A sodium hypophosphite-modified phosphorus-containing dendrimer with anti-inflammatory activity and a preparation method and application thereof

Abstract

The application relates to a sodium hypophosphite salt modified phosphorus-containing dendrimer with anti-inflammatory activity and a preparation method and application thereof, and a structural formula of the phosphorus-containing dendrimer is as follows: The phosphorus-containing dendrimer prepared by the application can realize intracellular delivery of various proteins, has the advantages of good biocompatibility and high cytoplasm delivery efficiency, and has potential application value in the field of developing protein drugs for intracellular disease targets.

Description

Technical Field

[0001] This invention belongs to the field of functional nanocarriers, and specifically relates to a phosphorus-containing dendritic macromolecule modified with sodium phosphite with anti-inflammatory activity, its preparation method, and its application. Background Technology

[0002] Proteins play crucial roles in biological processes such as signal transduction, cell metabolism, cell proliferation, and apoptosis. Currently, many protein drugs have been developed and widely used to treat diseases such as cancer, immunodeficiency, and bacterial infections, including insulin, monoclonal antibodies, cytokines, transcription factors, and functional proteases. Compared to gene therapy and chemotherapy drugs, protein drugs have high specificity, fewer side effects, and can avoid off-target effects and carcinogenic risks caused by permanent gene mutations and continuous expression. This makes proteins one of the most promising drug candidates in drug development and disease treatment. It has been reported that four of the top ten best-selling drugs globally in 2021 were monoclonal antibodies. Among them, Humira (adalimumab), a tumor necrosis factor blocker used to treat diseases such as rheumatoid arthritis and psoriasis, consistently ranked first in sales from 2017 to 2020, and its sales reached a staggering $20.7 billion in 2021. Keytruda (pembrolizumab) is estimated to become the best-selling drug in 2023, with sales reaching $21.6 billion (Nat. Rev. Drug Discovery, 2022, 21, 251), indicating a huge market demand and sales potential for protein drugs. However, most currently developed protein drugs target cell surface receptors or extracellular targets. This is because proteins are characterized by large molecular weight, hydrophilicity, poor membrane permeability, and susceptibility to enzymatic degradation, making it difficult for them to cross numerous biological barriers and act on intracellular disease targets. Therefore, developing simple and efficient intracellular protein delivery technologies to target the numerous intracellular disease targets is an important way to expand the use of protein drugs in disease treatment.

[0003] Among commonly used intracellular protein delivery carriers such as liposomes, exosomes, inorganic nanoparticles, nanogels, and polymers, dendritic macromolecules are a class of nanomaterials with a size of less than 10 nm. They possess characteristics such as good water solubility, high stability, and functionalizability, thus showing great promise for applications in nanomedicine and protein delivery. Existing literature reports that polyamidoamine (PAMAM) dendritic macromolecules modified with phenylboronic acid, guanidinyl groups, fluorinated chains, and natural polyphenols can effectively complex with proteins through physical interactions, serving as universal templates for intracellular delivery of different proteins (Sci. Adv. 2019, 5, eaaw8922; Nano Lett. 2017, 17, 1678-1684; Biomaterials 2018, 182, 167–175; CCSChem. 2022, 1-11). However, this protein delivery technology based on PAMAM dendrimers relies on the chemical modification of functional molecules, and the biosafety issues arising from the high surface potential of PAMAM dendrimers require further investigation. Furthermore, the use of large amounts of carriers can lead to adverse carrier metabolism and elimination, potentially causing toxicity. Considering these obstacles, it is hypothesized that if both the protein and the carrier possess the expected biological activity during the design of the protein delivery carrier, these problems could be effectively solved, and a synergistic therapeutic effect between the protein and the carrier could be achieved.

[0004] Phosphorus-containing dendritic macromolecules, obtained by introducing phosphorus-containing units into the core, branches, and surface of dendritic macromolecules, possess characteristics such as uniform molecular weight, high branching, strong rigidity, and absence of molecular structural defects. Importantly, previous studies have shown that phosphorus-containing dendritic macromolecules or canopy macromolecules modified with sodium phosphite can reduce the expression of pro-inflammatory cytokines such as tumor cytokines-α (TNF-α), interleukin (IL)-6, and IL-1β by inhibiting the NF-κB signaling pathway, and have the potential to treat inflammatory diseases such as rheumatoid arthritis and acute lung injury (Sci. Transl. Med. 2011, 3(81), 81ra35; Theranostics 2022, 12(7), 3407-3419). In addition, phosphorus-containing dendritic macromolecules also have the property of stimulating the proliferation of natural killer (NK) cells, and can achieve tumor immunotherapy by increasing the infiltration of NK cells in tumor sites (Adv. Mater. 2023, 35, 2208277). In addition to their inherent biological activity, these phosphorus-containing dendritic macromolecules possess numerous hydrophobic phenyl groups, which endow them with a rigid structure. Simultaneously, they can overcome the hydrophobic environment of the lipid bilayer and promote the escape of proteins from their endosomes, preventing degradation by proteases and maintaining protein bioactivity (Nano Lett. 2017, 17, 1678-1684; Adv. Funct. Mater. 2020, 30, 1910575). Furthermore, the other functional groups surrounding the phosphorus-containing dendritic macromolecules exhibit physical interactions with proteins, including electrostatic interactions, cation-p-ions, and hydrogen bonds, potentially leading to the formation of stable nanocomposites.

