Selenium-containing hyperbranched polymer as well as preparation method and application thereof

Abstract

The invention discloses a selenium-containing hyperbranched polymer as well as a preparation method and application thereof, and belongs to the technical field of preparation of intelligent biomimetic materials. The preparation method comprises the following steps: preparing a compound M1; preparing a hyperbranched polymer; the preparation method comprises the following steps: dissolving M1 in anhydrous dichloromethane, adding anhydrous DMF and oxalyl chloride, stirring, and removing a solvent and excessive oxalyl chloride under reduced pressure to obtain a reactant A; suspending the hyperbranched polymer and triethylamine in anhydrous tetrahydrofuran under the protection of nitrogen, and stirring to obtain a reactant B; dissolving the reactant A in anhydrous tetrahydrofuran, dropwise adding into the reactant B, heating to room temperature, stirring, removing the solvent, dissolving in anhydrous tetrahydrofuran, and dialyzing to obtain the selenium-containing hyperbranched polymer. The invention provides a selenium-containing hyperbranched polymer as well as a preparation method and application thereof. The selenium-containing hyperbranched polymer has excellent anticancer activity and can quickly change the pH value in cells and induce apoptosis of cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of intelligent bionic material preparation, and specifically relates to a selenium-containing hyperbranched polymer and a preparation method and application thereof. Background Art

[0002] Natural channel proteins, such as acid-sensing ion channels (ASICs), ATP-sensitive potassium channels (KATP), and human voltage-gated proton channels (Hv1), form channels on the cell membrane, control the transmembrane transport of species such as ions and water molecules, and are essential for maintaining the normal physiological functions of organisms. Pathological abnormalities of these channel proteins usually lead to a series of diseases such as cardiomyopathy and cystic fibrosis. The Hv1 channel is particularly important because it can effectively regulate the efflux of intracellular protons, affect the production of reactive oxygen species, and then regulate the normal operation of other physiological activities. Its dysfunction is associated with a variety of diseases including chronic pain.

[0003] Inspired by this, biomimetic chemists have been working on developing artificial transmembrane transport systems to mimic the high transport efficiency, significant ion selectivity, and gating properties of natural channel proteins. However, it is extremely challenging to develop artificial systems that simultaneously meet these characteristics, especially the selectivity and transport rate of artificial proton channels. As a three-dimensional macromolecule with an inner cavity and a large number of functional groups, hyperbranched polymers (HBPs) have attracted great interest and applications in drug delivery, nanomaterials, molecular sensing, and other fields. The three-dimensional structure and rich functional groups of HBPs make them potential in mimicking the selective proton transport of natural proton channels, and the introduction of selenides may affect ion transport by regulating the ability of HBPs to embed into phospholipid membranes, showing the potential to integrate multiple properties of natural channel proteins. Therefore, synthetic systems based on selenium-containing HBPs may efficiently and selectively transport protons across lipid bilayers and cell membranes while exhibiting controllable behaviors, providing a promising research direction for the development of new artificial proton channels. Summary of the invention

[0004] The present invention aims to provide a hyperbranched polymer and a preparation method and application thereof. The selenium-containing hyperbranched polymer has excellent anti-cancer activity, can quickly change the intracellular pH value, induce cancer cell apoptosis, and shows significant toxicity to two cancer cell lines, melanoma B16F10 and malignant glioma U87MG.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a selenium-containing hyperbranched polymer comprises the following steps:

[0007] S1. preparing compound M1;

[0008] S2, preparing a hyperbranched polymer;

[0009] S3, dissolving the compound M1 obtained in step S1 in anhydrous dichloromethane, adding anhydrous DMF and oxalyl chloride, heating and stirring overnight, and removing the solvent and excess oxalyl chloride under reduced pressure to obtain reactant A;

[0010] S4, under nitrogen protection, suspending the hyperbranched polymer obtained in step S2 and triethylamine in anhydrous tetrahydrofuran, stirring at 0° C. for 5 min, to obtain reactant B;

[0011] S5, dissolving the reactant A obtained in step S3 in anhydrous tetrahydrofuran, stirring evenly, and adding dropwise to the reactant B in step S4. After the addition is completed, the mixture is warmed to room temperature, stirred overnight, and the solvent is removed by a rotary evaporator, dissolved in anhydrous tetrahydrofuran, and dialyzed in a tetrahydrofuran-water mixed system with a volume ratio of 1:1 for 24 hours to obtain a selenium-containing hyperbranched polymer.

