Cascade enhanced anti-tumor supramolecular nanogel as well as preparation method and application thereof

Abstract

The invention discloses cascade enhanced anti-tumor supramolecular nanogel as well as a preparation method and application thereof, and relates to a preparation method and anti-cancer research of the cascade enhanced anti-tumor supramolecular nanogel. Research finds that the nanogel can effectively gather near mitochondria in cancer cells, can relieve intracellular hypoxia, and enhances chemical kinetics therapy through cascade reaction of ferrocene and glucose oxidase, thereby effectively optimizing hypoxia inhibition therapy effect in traditional chemical kinetics therapy, and having good application prospects. The biological safety and the like are realized.

Description

Technical Field

[0001] The invention relates to the fields of organic synthesis, nanometer self-assembly materials and cancer treatment. Background Art

[0002] In the emerging nanocatalytic medicine, chemodynamic therapy (CDT) is considered to be a tumor-specific treatment with minimal toxic side effects as an interesting alternative to traditional cancer treatments such as surgery, radiotherapy, and chemotherapy. Normal cells mainly produce energy through mitochondrial oxidative phosphorylation, while tumor cells produce a large amount of lactic acid through glycolysis even under aerobic conditions. This phenomenon is called the Warburg effect. This metabolic mode causes tumor cells to consume a large amount of glucose but produce less energy, while causing the tumor microenvironment to become slightly acidic. Glucose metabolism mediated by glucose oxidase can convert glucose into H 2 O 2 Therefore, in tumor cells, due to the vigorous glucose metabolism, the catalytic action of glucose oxidase can further increase H 2 O 2 The production of gluconic acid will further reduce the pH value of the tumor microenvironment, and the Fenton reaction based on catalysts such as ferrocene can efficiently produce -OH, thereby enhancing the therapeutic effect of CDT. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the difficulty of the hypoxic environment in cancer cells inhibiting the function of glucose oxidase, and use triphenylphosphine to target the non-anticancer drug atovaquone to the mitochondria and inhibit the respiration of mitochondria, so that oxygen is retained in the cells, and at the same time participates in the cascade Fenton reaction of ferrocene and glucose oxidase to form a three-step cascade reaction, thereby improving the effect of chemodynamic therapy. 2 O 2 Production, endogenous H 2 O 2 There are problems such as insufficient inhibition of the therapeutic effect of ferrocene and the high price of traditional chemotherapy drugs.

[0004] The PEI-Fc-GOx nanogel of the present invention comprises polyethyleneimine (PEI-Fc) modified with ferrocene, atovaquone (ATO), active oxygen response bond TK-linker, glucose oxidase (GOx), and triphenylphosphine modified DNA (DNA-TPP). ATO is encapsulated in PEI-Fc to form micelle PFA. TK-linker is then used to covalently modify GOx on PFA to form nanogel PFAG. Finally, DNA-TPP is loaded onto PFAG through electrostatic interaction to form the final nanogel PFAGD.

[0005] The PEI-Fc is formed by the condensation of ferrocene carboxylic acid and polyethyleneimine amide, and is obtained after dialysis purification and lyophilization.

[0006] The molecular formula of PEI-Fc is

[0007]

[0008] The TK-linker is obtained from the self-reaction of mercaptoacetic acid. First, TK-COOH is synthesized, then TK-OH is further synthesized, and finally TK-linker is synthesized. The molecular formula of TK-linker is

[0009]

[0010] The mitochondrion-targeted DNA-TPP molecule is obtained by condensing 5-carboxypentyl-triphenylphosphonium bromide with DNA having an amino group at the 5' end, followed by purification by high performance liquid chromatography; the nucleotide sequence is 5'-GCCACAACATCCTAA-3', and the molecular formula of DNA-TPP is:

[0011]

[0012] The PEI-Fc is formed by the amide condensation of ferrocene formic acid and PEI with a molecular weight of 1800, and is purified by dialysis. The preparation of PEI-Fc includes the following steps: Ferrocene formic acid (FCA) is dissolved in dimethyl sulfoxide (DMSO), and EDC·HCl and NHS are added and stirred to activate the carboxyl group. Polyethyleneimine (PEI 1800 ) is dissolved in DMSO and then added to the FCA solution, and the mixture is stirred and reacted. The crude product is dialyzed with a dialysis bag and lyophilized.