[0005] A search of domestic and international literature and patents has not yet yielded any reports on the application of sodium phosphite-modified phosphorus-containing dendritic macromolecules as intracellular protein delivery carriers. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a phosphorus-containing dendritic macromolecule modified with sodium phosphite with anti-inflammatory activity, its preparation method and application, wherein the phosphorus-containing dendritic macromolecule serves as an intracellular protein delivery carrier, wherein the carrier itself can also show therapeutic effects and has good biocompatibility.

[0007] This invention provides a phosphorus-containing dendritic macromolecule modified with sodium phosphite and possessing anti-inflammatory activity. The structural formula of the phosphorus-containing dendritic macromolecule is as follows: It is designated AK-137.

[0008] This invention also provides a method for preparing a phosphorus-containing dendritic macromolecule modified with sodium phosphite and possessing anti-inflammatory activity, comprising the following steps:

[0009] (1) Compound I was dissolved in triethyl phosphite, heated and stirred to react, and dried under vacuum. The crude product was dissolved in dichloromethane and precipitated with pentane to obtain compound II.

[0010] The structural formula of compound I is: The structural formula of compound II is:

[0011] (2) Dissolve compound II obtained in step (1) in anhydrous dichloromethane, cool the resulting solution to 0°C, add trimethylbromosilane dropwise, stir the reaction at room temperature, concentrate under reduced pressure, mix the resulting product with anhydrous methanol, stir at room temperature, filter, purify, and dry under vacuum.

[0012] (3) Dissolve the product obtained in step (2) in an alkaline solution, stir the reaction at room temperature, and remove the solvent to obtain compound III, which is a phosphorus-containing dendritic macromolecule modified with sodium phosphite with anti-inflammatory activity.

[0013] In step (1), the ratio of compound I to triethyl phosphite is 0.3-0.5 mmol: 20-40 mL.

[0014] The temperature of the stirring reaction in step (1) is 150-180℃, and the reaction time is 72-120h.

[0015] The volume ratio of dichloromethane to pentane in step (1) is 1:10-15.

[0016] In step (2), the ratio of compound II to anhydrous dichloromethane is 0.1-0.2 mmol: 50-100 mL; the molar ratio of trimethylbromosilane to compound II is 50-70:1.

[0017] The stirring reaction at room temperature in step (2) lasts for 48 to 96 hours.

[0018] The amount of anhydrous methanol used in step (2) is 50-60 mL, and the resulting white slurry is stirred at room temperature for 2-3 hours.

[0019] The purification process conditions in step (2) are as follows: purification is carried out by silica gel column chromatography with methanol / diethyl ether as the mobile phase, and the vacuum drying time is 8 to 12 hours.

[0020] The alkaline solution in step (3) is an aqueous sodium hydroxide solution with a concentration of 0.1-0.2 mol / L; the molar ratio of the product to the alkaline solution in step (2) is 20-25:1.

[0021] The stirring time at room temperature in step (3) is 30 to 60 minutes.

[0022] The purification process conditions in step (3) are: freeze drying.

[0023] The present invention also provides the application of a phosphorus-containing dendritic macromolecule modified with sodium phosphite with anti-inflammatory activity in the preparation of protein-carrying nanocomposites.

[0024] This invention also provides a method for preparing protein-loaded nanocomposites, comprising the following steps:

[0025] (1) Prepare aqueous solutions of bovine serum albumin (BSA) and phosphorus-containing dendritic macromolecules (AK-137). According to different mass ratios of AK-137 to BSA (4:1, 8:1, 12:1, 16:1), AK-137 is added dropwise to the BSA solution and stirred at room temperature for 2-4 hours to obtain AK-137@BSA nanocomposites.

[0026] (2) AK-137 was added dropwise to ribonuclease A (RNase A), ovalbumin (OVA) and fibronectin (FN) solutions according to the optimized mass ratio in step (1), and stirred at room temperature for 2-4 h to obtain AK-137@RNase A, AK-137@OVA and AK-137@FN nanocomposites respectively.

[0027] (3) The hydration kinetic diameter, surface potential and morphology of the four protein-carrying nanocomposites were characterized by a nanoparticle size analyzer and a transmission electron microscope.

[0028] The present invention also provides an application of a sodium phosphite-modified phosphorus-containing dendritic macromolecule for intracellular protein delivery.

[0029] (1) RAW 264.7 cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. The cytotoxicity of phosphorus-containing dendritic macromolecules was evaluated by the CCK-8 assay.

[0030] (2) RAW 264.7 cells, HeLa cells, DC and MH-S cells were seeded in 12-well plates and cultured at 37℃ and 5% CO2 for 24 h. Then, AK-137@BSA, AK-137@RNase A, AK-137@OVA and AK-137@FN nanocomplexes were added and co-incubated with the cells for 4 h. The cells were then collected, washed three times with PBS, and the phagocytosis of the four different nanocomplexes by the cells was detected by flow cytometry.

[0031] (3) RAW 264.7 cells were seeded in 12-well plates and cultured at 37°C and 5% CO2 for 24 h. Different endocytosis inhibitors were added and co-incubated with the cells for 2 h. Then, AK-137@BSA was added and co-incubated with the cells for 3 h. The cells were collected and washed three times with PBS. The changes in the phagocytosis of AK-137@BSA by cells pretreated with different endocytosis inhibitors were detected by flow cytometry.

