A selenium-containing hyperbranched polymer, its preparation method and application

Abstract

The present invention discloses a selenium-containing hyperbranched polymer and its preparation method and application, belonging to the technical field of the preparation of intelligent bionic materials. The preparation method comprises the following steps: preparing compound M1; preparing the hyperbranched polymer; dissolving M1 in anhydrous dichloromethane, adding anhydrous DMF and oxalyl chloride, stirring, and removing the solvent and excessive oxalyl chloride under reduced pressure to obtain reactant A; under nitrogen protection, suspending the hyperbranched polymer and triethylamine in anhydrous tetrahydrofuran, stirring to obtain reactant B; dissolving reactant A in anhydrous tetrahydrofuran, dropping it into reactant B, heating to room temperature, stirring, removing the solvent, dissolving in anhydrous tetrahydrofuran, and dialyzing to obtain the selenium-containing hyperbranched polymer. The present invention provides a selenium-containing hyperbranched polymer and its preparation method and application. The selenium-containing hyperbranched polymer exhibits excellent anti-cancer activity, can rapidly change the intracellular pH value, and induce apoptosis of cancer cells.

Description

Technical Field

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

[0002] Natural channel proteins, such as acid-sensing ion channels (ASIC), ATP-sensitive potassium channels (KATP), and human voltage-gated proton channels (Hv1), form channels on the cell membrane to control the transmembrane transport of species such as ions and water molecules, which is crucial for maintaining the normal physiological functions of organisms. The pathological disorders 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 related to various diseases including chronic pain.

[0003] Inspired by this, bionic chemists have been committed to developing artificial transmembrane transport systems to simulate the high transport efficiency, remarkable ion selectivity, and gating characteristics of natural channel proteins. However, it is extremely challenging to develop an artificial system that simultaneously meets these characteristics, especially the selectivity and transport rate of artificial proton channels. Hyperbranched polymers (HBPs), as three-dimensional macromolecules with inner cavities and a large number of functional groups, have attracted great interest and applications in the fields of drug delivery, nanomaterials, molecular sensing, etc. The three-dimensional structure and rich functional groups of HBPs make them have the 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 in the phospholipid membrane, showing the potential to integrate various characteristics of natural channel proteins. Therefore, a synthetic system based on selenium-containing HBPs may efficiently and selectively transport protons in the lipid bilayer and cell membrane, while showing controllable behavior, providing a promising research direction for the development of novel artificial proton channels. Summary of the Invention

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

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

[0006] A preparation method of a selenium-containing hyperbranched polymer, comprising the following steps:

[0007] S1. Prepare compound M1;

[0008] S2. Prepare hyperbranched polymer;

[0009] S3. Dissolve the compound M1 obtained in step S1 in anhydrous dichloromethane, add anhydrous DMF and oxalyl chloride, heat and stir overnight, remove the solvent and excess oxalyl chloride under reduced pressure to obtain reactant A;

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

[0011] S5. Dissolve the reactant A obtained in step S3 in anhydrous tetrahydrofuran, stir evenly, and drop it into the reactant B in step S4. After the dropping is completed, raise the temperature to room temperature, stir overnight, remove the solvent with a rotary evaporator, dissolve it in anhydrous tetrahydrofuran, and dialyze in a tetrahydrofuran-water mixed system with a volume ratio of 1:1 for 24 h to obtain a selenium-containing hyperbranched polymer.

[0012] Preferably, in step S1, the preparation method of the compound M1 includes the following steps:

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

[0014] A2. Dissolve 2-chloroethyl ester in anhydrous ethanol, stir evenly, add it to the mixed solution A obtained in step A1, and stir at 0 °C for 1 h to obtain mixture B;

[0015] A3. Add distilled water and anhydrous ethanol to the mixture obtained in step A2 in sequence, extract and separate the organic layer, wash it with distilled water and brine in sequence, retain the organic phase, remove the solvent under reduced pressure to obtain mixture C;

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

[0017] Preferably, in step S2, the preparation method of the hyperbranched polymer includes the following steps:

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

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

[0020] Preferably, in step S3, dissolve the compound M1 obtained in step S1 in anhydrous dichloromethane, add anhydrous DMF and oxalyl chloride, and under nitrogen protection, heat and reflux with stirring at 60 °C overnight.