[0013] The preparation of the TK-linker includes the following steps: Mercaptoacetic acid, acetone and p-toluenesulfonic acid are added to a round bottom flask, stirred at room temperature for 6 hours, cooled in an ice-water bath for 1 hour, then filtered by suction, and washed with cold n-hexane to obtain TK-COOH. TK-COOH is dissolved in anhydrous tetrahydrofuran, lithium aluminum hydride is added, and the mixture is heated under reflux for 4 hours. After the reaction is completed, sodium hydroxide solution and deionized water are added, and TK-OH is obtained by rotary evaporation under reduced pressure and column chromatography separation. TK-OH is dissolved in dichloromethane, then N,N'-carbonyldiimidazole (CDI) is added, and the mixture is stirred at room temperature for 8 hours. TK-linker is obtained by rotary evaporation under reduced pressure and column chromatography separation.

[0014] The preparation of DNA-TPP includes the following steps: 5-carboxypentyl-triphenylphosphonium bromide, EDC·HCl and NHS are loaded into a glass bottle, anhydrous DMF is added, and the mixture is stirred at room temperature for 1 hour to activate the carboxyl group. DNA-NH 2Dissolve it in CBS buffer, add the TPP-COOH solution, react overnight at room temperature, after the reaction is completed, dilute it with deionized water and purify it by high performance liquid chromatography.

[0015] Add the ATO solution to the PF solution, stir and then let it stand for 4 h, centrifuge and take the supernatant to obtain the PFA solution.

[0016] Further, add the TK-linker solution to the PFA solution, stir and then add the GOx solution, centrifuge and collect the supernatant, dialyze with a dialysis bag and freeze-dry to obtain the product PFAG.

[0017] Finally, add DNA-TPP to the PFAG solution, stir and let it stand overnight to obtain the PFAGD nanogel.

[0018] The present invention also provides the application of the nanogel in the anti-cancer effect, and verifies the role of the nanogel in alleviating the intracellular hypoxia effect.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] The present invention constructs a three-step cascaded nanoreactor and provides a synthesis method of PFAGD nanogel, and this nanogel exhibits excellent performance. Our research is expected to regulate the hypoxic microenvironment of cells, enhance the chemodynamic therapy effect based on ferrocene and glucose oxidase, and this treatment protocol does not contain traditional chemotherapeutic drugs, but instead uses non-anticancer drugs with lower toxicity, which will be beneficial to its biosafety. Description of the Drawings

[0021] Figure 1 is the characterization of the main synthetic products in the present invention; (a) 1H NMR characterization of PEI-Fc (600 MHz, DMSO-d 6 , 298 K); (b) 1H NMR characterization of TK-COOH (600 MHz, DMSO-d 6 , 298 K); (c) 1H NMR characterization of TK-OH (600 MHz, D 2 2O, 298 K); (d) 1H NMR characterization of TK-linker (600 MHz, DMSO-d 6 , 298 K); 20% PAGE gel electrophoresis (150 V, 55 min) and (f) time-of-flight mass spectrometry characterization of DNA-TPP.

[0022] Figure 2Characterization of nano-gels by transmission electron microscopy (TEM) and dynamic light scattering (DLS); TEM images of PF micelles (b) DLS results of PF micelles (c) TEM images of PFA micelles (d) DLS results of PFA micelles (e) TEM images of PFG nano-gels (f) DLS results of PFG nano-gels (g) TEM images of PFAG nano-gels (h) DLS results of PFAG nano-gels (i) TEM images of PFAGD nano-gels (j) DLS results of PFAGD nano-gels. The TEM results can directly show the morphology of the prepared micelles or nano-gels, while the DLS data can display the average particle size and distribution of the micelles or nano-gels.