[0012] Preferably, in step S1, the method for preparing compound M1 comprises the following steps:

[0013] A1. Dissolve dimethyl diselenide in anhydrous ethanol, stir evenly, slowly add sodium borohydride at 0°C, stir at 0°C for 30 minutes to obtain mixture A;

[0014] A2, dissolving 2-chloroethyl ester in anhydrous ethanol, stirring evenly, adding to the mixed solution A obtained in step A1, stirring at 0°C for 1h, to obtain a mixture B;

[0015] A3, sequentially distilling water and anhydrous ethanol into the mixture obtained in step A2, extracting and separating the organic layer, washing with distilled water and brine in sequence, retaining the organic phase, and removing the solvent under reduced pressure to obtain a mixture C;

[0016] A4. Dissolve the mixture C obtained in step A3 in 99% by mass ethanol, add KOH aqueous solution, stir at room temperature overnight, then add distilled water and ether in sequence, acidify to pH = 2 with concentrated hydrochloric acid, extract with ether, wash the organic layer with distilled water, dry with anhydrous Na2SO4, and remove the solvent on a rotary evaporator to obtain M1.

[0017] Preferably, in step S2, the method for preparing a hyperbranched polymer comprises the following steps:

[0018] B1. Under nitrogen protection, add anhydrous dichloromethane and boron trifluoride diethyl ether into a round-bottom flask to obtain a mixture D;

[0019] B2. Add 3-ethyl-3-(hydroxymethyl)-oxetane to the mixture D obtained in step B1 through a dropping funnel within 5 minutes, react at -20-30°C for 48-54 hours, quench the reaction with ethanol, add to ultrapure water, filter, and vacuum dry at 80°C for 180 minutes to obtain a hyperbranched polymer.

[0020] Preferably, in step S3, the compound M1 obtained in step S1 is dissolved in anhydrous dichloromethane, anhydrous DMF and oxalyl chloride are added, and the mixture is heated under reflux at 60° C. and stirred overnight under nitrogen protection.

[0021] Preferably, in step S5, the volume ratio of anhydrous tetrahydrofuran to distilled water in the tetrahydrofuran-water mixed system is 1:1.

[0022] Preferably, in step S5, during the dialysis process, the tetrahydrofuran-water mixed system is replaced every 3 hours, and the molecular weight cutoff of the dialysis membrane is 1000Da.

[0023] The invention also provides the selenium-containing hyperbranched polymer prepared by the preparation method.

[0024] The present invention also provides the selenium-containing hyperbranched polymer prepared by the preparation method or the use of the selenium-containing hyperbranched polymer in preparing drugs for treating and / or preventing anti-tumor.

[0025] The present invention also provides a pharmaceutical composition, comprising one or more selenium-containing hyperbranched polymers prepared by the above-mentioned preparation methods or the above-mentioned selenium-containing hyperbranched polymers.

[0026] Preferably, the pharmaceutical composition comprises the selenium-containing hyperbranched polymer as an active ingredient and a pharmaceutically acceptable carrier.

[0027] A pharmaceutical preparation comprises a therapeutically effective amount of any of the above selenium-containing hyperbranched polymers and a pharmaceutically acceptable excipient.

[0028] The pharmaceutical preparations include the following dosage forms: oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which can be used immediately by adding injection water before injection) and local preparations (such as ointments or solutions).

[0029] The carrier used in the pharmaceutical composition of the present invention is a common carrier available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for local preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically), and if some drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0030] Compared with the prior art, the present invention has the following advantages and technical effects:

[0031] (1) The present invention discloses a hyperbranched polymer, which can quickly and highly selectively transport protons at a rate comparable to that of natural gramicidin, and has extremely high ion selectivity, and can effectively distinguish protons from other ions such as potassium ions, sodium ions and chloride ions. By introducing selenide on the surface of the hyperbranched polymer, the selenium-containing hyperbranched polymer obtained not only maintains the efficient and selective proton transport ability, but also has the characteristics of redox regulation, and can achieve in-situ redox switching under the action of GSH and H2O2 to control the transport of protons.

[0032] (2) The selenium-containing hyperbranched polymer has excellent anticancer activity, can quickly change the intracellular pH value, induce cancer cell apoptosis, and shows significant toxicity to two cancer cell lines, melanoma B16F10 and malignant glioma U87MG. The technical effect of the present invention is not only reflected in its excellent proton transfer efficiency and selectivity, but also in its unique redox regulation mechanism and significant anticancer effect, which provides an important scientific basis and application prospect for the development of new intelligent bionic material drugs.