[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, replace the tetrahydrofuran-water mixed system every 3 h, and the cut-off molecular weight of the dialysis membrane is 1000 Da.

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

[0024] The present invention also provides the application of the selenium-containing hyperbranched polymer prepared by the described preparation method or the described selenium-containing hyperbranched polymer in the preparation of anti-tumor drugs for treatment and / or prevention.

[0025] The present invention also provides a pharmaceutical composition comprising one or more selenium-containing hyperbranched polymers prepared by the described preparation method or the described selenium-containing hyperbranched polymer.

[0026] Preferably, the described 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 the selenium-containing hyperbranched polymer described in any one of the above, and a pharmaceutically acceptable excipient.

[0028] The described pharmaceutical preparation includes 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 that can be used immediately after adding water for injection before injection); topical preparations (such as ointments or solutions).

[0029] The carriers for the pharmaceutical compositions of the present invention are common carriers available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorless agents, flavoring agents, etc. for oral preparations; preservatives, solubilizing agents, stabilizers, etc. for injectable preparations; matrices, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). If certain 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 rapidly and highly selectively transport protons at a rate comparable to that of natural gramicidin, and at the same time 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 obtained selenium-containing hyperbranched polymer 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 proton transport.

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

[0033] The technical solutions of the present invention will be further described in detail below with reference to the drawings and examples. Description of the Drawings

[0034] Figure 1 Chemical structures, synthesis routes, molecular weights (Mn) and degrees of branching (DB) of the hyperbranched polymers H1, H2, and H3 provided for Examples 1-3;

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

[0036] Figure 3 13C NMR spectrum of the hyperbranched polymer H1 provided for Example 1;

[0037] Figure 4 Gel permeation chromatography of the hyperbranched polymer H1 provided for Example 1;

[0038] Figure 5 1H NMR spectrum of the hyperbranched polymer H2 provided for Example 2;

[0039] Figure 6 13C NMR spectrum of the hyperbranched polymer H2 provided for Example 2;

[0040] Figure 7 Gel permeation chromatography of the hyperbranched polymer H2 provided for Example 2;

[0041] Figure 8 1H NMR spectrum of the hyperbranched polymer H3 provided for Example 3;

[0042] Figure 9 13C NMR spectrum of the hyperbranched polymer H3 provided for Example 3;

[0043] Figure 10 Gel permeation chromatography of the hyperbranched polymer H3 provided for Example 3;

[0044] Figure 11 Results of vesicle activity test, where, Figure 11 A in is a schematic diagram of the HPTS probe vesicle experiment, Figure 11 B in 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), Figure 11 C in is the EC 50 (half maximal effective concentration) value, Figure 11 D in is the EC 50 value of H1 and H2 provided in Examples 1 and 2;

[0045] Figure 12 Results of the lipid bilayer proton transport rate test of H3 provided for Example 3, where, Figure 12 A in is a schematic diagram of the planar lipid bilayer workstation, Figure 12 B in is the channel current signal of H3 at different voltages when both the cis and trans chambers are 0.25 M hydrochloric acid aqueous solution, Figure 12 C in is the current - voltage (I - V) curve used to measure the proton conductivity (γH + ) of H3, Figure 12 D in is the transmission stability result when the H3 channel remains open for more than 70 seconds, Figure 12 E in is the channel current signal of gA at different voltages when both the cis and trans chambers are 0.25 M hydrochloric acid aqueous solution, Figure 12 F in is the current - voltage (I - V) curve used to measure the proton conductivity (γH + ) of gA;