[0023] Figure 3 The biological safety and anti-cancer effect of nano-gels were detected by the MTT method; (a) normal 3T3 cells (b) 4T1 cells under normoxic conditions (c) 4T1 cells under hypoxic conditions.

[0024] Figure 4 Imaging of cancer cells by confocal laser scanning microscopy (CLSM) was used to judge the intracellular hypoxic condition, where Ru(ddp) 3 Cl 2 was used as a hypoxia probe. The higher the degree of intracellular hypoxia, the stronger the red fluorescence (scale bar is 20 μm). DAPI is a commonly used fluorescent dye for staining DNA in the nucleus. DAPI can bind to the A-T base pairs of DNA and produce blue fluorescence. In the figure, the DAPI-stained nuclei are shown in blue. Merge refers to merging the images of different fluorescence channels to show the distribution of multiple markers in the same cell. Detailed implementation mode

[0025] The invention will be further elaborated below through examples in combination with the attached drawings. The examples are only used to further explain the technical solutions of the present invention and should not be considered as limiting the protection scope of the present invention. Those skilled in the art make non-essential improvements or adjustments based on the above content of the present invention, which all fall within the protection scope of the present invention.

[0026] Example 1 Preparation of PFAGD nano-gels

[0027] (1) Synthesis of PEI-Fc:

[0028]

[0029] Experimental procedure: Ferrocene carboxylic acid (FCA, 115 mg, 0.5 mmol) was dissolved in 3 ml of DMSO, and (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC·HCl, 115 mg, 0.6 mmol) and N-hydroxysuccinimide (NHS, 73 mg, 0.6 mmol) were added and stirred for 1 h to activate the carboxyl group.

[0030] Polyethyleneimine (PEI 1800 , 900 mg, 0.5 mmol,) was dissolved in 3 ml of DMSO with a needle and injected into the FCA solution, and stirred for 48 h. The crude product was dialyzed with a dialysis bag with M.W. = 500 Da for 3 days and then freeze-dried to obtain PEI-Fc.

[0031] By controlling the molar ratio of amino groups in polyethyleneimine to ferrocene carboxylic acid, the grafting rate of Fc can be controlled, and it will affect the size of PEI-Fc (PF). Specifically as follows:

[0032] Table 1

[0033]

[0034] Subsequently, PEI-Fc (PF) with a size of 123.2 nm was selected for the experiment because it was considered that the volume of the subsequent assembly would increase and it would satisfy the enhanced permeability and retention effect (EPR effect) (<200 nm).

[0035] (2) Synthesis of TK-linker:

[0036]

[0037] Experimental procedure: Mercaptoacetic acid (5.52 g, 60 mmol), acetone (8.8 ml, 120 mmol) and p-toluenesulfonic acid (10 mg, 64 mmol) were added together to a 25 ml round-bottom flask. Stirred at room temperature for 6 h, placed in an ice-water bath for 1 h. After the reaction was completed, the system was filtered by suction to obtain a white solid, which was washed 3 times with cold n-hexane (25 ml) to obtain TK-COOH.

[0038] TK-COOH (896 mg, 4 mmol) was added to a 150 ml round-bottom flask and completely dissolved in 50 ml of anhydrous tetrahydrofuran. Under stirring conditions, a small amount of lithium aluminum hydride (0.61 g, 16 mmol) was repeatedly added, and a large number of bubbles were observed. Then it was heated under reflux at 70 °C for 4 h. After the reaction was completed, 1.9 mL of sodium hydroxide solution (w / w, 15%) and 1.9 mL of deionized water were added to the reaction solution in sequence, a large amount of heat was released, and a large number of bubbles were observed. The product was concentrated by rotary evaporation under reduced pressure and separated by column chromatography, and the eluent was dichloromethane:ethyl acetate = 1:1 to obtain a yellow oily liquid TK-OH.

