Preparation method and application of targeted fusobacterium nucleatum in-situ self-assembled photo-thermal nano material

Abstract

The invention relates to a preparation method and application of an in-situ self-assembled photo-thermal nano material capable of targeting fusobacterium nucleatum. The nano material is composed of BDP-GalNAc and nano gel PSBMA. The preparation method comprises the following steps: firstly, covalently linking a photo-thermal functional element molecule BDP and targeted fusobacterium nucleatum GalNAc-N3 to obtain BDP-GalNAc; and then assembling the BDP-GalNAc nanogel with the nanogel PSBMA to obtain the BDP-GalNAc nanogel which is stable in PBS and has a controllable particle size. An introduced Fn targeting group N-acetylgalactosamine (GalNAc) is specifically combined with Fap2 protein to inhibit the growth of Fn, and good antibacterial and anti-tumor effects are generated by utilizing the in-situ self-assembly morphology change of the material on the surface of bacteria and the photothermal enhancement effect initiated by 808nm laser, so that a new thought and method are provided for the treatment of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis technology and nanomedicine, and specifically relates to a method for preparing a targeted Fusobacterium nucleatum in-situ self-assembled photothermal nanomaterial, and an application of the targeted Fusobacterium nucleatum material in-situ self-assembled morphology and property changes in antibacterial and anti-tumor treatment. Background Art

[0002] Colorectal cancer (CRC) causes more than 1.85 million cases and an estimated 850,000 deaths each year, making it the second leading cause of cancer-related death and a common disease worldwide. The high abundance of Fusobacterium nucleatum (Fn) in CRC sites affects the tumor microenvironment and increases the difficulty of treatment. In recent years, a variety of antimicrobial strategies have been proposed to combat F. nucleatum. Fn exhibits high levels of localization in colorectal cancer tissues, which is due to the ability of the bacterial surface lectin Fap2 to specifically bind to Gal-GalNAc overexpressed on the surface of colorectal cancer cells, thereby forming a pro-inflammatory microenvironment and inducing immunosuppression. Targeting Fn and reducing its colonization in tumors has great potential in improving chemotherapy response and reducing the risk of cancer recurrence.

[0003] Boron fluoride fluorescent dye (BODIPY) is an organic photofunctional material with good biocompatibility. It has the advantages of large Stokes shift, tunable absorption / emission wavelength, easy chemical modification, low photobleaching and high fluorescence quantum yield. It has many applications in the biomedical field, such as bioluminescence imaging, photodynamic therapy and photothermal therapy. Moreover, under certain conditions, the molecular structure of this type of molecule can be orderly arranged, easily forming aggregates, and showing better photothermal conversion performance. There have been reports on the use of BODIPY as a photosensitizer / photothermal agent for anti-tumor research, but there are few reports on the antibacterial and anti-tumor research through the changes in photofunction and morphology before and after the in situ self-assembly of BODIPY to form aggregates. Summary of the invention

[0004] The present invention aims to design and synthesize a novel in situ self-assembled photothermal nanomaterial targeting Fusobacterium nucleatum, which is composed of BDP-GalNAc and nanogel PSBMA. First, the photothermal functional unit BDP and the targeting group GalNAc-N 3 Covalently linked to obtain BDP-GalNAc. Then the nanogel PSBMA and BDP-GalNAc were co-assembled to form a nanoassembly that was stable in water and had a controllable particle size. Compared with the BDP molecule, the photothermal conversion efficiency of the BDP-GalNAc molecule reached 54.3%, which could well target Fusobacterium nucleatum and aggregate in situ to form an assembly, thereby achieving targeted enhanced anti-Fusobacterium nucleatum infection CRC tumor treatment under laser excitation.

[0005] The BDP-GalNAc molecule provided by the present invention has a structural formula and a nuclear magnetic resonance hydrogen spectrum as shown in Figure 1 .

[0006] The PSBMA nanogel provided by the present invention is prepared according to the method of the reference literature, and its structural formula is as follows Figure 2 .

[0007] The technical purpose of the present invention is achieved through the following technical solutions.