[0046] Figure 13 The lipid bilayer H of H3 provided for Example 3 + / Na + , H + / K + and H + / Cl - selectivity test results, where Figure 13 A in is the channel current signal of H3 at different voltages when the cis chamber is 0.25 M hydrochloric acid aqueous solution and the trans chamber is 0.25 M potassium chloride aqueous solution, Figure 13 B in is the current-voltage (I-V) curve for measuring the H + / K + selectivity of H3, Figure 13 C in is the channel current signal of H3 at different voltages when the cis chamber is 0.25 M hydrochloric acid aqueous solution and the trans chamber is 0.25 M sodium chloride aqueous solution, Figure 13 D in is the current-voltage (I-V) curve for measuring the H+ / Na+ selectivity of H3, Figure 13 E in is the channel current signal of gA at different voltages when both the cis and trans chambers are 0.25 M hydrochloric acid aqueous solution, Figure 13 F in is the current-voltage (I-V) curve for measuring the proton conductivity (γH + ) of gA;

[0047] Figure 14 Schematic diagrams of the chemical structures of H1-H3 and H3-Se provided for Examples 1-3 and Example 4 respectively, and proton transport and induction of cancer cell apoptosis, where Figure 14 A in is the chemical structures, synthetic routes, molecular weights (Mn) and degrees of branching (DB) of H1, H2 and H3 provided for Examples 1-3, Figure 14 B in is the chemical structure of H3-Se provided for Example 4 and the structural changes regulated by redox, Figure 14 C in is the high-selectivity proton transport of H3 through multiple paths, Figure 14 D in is the schematic diagram of redox-regulated proton transport of H3-Se provided for Example 4, Figure 14 E in is the process of H3-induced cancer cell apoptosis provided for Example 3;

[0048] Figure 15 Synthetic routes of M1 and H3-Se in Example 4;

[0049] Figure 16 1H NMR spectrum of M1 provided for Example 4;

[0050] Figure 17 13C NMR spectrum of M1 provided for Example 4;

[0051] Figure 18 1H NMR spectrum of M1 provided for Example 4;

[0052] Figure 19 Mass spectrum of M1 provided for Example 4;

[0053] Figure 20 1H NMR spectrum of H3-Se provided for Example 4;

[0054] Figure 21 77Se NMR spectrum of H3-Se provided for Example 4;

[0055] Figure 22 77Se and 13C NMR spectra for redox regulation of M1, EC 50 value of H3-Se and proton transport diagram for redox regulation, where Figure 22 A in is the 77 Se NMR spectrum of M1, Figure 22 B in is the 13 C NMR spectrum of M1, Figure 22 C in is the EC 50 value of H3-Se, Figure 22 D in is the change in relative fluorescence intensity of HPTS probe after adding different amounts of H2O2 to H3-Se (3.5 μM) and then adding different amounts of GSH, Figure 22 E in is the change in transport activity of H3-Se (3.5 μM) upon cyclic addition of H2O2 and GSH;

[0056] Figure 23 Cytotoxicity and apoptosis induction diagrams of H3, where Figure 23 A in is the dose-dependent curve of cell viability of B16F10 and U87MG cancer cells after culturing with H3 for 24 hours, Figure 23 B in is the result of flow cytometry analysis of U87MG cells cultured with H3 or DMSO for 24 hours, followed by staining with Annexin V and propidium iodide (PI). Detailed implementation mode

[0057] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0058] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

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

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

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

[0062] B2. 11.6 mL of 0.1 mol of 3-ethyl-3-(hydroxymethyl)-oxetane is added dropwise into the mixture D obtained in step B1 through a dropping funnel within 5 minutes, and the reaction is carried out at -20 °C for 54 h. The reaction is quenched with ethanol, added to ultrapure water, filtered, and dried in vacuo at 80 °C for 180 min to obtain the hyperbranched polymer H1, and the yield is 83%.

[0063] Example 2 This example provides a hyperbranched polymer H2, and the preparation method is the same as that of Example 1. The difference lies in that in step B2, the reaction is carried out at -20 °C for 48 h, 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. The difference lies in that in step B2, the reaction is carried out at 30 °C for 48 h, and the yield of the hyperbranched polymer H3 is 91%.

