Pillyarene WP5-PEG-DNS as well as preparation method and application thereof

Abstract

The invention discloses pillararene WP5-PEG-DNS and a preparation method and application thereof.The preparation method comprises the steps that under the protection of inert gas, HATU serves as a condensing agent, diisopropylethylamine serves as alkali, DMF serves as a solvent, a compound 1, amino polyethylene glycol, HATU, diisopropylethylamine and DMF are subjected to an amidation reaction, and an intermediate WP5-PEG-NH2 is obtained; the method comprises the following steps: adding an intermediate WP5-PEG-NH2 and 2, 4-dinitrobenzene sulfonyl chloride into a single-neck round-bottom flask, and reacting at room temperature by taking diisopropylethylamine as alkali and CH2Cl2 as a solvent to obtain pillararene WP5-PEG-DNS; the pillararene WP5-PEG-DNS can be used for constructing a nano-drug carrier with excellent water solubility, a chemotherapy drug DOX can be further loaded, combined treatment of chemotherapy and SO2 gas is realized, and the tumor treatment effect is further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a pillar aromatic hydrocarbon WP5-PEG-DNS and a preparation method and application thereof. Background Art

[0002] At present, cancer is still one of the major diseases threatening human health. Some traditional treatment methods such as chemotherapy, radiotherapy and surgery are still used clinically. These traditional treatment methods have the disadvantages of large side effects and high cancer recurrence rate. Therefore, we have been constantly exploring some new treatment methods. Nano gas therapy is a new treatment method that has developed rapidly in recent years. Some gaseous molecules such as SO2 can cause oxidative stress-induced damage to biomacromolecules (such as proteins, lipids and DNA) and kill tumor cells. However, some inherent characteristics of tumor cells such as deep penetration and complex location often lead to unsatisfactory treatment effects of single treatment methods. Therefore, in recent years, the treatment method combining SO2 gas therapy with traditional chemotherapy can not only give play to the advantages of SO2 gas therapy and traditional chemotherapy, but also bring synergistic tumor treatment effects in some cases. How to construct excellent SO2 donor molecules and controllably release SO2 gas molecules in the tumor microenvironment is the key to developing such tumor treatment methods.

[0003] As a new generation of macrocyclic molecules, amphiphilic pillararenes have been widely used in the construction of multifunctional nanodrugs. Their diverse structures and good water solubility have gradually attracted people's favor. However, as of now, most pillararenes only have the function of constructing nanodrug carriers, and there are only a handful of amphiphilic pillararenes that can exert therapeutic effects themselves. Therefore, designing multifunctional amphiphilic pillararenes with both drug delivery and tumor treatment capabilities has practical application value.

[0004] Therefore, the design and preparation of nanomedicines based on columnar aromatics for combined chemotherapy and SO2 gas therapy still requires more exploration and research. Summary of the invention

[0005] Technical issues solved:

[0006] The present application aims at the deficiencies in the prior art and solves the technical problems of low SO2 release efficiency and single function of the existing sulfur dioxide donors, and provides a pillar aromatic hydrocarbon WP5-PEG-DNS and a preparation method and application thereof. The pillar aromatic hydrocarbon WP5-PEG-DNS is an amphiphilic pillar aromatic hydrocarbon with controllable SO2 release capability. It is applied to a novel nanomedicine for combined chemotherapy and SO2 gas therapy through molecular self-assembly and loading of the chemotherapy drug doxorubicin (DOX), thereby providing a novel therapeutic drug for tumor treatment.

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] A kind of pillar aromatic hydrocarbon WP5-PEG-DNS, the chemical structure of the pillar aromatic hydrocarbon WP5-PEG-DNS is:

[0010] A method for synthesizing pillar aromatic hydrocarbon WP5-PEG-DNS, comprising the following steps:

[0011] Step 1: Under the protection of inert gas, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU is used as a condensation agent, diisopropylethylamine is used as a base and DMF is used as a solvent to react compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF through an amidation reaction to obtain the intermediate WP5-PEG-NH2;

[0012] Step 2: Add the intermediate WP5-PEG-NH2 and 2,4-dinitrobenzenesulfonyl chloride into a single-necked round-bottom flask, use diisopropylethylamine as a base and CH2Cl2 as a solvent, and react at room temperature to obtain the columnar aromatic hydrocarbon WP5-PEG-DNS.

[0013] Furthermore, in the first step, the mass volume ratio of compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF is 100 mg of compound 1: 600 mg of aminopolyethylene glycol: 114 mg of HATU: 60 μL of diisopropylethylamine: 3 mL of DMF.

[0014] Furthermore, the amidation reaction in the first step is specifically to add compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF into a three-necked round-bottom flask, stir at room temperature for 24 hours, and after the reaction is completed, remove the solvent by rotary evaporation, dissolve the mixture in deionized water and place it in a dialysis bag for dialysis purification, remove insoluble impurities by centrifugation after the dialysis is completed, and freeze-dry the supernatant to obtain the intermediate WP5-PEG-NH2.

