Fluorine-containing macrocyclic polyamine polymer based on valerolactone ring-opening polymerization as well as preparation method and application of fluorine-containing macrocyclic polyamine polymer

Abstract

The invention discloses a fluorine-containing macrocyclic polyamine polymer based on valerolactone ring-opening polymerization as well as a preparation method and application of the fluorine-containing macrocyclic polyamine polymer. The fluorine-containing macrocyclic polyamine polymer based on valerolactone ring-opening polymerization is mainly prepared and synthesized by nucleophilic substitution, ring-opening polymerization, esterification reaction and click reaction. The oxygen carrying capacity is as high as 15 mg / L; singlet oxygen can be efficiently generated; siRNA can be effectively condensed; after the LipoTMRNAiMAX is compounded with auxiliary lipid (DSPE-PEG-iRGD) to form nano-particles, the transfection efficiency of the LipoTMRNAiMAX is 3 times that of LipoTMRNAiMAX after illumination under the condition of 10% serum; by delivering oxygen and HIF-1alpha, proliferation of 4T1 cells in a mouse body is remarkably inhibited, and the tumor inhibition rate reaches up to 87%. The fluorine-containing macrocyclic polyamine polymer based on valerolactone ring opening polymerization can be used for preparing a non-viral gene vector, serving as a photosensitizer and preparing a carrier for delivering oxygen and HIF-1alpha in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of polymers, and particularly relates to a fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone, and a preparation method and application thereof. Background Art

[0002] RNA interference can precisely trigger and guide the degradation of targeted mRNA, thereby achieving specific silencing of gene expression. Its fineness and efficiency in the field of gene activity regulation have increasingly become a key force in breaking through the limitations of traditional therapies in the field of tumor treatment. However, when traditional carriers deliver small interfering RNA (siRNA) to tumor cells, they often face the obstacle of lysosomal capture, which limits the exertion of their gene silencing effect. Therefore, enhancing the lysosomal escape ability of the carrier is crucial for realizing the RNAi regulation function. As an emerging strategy to promote lysosomal escape, photochemical internalization (PCI) destroys the lysosomal membrane through singlet oxygen generated by photodynamic therapy (PDT), effectively promoting the intracellular delivery of siRNA. Given the minimally invasive and anti-drug resistance advantages of PDT, developing a synergistic photosensitizer-based therapeutic delivery carrier by combining PCI technology has become a current research frontier hotspot.

[0003] However, the unique microenvironment of solid tumors, including factors such as abnormal blood vessels, hypoxia, and high levels of glutathione, severely restricts the clinical application effect of PDT. Therefore, there is an urgent need to develop innovative strategies that can reshape the tumor microenvironment and enhance the PDT efficacy. Among many strategies, directly delivering exogenous oxygen is directly effective, but the problem of oxygen leakage cannot be ignored; while indirectly inhibiting the HIF-1 signaling pathway is persistent but limited in efficiency. Based on this, designing a polymer that integrates photodynamic activation, fluorinated oxygen delivery, and HIF-1α siRNA delivery has very important research value. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or defects, and provide at least the advantages described hereinafter.

[0005] Another object of the present invention is to provide a fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone, which is a novel compound with an oxygen-carrying capacity of up to 15 mg / L and can efficiently generate singlet oxygen; it can effectively condense siRNA, and after being compounded with an auxiliary lipid (DSPE-PEG-iRGD) to form nanoparticles, under 10% serum conditions, after illumination, the transfection efficiency is 3 times that of Lipo TM RNAiMAX; this polymer significantly inhibits the proliferation of 4T1 cells in mice by delivering oxygen and HIF-1α, and the tumor inhibition rate is as high as 87%.

[0006] Another object of the present invention is to provide a method for preparing a fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone, which uses BODIPY as an initiator and a valerolactone derivative as a monomer to carry out ring-opening polymerization, and through stepwise click reactions before and after removal by tetramethylsilane, glutathione-responsive

[12] aneN3 and perfluorocarbon chains of different lengths are introduced to form an amphiphilic polymer.

[0007] Another object of the present invention is to provide the application of the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone in the preparation of non-viral gene vectors, as photosensitizers, or in the preparation of oxygen and HIF-1α carriers for tumor treatment.