[0065] 1. The hyperbranched polymers H1, H2, and H3 obtained in the above Examples 1-3 are characterized by nuclear magnetic resonance and gel permeation chromatography. The results are as Figures 1 - 10 .

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

[0067] H1: 1 H NMR (500 MHz, DMSO-d6) δ 4.15 (s, 1H), 3.30–3.03 (m, 3H), 1.26 (t, J = 11.8 Hz, 1H), 0.80 (t, J = 7.4 Hz, 2H). 13 C NMR (500 MHz, DMSO-d6) δ 72.08, 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. Figure 11 .

[0071] like Figure 11As shown in A in [reference], the buffer outside the vesicles is 10 mM HEPES, pH = 6.4. Affected by the pH gradient, protons outside are transported from outside the vesicles into the vesicles. The transport efficiency of protons can be monitored by the change in fluorescence intensity of the HPTS probe inside the vesicles. As Figure 11 shown in B in [reference], at the same concentration (3.18 μM), H3 shows higher proton transport activity and reaches 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 the degree of branching can improve the transport activity. Finally, we used the Hill equation (Y = 1 / (1 + EC 50 / [C]n)) to determine its EC 50 (half-maximal effect concentration) value. As can be seen from Figure 11 C in [reference] and Figure 11 D in [reference], the EC 50 values of gA, H3, H2, and H1 are 0.07 μM, 1.43 μM, 2.36 μM, and 2.60 μM respectively. This shows that the proton transport activity of H3 is extremely high, comparable to that of natural gramicidin (≈1 / 20). Currently, artificial proton channels can hardly reach such high activity.

[0072] 3. Lipid bilayer transport mechanism, transport rate, and ion selectivity test: To determine whether this 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 well and divide them into 10 glass bottles in equal volumes. Then use an oil pump to evacuate for 4 hours to remove the solvent, and a thin film can also be seen. The liposomes aliquoted 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 for dissolution. At this time, if the unused solution is also stored in a -20 °C refrigerator and used within 7 days. Before the test, coat with 0.5 μL of the prepared solution. The test effect is best when the capacitance value is about 80 pF. To measure the transport rate, add 0.25 M hydrochloric acid (HCl) aqueous solution to both the cis and trans compartments. The results are as shown in Figure 12 and Figure 13 .

[0073] As can be seen from Figure 12 A in [reference], an obvious square proton channel signal can be detected, indicating that H3 transports protons through a channel mechanism rather than a carrier mechanism. Figure 12 B in [reference], Figure 12 C in [reference], Figure 12 E in [reference], and Figure 12 F in [reference], by changing different voltages and measuring the corresponding current values, it can be found that the conductance value of H3 (γH+ ) was 180.5 ± 4.4 pS. The conductance value of gA was 213.0 ± 4.1 pS, indicating that H3 transports protons very fast and is of the same order of magnitude as gA. As can be seen from Figure 12 D in [reference], occasionally, extremely long and stable channel current signals were observed in this experiment, indicating that H3 can stably transport protons. The H3 channel remained open continuously for more than 70 seconds and is one of the most stable synthetic channels known so far.

[0074] As can be seen from Figure 13 A in [reference] and Figure 13 B in [reference], finally, in the asymmetric cell, the cis cell contained 0.25 M hydrochloric acid aqueous solution and the trans cell contained 0.25 M potassium chloride aqueous solution. The H + / K + selectivity (PH + / PK + ) of H3 was determined using the simplified Goldman - Hodgkin - Katz (GHK) equation. According to the recorded I - V curve and the obtained reversal potential (εrev = - 112.0 mV), the calculated PH + / PK + was 78.4, indicating that H3 has a high H⁺ / K⁺ selectivity. As can be seen from Figure 13 C in [reference] and Figure 13 D in [reference], by replacing 0.25 M KCl with 0.25 M NaCl in the trans cell, the selectivity of H + for Na + (PH + / PNa + ) was investigated. According to the I - V curve and the reversal potential (εrev = - 140.0 mV) obtained in the same way, the calculated PH + / PNa + was 233.2. As can be seen from Figure 13 E in [reference] and Figure 13 F in [reference], finally, the H + / Cl - selectivity (PH + / PCl - ) of H3 was measured in an asymmetric hydrochloric acid aqueous solution (cis cell = 0.25 M hydrochloric acid, trans cell = 0.1 M hydrochloric acid). Considering the proton gradient from 0.1 M to 0.25 M and the reversal potential (εrev = - 46.7 mV) obtained from the I - V curve, the H + / Cl - selectivity value of H3 reached 167.8. These results finally strongly confirmed that H3 can not only transport protons rapidly but also has remarkable H + / K + , H + / Na+ and H + / Cl - Selectivity