[0015] Furthermore, in the first step, the dialysis bag has a molecular weight cut-off of 2000.

[0016] Furthermore, in the second step, the mass volume ratio of WP5-PEG-NH2, 2,4-dinitrobenzenesulfonyl chloride, diisopropylethylamine and CH2Cl2 is WP5-PEG-NH2 75 mg: 2,4-dinitrobenzenesulfonyl chloride 67 mg: diisopropylethylamine 10 μL: CH2Cl2 3 mL.

[0017] Furthermore, the reaction at room temperature in the second step is specifically to mix WP5-PEG-NH2, 2,4-dinitrobenzenesulfonyl chloride, diisopropylethylamine and CH2Cl2 evenly and then stir at room temperature overnight. After the reaction is completed, the reaction mixture is placed in a dialysis bag and dialyzed with distilled water for 48 hours to purify the columnar aromatic hydrocarbon WP5-PEG-DNS.

[0018] Furthermore, in the second step, the dialysis bag has a molecular weight cut-off of 3500.

[0019] Furthermore, the synthesis process of the pillar aromatic hydrocarbon WP5-PEG-DNS is as follows:

[0020]

[0021] The present application also discloses the application of pillar aromatic hydrocarbon WP5-PEG-DNS in nanomedicine for combined treatment of tumor chemotherapy and SO2 gas.

[0022] Pillar aromatic hydrocarbon WP5-PEG-DNS was used to prepare nanodrugs through molecular self-assembly and loading of the chemotherapy drug doxorubicin DOX.

[0023] Furthermore, the specific application steps are:

[0024] S1. 5-10 mg of pillar aromatic hydrocarbon WP5-PEG-DNS was dissolved in 10-25 mL of deionized water to prepare a pillar aromatic hydrocarbon WP5-PEG-DNS aqueous solution;

[0025] S2, 1-2 mg of the chemotherapy drug doxorubicin DOX was dissolved in 1-2 mL of N,N-dimethylformamide, and then added dropwise into the aqueous solution of the pillar aromatic hydrocarbon WP5-PEG-DNS;

[0026] S3, stirring the mixture at a speed of 300-800 r / min for 2-6 hours, adding the mixture into a dialysis bag for dialysis to obtain a nano drug aqueous solution,

[0027] The molecular weight cut-off of the dialysis bag is 10,000, and the dialysis is performed for 48 hours.

[0028] Principle explanation: The 2,4-dinitrobenzenesulfonamide group contained in the pillar aromatic hydrocarbon WP5-PEG-DNS can release SO2 by reacting with glutathione GSH, making it an excellent SO2 donor. At the same time, through the interaction between the hydrophobic and hydrophilic parts of the pillar aromatic hydrocarbon, it can self-assemble into uniform spherical nanocarriers in water. The nanocarriers can further load anti-cancer chemotherapy drugs, realizing the combined treatment of chemotherapy and SO2 gas, and further improving the tumor treatment effect.

[0029] Beneficial effects:

[0030] The present application provides a pillar aromatic hydrocarbon WP5-PEG-DNS and a preparation method and application thereof, which, compared with the prior art, has the following advantages:

[0031] Beneficial effects:

[0032] 1. The novel pillar aromatic hydrocarbon WP5-PEG-DNS in this application has both drug-carrying and tumor-treating capabilities, thus greatly reducing the complexity of drugs and the dosage of drugs used;

[0033] 2. Pillar aromatic hydrocarbon WP5-PEG-DNS can release SO2 by reacting with glutathione GSH, with a release efficiency of 88%. SO2 can be released in a controllable manner under the action of intracellular GSH, achieving excellent SO2 gas treatment ability;

[0034] 3. The new pillar aromatic hydrocarbon WP5-PEG-DNS in this application can be used to construct a nano drug carrier with excellent water solubility, which can be further loaded with the chemotherapy drug DOX, realizing the combined treatment of chemotherapy and SO2 gas, and further improving the tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope image of the nano drug WP5-PEG-DNS / DOX of the present application;

[0036] Figure 2 It is the ultraviolet absorption spectrum of the SO2 indicator of the present application;

[0037] Figure 3 It is the color change diagram of the SO2 indicator of the present application;

[0038] Figure 4 This is the intracellular SO2 detection diagram of this application;

[0039] Figure 5 This is a graph showing the lethality of the nanomedicine WP5-PEG-DNS / DOX in this application to HeLa cells. DETAILED DESCRIPTION

[0040] In this section, the specific embodiments of this patent application will be described in detail in conjunction with the accompanying drawings, aiming to provide a clear and comprehensive description of the technical solution. It should be clear that the embodiment described here only represents one of the possible applications of this patent and does not represent all possible implementation methods. Based on the content of this document, a person of ordinary skill in the art can derive all other potential embodiments covered by this application without performing any creative work. Therefore, these embodiments should all be deemed to be within the scope of protection of this patent.

