are imaged and photographed under CLSM, one image is taken every 45 seconds. After taking 5 to 6 images, the buffer containing the molecule is added to the chamber containing the cells and the images are continued. The monitoring starts from the addition of the molecule for two hours. The final concentration of G4C7 is 64μM.

[0108] G4C7 induces the intracellular potassium ion effect of U87 MG Fig. 9 As shown, Fig. 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 after exposure to G3C7 or G4C7 was assessed by CellCounting Kit-8 (CCK-8) assay. Briefly, 10,000 cells in 100 μL DMEM medium (containing 10% FBS) were added to 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 solution containing different concentrations of G3C7 or G4C7 (0.25-64 μM, containing 1 wt% DMSO) was added. Cells not exposed to channel molecules served as the control group. After 24 hours of culture, the medium was removed and the cells were cultured with 100 μL of DMEM medium (containing 10% FBS) solution containing CCK-8 (10% v / v) for 2-3 hours. The absorbance of each well was measured at a wavelength of 450 nm using a Tecan Spark multifunctional 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 results of the anticancer activity test of GnC7 are as follows Fig.10 shown. Fig.10 The left figure in the 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 (IC 50 ) is 25.3μM. Fig.10 The right figure in the 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 the IC 50 The values ​​were 7.6 μM and 17.7 μM, respectively, which were highly toxic.

[0113] The results of CCK-8 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 value of 7.6 μM. 50 Effectively induces apoptosis of 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 principle of the present invention. These improvements and modifications should also be regarded as 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 formula 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: The dendrimer POPAM having a structure shown in formula a or formula b is subjected to isocyanate reaction with di-tert-butyl tricarbonate to obtain isocyanate-modified POPAM; The isocyanate-modified POPAM undergoes a nucleophilic addition reaction with benzo-21-crown-7 having an amino group and having a structure shown in formula c, thereby obtaining a butterfly-shaped potassium ion channel molecule having a structure shown in formula G3C7 or formula G4C7 and having dual mechanical and pH control.

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 precooled heptane, and the obtained solid product is purified by Al2O3 chromatography column to obtain a pure butterfly 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, which in turn causes the vesicle membrane tension to change, thereby regulating the potassium ion transmission rate of the butterfly potassium channel molecules.

8. 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: Mixing benzylamine hexafluorophosphate, the 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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