[0008] We first synthesized BDP molecules as photothermal functional units and used click chemistry to react with the hydrophilic Fusobacterium nucleatum targeting group GalNAc-N 3 BDP-GalNAc was formed by covalent linkage, and the nanogel assembly BDP-GalNAc NGs was prepared under the action of nanogel PSBMA.

[0009] The transmission electron microscope detected that the morphology was spherical. The average particle size of the nanogel assembly was 285nm. After being placed in PBS for 5 days, the particle size did not change significantly, and it had good stability.

[0010] Preparation method of nanogel assembly BDP-GalNAc NGs: BDP-GalNAc and zwitterionic hydrogel PSBMA are dissolved in acetonitrile solution, stirred at room temperature for 8 hours, and the acetonitrile solution is slowly dripped into water under ultrasonic conditions and stirred for 30 minutes, and then the acetonitrile is removed by rotary evaporation to obtain BDP-GalNAc NGs.

[0011] The BDP-GalNAc NGs nanogel proposed in the present invention has the following outstanding advantages: (1) BDP-GalNAc NGs nanogel has excellent stability in aqueous solution; (2) BDP-GalNAc NGs nanogel has excellent biocompatibility, and the nanogel PSBMA can be degraded in response to glutathione, releasing BDP-GalNAc, recognizing Fap2 on the surface of Fusobacterium nucleatum, and self-assembling in situ on the bacterial surface to form larger aggregates with morphological changes, while the photothermal conversion performance is improved; (3) BDP-GalNAc NGs can prevent BDP-GalNAc from being targeted to the liver when encapsulated in the nanogel PSBMA.

[0012] In summary, the preparation and application of the in situ self-assembled photothermal nanomaterials targeting Fusobacterium nucleatum proposed in the present invention have the following beneficial effects: under the encapsulation of PSBMA with good biocompatibility, BDP-GalNAc NGs can be applied to in vivo and in vitro experiments; PSBMA depolymerizes in response to high GSH concentration in the tumor site, releases BDP-GalNAc and targets the surface of Fusobacterium nucleatum, self-assembles in situ to form aggregates to inhibit bacterial growth, and exhibits enhanced photothermal effect under 808 nm laser irradiation, which plays a role in treating colorectal cancer infected with Fusobacterium nucleatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The molecular structure and H-NMR spectrum of the compound synthesized in the present invention are shown in FIG.

[0014] Figure 2 The molecular structure formula of the nanogel PSBMA provided by the present invention.

[0015] Figure 3 The following is a synthetic route for the molecules designed in the present invention.

[0016] Figure 4 The stability of the nanoassembly prepared in the present invention in PBS.

[0017] Figure 5 This is the morphological characterization of the nanoassembly prepared in the present invention in response to GSH.

[0018] Figure 6 The present invention provides morphological characterization of the targeting effect and photothermal killing of the nanoassembly prepared in the present invention on Fusobacterium nucleatum.

[0019] Figure 7 The nanoassembly prepared in the present invention kills CT26 cells with or without Fusobacterium nucleatum infection.

[0020] Figure 8 These are the changes in the volume of the proximal and distal tumors in tumor-bearing mice in different experimental groups after one dose of photothermal therapy via the tail vein, as well as comparison photos of the tumors obtained by dissection after the treatment of tumor-bearing mice in different groups. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below by way of examples. The examples are intended to describe the present invention in detail, but the protection scope of the present invention is not limited to the following examples.