[0075] Example 4 This example provides a selenium-containing hyperbranched polymer, and the preparation method includes the following steps:

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

[0077] A2. Dissolve 1.23 g (10 mmol) of 2-chloroethyl ester in 5 mL of absolute ethanol, stir evenly, add it to the mixed solution A obtained in step A1, and stir at 0 °C for 1 h to obtain mixture B;

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

[0079] A4. Dissolve the mixture C obtained in step A3 in 10 mL of ethanol with a mass fraction of 99%, add 10 mL of 8 mol / L KOH aqueous solution, stir overnight at room temperature, then add 30 mL of distilled water and 30 mL of diethyl ether in sequence, acidify with concentrated hydrochloric acid to pH = 2, extract with 30 mL of diethyl ether, wash the organic layer with 30 mL of distilled water, dry with anhydrous Na2SO4, and remove the solvent with a rotary evaporator to obtain M1 (0.43 g, 70%).

[0080] S2. Prepare the hyperbranched polymer: The preparation method is the same as that in Example 3 to obtain the hyperbranched polymer H3;

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

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

[0083] S5. Dissolve the reactant A obtained in step S3 in 2 mL of anhydrous tetrahydrofuran, stir evenly, and add dropwise to the reactant B in step S4. After the addition is completed, warm up to room temperature, stir overnight, remove the solvent with a rotary evaporator, dissolve in 1 mL of anhydrous tetrahydrofuran, and dialyze in a tetrahydrofuran-water mixed system (the volume ratio of anhydrous tetrahydrofuran to distilled water is 1:1) for 24 h. Replace the tetrahydrofuran-water mixed system every 3 h during the dialysis process. The cut-off molecular weight of the dialysis membrane is 1000 Da to obtain the selenium-containing hyperbranched polymer H3-Se (23.2 mg, 50%).

[0084] Characterize the compound M1 and the selenium-containing hyperbranched polymer H3-Se obtained in Example 4 above by nuclear magnetic resonance and gel chromatography.

[0085] Figure 14 Schematic diagrams of the chemical structures of H1-H3 and H3-Se and the transport of protons and induction of apoptosis in cancer cells.

[0086] Figure 15 Synthesis routes of M1 and H3-Se in Example 4

[0087] The results of M1 are as Figures 16 - 19 .

[0088] From Figures 16 - 19 it can be seen that M1 was successfully synthesized.

[0089] M1: 1 H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 3.17–3.08 (m, 2H), 2.15–2.05 (m, 3H).

[0090] 13 C NMR (500 MHz, DMSO-d6) δ 172.91, 24.35, 5.68.

[0091] 77 Se NMR (500 MHz, DMSO-d6) δ 114.53.

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

[0093] For the selenium-containing hyperbranched polymer H3-Se, the nuclear magnetic resonance characterization data are as Figures 20 - 21 .

[0094] From Figures 20 - 21 it can be seen that

[0095] H3-Se: 11H NMR (500 MHz, DMSO-d6) δ 3.34 (s, 3H), 2.98 (s, 2H), 1.29 (dd, J = 39.8, 20.2 Hz, 1H), 1.13 (s, 2H), 0.78 (s, 2H).

[0096] 77 77Se NMR (500 MHz, DMSO-d6) δ 123.20.