[0032] (4) DCs were seeded in 12-well plates and cultured at 37°C and 5% CO2 for 24 h. Then, AK-137, OVA, and AK-137@OVA nanocomplex were added and co-incubated with the cells for 24 h. The cells were collected, stained with CD86 and CD80 fluorescent antibodies, washed, and the effect of AK-137@OVA nanocomplex on DC cell maturation was detected by flow cytometry.

[0033] (5) HeLa cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Then, different concentrations of RNase A and AK-137@RNase A nanocomplex were added and incubated for 24 h. The old culture medium was discarded, washed three times with PBS, and replaced with fresh serum-free culture medium containing 10% CCK-8 solution for 3 h. Cell viability was then measured by a multi-functional microplate reader.

[0034] (6) MH-S cells were seeded in 12-well plates and cultured at 37°C and 5% CO2 for 24 h. After stimulation with LPS for 24 h, the medium was replaced with medium containing AK-137@FN nanocomplex and co-incubated with the cells for 24 h. Cells were then collected, stained with CD206 and CD86 fluorescent antibodies, washed with PBS, and the effect of AK-137@FN nanocomplex on macrophage polarization was detected by flow cytometry.

[0035] (7) MH-S cells were seeded in 12-well plates and cultured at 37°C and 5% CO2 for 24 h. After stimulating with LPS for 24 h, the culture medium was replaced with one containing AK-137@FN nanocomposite and co-incubated with the cells for 24 h. The cells were collected and co-incubated with the ROS fluorescent probe (DCFH-DA) for 20 min. After washing with PBS, the ROS level was detected by flow cytometry.

[0036] (8) MH-S cells were seeded in a laser confocal microscope culture dish and cultured at 37°C and 5% CO2 for 24 h. LPS was added for stimulation for 24 h. The culture medium was replaced with one containing AK-137@FN nanocomposite and co-incubated with the cells for 24 h. The cells were washed with PBS and co-incubated with the ROS fluorescent probe (DCFH-DA) for 20 min. The cells were washed with PBS, fixed with glutaraldehyde, stained with DAPI, and ROS fluorescence was observed by laser confocal microscope.

[0037] (9) MH-S cells were seeded in 12-well plates and cultured at 37℃ and 5% CO2 for 24 h. After stimulating with LPS for 24 h, the culture medium was replaced with fresh medium containing AK-137@FN nanocomposite and co-incubated with the cells for 24 h. The cells were collected and the effect of AK-137@FN on the mRNA expression of inflammatory factors (TNF-α, IL-1β, IL-6) in MH-S cells was detected by real-time quantitative PCR (RT-PCR).

[0038] (10) PBS, AK-137, FN, AK-137@FN, and AK-137@BSA were administered to the lungs of mice with acute lung injury via bronchial nebulization, respectively. Normal mice were treated with PBS. The treatment period was 24 hours. After treatment, bronchoalveolar lavage fluid from each experimental group was collected and the expression of inflammatory factors was detected by ELISA. Lung tissue from each experimental group was ground to obtain single-cell suspensions, and the infiltration of centrifugal cells in the lung tissue was analyzed by flow cytometry. Additionally, lung tissue from each experimental group was collected, cleaned, and soaked in 4% paraformaldehyde. After tissue sectioning, the lung tissue structure was analyzed by hematoxylin / eosin (H&E) staining.

[0039] (11) Before constructing the acute gout model, PBS, AK-137, FN, AK-137@FN, and AK-137@BSA were injected into the ankle joint cavity of mice for pretreatment. Subsequently, monosodium urate (MSU) crystals were injected into the ankle joint cavity to construct the acute gout model. Normal mice were treated with PBS for 24 hours. After treatment, the paw status was recorded by camera, and the degree of ankle swelling of each experimental mouse was recorded by caliper. In addition, ankle joint tissues of mice in each experimental group were taken, cleaned, soaked in 4% paraformaldehyde, and the joint tissue structure was analyzed by hematoxylin / eosin (H&E) staining after tissue sectioning.

[0040] This invention uses proton, carbon, and phosphorus nuclear magnetic resonance spectra to characterize the prepared phosphorus-containing dendritic macromolecules. The physical and chemical properties of the prepared phosphorus-containing dendritic macromolecule / protein nanocomposites are characterized using zeta potential and dynamic light scattering analysis (DLS), fluorescence spectrophotometer, and transmission electron microscope (TEM). The cytotoxicity of phosphorus-containing dendritic macromolecules was evaluated using CCK-8 assay; the effects of phosphorus-containing dendritic macromolecules on the intracellular delivery of four different proteins and the endocytic pathway were detected using flow cytometry; the effect of AK-137@OVA on DC maturation was evaluated using flow cytometry; the effect of AK-137@RNase A on HeLa cell viability was evaluated using CCK-8 assay; the effect of AK-137@FN on intracellular ROS levels was detected using a reactive oxygen species (ROS) assay kit; the effect of AK-137@FN on MH-S cell polarization was evaluated using flow cytometry; the effect of AK-137@FN on the expression of intracellular inflammatory factors TNF-α, IL-1β, and IL-6 in MH-S cells was detected using RT-PCR; finally, acute lung injury and acute gout models were established to further evaluate the anti-inflammatory properties and therapeutic effects of AK-137@FN.