[0022] Example 1. Preparation method of in situ self-assembly photothermal material BDP-GalNAc targeting Fusobacterium nucleatum, the synthesis route is shown in Figure 3 , including the following steps:

[0023] (1) Dissolve 1.2 mL of 4-pyridinecarboxaldehyde and 2.7 mL of 2,4-dimethylpyrrole in 250 mL of anhydrous dichloromethane solution, add 0.1 mL of trifluoroacetic acid, and stir for 8 h at room temperature under nitrogen protection. Add 2.7 g of 2,3-dichloro-5,6-dicyanobenzoquinone, continue stirring for 4 h, and then slowly add 18 mL of triethylamine. After continuing stirring for 15 min, add 27 mL of boron trifluoride ether solution at 0°C, and stir at room temperature for 3 h. After washing and extracting with saturated sodium bicarbonate aqueous solution, column chromatography purification is performed to prepare compound 1; 200 mg of compound 1, 0.4 g of iodine particles and 0.2 g of iodic acid are dissolved in ethanol, heated at 60°C for 40 min, and the reaction is monitored by TLC. After the reaction is completed, the reaction solution is poured into 200 mL of saturated sodium thiosulfate solution and stirred at room temperature for 30 min. The compound 2 was obtained after washing and extraction with saturated brine; anhydrous acetonitrile was used as solvent, 4 g of N-ethylaniline and 3.66 mL of bromopropyne were used as raw materials, and 13.6 g of potassium carbonate was used as an acid-binding agent. The reaction was refluxed for 24 h under a nitrogen atmosphere, the solvent was removed under reduced pressure, the mixture was extracted and dried, and the compound 3 was purified by column chromatography; DMF was used as solvent, and according to the Vilsmeier-Haack reaction mechanism, 3.5 g of compound 3 was reacted with 14.5 g of phosphorus oxychloride to purify the compound 4; 210 mg of compound 2 and 260 mg of compound 4 were heated at 120°C for reaction under the action of acetic acid and piperidine, and then the photothermal functional element BDP compound was obtained by column chromatography;

[0024] (2) 2.5 g of N-acetyl-d-galactosamine and acetic anhydride were used as raw materials, iodine was used as a catalyst, and the mixture was stirred at room temperature until the solid was completely dissolved. A saturated sodium sulfite solution, a saturated sodium bicarbonate solution, and a saturated saline solution were used for extraction and drying to obtain compound 5; 5 g of compound 5 was used as raw material, 3.75 mL of trimethylsilyl trifluoromethanesulfonate (TMSOTf) was used as a catalyst, and dichloromethane was used as a solvent. The mixture was stirred at 40° C. for 24 h. The reaction was quenched with triethylamine. Compound 6 was obtained after extraction and separation; 1.3 g of compound 6 and 2.07 g of 2-[2-(2-azidoethoxy)ethoxy]ethanol were used as raw materials, 2.75 g of camphorsulfonic acid (CSA) was used as a catalyst, and DMF was used as a solvent. The mixture was stirred at room temperature for 8 h and at 45 ° C for 24 h. After the reaction was completed, triethylamine was used to neutralize the mixture, and compound 7 was obtained after extraction and separation; 1 g of compound 7 was used as a raw material, sodium methoxide was used as a deacetylation agent, and methanol was used as a solvent. The mixture was stirred at room temperature for 12 h. After the reaction was completed, an ice-cold HCl solution was added to acidify the mixture to pH = 7.0, and the solvent was evaporated to prepare a target Fusobacterium nucleatum group GalNAc-N 3 .

[0025] (3) 0.28 g of BDP compound and 0.25 g of GalNAc-N 3As raw material, 10 mg CuBr and 26 mg PMDETA were used as catalysts, DMF was used as solvent, heated and stirred at 50°C for 24 h, the solvent was removed by rotary evaporation, and the final product BDP-GalNAc was prepared by column chromatography.

[0026] Example 2. Preparation of BDP-GalNAc nanogels. 100 μL of 1 mg / mL BDP-GalNAc acetonitrile solution and 200 μL of freshly reacted zwitterionic PSBMA acetonitrile solution were mixed, stirred overnight at room temperature, the acetonitrile solution was slowly added dropwise to water under ultrasonic conditions, and the acetonitrile was removed by vacuum distillation to obtain BDP-GalNAc NGs.

[0027] Example 3. Characterization of BDP-GalNAc nanogel morphology and particle size with and without GSH response. The prepared BDP-GalNAc nanogel was observed to have a spherical morphology under TEM and had good stability in PBS. In response to GSH, the process of disaggregation and formation of aggregates of the nanogel was observed as follows: Figure 4-5 shown.