[0033] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The chemical structure, synthesis route, molecular weight (Mn) and degree of branching (DB) of the hyperbranched polymers H1, H2 and H3 provided in Examples 1-3;

[0035] Figure 2 The H NMR spectrum of the hyperbranched polymer H1 provided in Example 1;

[0036] Figure 3 The NMR carbon spectrum of the hyperbranched polymer H1 provided in Example 1;

[0037] Figure 4 The gel chromatogram of the hyperbranched polymer H1 provided in Example 1;

[0038] Figure 5 The H NMR spectrum of the hyperbranched polymer H2 provided in Example 2;

[0039] Figure 6 The NMR carbon spectrum of the hyperbranched polymer H2 provided in Example 2;

[0040] Figure 7 Gel chromatogram of the hyperbranched polymer H2 provided in Example 2;

[0041] Figure 8 The H NMR spectrum of the hyperbranched polymer H3 provided in Example 3;

[0042] Fig. 9 The NMR carbon spectrum of the hyperbranched polymer H3 provided in Example 3;

[0043] Fig.10 The gel chromatogram of the hyperbranched polymer H3 provided in Example 3;

[0044] Fig.11 is the result of vesicle activity test, among which, Fig.11 A in the figure is a schematic diagram of the HPTS probe vesicle experiment. Fig.11 B in FIG. 1 is a comparison of the proton transport activities of H1-H3 and gA provided in Examples 1-3 at the same concentration (3.18 μM). Fig.11 C in the equation is the EC of H3 and gA provided in Example 3 50 (half maximal effect concentration) value, Fig.11 D in the example 1 and the example 2 provided H1 and H2 EC 50 value;

[0045] Fig.12 The lipid bilayer proton transfer rate test results of H3 provided in Example 3, wherein: Fig.12 A in the figure is a schematic diagram of a planar lipid bilayer workstation. Fig.12 B in the figure is the channel current signal of H3 at different voltages when both the cis and trans chambers are filled with 0.25M hydrochloric acid aqueous solution. Fig.12 C is used to measure the proton conductivity of H3 (γH + )’s current-voltage (IV) curve, Fig.12 D in the figure is the result of transmission stability when the H3 channel is continuously open for more than 70 seconds. Fig.12 E in the figure is the channel current signal of gA at different voltages when both the cis and trans chambers are filled with 0.25M hydrochloric acid aqueous solution. Fig.12 F is the current-voltage (IV) curve used to determine the proton conductivity (γH + );

[0046] Fig.13 The lipid bilayer H of H3 provided in Example 3 + / Na + ,H + / K + and H + / Cl - Selective test results, among which, Fig.13 A in the figure is the channel current signal of H3 at different voltages when the cis-sink is 0.25M hydrochloric acid aqueous solution and the trans-sink is 0.25M potassium chloride aqueous solution. Fig.13 B in the figure is H used to determine H3 + / K + Selective current-voltage (IV) curves, Fig.13 C in the figure is the channel current signal of H3 at different voltages when the cis-sink is 0.25M hydrochloric acid aqueous solution and the trans-sink is 0.25M sodium chloride aqueous solution. Fig.13 D in is the current-voltage (IV) curve used to determine the H+ / Na+ selectivity of H3, Fig.13 E in the figure is the channel current signal of gA at different voltages when both the cis and trans chambers are filled with 0.25M hydrochloric acid aqueous solution. Fig.13 F is the proton conductivity (γH + )’s current-voltage (IV) curve;

[0047] Fig.14 The chemical structures of H1-H3 and H3-Se provided in Examples 1-3 and 4, respectively, and schematic diagrams of transporting protons and inducing apoptosis of cancer cells, wherein: Fig.14 A in the formula (A) is the chemical structure, synthesis route, molecular weight (Mn) and degree of branching (DB) of H1, H2 and H3 provided in Examples 1-3, Fig.14 B in the figure is the chemical structure of H3-Se and the redox regulated structural changes provided in Example 4. Fig.14 The C in H3 transfers protons through multiple pathways with high selectivity. Fig.14 D in FIG. 4 is a schematic diagram of H3-Se redox regulation of proton transport provided in Example 4, Fig.14 E in the figure is the process of H3 inducing cancer cell apoptosis provided in Example 3;

[0048] Fig.15 The synthetic route of M1 and H3-Se in Example 4;

[0049] Fig.16 The H NMR spectrum of M1 provided in Example 4;

[0050] Fig.17 The NMR carbon spectrum of M1 provided in Example 4;