[0039] Dissolve TK-OH (300 mg, 1.5 mmol) in a 50-ml round-bottom flask. After adding 20 ml of DCM to completely dissolve it, add N,N'-carbonyldiimidazole (CDI, 584 mg, 3.6 mmol). Stir the reaction at room temperature for 8 h. The product is concentrated by rotary evaporation under reduced pressure and separated by column chromatography. The eluent is dichloromethane:ethyl acetate = 1:1 to obtain a yellow oily liquid TK-linker.

[0040] (3) Synthesis of DNA-TPP:

[0041]

[0042] (4) Experimental procedure: Dissolve 5-carboxypentyl-triphenylphosphonium bromide (TPP-COOH, 22.87 mg, 0.05 mmol) in a 10-ml glass bottle, and then add EDC·HCl (19.17 mg, 0.1 mmol) and NHS (23.02 mg, 0.2 mmol) respectively. Finally, add 5 ml of anhydrous DMF to the above system with a syringe and stir at room temperature for 1 h to fully activate the carboxyl group. The single-stranded oligonucleotide with an amino group at the 5'-end (DNA-NH 2 ) is 10 nmol / tube. Take two tubes and dissolve each tube with 20 μL of CBS buffer. Add 20 μL of the TPP-COOH solution to each tube and incubate overnight (about 10 - 12 h) at 25 °C. Shake the centrifuge tube every 45 min during the first three hours of the reaction. After the reaction, dilute the liquid in each tube to 400 μL with deionized water, and then purify it by high-performance liquid chromatography with an injection volume of 100 μL each time. Collect all the products and concentrate them with a DNA concentrator until the liquid completely disappears. Use denaturing gel electrophoresis and time-of-flight mass spectrometry to verify the successful acquisition of DNA-TPP.

[0043] The final DNA-TPP has the function of mitochondrial targeting. The sequence of the single-stranded oligonucleotide is 5'-GCCACAACATCCTAA-3', which is purchased from Huzhou Hippo Biotechnology Co., Ltd.

[0044] (5) Synthesis and characterization of nano-gel PFAGD:

[0045] Add 80 μL of 0.6 mg / ml atovaquone (ATO) solution to 1 mg / ml PEI-Fc (PF) solution, stir for 10 min, then let it stand for 4 h, centrifuge at 4000 rpm for 5 min, and take the supernatant, which is the PFA solution. Encapsulate ATO in PEI-Fc to form the micelle PFA.

[0046] 10 μL of 1 M TK-linker solution was added to 2 mL of 1 mg / mL PFA solution and stirred for 10 min. Then 80 μL of 100 U / mL glucose oxidase (Gox) solution was added and stirred for another 10 min. After centrifugation at 8000 rpm for 10 min, the supernatant was collected, dialyzed with a 3500 Da dialysis bag for 2 days, and freeze-dried to obtain the product. That is, GOx was covalently modified on PFA using TK-linker to form the nanogel PFAG. 20 nmol of DNA-TPP was added to 1 mL of 1 mg / mL PFAG solution and stirred for 10 min. After standing overnight, the PFAGD nanogel was obtained. That is, DNA-TPP was loaded onto PFAG through electrostatic interaction to form the final nanogel PFAGD. The size of the nanogel was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0047] Example 2 Detection of Biosafety and Anticancer Effect:

[0048] PEI-Fc (PF) solutions with different Fc concentrations were prepared according to the method of Example 1. Then, different concentrations of atorvastatin (ATO) and glucose oxidase (Gox) were added during the preparation of PFA, PFAG, and PFAGD, so that the contents of ferrocene, glucose oxidase, and atorvastatin in the PFAGD nanogel obtained in step (5) were: (Fc: 0 μmol / L, 2.5 μmol / L, 5 μmol / L, 7.5 μmol / L, 10 μmol / L, 12.5 μmol / L, 15 μmol / L), (Gox: 0 mU / mL, 20 mU / mL, 40 mU / mL, 60 mU / mL, 80 mU / mL, 100 mU / mL, 120 mU / mL), (ATO: 0 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1.0 μg / mL, 1.2 μg / mL).