[0041] The sodium phosphite-modified phosphorus-containing dendritic macromolecules provided by this invention can effectively deliver a variety of proteins into cells, greatly improving the bioavailability and biological function of proteins, enabling a variety of proteins to act on intracellular disease targets, thus showing great application potential in the fields of anti-tumor, anti-inflammatory and immunotherapy.

[0042] Beneficial effects

[0043] (1) The process of this invention is simple, the cost is low, and it is easy to operate and separate, and it has good development prospects.

[0044] (2) The nanoplatform prepared by this invention has good stability, water solubility and biocompatibility, providing a new idea for constructing safe and efficient nanomedicine carriers.

[0045] (3) The sodium phosphite-modified phosphorus dendritic macromolecules prepared in this invention can effectively combine with a variety of proteins through electrostatic forces, hydrogen bonds, cation-Π, and hydrophobic interactions, overcome the cell membrane barrier, and promote the intracellular delivery of different proteins (BSA, OVA, RNase A, FN). It can be used as a universal template for intracellular protein delivery for anti-tumor, anti-inflammatory and immunotherapy.

[0046] (4) The sodium phosphite-modified phosphorus-containing dendritic macromolecules prepared in this invention possess certain anti-inflammatory activity on the one hand, and enhance the biological function of proteins by promoting intracellular protein delivery and enabling proteins to act on intracellular targets. Among them, the phosphorus-containing dendritic macromolecule protein delivery carrier, when combined with fibronectin, exhibits significant anti-inflammatory therapeutic effects at both the cellular and animal levels, and has potential clinical application value. Attached Figure Description

[0047] Figure 1 A schematic diagram (a) of the synthesis of the phosphorus-containing dendritic macromolecule prepared in this invention and a schematic diagram (b) of the preparation and application of the AK-137@Protein nanocomposite;

[0048] Figure 2 Hydration dynamics (a) and surface potential (b) of AK-137 and BSA co-incubated in different proportions;

[0049] Figure 3 The fluorescence emission spectra of AK-137 and Cy5.5-BSA co-incubated in different proportions are shown.

[0050] Figure 4 TEM image (a) of the AK-137@BSA nanocomposite prepared for this invention and curve (b) showing the hydrodynamic diameter of the nanocomposite in water, PBS and DMEM culture medium as a function of time.

[0051] Figure 5 Cell viability diagram of RAW 264.7 cells after co-incubation of the phosphorus-containing dendritic macromolecule AK-137 prepared for this invention with RAW 264.7 cells for 24 hours;

[0052] Figure 6 Flow cytometry (a) and quantitative fluorescence intensity (b) plots of the BSA and AK-137@BSA nanocomposites prepared for this invention after co-incubation with RAW 264.7 cells for 4 hours.

[0053] Figure 7 Flow cytometry (a) and average fluorescence intensity quantification (b) of RAW264.7 cells pretreated with different endocytosis inhibitors after 3 hours of co-incubation of the AK-137@BSA nanocomposite prepared for this invention.

[0054] Figure 8 Flow cytometry (a) and quantitative fluorescence intensity (b) of the OVA and AK-137@OVA nanocomposites prepared for this invention after co-incubation with dendritic cells for 4 hours.

[0055] Figure 9Flow cytometry analysis (a) and quantitative graph (b) of the expression levels of CD80 and CD86 in dendritic cells after co-incubation of AK-137, OVA, AK-137@OVA and positive control LPS prepared for this invention with dendritic cells for 24 h.

[0056] Figure 10 Flow cytometry and quantitative fluorescence intensity diagrams of RNase A and AK-137@RNase A prepared for this invention after co-incubation with HeLa cells for 4 h;

[0057] Figure 11 Cell viability graphs of HeLa cells after co-incubation of RNase A and AK-137@RNase A prepared for this invention with HeLa cells for 24 hours;

[0058] Figure 12 Flow cytometry (a) and quantitative fluorescence intensity (b) of FN and AK-137@FN prepared for this invention after co-incubation with MH-S cells for 4 h;

[0059] Figure 13 Flow cytometry analysis of the expression levels of CD86 and CD206 in LPS-activated MH-S cells after co-incubation of AK-137, FN, AK-137@FN and AK-137@BSA prepared for this invention with LPS activated MH-S cells for 24 h (a), percentage content of M1 macrophages (b) and M2 macrophages (c).

[0060] Figure 14 The flow cytometry analysis (a), relative fluorescence intensity quantification (b), and laser confocal microscopy image (c) of the reactive oxygen species expression levels in LPS-activated MH-S cells after co-incubation of AK-137, FN, AK-137@FN, and AK-137@BSA prepared for this invention with LPS-activated MH-S cells for 24 h (scale bar in the figures is 10 μm).

[0061] Figure 15 The relative mRNA expression levels of TNF-α(a), IL-6(b) and IL-1β(c) in LPS-activated MH-S cells were determined after co-incubating AK-137, FN, AK-137@FN and AK-137@BSA prepared for this invention with LPS-activated MH-S cells for 24 h.

[0062] Figure 16 The figure shows the ELISA test results of pro-inflammatory cytokines (a) TNF-α, (b) IL-6, (c) IL-1β and (d) IL-10 in the bronchoalveolar lavage fluid of mice in each experimental group in Example 16.