[0008] To achieve these and other advantages in accordance with the present invention, there is provided a fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone, which has the structure of the following formula (I): (I) Wherein, R is a fluorine-containing substituent, R1 is a fluorinated carbon chain, R2 is a substituent containing a macrocyclic polyamine ring, and m and n are positive integers.

[0009] Preferably, m = 20 and n = 20.

[0010] Preferably, R is , wherein a is 2 or 6.

[0011] Preferably, R1 is .

[0012] Preferably, R2 is .

[0013] The object of the present invention can also be further achieved by a method for preparing a fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone, including the following steps: 1) Prepare an intermediate containing a macrocyclic polyamine ring through a condensation reaction; 2) Carry out a ring-opening polymerization reaction on propargyl valerolactone and propargyl valerolactone protected by tetramethylsilane to form an intermediate of ring-opening polymerization of valerolactone; 3) Carry out a click reaction between the intermediate containing a macrocyclic polyamine ring and the intermediate of ring-opening polymerization of valerolactone to form an intermediate of ring-opening polymerization of valerolactone with a macrocyclic polyamine ring substituent; 4) Carry out a click reaction between the intermediate of ring-opening polymerization of valerolactone with a macrocyclic polyamine ring substituent and a fluorinated carbon chain to prepare the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of formula (I).

[0014] Preferably, in step 2), diphenyl phosphate is used as the catalyst for the ring-opening polymerization, and the molar ratio of propargyl valerolactone, propargyl valerolactone protected by tetramethylsilane to diphenyl phosphate is 20﹕20﹕1.

[0015] The object of the present invention can also be further achieved by the application of the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone in the preparation of non-viral gene vectors.

[0016] The object of the present invention can also be further achieved by the application of the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone as a photosensitizer.

[0017] The object of the present invention can also be further achieved by the application of the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone in the preparation of carriers for delivering oxygen and HIF-1α in tumor treatment.

[0018] The present invention at least includes the following beneficial effects: The fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone in the present invention is a novel compound, its oxygen-carrying capacity is as high as 15 mg / L, and it can efficiently generate singlet oxygen; it can effectively condense siRNA, and can form nanoparticles after being compounded with the auxiliary lipid (DSPE-PEG-iRGD); it can be used as a non-viral gene vector. Among them, after the polymer BFN2 is compounded with the auxiliary lipid (DSPE-PEG-iRGD) to form nanoparticles, under 10% serum conditions, after light irradiation, the transfection efficiency is 3 times that of Lipo TM RNAiMAX; by delivering oxygen and HIF-1α, the proliferation of 4T1 cells in mice is significantly inhibited, and the tumor inhibition rate is as high as 87%. The fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone in the present invention can be used to prepare non-viral gene vectors, as a photosensitizer, and to prepare carriers for delivering oxygen and HIF-1α in tumor treatment.

[0019] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and partially will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1H NMR spectrum of polymers 1-12 prepared in Example 1 of the present invention; Figure 2 1H NMR spectrum of polymers 1-13 prepared in Example 1 of the present invention; Figure 3 1H NMR spectrum of polymers 1-14 prepared in Example 1 of the present invention; Figure 4 1H NMR spectrum of polymers 1-15 prepared in Example 1 of the present invention; Figure 5 1H NMR spectrum of Polymer 1-16 prepared in Example 1 of the present invention; Figure 6 1H NMR spectrum of Polymer BFN1 prepared in Example 1 of the present invention; Figure 7 1H NMR spectrum of Polymer BFN2 prepared in Example 1 of the present invention; Figure 8 Oxygen-carrying capacity diagram of Polymer BFN1 and Polymer BFN2 in Example 2 of the present invention; Figure 9 Singlet oxygen generation diagram of Polymer BFN1 and Polymer BFN2 in Example 3 of the present invention; Figure 10 Agarose gel retardation assay diagram of Polymer BFN1 and Polymer BFN2 on siRNA in Example 4 of the present invention; Figure 11 Knockout result diagram of GFP gene by Polymer BFN1 and Polymer BFN2 delivering GFP siRNA in HeLa-GFP cells in Example 5 of the present invention; Figure 12 Diagram of nanoparticles formed by Polymer BFN2 complexed with auxiliary lipid (DSPE-PEG-iRGD) and siRNA in Example 6 of the present invention; Figure 13 Knockout result diagram of GFP gene by Polymer BFN2 delivering GFP siRNA in HeLa-GFP cells under different serum concentration conditions after light treatment in Example 7 of the present invention; Figure 14 Inhibitory effect diagram of Polymer BFN2 on 4T1 tumors in mice by delivering oxygen and HIF-1α in Example 8 of the present invention. Detailed implementation manners

[0023] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0024] It should be understood that the terms such as "having", "comprising" and "including" used herein do not preclude the presence or addition of one or more other elements or their combinations.