[0041] Compound 1 was synthesized according to the literature method B.Xia, M.Xue, Chem. Commun., 2014, 50, 1021.

[0042] Example 1

[0043] A method for preparing pillar aromatic hydrocarbon WP5-PEG-DNS, the steps are as follows:

[0044] Step 1: Under the protection of inert gas, add 1100 mg of compound, 600 mg of amino polyethylene glycol, 114 mg of HATU, 60 μL of diisopropylethylamine and 3 mL of DMF into a three-necked round-bottom flask and stir at room temperature for 24 hours. After the reaction is completed, the solvent is removed by rotary evaporation, the mixture is dissolved in deionized water and placed in a dialysis bag (molecular weight cutoff: 2000) for dialysis, and after the dialysis is completed, the excess insoluble impurities are removed by multiple centrifugation. The supernatant is lyophilized to obtain the compound WP5-PEG-NH2. ;

[0045] Step 2: Add 75 mg of WP5-PEG-NH2, 67 mg of 2,4-dinitrobenzenesulfonyl chloride, 10 μL of diisopropylethylamine and 3 mL of CH2Cl2 to a single-necked round-bottom flask. Stir the mixture overnight at room temperature. After the reaction is completed, place the reaction mixture in a dialysis bag (molecular weight cutoff: 3500), and WP5-PEG-DNS is purified by dialysis with distilled water for 48 hours.

[0046] 1 H NMR (400MHz, CDCl3) δ: 8.56 (s, 1H), 8.48 (d, J = 8.4Hz, 2H), 8.28 (d, J = 8.6Hz, 2H), 6.99 –6.64(m,10H),4.47(bs,2H),4.03(bs,2H),3.86–3.54(m,220H),1.35–1.17(m,27H).

[0047] Example 2

[0048] Study on SO2 release ability of pillar aromatic hydrocarbon WP5-PEG-DNS:

[0049] Step 1: Dissolve 1.5 mg of WP5-PEG-DNS in 10 mL of water to prepare a 50 μM WP5-PEG-DNS aqueous solution. Dissolve 1.5 mg of GSH in 20 mL of water to prepare a 250 μM GSH aqueous solution.

[0050] Step 2: Add 10 μM SO2 indicator, 50 μM WP5-PEG-DNS aqueous solution and 250 μM GSH aqueous solution into the quartz cuvette;

[0051] Step 3: Measure the ultraviolet absorption spectrum of the SO2 indicator every ten minutes and observe the color change of the SO2 indicator.

[0052] like Figure 2 As shown, the absorption of the indicator at 540 nm continued to decrease over time, confirming that WP5-PEG-DNS can release SO2 under the action of GSH. Figure 3 The gradual disappearance of the indicator color also confirmed the generation of SO2. The release efficiency of WP5-PEG-DNS reached 88% through calculation.

[0053] Example 3

[0054] Application of pillar aromatic hydrocarbon WP5-PEG-DNS in nanomedicine for combined treatment of tumor chemotherapy and SO2 gas, pillar aromatic hydrocarbon

[0055] WP5-PEG-DNS was prepared into nanomedicine by molecular self-assembly and loading of chemotherapy drug doxorubicin DOX. The specific steps are as follows:

[0056] S1. Dissolve 5 mg WP5-PEG-DNS in 10 mL deionized water to prepare a WP5-PEG-DNS aqueous solution;

[0057] S2, 1.0 mg of the chemotherapy drug doxorubicin DOX was dissolved in 1 mL of N,N-dimethylformamide, and then the obtained solution was added dropwise into the aqueous solution of pillar aromatic hydrocarbon WP5-PEG-DNS;

[0058] S3. After reacting at a speed of 400 r / min for 4 hours, it was added into a dialysis bag for dialyzing to obtain a nano drug aqueous solution. The molecular weight cutoff of the dialysis bag was 10,000, and the dialysis was performed for 48 hours.

[0059] Example 4

[0060] Detection of SO2 release in cells by nanodrug WP5-PEG-DNS / DOX:

[0061] HeLa cells were plated at 20 × 10 4 The cells were seeded in a 6-well plate at a density of 1 mL per well and cultured in a 5% CO2 and 37°C incubator for 24 h. Nanodrug WP5-PEG-DNS / DOX (100 μg / mL) was added and cultured for 4 h. After staining with SO2 fluorescent probe DEACA (5 μM) for 30 min, the cells were rinsed three times with PBS buffer and the cell fluorescence was observed under a fluorescence microscope. Figure 4 As shown, HeLa cells incubated with nanomedicine showed obvious green fluorescence, confirming that nanomedicine can release SO2 under the action of intracellular GSH.