[0063] Figure 17The flow cytometry analysis (a) and quantitative results (b) of the number of neutrophils in the lung tissue of mice in each experimental group in Example 17, and the expression level of myeloperoxidase in the bronchoalveolar lavage fluid of mice in each experimental group (c);

[0064] Figure 18 Histological section analysis of lung tissue from mice in each experimental group in Example 18. Black arrows indicate alveolar wall congestion, and gray arrows indicate inflammatory cells (scale bar in the figure is 50 μm);

[0065] Figure 19 The images show the paws (a), ankle swelling (b), and histological section analysis (c) of the mice in each experimental group after treatment in Example 19 (the scale bars in the figures are 200 μm and 50 μm, respectively). Detailed Implementation

[0066] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0067] Unless otherwise specified, all chemical reagents were commercially available and ready for use without further purification. Bovine serum albumin and ovalbumin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ribonuclease A was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Fibronectin was from Shanghai Fibronectin Biotechnology Co., Ltd. (Shanghai, China). RAW264.7, HeLa, DC, and MH-S cells were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin antibiotics, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou, China). Cell Counting Kit-8 (CCK-8) and ROS detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Monoclonal antibodies including Anti-CD80-PE, Anti-CD86-FITC, Anti-CD86-PE, Anti-CD206-FITC, Anti-Ly6G-FITC, and Anti-CD11b-PE were purchased from Thermo Fisher Scientific (Waltham, MA). BALB / c mice and CD-1 mice were purchased from Shanghai Slack Laboratory Animal Center (Shanghai, China). All water with a resistivity higher than 18.2 MΩ·cm used in the experiments was purified using a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0068] Example 1

[0069] (1) Weigh 0.42 mmol of compound I (synthesized according to reference: J. Org. Chem. 2022, 87, 3433-3441) and dissolve it in 30 mL of triethyl phosphite. The mixture was stirred at 165 °C for 96 h under an argon atmosphere. After the reaction, volatiles were removed by vacuum. The obtained crude product was dissolved in dichloromethane and added dropwise to pentane, wherein the volume ratio of dichloromethane to pentane was 1:10. The precipitate was collected. This process was repeated three times to obtain compound II.

[0070] (2) Weigh 0.19 mmol of compound II obtained in the above steps and dissolve it in 80 mL of anhydrous dichloromethane. Cool the resulting solution to 0 °C and add trimethylbromosilane dropwise (the molar ratio of trimethylbromosilane to compound II is 60:1). Stir the resulting reaction mixture at room temperature for 72 h. After the reaction, remove all volatiles under vacuum. Mix the resulting white foamy crude product with 60 mL of anhydrous methanol, stir at room temperature for 2 h, filter, and purify by silica gel column chromatography with methanol / diethyl ether as the mobile phase. Dry under high vacuum for 8–12 h, and seal and store the resulting product at -20 °C.

[0071] (3) The product obtained after drying in the above steps was dissolved in a 0.1-0.2 mol / L sodium hydroxide aqueous solution, stirred at room temperature for 60 minutes, and then the solvent was removed to obtain compound III (AK-137).

[0072] This invention uses a nuclear magnetic resonance spectrometer to perform proton NMR spectroscopy (NMR). 1 H NMR), phosphorus spectrum ( 31 P NMR), carbon spectrum ( 13 The results of the C NMR test are as follows:

[0073] Compound II:

[0074]

[0075] Compound II, white non-crystalline solid, C 240 H 366 N3O 90 P 27 M r = 5569.79 g / mol. Yield 85%. 1 HNMR(400MHz,CDCl3)δ(ppm):7.36(6H,s, Ar C H ),7.16(12H,s, Ar C H ),6.86(12H,s,Ar C H ),6.71(24H,s, Ar C H ), 4.84 (24H,s,OC) H2 ), 4.06–3.88 (96H,m,POC) H 2CH3), 3.03(48H,d, 2 J HP =21.7Hz,C H 2P), 1.21(144H,t, 3 J HH =7.1Hz, POCH2C H 3). 13 C{ 1 H}NMR(101MHz,CDCl3)δ(ppm):158.6( Ar C -OCH2),150.9(PO- C Ar ),139.1( Ar C -CH2),133.3( Ar C -CH2),124.0( Ar CH), 123.4( Ar CH), 118.9 ( Ar CH), 114.8( Ar CH), 68.9(O C H2-C Ar ),62.1(d, 2 J CP =6.7Hz, PO C H2CH3), 33.5(d, 1 J CP =137.5Hz, Ar C- C H2P), 16.4 (d, 3 J CP =6.0Hz, PO C H2CH3). 31 P{ 1 H}NMR (162MHz, CDCl3) δ (ppm): 26.1 (P=O), 7.32 (P3N3).

[0076] Compound III:

[0077]

[0078] Compound III, a pale yellow solid, C 144 H150 N3Na 24 O 90 P 27 ,M r = 4750.76 g / mol. Yield 87%. 1 1H NMR (400 MHz, D2O) δ (ppm): 7.17 (6H, s, Ar C H ), 7.02 (12H, s, Ar C H ), 6.80 (12H, s, Ar C H ), 6.74 (24H, s, Ar C H ), 4.75 (24H, s, OC H2 ), 2.94 (48H, d, 2 J HP = 21.5 Hz, C H 2P). 13 13C{ 1 1H} NMR (101 MHz, D2O) δ (ppm): 157.7( Ar C -OCH2), 149.9 (P-O- C Ar ), 139.1 ( Ar C -CH2), 136.3 ( Ar C -CH2), 123.8 ( Ar CH), 123.0 ( Ar CH), 119.1 ( Ar CH), 113.8 ( Ar CH), 68.6 (O C H2-C Ar ), 33.6 (d, 1 J CP = 127.8 Hz, Ar C- C H2P). 31 31P{ 1 1H} NMR (162 MHz, D2O) δ (ppm): 24.5 (P=O), 8.72 (P3N3).