[0025] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0026] Major chemical reagents: diphenyl phosphate (DPP, analytical grade, Shanghai Macklin Biochemical Co., Ltd.), triethylamine (analytical grade, Shanghai Macklin Biochemical Co., Ltd.), sodium ascorbate (analytical grade, Beijing Innochem Science & Technology Co., Ltd.), copper sulfate pentahydrate (analytical grade, Sigma-Aldrich Trading Co., Ltd.), tetrabutylammonium fluoride (TBAF, Shanghai Macklin Biochemical Co., Ltd.), HCl / EA (analytical grade, Anhui Zesheng Technology Co., Ltd.), 2-hydroxyethyldisulfide (analytical grade, Shanghai Macklin Biochemical Co., Ltd.), p-toluenesulfonyl chloride (analytical grade, Anhui Zesheng Technology Co., Ltd.), 4-dimethylaminopyridine (DMAP, analytical grade, Shanghai Macklin Biochemical Co., Ltd.).

[0027] Major equipment: portable dissolved oxygen meter (AR8010, Simma UK Limited), ultraviolet-visible spectrophotometer (Hitachi U-3900 UV-visible spectrophotometer, Hitachi, Ltd., Japan), MVP EC3 gel imaging system (MVP EC3 gel imaging system, USA, Tianmei (Shanghai) Instrument & Equipment Co., Ltd.), flow cytometer (Beckman CytoFLEX flow cytometer (USA), Beckman Coulter, Inc.), scanning electron microscope (Hitachi S-4800 cold field emission scanning electron microscope (Japan), Hitachi, Ltd., Japan), LED lamp (PLS-LED 100 high-power LED light source, Beijing Perfect Light Technology Co., Ltd.).

[0028] Example 1 A fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone, having the structure of the following formula (I): (I) Wherein, R is a fluorinated substituent, R1 is a fluorinated carbon chain, R2 is a substituent containing a macrocyclic polyamine ring, and m and n are positive integers.

[0029] Wherein, when the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone is a compound with the following structure: When R is , where a = 2, m = n = 20, and the formed fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone is denoted as BFN1; When R is , where a = 6, m = n = 20, and the formed fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone is denoted as BFN2.

[0030] The synthesis route is as follows: The specific synthesis steps are as follows: Synthesis of Compound 1-5: Place 2-hydroxyethyl disulfide (5.00 g, 32.41 mmol) in a pressure-resistant bottle, add 50 mL of THF, and while stirring, add p-toluenesulfonyl chloride (4.94 g, 25.92 mmol) and triethylamine (9.84 g, 97.23 mmol). Heat to 75 °C and react for 5 h. Post-treatment: Filter the white solid through ordinary filter paper, remove the solvent by rotary evaporation under reduced pressure, and directly use it for the next reaction without other purification methods.