[0049] In addition, to explore whether ATO can play a role in relieving hypoxia and enhancing the cascade reaction in cells, a nanogel PFG without ATO was prepared. Specifically, 10 μL of 1 M TK-linker solution was added to 2 mL of 1 mg / mL PF solution and stirred for 10 min. Then 80 μL of 100 U / mL glucose oxidase (Gox) solution was added and stirred for another 10 min. After centrifugation at 8000 rpm for 10 min, the supernatant was collected, dialyzed with a 3500 Da dialysis bag for 2 days, and freeze-dried to obtain the product. That is, the nanogel PFG without ATO was obtained.

[0050] 3T3 cells, also known as NIH / 3T3 cells, are a mouse embryonic fibroblast cell line commonly used in cell biology and cancer research.

[0051] 3T3 cells were first seeded in a 96-well plate (1000 cells per well) and cultured for 24 h. Then, the culture medium was replaced with fresh medium containing different concentrations of PBS, free GOx, PFA, PFG, PFAG, and PFAGD. After incubation for 24 h, cell viability was measured using the MTT method.

[0052] As Figure 3 shown in a of [reference], the toxicity of each group to normal 3T3 cells was relatively low. Even in the group with the highest concentration, the cell survival rate of PFAGD could still reach 75%, demonstrating its good biosafety.

[0053] As Figure 3 shown in b and c of [reference], under normoxic and hypoxic conditions respectively, 4T1 cells were first seeded in a 96-well plate (1000 cells per well) and cultured for 24 h. Then, the culture medium was replaced with fresh medium containing different concentrations of PBS, free GOx, PFA, PFG, PFAG, and PFAGD. After incubation for 24 h, cell viability was measured using the MTT method. 4T1 cells are a murine breast cancer cell line commonly used in breast cancer-related research.

[0054] In terms of the anti-cancer effect, the effect of PFAGD under normoxic and hypoxic conditions was significantly better than that of other groups. When Fc was 15 μmol / L, GOx was 120 mU / mL, and ATO was 1.2 μg / mL, the killing effect of PFAGD on cancer cells 4T1 was the best.

[0055] Example 3 Detection of intracellular hypoxia in cells:

[0056] Under hypoxic conditions, 4T1 cells were first seeded in a 6-well plate (1000 cells per well) and cultured for 12 h. Then, the culture medium was replaced with fresh medium containing PBS, free GOx, PFA, PFG, PFAG, and PFAGD (where Fc was 15 μmol / L, GOx was 120 mU / mL, and ATO was 1.2 μg / mL). After incubation for 6 h, the hypoxic probe Ru(ddp) 3 Cl 2 was used to detect the intracellular hypoxia situation.

[0057] As Figure 4As shown, ATO can be transported into cells to relieve cell hypoxia. Among them, PFA has the most obvious effect in relieving hypoxia. However, through the anti-cancer effect experiment in Example 2, it can be seen that the anti-cancer effect of PFA is not ideal. This is because even after relieving the hypoxia condition, there are no other factors to restrict the physiological activities of cancer cells. After adding Fc and GOx, under hypoxic conditions, ATO effectively relieves cell hypoxia, causing the oxygen originally used for mitochondrial respiration to remain in the cells, and this retained oxygen just makes up for the problem of the GOx-catalyzed glucose production of H 2 O 2 that requires oxygen, thus realizing the three-step cascade reaction of oxygen retention, GOx-catalyzed glucose production of H 2 O 2 and Fc-catalyzed H 2 O 2 to produce highly toxic ROS, and enhancing chemodynamic therapy thereby. Combining with Example 2, the conclusion can be drawn that PFAGD can relieve cell hypoxia and enhance chemodynamic therapy simultaneously.