[0079] Example 2

[0080] BSA was used as a model protein to verify the binding ability of AK-137 to proteins. BSA and AK-137 prepared in Example 1 were dissolved separately in aqueous solutions. AK-137 was added dropwise to the BSA solution at different mass ratios (AK-137:BSA = 4:1, 8:1, 12:1, 16:1), and stirred at room temperature for 2–4 hours until the solution turned milky white. The mixture was then transferred to a centrifuge tube, centrifuged at 12000 rpm for 15 minutes to collect the precipitate, resuspended in an appropriate amount of ultrapure water, and centrifuged again. Finally, the precipitate was redissolved in 1 mL of ultrapure water to obtain the nanocomposite AK-137@BSA, which was used to determine the surface potential, hydrodynamic diameter, and polymer dispersibility index (PDI) of AK-137@BSA at different mass ratios. Figure 2 As shown in ab, the hydration kinetic diameter of AK-137 is 103.2 nm, while the hydration size of BSA alone is 4.8 nm. When AK-137 and BSA are mixed, the hydration kinetic size of the composite increases compared to BSA, and the surface potential decreases further, indicating that BSA and AK-137 form a nanocomposite. In particular, at a mass ratio of 12:1, the AK-137@BSA nanocomposite has a Zeta potential of -36.1 mV and exhibits a smaller hydration kinetic size (209.4 nm) and a PDI (0.15).

[0081] Example 3

[0082] BSA was used as a model protein to further verify the binding behavior of AK-137 to the protein. The fluorescent dye Cy5.5-NHS was added dropwise to an aqueous BSA solution at a molar ratio of 3:1. After stirring at room temperature in the dark for 24 hours, the solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed with ultrapure water for three days. The resulting lyophilized solution yielded Cy5.5-labeled BSA (Cy5.5-BSA). Subsequently, AK-137 was added dropwise to the Cy5.5-BSA solution at different mass ratios (final Cy5.5-BSA concentration 0.1 mg / mL). After mixing, the changes in the fluorescence emission spectrum of the samples were immediately detected using a fluorescence spectrometer (excitation wavelength 678 nm). Figure 3As shown, the fluorescence intensity gradually decreased with increasing AK-137 to Cy5.5-BSA mass ratio. This quenching effect may be due to changes in the microenvironment surrounding the fluorescent molecules when AK-137 binds to the protein. The fluorescence intensity reached its minimum when the AK-137 to BSA mass ratio was 12, further verifying that within this experimental range, AK-137 exhibits the best binding ability to the protein at a mass ratio of 12:1. Therefore, a 12:1 AK-137 to protein mass ratio was selected, and AK-137@OVA, AK-137@RNase A, and AK-137@FN nanocomposites were prepared according to the method in Example 2 for subsequent experiments.

[0083] Example 4

[0084] Following the method in Example 2, the prepared AK-137@BSA nanocomposite was formulated into a protein concentration of 0.1 mg / mL, dropped onto the surface of a copper mesh with a carbon film, and allowed to dry at room temperature to obtain the sample. The sample was then examined using a JEM-2010F transmission electron microscope. TEM images of AK-137@BSA are shown below. Figure 4 As shown in Figure a, its morphology is a uniform sphere with a size of approximately 110 nm. When 0.1 mg of AK-137@BSA was dispersed in 1 mL of H2O, PBS, and DMEM medium, the hydrodynamic size of AK-137@BSA in each solution did not change significantly within one week (e.g., ...). Figure 4 As shown in b), the prepared AK-137@BSA has good colloidal stability, further proving that AK-137 can form a stable and uniform nanocomposite with proteins.

[0085] Example 5

[0086] The effect of the phosphorus-containing dendritic macromolecule AK-137 on the viability of RAW 264.7 cells was evaluated using the CCK-8 assay. RAW 264.7 cells in logarithmic growth phase were collected and analyzed at a concentration of 1×10⁻⁶ cells / cell. 4 Cells were seeded at a density of 100 cells per well in a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 24 hours. Fresh medium was then added, and AK-137 (concentrations of 0, 30, 60, 120, 240, and 480 μg / mL) was added and incubated with the cells for another 24 hours. The original medium was discarded, and the cells were washed three times with PBS. Fresh medium containing 10% (v / v) CCK-8 was added, and the cells were incubated for another 3 hours. The absorbance of each well was measured at 450 nm using a multi-mode microplate reader. Cells treated with PBS served as a blank control, and cell viability was recorded as 100%. Results are as follows: Figure 5As shown, within the experimental concentration range, the viability of RAW 264.7 cells treated with AK-137 was above 90%, indicating that AK-137 has good cell compatibility and can be used as a safe intracellular protein delivery carrier.

[0087] Example 6

[0088] RAW 264.7 cells were used as a cell model to validate intracellular BSA delivery. Cells in the logarithmic growth phase were collected and delivered at a rate of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells per well in 12-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was then replaced with fresh serum-free DMEM, and BSA and AK-137@BSA were added and co-incubated with the cells for 4 hours (using Cy5.5-labeled BSA at a concentration of 10 μg / mL). The control group was treated with PBS. The culture medium was discarded, and the cells were trypsinized, centrifuged, and washed three times with PBS. Intracellular fluorescence intensity was then detected by flow cytometry. Figure 6 As shown in a and 6b, the fluorescence intensity of AK-137@BSA was significantly higher than that of the BSA and PBS groups alone, verifying that AK-137 has a strong intracellular protein delivery efficiency.