[0031] Synthesis of Compound 1-6: Dissolve Compound 1-5 in 50 mL of DMF, and while stirring, slowly add sodium azide (2.52 g, 38.76 mmol). Heat to 70 °C and react for 12 h. Post-treatment: Add 200 mL of a large amount of saturated brine, extract three times with 200 mL of ethyl acetate. Collect the organic phase, add anhydrous sodium sulfate for drying. Filter the anhydrous sodium sulfate through ordinary filter paper, and remove the solvent by rotary evaporation under reduced pressure. The product is purified by column chromatography (the eluent ratio is PE:EA = 5:1) to finally obtain 1.9 g of a yellow oil, which is Compound 1-6. The yield is 34%. 1 H NMR (CDCl3, 600 MHz) δ 3.82(t, 2H), 3.55 (t, 2H), 2.82 (td, 4H). 13 C NMR (CDCl3, 151 MHz) δ 60.31, 50.00,41.37, 37.49. Synthesis of Compound 1-7: Dissolve Compound 1-4 (101.60 mg, 0.23 mmol) in anhydrous THF, add EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (54.40 mg, 0.27 mmol) and DMAP (4-dimethylaminopyridine) (34.70 mg, 0.23 mmol). After stirring for 30 min, add Compound 1-6 (42.40 mg, 0.23 mmol). React at room temperature for 24 h. Post-treatment: Filter the solid through ordinary filter paper, and remove the solvent from the filtrate by vacuum distillation. The product is purified by column chromatography (the eluent ratio is PE:EA = 5:1) to finally obtain 72.7 mg of a yellow oil, which is Compound 1-7. The yield is 52%. 1 H NMR (CDCl3, 600 MHz) δ 4.44 – 4.27 (m, 2H), 3.67 – 3.55(m, 2H), 3.47 – 3.29 (m, 10H),3.04 – 2.91 (m, 2H), 2.90 – 2.82 (m, 2H), 2.63(s, 4H), 1.95 – 1.85 (m, 3H), 1.82 –1.74 (m, 3H), 1.45 (s, 18H). 13 C NMR(CDCl3, 151 MHz) δ 170.76, 155.83, 78.83, 61.55, 53.37, 50.74, 49.56, 45.16,43.11, 37.22,36.89, 31.02, 29.71, 29.20, 28.09, 28.04. ESI–MS: calcd forC 25 H 46 N6O6S2: 590.30 (M + H + ), found: 591.30 (M + H + ). Among them, the synthesis of Compound 1-4 is the same as that in the reference: Syntheses of

[12] aneN3–oligopeptide conjugates as effective DNA condensation agents. Biorg. Med. Chem. ,2012, 20,2897-2904. Synthesis of Polymer 1-12: Inside the glove box, compound 1-2 (100.0 mg, 0.47 mmol, 20 equiv.), 1-11 (14.42 mg, 0.23 mmol, 1.0 equiv.), and diphenyl phosphate DPP (5.88 mg, 0.023 mmol, 1.0 equiv.) were added to a 10 mL eggplant-shaped flask, and 0.3 mL of anhydrous toluene was added. The mixture was stirred and reacted at room temperature for 24 h. A sample was taken for NMR detection, and the monomer conversion reached about 95%. Then, compound 1-1 (64.89 mg, 0.47 mmol, 20 equiv.) was added, and the catalyst DPP (5.88 mg, 0.023 mmol, 1.0 equiv.) and 100 μL of anhydrous toluene were replenished. The reaction continued for 24 h, and then a sample was taken for NMR detection with a conversion of 95%. The reaction flask was taken out of the glove box, and the reaction was quenched with triethylamine. The solvent was rotary evaporated. Dialysis was carried out for 24 h using a dialysis bag (cut-off molecular weight 6000) with ethanol as the dialysis medium to remove small molecular weight impurities to obtain Polymer 1-12. The 1H NMR spectrum of Polymer 1-12 (600 MHz, CDCl3) is as shown in Figure 1 shown below.

[0032] Among them, the synthesis of compound 1-1 is the same as the reference: Peg- and Peptide-Grafted Aliphatic Polyesters by Click Chemistry. Journal of the American Chemical Society , 2005, 127 , 7404 - 7410. The synthesis of compound 1-2 is the same as the reference: Sequential and Localized Grafting on Aliphatic Polyester Diblock Copolymers Using Alkyne Deprotection and Click Cycloaddition. J. Polym. Sci., Part A: Polym. Chem. , 2009, 47 , 7054 - 7065. The synthesis of compound 1-10 is the same as the reference: Self-Destructive Peg-Bodipy Nanomaterials for Photodynamic and Photothermal Therapy. J Mater Chem B , 2019, 7, 4655 - 4660. The synthesis of compound 1-11 is the same as the reference: Self-Destructive Peg-Bodipy Nanomaterials for Photodynamic and Photothermal Therapy.J Mater Chem B , 2019, 7, 4655 - 4660. Synthesis of Polymer 1 - 13: In a two - necked round - bottom flask, Polymer 1 - 12 (104.90 mg, 1.0 equiv.) and Compound 1 - 7 (196.60 mg, 24.0 equiv.) were dissolved in 10 mL of anhydrous THF. The flask was purged with argon three times until all the air was replaced by argon. Sodium ascorbate (16.50 mg, 6.0 equiv.) and copper(II) sulfate pentahydrate (10.50 mg, 3.0 equiv.) were dissolved in 1 mL of water and quickly added to the reaction system via a syringe. The reaction was carried out at room temperature for 12 h. The next day, THF was removed by rotary evaporation under reduced pressure. The crude product was dissolved in 20 mL of DCM and washed three times with 20 mL of saturated sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered through ordinary filter paper to remove anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. Dialysis was carried out for 24 h using a dialysis bag (cut - off molecular weight 14000) with ethanol as the dialysis medium to remove small - molecular - weight impurities, obtaining Polymer 1 - 13. The 1H NMR spectrum of Polymer 1 - 13 (600 MHz, CDCl3) is as Figure 2 shown.