[0051] Fig.18 The NMR selenium spectrum of M1 provided in Example 4;

[0052] Fig.19 The mass spectrum of M1 provided in Example 4;

[0053] Fig. 20 The H NMR spectrum of H3-Se provided in Example 4;

[0054] Fig.21 The NMR selenium spectrum of H3-Se provided in Example 4;

[0055] Fig. 22 The selenium and carbon spectra of M1 redox regulation, EC of H3-Se 50 Values ​​and redox-regulated proton transport diagram, where Fig. 22 A in M1 77 Se NMR spectrum, Fig. 22 B in M1 13 C NMR spectrum, Fig. 22 The C in the equation is the EC of H3-Se. 50 value, Fig. 22 D in the figure is the change of relative fluorescence intensity of HPTS probe after adding different amounts of H2O2 and then different amounts of GSH to H3-Se (3.5 μM). Fig. 22 E in the figure shows the change of H3-Se (3.5 μM) transport activity after cyclic addition of H2O2 and GSH;

[0056] Fig.23 The cytotoxicity and apoptosis induction diagram of H3, Fig.23 A in the figure is the dose-dependent curve of cell viability of B16F10 and U87MG cancer cells after being cultured with H3 for 24 hours. Fig.23 B shows the results of flow cytometry analysis of U87MG cells cultured with H3 or DMSO for 24 hours, followed by staining with Annexin-5 (antibody) (Annexin V) and propidium iodide (PI). DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0058] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0059] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0060] Embodiment 1 This embodiment provides a hyperbranched polymer H1, and the preparation method comprises the following steps:

[0061] B1. Under nitrogen protection, add 80 mL of anhydrous dichloromethane and 6.4 mL of 0.05 mol boron trifluoride diethyl ether into a round-bottom flask to obtain a mixture D;

[0062] B2. Add 11.6 mL 0.1 mol 3-ethyl-3-(hydroxymethyl)-oxetane to the mixture D obtained in step B1 through a dropping funnel within 5 min, react at -20 ° C for 54 h, quench the reaction with ethanol, add to ultrapure water, filter, and vacuum dry at 80 ° C for 180 min to obtain a hyperbranched polymer H1 with a yield of 83%.

[0063] Example 2 This example provides a hyperbranched polymer H2, and the preparation method is the same as that of Example 1, except that in step B2, the reaction is carried out at -20°C for 48 hours, and the yield of the hyperbranched polymer H2 is 85%.

[0064] Example 3 This example provides a hyperbranched polymer H3, and the preparation method is the same as that of Example 1, except that in step B2, the reaction is carried out at 30° C. for 48 hours, and the yield of the hyperbranched polymer H3 is 91%.

[0065] 1. The hyperbranched polymers H1, H2, and H3 obtained in Examples 1-3 were characterized by NMR and Gel chromatography. Figure 1-10 .

[0066] Depend on Figure 1-Figure 10 It can be seen that the polymers (Mn) of H1, H2 and H3 are 6273, 3129 and 3057, respectively, while the corresponding branching degree (DB) values ​​are 23.5%, 20.1% and 45.7%, respectively.

[0067] H1: 1 H NMR (500MHz, DMSO-d6) δ4.15 (s, 1H), 3.30–3.03 (m, 3H), 1.26 (t, J = 11.8Hz, 1H), 0.80 (t, J = 7.4Hz, 2H). 13 C NMR (500MHz, DMSO-d6) δ72.08, 62.62, 43.77, 26.58, 23.07, 22.49, 8.08. GPC (THF): Mn = 6273, PDI = 1.80. DB = 23.5%.

[0068] H2: 1H NMR (500MHz, DMSO-d6) δ4.22–4.11(m,1H),3.31–3.02(m,3H),1.37–1.18(m,1H),0.79(dd,J=9.6,5.2Hz,2H). 13 C NMR (500MHz, DMSO-d6) δ72.10, 62.51, 43.78, 23.66, 23.08, 22.49, 8.09. GPC (THF): Mn = 3129, PDI = 1.79. DB = 20.1%.

[0069] H3: 1 H NMR (500MHz, DMSO-d6) δ4.15 (s, 1H), 3.32–2.98 (m, 3H), 1.25 (dd, J = 12.2, 4.6Hz, 1H), 0.79 (d, J = 4.0Hz, 2H). 13 C NMR (500MHz, DMSO-d6) δ72.11, 62.52, 43.78, 23.39, 23.07, 22.49, 8.04. GPC (THF): Mn = 3057, PDI = 1.60. DB = 45.7%.