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

[0098] As can be seen from Figure 22 A in 77 the 77Se NMR spectrum shows that the chemical shift of M1 containing selenoether is 114.53 ppm. After M1 was oxidized by H2O2, the chemical shift shifted to 1047.12 ppm, indicating that the selenoether was converted to a selenium oxide group. After adding GSH, the chemical shift returned to 114.53 ppm. Figure 22 As can be seen from 13 B in

[0099] Figure 22 the 13C NMR spectrum shows that after adding H2O2, significant chemical shifts occurred in three different peaks (C1, C2, and C3) of M1. 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 a reversible transformation between selenoether and selenium oxide. 50 As can be seen from Figure 22 C in Figure 22 the EC 50 value of H3-Se is 1.41 μM, and its activity is comparable to that of H3. To measure the redox-regulated proton transport activity of H3-Se, it was pre-incubated with different concentrations of H2O2 for 1 minute before adding it to the LUVs solution. Figure 22 As can be seen from Figure 22 D in Figure 22 the relative fluorescence intensity of the HPTS probe increased with the increasing concentration of added H2O2, which means that the proton transport activity decreased accordingly. When the concentration of H2O2 was 180 μM, the proton transport activity of H3-Se was extremely low, comparable to the DMSO control group. Subsequently, different concentrations of GSH were added to the H2O2-treated H3-Se solution (pre-cultured for 1 min), and the proton transport ability was restored. As can be seen from Figure 22As can be seen from E in [reference], the proton transport activity of H3-Se can be cycled multiple times. It is inferred that hydrophobic selenide molecules containing H3-Se can integrate into the hydrophobic phospholipid bilayer, while the conversion to hydrophilic selenium oxide moieties hinders this process.

[0100] After confirming the rapid proton transport ability of H3 in the liposome model, its potential anti-cancer properties were investigated. First, the CCK-8 (Cell Counting Kit-8) assay was used to evaluate the cytotoxicity of H3 against two cancer cell lines, melanoma B16F10 and glioblastoma U87MG. The results are as Figure 23 .

[0101] As Figure 23 can be seen from A in [reference], the half-maximal inhibitory concentration (IC50) of H3 against B16F10 cells is 1.04 μM, and the half-maximal inhibitory concentration (IC 50 ) against U87MG cells is 0.23 μM, indicating significant toxicity to 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. As Figure 23 can be seen from B in [reference], after culturing with H3 for 24 hours, the percentages of early and late apoptotic cells are 5.45% and 89.8% respectively, which are 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 cell pH value, and ultimately leads to cancer cell apoptosis.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements do not make the modified technical solutions deviate from the spirit and scope of the technical solutions 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, the reactant A obtained in step S3 is dissolved in anhydrous tetrahydrofuran, stirred evenly, and added dropwise to the reactant B in step S4, and the addition is completed, the temperature is raised to room temperature, stirred overnight, the solvent is removed by a rotary evaporator, and the reactant is 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; 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, dissolving the mixture C obtained in step A3 in 99% by mass ethanol, adding KOH aqueous solution, stirring at room temperature overnight, then adding distilled water and ether in sequence, acidifying with concentrated hydrochloric acid to pH = 2, extracting with ether, washing the organic layer with distilled water, drying with anhydrous Na2SO4, and removing the solvent with a rotary evaporator to obtain M1; 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.

2. 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.

3. 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.

4. 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 1000 Da.

5. The selenium-containing hyperbranched polymer prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the selenium-containing hyperbranched polymer prepared by the preparation method according to any one of claims 1 to 4 or the selenium-containing hyperbranched polymer according to claim 5 in the preparation of therapeutic and / or preventive anti-tumor drugs.

7. A pharmaceutical composition comprising one or more selenium-containing hyperbranched polymers prepared by the preparation method according to any one of claims 1 to 4 or the selenium-containing hyperbranched polymer according to claim 5.

8. The pharmaceutical composition according to claim 7, characterized in that The method comprises the selenium-containing hyperbranched polymer as an active ingredient and a pharmaceutically acceptable carrier.

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