[0033] Synthesis of Polymer 1 - 14: Polymer 1 - 13 (92.9 mg, 1.0 equiv.) was placed in a 50 - mL round - bottom flask and dissolved in 30 mL of anhydrous THF. The reaction flask was placed in a low - temperature bath at 0 °C, and a THF solution (20 mL) of TBAF (tetrabutylammonium fluoride) (100.0 mg, 80.0 equiv.) was added dropwise within one hour. The reaction was carried out for 3 h. Work - up: The reaction was quenched by adding 30 mL of saturated ammonium chloride solution. THF was removed by rotary evaporation under reduced pressure. The crude product was extracted three times with 50 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered through ordinary filter paper to remove anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. Dialysis was carried out for 24 h using a dialysis bag (cut - off molecular weight 14000) with ethanol as the dialysis medium to remove small - molecular - weight impurities, obtaining Polymer 1 - 14. The 1H NMR spectrum of Polymer 1 - 13 (600 MHz, CDCl3) is as Figure 3As shown below. Synthesis of Polymer 1-15 and Polymer 1-16: In a two-necked round-bottom flask, Polymer 1-14 (340.0 mg, 1.0 equiv.) and Compound 1-8 / 1-9 (24.0 equiv.) were dissolved in 10 mL of anhydrous THF, and the system was purged with argon three times. Sodium ascorbate (22.5 mg, 6.0 equiv.) and copper(II) sulfate pentahydrate (14.0 mg, 3.0 equiv.) were dissolved in 1 mL of water and quickly added to the reaction system via syringe. The reaction was carried out at room temperature for 12 h. THF was removed by distillation under reduced pressure. The crude product was dissolved in 20 mL of DCM, washed three times with 20 mL of saturated sodium chloride solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered through ordinary filter paper to remove anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Dialysis was carried out for 24 h using a dialysis bag (molecular weight cut-off 20,000) with ethanol as the dialysis medium to remove small molecular weight impurities, obtaining Polymer 1-15 and Polymer 1-16. The 1H NMR spectrum of Polymer 1-15 (600 MHz, CDCl3) is as shown in Figure 4 As shown below. The 1H NMR spectrum of Polymer 1-16 (600 MHz, CDCl3) is as shown in Figure 5 As shown below.

[0034] Among them, the synthesis of Compound 1-8 is the same as that in the reference Multivalency for Modularity: A Versatile Adhesive with Cooperatively Activated Fast Dismantlability. ACS Applied Polymer Materials , 2022, 4 , 6812 - 6816. The synthesis of Compound 1-9 is the same as that in the reference: Self-Adapting Peripherally Heterofunctionalized Hyperbranched Polymers: Formation of Janus and Tripodal Structures. Langmuir , 2013, 29 , 1245 - 1257. Synthesis of Polymer BFN1 and BFN2: Under an ice bath, Polymer 1-15 / 1-16 (100.0 mg) was added to a 25 mL eggplant-shaped flask, 5 mL of HCl / EA and 10 mL of DCM were added, and the mixture was stirred at room temperature for 15 min. Then 5 mL of ether was added and sonicated for 5 min. The solid was obtained by filtration through ordinary filter paper, which is the target polymer.