[0070] 2. Vesicle activity test: In order to evaluate the ion transport activity of H1-H3 provided in Examples 1-3, large unilamellar vesicles (LUVs) containing a pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) probe were used for the experiment. The preparation process of LUVs is as follows: 11 mg of egg yolk L-α-phosphatidylcholine (EYPC) purchased from Sigma was dissolved in 1 mL of chloroform, and then vacuumed with an oil pump for 3 hours, at which time a clear white film can be seen. Subsequently, 1 mL of 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) buffer solution (10 mM HEPES, pH = 7.0) and HPTS probe (1 mM) were added and hydrated in a 37°C water bath for 3 hours. The solution was then repeatedly frozen and thawed using liquid nitrogen and a constant temperature water bath (37°C), and the entire freeze-thaw process was cycled 10 times. The solution was then filtered through a 0.22 μm polycarbonate membrane 10 times, and finally purified using a Sephadex G-50 glucose column using a HEPES buffer solution without the HPTS probe as the eluent to obtain a LUVs solution containing the HPTS probe. The vesicles were stored at 4°C and used within two days to ensure uniform size of the vesicles. Fig.11 .

[0071] like Fig.11As shown in A: The buffer outside the vesicle is 10mM HEPES, pH = 6.4. Under the influence of the pH gradient, the external protons are transferred from the outside of the vesicle to the inside of the vesicle. The proton transfer efficiency can be monitored by the change in the fluorescence intensity of the HPTS probe inside the vesicle. Fig.11 As shown in Figure B: At the same concentration (3.18 μM), H3 exhibits higher proton transport activity, reaching about 80% of gA within 300 seconds. The transport activity of H2 is approximately equal to that of H1, indicating that the polymer has little effect on the transport activity, while the increase in branching can improve the transport activity. Finally, we use the Hill equation (Y = 1 / 1 + EC 50 / [C]n) to determine its EC 50 (half-maximal effect concentration) value, given by Fig.11 C and Fig.11 From D in the figure, we can see that the EC of gA, H3, H2 and H1 50 The values ​​were 0.07μM, 1.43μM, 2.36μM and 2.60μM, respectively. This indicates that the proton transport activity of H3 is particularly high, comparable to that of natural gramicidin (≈1 / 20). Currently, artificial proton channels can hardly achieve such high activity.

[0072] 3. Lipid bilayer transport mechanism, transport rate and ion selectivity test: In order to determine whether the proton transport system transports protons in the form of a channel or a carrier, a planar lipid bilayer experiment was used for verification. The preparation process of the phospholipids used in the lipid bilayer is as follows: First, weigh glycerol monooleate (GMO, 15 mg) and cholesterol (15 mg) and dissolve them in 20 mL of chloroform, mix them thoroughly and divide them into 10 glass bottles with equal volumes. Then use an oil pump to vacuum for 4 hours to remove the solvent, and a thin film can also be seen. The liposomes in these vials are stored in a -20°C refrigerator. Before each use, one of the vials is restored to room temperature, and 50 μL of n-decane is added to dissolve it for use. At this time, if the unused solution is also stored in a -20°C refrigerator and used within 7 days. Before the test, 0.5 μL of the prepared solution is used to coat the film, and the test effect is best when the capacitance value is about 80 pF. In order to determine the transport rate, 0.25M hydrochloric acid (HCl) aqueous solution is added to both the cis and trans tanks. The results are as follows Fig.12 and Fig.13 .

[0073] Depend on Fig.12 As can be seen from A in the figure, an obvious square proton channel signal can be detected, indicating that H3 transports protons via a channel mechanism rather than a carrier mechanism. Fig.12 B in Fig.12 C in Fig.12 E and Fig.12 From the F in the figure, we can know that by changing the voltage and measuring the corresponding current value, we can find the conductivity value of H3 (γH+ ) is 180.5±4.4pS. The conductivity of gA is 213.0±4.1pS, indicating that the rate of H3 proton transport is very fast, which is of the same order of magnitude as gA. Fig.12 As can be seen from D in the figure, unusually long and stable channel current signals were occasionally observed in this experiment, indicating that H3 can stably transport protons. The H3 channel was continuously open for more than 70 seconds, making it one of the most stable synthetic channels known to date.