[0028] Example 4. Targeted adhesion and photothermal killing of Fusobacterium nucleatum by BDP-GalNAc. Co-incubation of BDP-GalNAc nanogel with Fusobacterium nucleatum showed that BDP-GalNAc had a good targeting effect on Fusobacterium nucleatum and had the function of photothermal killing of bacteria under 808 nm laser irradiation. Figure 6 shown.

[0029] Example 5. Killing effect of BDP-GalNAc nanogel on CT26 cells infected with or without Fusobacterium nucleatum. BDP-GalNAc nanogel was co-incubated with CT26 cells infected with or without Fusobacterium nucleatum, and photothermal treatment was induced under 808 nm laser irradiation. As shown in the figure, BDP-GalNAc nanogel has good antibacterial and anti-tumor cell effects, as shown in the figure. Figure 7 shown.

[0030] Example 6. Application of BDP-GalNAc nanogel in in vivo anti-tumor study: The BDP-GalNAc nanogel of the present invention was injected into subcutaneous colorectal cancer tumor-bearing mice through the tail vein. After a photothermal treatment (808 nm, 0.8 W / cm 2 , 10min) treatment can inhibit tumor growth and achieve therapeutic effects, such as Figure 8 shown.

[0031] References

[0032] [1] Yongzhi Men, Shaojun Peng, Peng Yang, Qin Jiang, Yanhui Zhang, BinShen, Pin Dong, Zhiqing Pang, and Wuli Yang. ACS Applied Materials & Interfaces 2018 10(28), 23509-23521.

Claims

1. A method for preparing in-situ self-assembled photothermal nanomaterials targeting Fusobacterium nucleatum, characterized in that: The method for preparing the photothermal functional element BDP comprises the following steps: (1) Using 4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole as raw materials, dichloromethane as solvent, trifluoroacetic acid and 2,3-dichloro-5,6-dicyanobenzoquinone as catalysts, triethylamine and boron trifluoride ethyl ether as complexing agents, purifying and preparing compound 1; the molar ratio of 4-pyridinecarboxaldehyde, 2,4-dimethylpyrrole as raw materials, 2,3-dichloro-5,6-dicyanobenzoquinone, triethylamine, and boron trifluoride ethyl ether is 1:2-3:1-3:10-30:10-30; (2) Compound 1, iodine particles and iodic acid are used as raw materials, ethanol is used as solvent, and the mixture is heated to obtain compound 2; the molar ratio of iodine particles to iodic acid is 1:2-3:1-3; (3) using anhydrous acetonitrile as solvent, N-ethylaniline and propyne bromide as raw materials, and potassium carbonate as an acid-binding agent to purify and obtain compound 3; the molar ratio of N-ethylaniline, propyne bromide, and potassium carbonate is 1:1-3:2-5; (4) using DMF as solvent, reacting compound 3 with phosphorus oxychloride according to the Vilsmeier-Haack reaction mechanism, and purifying to obtain compound 4; the molar ratio of compound 3 to phosphorus oxychloride is 1:3-5; (5) Under the action of acetic acid and piperidine, compound 2 and compound 4 are heated in toluene to react to obtain a photothermal functional element BDP compound; the molar ratio of acetic acid, piperidine, compound 2 and compound 4 is 1-20:1-20:1:2-5, the reaction temperature is 120°C, and the reaction time is 3-4 hours.