[0035] Among them, the 1H NMR spectrum of Polymer BFN1 (600 MHz, DMSO-d 6) As Figure 6 shown. The 1H NMR spectrum (600 MHz, DMSO- d 6) As Figure 7 shown.

[0036] Example 2 Oxygen-carrying capacity of the polymers BFN1 and BFN2 prepared in Example 1: The oxygen-carrying capacity of the polymers BFN1 and BFN2 prepared in Example 1 was detected using a portable dissolved oxygen meter. Specifically, 5 mL of the polymer BFN1 solution (5 mg / mL) and the polymer BFN2 solution (5 mg / mL) were placed in a sterile oxygen chamber, and O2 was introduced at a constant flow rate of 5 L / min for 30 min to prepare an oxygen-saturated solution. Subsequently, this oxygen-saturated solution was added to 15 mL of deoxygenated water, and the liquid surface was sealed with paraffin oil to isolate air. The change in the oxygen concentration in the deoxygenated water under closed conditions was measured using a portable dissolved oxygen meter. The test results are as Figure 8 shown. It can be Figure 8 concluded that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention has good oxygen-carrying capacity, and the oxygen-carrying capacity is as high as 15 mg / L.

[0037] Example 3 Singlet oxygen generation ability of the polymers BFN1 and BFN2 prepared in Example 1: Prepare 2 mL of a test solution, which is an aqueous solution containing 20 μg / mL of polymer BFN1 or BFN2 and 100 μM of singlet oxygen fluorescent probe ABDA. Place this test solution under a LED white light source of 50 mW / cm 2 and irradiate it, and record the change in the absorption intensity of this test solution at 378 nm using a UV-visible spectrophotometer. The test results are as Figure 9 shown. It can be Figure 9 concluded from (A) and (B) therein that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention has good singlet oxygen generation ability.

[0038] Example 4 Aggregation siRNA imaging of the polymers BFN1 and BFN2 prepared in Example 1: Mix the polymers BFN1 and BFN2 prepared in Example 1 with 5 pmol of siRNA at different mass ratios in DEPC water. After incubating at 37 °C for 30 min, load it onto a 0.7% agarose gel containing Gold View dye and perform electrophoresis at 85 V and 100 mA for 20 min. Analyze by photographing with a MVP EC3 gel imaging system. The test results are as Figure 10 shown. It can beFigure 10 From (A) and (B), it can be concluded that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention can effectively condense siRNA.

[0039] Example 5 Cell transfection of the polymers BFN1 and BFN2 prepared in Example 1: HeLa-GFP cells (1×10 5 cells / well) were seeded in a 6-well plate and cultured overnight until the confluence rate reached 70%. Complexes formed by condensing polymer BFN1 or BFN2 with GFP siRNA were added at different weight ratios. The dose of GFP siRNA was fixed at 0.33 μg / well (83 nM). After incubation for 6 h, the old medium was removed and replaced with fresh DMEM medium containing 10% FBS, and the cells were cultured for another 42 h. The fluorescence intensity in the FITC channel was detected using a cell flow cytometer to quantify the gene silencing effect. The test results are as Figure 11 shown. From Figure 11 it can be concluded that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention can effectively transfect HeLa-GFP cells. The transfection efficiencies of BFN1 and BFN2 are 1.4 times and 1.7 times that of Lipo TM RNAiMAX, respectively, and can be used to prepare non-viral gene vectors or as photosensitizers. The transfection efficiency of BFN2 is better than that of BFN1, and BFN2 with better transfection efficiency was used in subsequent performance experiments.

[0040] In Example 6, BFN2-DR / siRNA NPs were prepared by a co-assembly method: DOPE (D) and DSPE-PEG-iRGD (R) were dissolved in CDCl3 at a molar ratio of 2:0.19. The solvent was removed by rotary evaporation and the residue was dried in a vacuum drying oven for 12 h to obtain a DOPE / DSPE-PEG-iRGD film. 500 μL of a BFN2 / siRNA complex solution (5 nmol siRNA) with a mass ratio of 30:1 was added to the DOPE / DSPE-PEG-iRGD film, and the mixture was stirred at room temperature for 12 h and then sonicated for 2 h to obtain BFN2-DR / siRNA NPs. From Figure 12 it can be concluded that spherical nanoparticles can be formed after self-assembly of the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention with DSPE-PEG-iRGD as observed by scanning electron microscopy.