[0074] Depend on Fig.13 A and Fig.13 As can be seen from B in the figure, in the asymmetric tank, the cis tank contains 0.25M hydrochloric acid aqueous solution, and the trans tank contains 0.25M potassium chloride aqueous solution. The simplified Goldman-Hodgkin-Katz (GHK) formula is used to determine the H of H3 + / K + Selectivity (PH + / PK + ). Based on the recorded IV curve and the obtained reversal potential (εrev=-112.0mV), the pH + / PK + is 78.4, indicating that H3 has high H+ / K+ selectivity. Fig.13 C and Fig.13 As shown in D, by replacing 0.25M KCl with 0.25M NaCl in the trans tank, the H + To Na + Selectivity (PH + / PNa + ). Based on the IV curve and reversal potential (εrev=-140.0mV) obtained by the same method, the pH was calculated. + / PNa + is 233.2. Fig.13 E and Fig.13 Finally, the H was measured in an asymmetric hydrochloric acid solution (cis tank = 0.25 M hydrochloric acid, trans tank = 0.1 M hydrochloric acid). + / Cl - Selectivity (PH + / PCl - Considering the proton gradient from 0.1M to 0.25M and the reversal potential (εrev = -46.7 mV) obtained from the IV curve, the H + / Cl - The selectivity value reached 167.8. These results finally and strongly confirmed that H3 not only can quickly transport protons, but also has a significant + / K + , H + / Na+ and H + / Cl - Selective.

[0075] Embodiment 4 This embodiment provides a selenium-containing hyperbranched polymer, and the preparation method comprises the following steps:

[0076] S1. Preparation of compound M1: A1. Dissolve 0.75 g (4 mmol) of dimethyl diselenide in 30 mL of anhydrous ethanol, stir evenly, slowly add 454 mg (12 mmol) of sodium borohydride at 0°C, stir at 0°C for 30 min, the solution changes from yellow to colorless, and obtains mixture A;

[0077] A2, 1.23 g (10 mmol) of 2-chloroethyl ester was dissolved in 5 mL of anhydrous ethanol, stirred evenly, added to the mixed solution A obtained in step A1, stirred at 0°C for 1 h, to obtain a mixture B;

[0078] A3, add 50 mL of distilled water and 50 mL of anhydrous ethanol to the mixture obtained in step A2, extract and separate the organic layer, wash with 50 mL of distilled water and 50 mL of brine, retain the organic phase, and remove the solvent under reduced pressure to obtain a mixture C;

[0079] A4. The mixture C obtained in step A3 was dissolved in 10 mL of 99% ethanol by mass, 10 mL of 8 mol KOH aqueous solution was added, and the mixture was stirred at room temperature overnight. Then 30 mL of distilled water and 30 mL of ether were added in sequence, and the mixture was acidified to pH = 2 with concentrated hydrochloric acid. The mixture was extracted with 30 mL of ether, and the organic layer was washed with 30 mL of distilled water, dried over anhydrous Na2SO4, and the solvent was removed by a rotary evaporator to obtain M1 (0.43 g, 70%).

[0080] S2, preparation of hyperbranched polymer: the preparation method is the same as that of Example 3, to obtain a hyperbranched polymer H3;

[0081] S3, dissolving 224 mg (1.46 mmol) of the compound M1 obtained in step S1 in 10 mL of anhydrous dichloromethane, adding 10 μL of anhydrous DMF and 1.85 g (14.6 mmol) of oxalyl chloride, heating under reflux at 60° C. with stirring overnight under nitrogen protection, removing the solvent and excess oxalyl chloride under reduced pressure to obtain reactant A;

[0082] S4, under nitrogen protection, 20 mg (0.007 mmol) of the hyperbranched polymer obtained in step S2 and 6.83 mg (0.07 mmol) of triethylamine were suspended in 5 mL of anhydrous tetrahydrofuran, and stirred at 0° C. for 5 min to obtain reactant B;

[0083] S5. The reactant A obtained in step S3 was dissolved in 2 mL of anhydrous tetrahydrofuran, stirred evenly, and added dropwise to the reactant B in step S4. After the addition was completed, the temperature was raised to room temperature, stirred overnight, and the solvent was removed by a rotary evaporator. The reactant was dissolved in 1 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran-water mixed system (the volume ratio of anhydrous tetrahydrofuran to distilled water was 1:1) was dialyzed for 24 h. During the dialysis process, the tetrahydrofuran-water mixed system was replaced every 3 h. The molecular weight cutoff of the dialysis membrane was 1000 Da, and selenium-containing hyperbranched polymer H3-Se (23.2 mg, 50%) was obtained.