[0089] Example 7

[0090] The pathway of the nanocomplex into cells was verified using the model protein BSA. RAW264.7 cells in logarithmic growth phase were collected and analyzed at a ratio of 1×10⁻⁶ cells. 5 Cells were seeded at a density of 100 cells per well in 12-well plates and cultured at 37°C for 24 h in 5% CO2. The medium was then replaced with fresh medium, and different endocytosis inhibitors (methyl-β-cyclodextrin (10 mM), chlorpromazine (20 μM), amiloride (100 μM), or genistein (700 μM)) were added and co-incubated with the cells for 2 h. The normal control group was treated with PBS. The old medium was discarded and replaced with fresh serum-free DMEM medium containing AK-137@BSA (Cy5.5-labeled BSA at a concentration of 10 μg / mL). The normal control group and the inhibitor-pretreated group were cultured at 37°C, while the negative control group was cultured at 4°C. After co-incubating the nanocomposite with the inhibitor-pretreated cells for 3 h, the cells were collected and resuspended in 300 μL of PBS, transferred to flow cytometry tubes, and the fluorescence intensity changes of each group were detected by flow cytometry. The results are as follows: Figure 7As shown in ab, the intracellular fluorescence intensity of the methyl-β-cyclodextrin and genistein pretreatment groups was slightly lower than that of PBS, indicating that a very small number of nanocomplexes may enter the cells via lipid raft-mediated endocytosis. In contrast, the phagocytic capacity of AK-137@BSA in cells pretreated with chlorpromazine was greatly reduced, which suggests that the nanocomplexes mainly rely on the classical endocytosis pathway mediated by clathrin to enter the cells.

[0091] Example 8

[0092] Ovalbumin, as a model antigen, can induce dendritic cell maturation through the Toll-like receptor 4 (TLR4)-mediated signaling pathway, thereby achieving efficient antigen cross-presentation. First, dendritic cells (DCs) were used as a cell model to verify the ability of AK-137 to promote intracellular OVA delivery. Cells in logarithmic growth phase were collected and processed at a ratio of 1 × 10⁻⁶ cells / cell. 5 Cells were seeded at a density of 100 cells per well in 12-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was then replaced with fresh serum-free DMEM, and OVA and AK-137@OVA were added and co-incubated with the cells for 4 hours (FITC-labeled OVA at a concentration of 10 μg / mL). The control group was treated with PBS. The culture medium was discarded, and the cells were trypsinized, centrifuged, and washed three times with PBS. Intracellular fluorescence intensity was then detected by flow cytometry. Figure 8 a and Figure 8 As shown in b, the fluorescence intensity of the AK-137@OVA group was significantly higher than that of the OVA group, verifying that the AK-137@OVA nanocomposite can also promote the delivery of OVA in dendritic cells.

[0093] Example 9

[0094] The efficient cytoplasmic delivery of OVA promotes dendritic cell maturation, which is crucial for initiating subsequent anti-tumor immune responses. To examine the effect of intracellular delivery of AK-137@OVA on dendritic cell maturation, dendritic cells in logarithmic growth phase were collected and processed at a rate of 2 × 10⁻⁶ cells / cells. 5Cells were seeded at a density of 1000 cells per well in 6-well cell culture plates and cultured at 37°C with 5% CO2 for 24 h. The medium was then replaced with serum-free DMEM containing OVA or AK-137@OVA (OVA concentration 10 μg / mL) and co-incubated for 4 h. The medium was then replaced with serum-containing DMEM and cultured at 37°C with 5% CO2 for 20 h. The positive control group was treated with lipopolysaccharide (LPS) (LPS concentration 2 μg / mL). The medium was then removed, and the cells were washed three times with PBS. 1 mL of trypsin was added to each well to digest the cells, and digestion was terminated. The cells were collected by centrifugation. 200 μL of PBS was added to each well to resuspend the cell pellet, and then Anti-CD80-PE and Anti-CD86-FITC antibodies were added and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the DCs were resuspended in 300 μL of PBS and transferred to flow cytometry tubes. The expression of CD80 and CD86 in the DCs was detected by flow cytometry. Figure 9 a and Figure 9 As shown in b, OVA, as a model antigen, can promote dendritic cell maturation, while OVA alone, due to its poor intracellular delivery efficiency, cannot significantly stimulate dendritic cell maturation. The combination of AK-137 and OVA forms a stable nanocomplex that can significantly stimulate the expression of CD80 and CD86 in dendritic cells (DCs), thereby promoting dendritic cell maturation. This is attributed to the enhanced intracellular delivery of OVA induced by the AK-137@OVA complex, and can be used for subsequent tumor immunotherapy.