Claims

1. A cascade-enhanced anti-tumor supramolecular nanogel, characterized in that: The invention comprises DNA-TPP loaded on a nanogel PFAG by electrostatic action, wherein the nanogel PFAG is formed by modifying glucose oxidase on PEI-Fc by TK-linker, atovaquone is encapsulated in PEI-Fc, and PEI-Fc is polyethyleneimine connected to the carboxyl group of ferrocenecarboxylic acid by peptide bond; The DNA-TPP is a single-stranded oligonucleotide modified with triphenylphosphine; The molecular formula of the TK-linker is 2. The nanogel according to claim 1, characterized in that The sequence of the single-stranded oligonucleotide is 5'-GCCACAACATCCTAA-3'.

3. The nanogel according to claim 1, characterized in that The molar ratio of the amino group on the PEI-Fc to the grafted ferrocene is 16:

1.

4. The nanogel according to claim 1, characterized in that The molecular formula of the DNA-TPP is:

5. The method for preparing the nanogel according to claim 1, characterized in that: The following steps are involved: S1. Grafting ferrocenecarboxylic acid onto polyethyleneimine to obtain PEI-Fc; S2. Synthesis of TK-linker; S3. Synthesize DNA-TPP; S4. adding the atovaquone solution to the PEI-Fc solution, stirring and then allowing to stand, and centrifuging to obtain the supernatant to obtain the PFA solution; S5. Add TK-linker solution to the above PFA solution, stir and then add glucose oxidase solution, continue stirring, collect the supernatant after centrifugation, dialyze with a dialysis bag and freeze-dry to obtain the product PFAG; S6. Add DNA-TPP to the PFAG solution, stir and let stand overnight to obtain PFAGD nanogel.

6. The preparation method according to claim 5, characterized in that: Step S1 includes the following steps: Dissolve ferrocenecarboxylic acid in dimethyl sulfoxide, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and stir to activate the carboxyl group; dissolve polyethyleneimine in DMSO and add it to the activated FCA solution, stir to react; after the reaction is completed, dialyze the crude product with a dialysis bag and freeze-dry to obtain a PEI-Fc compound.

7. The preparation method according to claim 5, characterized in that: Step S2 includes the following steps: Add mercaptoacetic acid, acetone and p-toluenesulfonic acid into a round-bottom flask, stir at room temperature, cool in an ice-water bath, filter, and wash with cold n-hexane to obtain TK-COOH; TK-COOH was dissolved in anhydrous tetrahydrofuran, lithium aluminum hydride was added, and the mixture was heated to reflux. After the reaction, sodium hydroxide solution and deionized water were added, and TK-OH was obtained by vacuum rotary evaporation and column chromatography separation. TK-OH was dissolved in dichloromethane, and N,N'-carbonyldiimidazole was added, and the mixture was stirred at room temperature. TK-linker was obtained by vacuum rotary evaporation and column chromatography separation.

8. The preparation method according to claim 5, characterized in that: Step S3 includes the following steps: 5-Carboxypentyl-triphenylphosphonium bromide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and n-hydroxysuccinimide were placed in a glass bottle, anhydrous N,N-dimethylformamide was added, and the mixture was stirred at room temperature for 1 hour to activate the carboxyl group; Dissolve the oligonucleotide single strand with an amino group at the 5' end in CBS buffer, add the activated TPP-COOH solution, and react overnight at room temperature; After the reaction was completed, the mixture was diluted with deionized water and purified by high performance liquid chromatography to obtain a DNA-TPP compound. The sequence of the single-stranded oligonucleotide was 5'-GCCACAACATCCTAA-3'.

9. The preparation method according to claim 5, characterized in that: The contents of ferrocene, glucose oxidase and atovaquone in the PFAGD nanogel obtained in S6 are: ferrocene 15 μmol / L, glucose oxidase 120 mU / mL, and atovaquone 1.2 μg / mL, respectively.

10. Use of the nanogel according to claim 1 in preparing anti-tumor drugs for chemodynamic therapy.

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