[0084] The compound M1 and the selenium-containing hyperbranched polymer H3-Se obtained in the above Example 4 were characterized by NMR and Gel chromatography.

[0085] Fig.14 The chemical structure of H1-H3 and H3-Se and the schematic diagram of transporting protons and inducing apoptosis of cancer cells.

[0086] Fig.15 This is the synthesis route of M1 and H3-Se in Example 4.

[0087] M1 results are as follows Figure 16-19 .

[0088] Depend on Figure 16-19 It can be seen that M1 was successfully synthesized.

[0089] M1: 1 H NMR (500MHz, DMSO-d6) δ12.31(s,1H),3.17–3.08(m,2H),2.15–2.05(m,3H).

[0090] 13 C NMR(500MHz,DMSO-d6)δ172.91,24.35,5.68.

[0091] 77 Se NMR(500MHz,DMSO-d6)δ114.53.

[0092] HR-MS(ESI,m / z):calculated for C3H6O2Se[MH]-:152.9449; found,152.9449.

[0093] Selenium-containing hyperbranched polymer H3-Se, NMR characterization data such as Figure 20-21 .

[0094] Depend on Figure 20-21 It can be seen that

[0095] H3-Se: 1H NMR (500MHz, DMSO-d6) δ3.34 (s, 3H), 2.98 (s, 2H), 1.29 (dd, J = 39.8, 20.2Hz, 1H), 1.13 (s, 2H), 0.78 (s, 2H).

[0096] 77 Se NMR(500MHz,DMSO-d6)δ123.20.

[0097] The redox behavior of monomer 1 (M1) was analyzed by nuclear magnetic resonance (NMR) spectroscopy, and the transport activity and “ON-OFF” switching characteristics of H3-Se were evaluated using LUVs-based HPTS experiments (intravesicular solution: 10 mM HEPES, pH 7.0; extravesicular solution: 10 mM HEPES, pH 7.0). Fig. 22 .

[0098] Depend on Fig. 22 From A in the figure, we can see that 77 Se NMR spectrum showed that the chemical shift of M1 containing selenoether was 114.53 ppm. After M1 was oxidized by H2O2, the chemical shift shifted to 1047.12 ppm, indicating that the selenoether was converted into selenoxide groups. After adding GSH, the chemical shift returned to 114.53 ppm. Fig. 22 From B in the figure, we can see that 13 C NMR spectrum showed that after adding H2O2, three different peaks of M1 (C1, C2 and C3) underwent obvious chemical shifts, for example, the chemical shift of C3 changed from 172.95 ppm to 161.40 ppm. After adding DTT, the chemical shifts of these three peaks returned to their original positions, indicating that a reversible transformation occurred between selenoether and selenoxide.

[0099] Fig. 22 From the C in the figure, we can see that the EC of H3-Se 50 The value was 1.41 μM, which was comparable to that of H3. To measure the proton transport activity of redox-regulated H3-Se, H3-Se was preincubated with different concentrations of H2O2 for 1 min before adding it to the LUVs solution. Fig. 22 As shown in D, the relative fluorescence intensity of the HPTS probe increases with the increase of the added H2O2 concentration, which means that the proton transport activity decreases accordingly. When the H2O2 concentration is 180 μM, the proton transport activity of H3-Se is extremely low, which is equivalent to the DMSO control group. Subsequently, different concentrations of GSH were added to the H3-Se solution treated with H2O2 (pre-cultured for 1 min), and the proton transport capacity was restored. Fig. 22As shown in E, the proton transport activity of H3-Se can be operated in multiple cycles. It is inferred that the hydrophobic selenide molecules containing H3-Se can be integrated into the hydrophobic phospholipid bilayer, while the conversion into hydrophilic selenoxide partially hinders this process.

[0100] After confirming the rapid proton transport ability of H3 in the liposome model, its potential anticancer properties were investigated. The cytotoxicity of H3 against two cancer cell lines, melanoma B16F10 and glioblastoma U87MG, was first evaluated using the CCK-8 (cell counting kit-8) assay. Fig.23 .