[0041] Example 7 HeLa-GFP cells (1×10 5Cells / well) were seeded in 6-well plates and cultured overnight until the confluence rate reached 70%. BFN2-DR / siRNA (siRNA at 0.33 μg / well and BFN2-DR at 9.9 μg / well) was dissolved in 1640 medium containing 10% FBS and administered. After 6 h of administration, the old medium was removed and replaced with fresh DMEM medium containing 10% FBS. Irradiation was performed with white light LED (50 mW / cm 2 ) for 0.5 min, and the culture was continued for 42 h. The fluorescence intensity in the FITC channel was detected using a flow cytometer to quantify the gene silencing effect. It can be concluded from Figure 13 that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention can effectively transfect HeLa-GFP cells under 10% serum conditions. After light irradiation, the transfection efficiency of BFN2 was increased to 3 times that of Lipo TM RNAiMAX.

[0042] Example 8 4T1 cells were inoculated into the axilla of immunodeficient mice seven days in advance. Then, the mice were divided into six groups for treatment: Group 1: injected with PBS; Group 2: injected with BFN2-DR NPs without irradiation; Group 3: injected with BFN2-DR NPs and irradiated; Group 4: injected with BFN2-DR@O2 NPs and irradiated; Group 5: injected with BFN2-DR / HIF-1α siRNA NPs and irradiated; Group 6: injected with BFN2-DR / HIF-1α siRNA@O2 NPs and irradiated (siRNA dose was 600 μg / kg). Each group contained three mice. Nine hours after injection, the tumor area was irradiated with a white LED lamp (70 mW / cm²) for 20 min for treatment. The body weight and tumor volume of the mice were measured regularly (tumor volume = length × width 2 / 2). It can be concluded from Figure 14 that the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention can achieve 87% inhibition of 4T1 tumors in mice by delivering oxygen and HIF-1α. Therefore, the fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone of the present invention can be used to prepare carriers for delivering oxygen and HIF-1α in tumor treatment for tumor treatment.

[0043] Although the embodiments of the present invention have been disclosed as above, they are not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone, characterized in that, It has the structure of the following formula (I): （I） Wherein, R is a fluorine-containing substituent, R1 is a fluorinated carbon chain, R2 is a substituent containing a macrocyclic polyamine ring, and m and n are positive integers.

2. The fluorine-containing macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone according to claim 1, wherein m = 20, n = 20.

3. The fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone according to claim 2, wherein, R is , where a is 2 or 6.

4. The fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone according to claim 3, characterized in that, R1 is 。 5. The fluorine-containing macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone according to claim 4, characterized in that, R2 is 。 6. A method for preparing a fluorinated macrocyclic polyamine polymer based on the ring-opening polymerization of valerolactone as described in any one of claims 1 to 5, characterized in that, It includes the following steps: 1) Prepare an intermediate containing a macrocyclic polyamine ring through a condensation reaction; 2) Conduct a ring-opening polymerization reaction on propargyl valerolactone and tetramethylsilane-protected propargyl valerolactone to form an intermediate of ring-opening polymerization of valerolactone; 3) Conduct a click reaction between the intermediate containing a macrocyclic polyamine ring and the intermediate of ring-opening polymerization of valerolactone to form an intermediate of ring-opening polymerization of valerolactone substituted with a macrocyclic polyamine ring; 4) Conduct a click reaction between the intermediate of ring-opening polymerization of valerolactone substituted with a macrocyclic polyamine ring and a fluorinated carbon chain to prepare a fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone of formula (I).

7. The method according to claim 6, wherein In step 2), the ring-opening polymerization uses diphenyl phosphate as a catalyst, and the molar ratio of propargyl valerolactone, tetramethylsilane-protected propargyl valerolactone to diphenyl phosphate is 20﹕20﹕1.

8. Use of the fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone according to any one of claims 1 to 5 in the preparation of a non-viral gene vector.

9. Use of the fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone according to any one of claims 1 to 5 as a photosensitizer.

10. Use of the fluorinated macrocyclic polyamine polymer based on ring-opening polymerization of valerolactone according to any one of claims 1 to 5 in the preparation of a carrier for delivering oxygen and HIF-1α in tumor treatment.

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