2. The method for preparing a photothermal nanomaterial targeting in situ self-assembly of Fusobacterium nucleatum according to claim 1, characterized in that: The method for preparing the targeted Fusobacterium nucleatum group GalNAc-N3 comprises the following steps: (1) Using N-acetyl-d-galactosamine and acetic anhydride as raw materials and iodine as a catalyst, compound 5 was prepared; the molar ratio of N-acetyl-d-galactosamine to iodine was 20-30:1-3; (2) Compound 5 is used as a raw material, trimethylsilyl trifluoromethanesulfonate (TMSOTf) is used as a catalyst, and dichloromethane is used as a solvent to prepare compound 6; the molar ratio of compound 5 to trimethylsilyl trifluoromethanesulfonate (TMSOTf) is 1:2-5; (3) Compound 7 was prepared using compound 6 and 2-[2-(2-azidoethoxy)ethoxy]ethanol as raw materials, camphorsulfonic acid (CSA) as a catalyst, and DMF as a solvent; the molar ratio of compound 6, 2-[2-(2-azidoethoxy)ethoxy]ethanol, and camphorsulfonic acid was 1:3-5:3-5; (4) Using compound 7 as raw material, sodium methoxide as deacetylation agent, and methanol as solvent, the target Fusobacterium nucleatum group GalNAc-N3 was prepared.

3. The method for preparing a photothermal nanomaterial targeting in situ self-assembly of Fusobacterium nucleatum according to claim 1, characterized in that: The preparation method of the in situ self-assembled photothermal nanomaterial BDP-GalNAc targeting Fusobacterium nucleatum includes the following steps: using BDP compound and GalNAc-N3 as raw materials, CuBr and PMDETA as catalysts, and DMF as solvent to synthesize and prepare the final product BDP-GalNAc; the molar ratio of BDP, GalNAc-N3, CuBr, and PMDETA is 1:2-5:0.2-1:0.2-1.

4. A method for preparing nanogel assembly BDP-GalNAc NGs, characterized in that: BDP-GalNAc and zwitterionic hydrogel PSBMA were dissolved in acetonitrile solution and stirred at room temperature overnight. The acetonitrile solution was slowly added dropwise to water under ultrasonic conditions, and the acetonitrile was removed under reduced pressure to obtain BDP-GalNAc NGs.

5. The in-situ self-assembled photothermal nanomaterial targeted to Fusobacterium nucleatum obtained by the preparation method according to claim 2, characterized in that: The zwitterionic hydrogel PSBMA is a freshly reacted acetonitrile solution, and BDP-GalNAc is a 1-2 mg / mL acetonitrile solution.

6. The in-situ self-assembled photothermal nanomaterial targeted to Fusobacterium nucleatum obtained by the preparation method according to claim 2 has the function that the nanomaterial can stably exist in PBS solution for 5 days and can depolymerize and release BDP-GalNAc in response to GSH.

7. The in situ self-assembly photothermal nanomaterial targeted to Fusobacterium nucleatum obtained by the preparation method according to claim 2 has the function that after the nanomaterial is depolymerized, BDP-GalNAc can target Fusobacterium nucleatum to self-assemble in situ to form aggregates, and exhibit an enhanced photothermal effect, with a photothermal conversion efficiency of 54.3%. In a 96-well plate, 10 μM BDP-GalNAcNGs was mixed with 1 x 10 8 Fusobacterium nucleatum was incubated for 4 h, and the excess material was washed off at 0.8 W / cm 2 After 808nm laser irradiation for 10 minutes, the culture was continued for 12 hours. Compared with the control group, the survival inhibition rate of bacteria in the group treated with BDP-GalNAc NGs reached 80%.

8. The in-situ self-assembled photothermal nanomaterial targeted to Fusobacterium nucleatum obtained by the preparation method according to claim 2 is used to generate 5 x 10 3 CT26 cells were cultured with 5 x 10 5 After incubation with Fusobacterium nucleatum for 4 h, 10 μM nanomaterials were added for 3 h and then the excess materials were washed away. 2 , 808nm laser irradiation for 10 minutes can produce more than 90% killing effect on cells.

9. The in situ self-assembled photothermal nanomaterials targeted to Fusobacterium nucleatum obtained by the preparation method according to claim 2, wherein the nanomaterials can be used for in vivo treatment. 100 μL of 10 μM BDP-GalNAc NGs was injected into the tail vein of mice. After 8 hours of systemic circulation, the materials were enriched in the tumor site. The nanomaterials were treated with 0.8 W / cm 2 , 808nm laser was used to irradiate the tumor site of mice for 10 minutes, and the photothermal effect had an inhibition rate of 69% on the tumor.

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