[0101] Depend on Fig.23 As shown in A, the half-maximal inhibitory concentration (IC50) of H3 for B16F10 cells is 1.04 μM, and the half-maximal inhibitory concentration (IC 50 ) was 0.23 μM, indicating significant toxicity against both cancer cell lines. To determine whether H3 causes cell death through an apoptotic mechanism, we performed flow cytometry experiments in combination with Annexin V and propidium iodide (PI) staining. Fig.23 As shown in Figure B, after 24 hours of culture with H3, the percentages of early and late apoptotic cells were 5.45% and 89.8%, respectively, which were much higher than those of the DMSO-treated control group (1.02% and 0.34%). These experiments clearly show that H3 efficiently transports protons, changes the pH value of cells, and ultimately leads to apoptosis of cancer cells.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a selenium-containing hyperbranched polymer, characterized in that: The following steps are involved: S1. preparing compound M1; S2, preparing a hyperbranched polymer; S3, dissolving the compound M1 obtained in step S1 in anhydrous dichloromethane, adding anhydrous DMF and oxalyl chloride, heating and stirring overnight, and removing the solvent and excess oxalyl chloride under reduced pressure to obtain reactant A; S4, under nitrogen protection, suspending the hyperbranched polymer obtained in step S2 and triethylamine in anhydrous tetrahydrofuran, stirring at 0° C. for 5 min, to obtain reactant B; S5, dissolving the reactant A obtained in step S3 in anhydrous tetrahydrofuran, stirring evenly, and adding dropwise to the reactant B in step S4. After the addition is completed, the mixture is warmed to room temperature, stirred overnight, and the solvent is removed by a rotary evaporator, dissolved in anhydrous tetrahydrofuran, and dialyzed in a tetrahydrofuran-water mixed system with a volume ratio of 1:1 for 24 hours to obtain a selenium-containing hyperbranched polymer.

2. The preparation method according to claim 1, characterized in that: In step S1, the method for preparing compound M1 comprises the following steps: A1. Dissolve dimethyl diselenide in anhydrous ethanol, stir evenly, slowly add sodium borohydride at 0°C, stir at 0°C for 30 minutes to obtain mixture A; A2, dissolving 2-chloroethyl ester in anhydrous ethanol, stirring evenly, adding to the mixed solution A obtained in step A1, stirring at 0°C for 1h, to obtain a mixture B; A3, sequentially distilling water and anhydrous ethanol into the mixture B obtained in step A2, extracting and separating the organic layer, washing with distilled water and brine in sequence, retaining the organic phase, and removing the solvent under reduced pressure to obtain a mixture C; A4. Dissolve the mixture C obtained in step A3 in 99% by mass ethanol, add KOH aqueous solution, stir at room temperature overnight, then add distilled water and ether in sequence, acidify to pH = 2 with concentrated hydrochloric acid, extract with ether, wash the organic layer with distilled water, dry with anhydrous Na2SO4, and remove the solvent on a rotary evaporator to obtain M1.

3. The preparation method according to claim 1, characterized in that: In step S2, the method for preparing a hyperbranched polymer comprises the following steps: B1. Under nitrogen protection, add anhydrous dichloromethane and boron trifluoride diethyl ether into a round-bottom flask to obtain a mixture D; B2. Add 3-ethyl-3-(hydroxymethyl)-oxetane to the mixture D obtained in step B1 through a dropping funnel within 5 minutes, react at -20-30°C for 48-54 hours, quench the reaction with ethanol, add to ultrapure water, filter, and vacuum dry at 80°C for 180 minutes to obtain a hyperbranched polymer.

4. The preparation method according to claim 1, characterized in that: In step S3, the compound M1 obtained in step S1 is dissolved in anhydrous dichloromethane, anhydrous DMF and oxalyl chloride are added, and the mixture is heated under reflux at 60° C. and stirred overnight under nitrogen protection.

5. The preparation method according to claim 1, characterized in that: In step S5, the volume ratio of anhydrous tetrahydrofuran to distilled water in the tetrahydrofuran-water mixed system is 1:

1.

6. The preparation method according to claim 1, characterized in that: In step S5, during the dialysis process, the tetrahydrofuran-water mixed system is replaced every 3 hours, and the molecular weight cutoff of the dialysis membrane is 1000Da.

7. The selenium-containing hyperbranched polymer prepared by the preparation method as described in claims 1-6.

8. Use of the selenium-containing hyperbranched polymer prepared by the preparation method according to any one of claims 1 to 6 or the selenium-containing hyperbranched polymer according to claim 7 in the preparation of therapeutic and / or preventive anti-tumor drugs.

9. A pharmaceutical composition comprising one or more selenium-containing hyperbranched polymers prepared by the preparation method according to any one of claims 1 to 6 or the selenium-containing hyperbranched polymer according to claim 7.

10. The pharmaceutical composition according to claim 9, characterized in that The method comprises the selenium-containing hyperbranched polymer as an active ingredient and a pharmaceutically acceptable carrier.

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