[0062] Example 5

[0063] The therapeutic effect of nanodrug WP5-PEG-DNS / DOX on HeLa cells:

[0064] HeLa cells were plated at 30 × 10 4 The cells were inoculated in a 6-well plate at a density of 1 mL per well and cultured in a 5% CO2 and 37°C incubator for 24 hours. Different concentrations of nanodrug WP5-PEG-DNS / DOX were added and cultured for 24 hours. The cell survival rate was calculated. Figure 5 As shown, the viability of HeLa cells was less than 10%, confirming that the nanomedicine had excellent antitumor activity.

[0065] The above description has fully disclosed the specific embodiments of the present invention. It should be pointed out that any changes made by those skilled in the art to the specific embodiments of the present invention do not deviate from the scope of the claims of the present invention. Accordingly, the scope of the claims of the present invention is not limited to the above specific embodiments.

Claims

1. A pillar aromatic hydrocarbon WP5-PEG-DNS, characterized in that The chemical structure of the pillar aromatic hydrocarbon WP5-PEG-DNS is:

2. A method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 1, characterized in that: Here are the steps: Step 1: Under the protection of inert gas, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU is used as a condensation agent, diisopropylethylamine is used as a base and DMF is used as a solvent to react compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF through an amidation reaction to obtain the intermediate WP5-PEG-NH2; Step 2: Add the intermediate WP5-PEG-NH2 and 2,4-dinitrobenzenesulfonyl chloride into a single-necked round-bottom flask, use diisopropylethylamine as a base and CH2Cl2 as a solvent, and react at room temperature to obtain the pillar aromatic hydrocarbon WP5-PEG-DNS 3. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 2, characterized in that: In the first step, the mass volume ratio of compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF is 100 mg of compound 1: 600 mg of aminopolyethylene glycol: 114 mg of HATU: 60 μL of diisopropylethylamine: 3 mL of DMF.

4. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 2, characterized in that: In the first step, the amidation reaction is specifically to add compound 1, aminopolyethylene glycol, HATU, diisopropylethylamine and DMF into a three-necked round-bottom flask, stir at room temperature for 24 hours, and after the reaction is completed, remove the solvent by rotary evaporation, dissolve the mixture in deionized water and place it in a dialysis bag for dialyzation and purification. After the dialysis is completed, remove insoluble impurities by centrifugation, and freeze-dry the supernatant to obtain the intermediate WP5-PEG-NH2.

5. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 4, characterized in that: In the first step, the molecular weight cut-off of the dialysis bag was 2000.

6. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 2, characterized in that: In the second step, the mass volume ratio of WP5-PEG-NH2, 2,4-dinitrobenzenesulfonyl chloride, diisopropylethylamine and CH2Cl2 was WP5-PEG-NH2 75 mg: 2,4-dinitrobenzenesulfonyl chloride 67 mg: diisopropylethylamine 10 μL: CH2Cl2 3 mL.

7. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 2, characterized in that: In the second step, the reaction at room temperature is specifically to mix WP5-PEG-NH2, 2,4-dinitrobenzenesulfonyl chloride, diisopropylethylamine and CH2Cl2 evenly and then stir at room temperature overnight. After the reaction is completed, the reaction mixture is placed in a dialysis bag and dialyzed with distilled water for 48 hours to purify the columnar aromatic hydrocarbon WP5-PEG-DNS.

8. The method for preparing the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 7, characterized in that: In the second step, the molecular weight cut-off of the dialysis bag was 3500.

9. An application of the pillar aromatic hydrocarbon WP5-PEG-DNS according to claim 1 in a nanomedicine for combined treatment of tumor chemotherapy and SO2 gas, characterized in that: Pillar aromatic hydrocarbon WP5-PEG-DNS was used to prepare nanodrugs through molecular self-assembly and loading of the chemotherapy drug doxorubicin DOX.

10. The use according to claim 9, characterized in that: The specific application steps are: S1, 5-10 mg of pillar aromatic hydrocarbon WP5-PEG-DNS was dissolved in 10-25 mL of deionized water to prepare a pillar aromatic hydrocarbon WP5-PEG-DNS aqueous solution; S2, 1-2 mg of the chemotherapy drug doxorubicin DOX was dissolved in 1-2 mL of N,N-dimethylformamide, and then added dropwise into the aqueous solution of the pillar aromatic hydrocarbon WP5-PEG-DNS; S3. After stirring and reacting at a speed of 300 to 800 r / min for 2 to 6 hours, the mixture is added to a dialysis bag and dialyzed to obtain a nano drug aqueous solution. The dialysis bag has a molecular weight cutoff of 10,000 and the dialysis is performed for 48 hours.

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