[0095] Example 10

[0096] RNase A is a cytotoxic protein that induces apoptosis by degrading intracellular RNA; however, its low intracellular delivery efficiency limits its application in antitumor therapy. We used HeLa cells as a model to verify the effect of AK-137 on promoting intracellular delivery of RNase A. Cells in logarithmic growth phase were collected and processed at a rate of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells per well in 12-well plates and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with fresh serum-free DMEM containing RNase A or AK-137@RNase A and incubated for 4 h (using FITC-labeled RNase A at a concentration of 10 μg / mL). The control group was treated with PBS. The culture medium was discarded, and the cells were washed three times with PBS. 1 mL of trypsin was added to each well to digest the cells, and digestion was terminated. The cells were collected by centrifugation. The cell pellet was resuspended in 300 μL of PBS and transferred to flow cytometry tubes. The fluorescence intensity of each group was detected by flow cytometry. The results are as follows: Figure 10As shown, the RNase A group did not show significant fluorescence intensity because RNase A alone is difficult to enter HeLa cells, while the fluorescence intensity of the AK-137@RNase A group was significantly higher than that of the RNase A group, indicating that the nanocomposite can also promote the intracellular delivery of the cytotoxic protein RNase A.

[0097] Example 11

[0098] To verify the effect of AK-137@RNase A on HeLa cell viability, cells in the logarithmic growth phase were collected and processed at a ratio of 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with fresh DMEM containing different concentrations of RNase A or AK-137@RNase A (RNase A concentrations of 0, 2, 4, 8, 16, and 32 μg / mL) and incubated for another 24 h. The control group was treated with PBS. The medium was then discarded, and the cells were washed three times with PBS. 100 μL of fresh serum-free DMEM containing 10% (v / v) CCK-8 solution was added to each well and incubated for 3 h. The absorbance of each well was measured at 450 nm using a multi-mode microplate reader. Cells treated with PBS served as a blank control, and cell viability was recorded as 100%. Within the experimental concentration range, RNase A alone is difficult to enter cells. Therefore, at an RNase A concentration of 32 μg / mL, the viability of HeLa cells remained above 80%. However, at the same concentration, the viability of HeLa cells in the AK-137@RNase A group decreased to 32%. This demonstrates that the phosphorus-containing dendritic macromolecule AK-137 can effectively deliver RNase A into cells and maintain its enzymatic activity, successfully inducing apoptosis in HeLa cells, showing great potential in anti-tumor applications. Figure 11 ).

[0099] Example 12

[0100] Fibronectin is a major non-collagenous glycoprotein in the extracellular matrix, playing a well-known key role in mediating cell surface interactions and participating in numerous biological processes such as cell differentiation, growth, and adhesion. Fibronectin can be used as a therapeutic protein to treat inflammatory diseases by inducing macrophage polarization and reducing the expression of inflammatory factors. However, its low intracellular delivery efficiency hinders its biological activity. Therefore, AK-137 can be used to promote intracellular delivery of fibronectin and exert its biological function.

[0101] The effect of AK-137 on promoting intracellular FN delivery was verified using MH-S cells as a model. Cells in logarithmic growth phase were collected and processed at a ratio of 1×10⁻⁶ cells. 5Cells were seeded at a density of 100 cells per well in 12-well plates and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with fresh serum-free RPMI-1640 medium containing FN or AK-137@FN and incubated for 4 h (using Cy5.5-labeled FN at a concentration of 20 μg / mL). The control group was treated with PBS. The medium was discarded, and the cells were washed three times with PBS. 1 mL of trypsin was added to each well to digest the cells, and digestion was terminated. Cells were collected by centrifugation. The cell pellet was resuspended in 300 μL of PBS and transferred to flow cytometry tubes. The fluorescence intensity of each group was detected by flow cytometry. Results are as follows: Figure 12 As shown in ab, the fluorescence intensity of the AK-137@FN group was significantly higher than that of the free FN group, indicating that AK137 can also promote the delivery of FN in alveolar macrophages, and AK-137 can serve as a universal carrier for the intracellular delivery of different proteins.

[0102] Example 13

[0103] To verify the effect of the AK-137@FN nanocomposite on macrophage polarization, MH-S cells in logarithmic growth phase were collected and polarized at a ratio of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells per well in 12-well plates and cultured at 37°C with 5% CO2 for 24 h. The medium was then discarded and replaced with RPMI-1640 medium containing 2 μg / mL LPS for 24 h. PBS was added to the control group. Subsequently, the medium was replaced with fresh serum-free RPMI-1640 medium containing AK-137, FN, AK-137@FN, or AK-137@BSA (BSA or FN concentration 20 μg / mL) for 4 h. The old medium was discarded, and serum-containing RPMI-1640 medium was added for co-incubation for 20 h. The medium was then discarded, and the cells were washed twice with PBS. After trypsin digestion and cell collection, the cell pellet was resuspended in 200 μL PBS. Anti-CD206-FITC and Anti-CD86-PE antibodies were added, and the cells were co-incubated at 4°C in the dark for 30 min. After washing three times with PBS to remove unbound antibodies, the cell pellet was resuspended in 300 μL PBS and transferred to flow cytometry tubes. The effect of nanomaterials on macrophage polarization was evaluated by detecting changes in the expression levels of CD86 and CD206 in the cells. Figure 13As shown in Figure ac, compared with the LPS group, the AK-137 group showed decreased CD86 expression and increased CD206 expression. The number of M2 macrophages was higher in the AK-137 group than in the LPS group, while the number of M1 macrophages was lower. This indicates that AK-137 itself can inhibit the LPS-induced tendency of macrophages to polarize towards M1. Although FN alone is difficult to enter the cell, it still exhibits the ability to induce macrophage polarization towards M2, possibly because FN binds to macrophage surface receptors and participates in regulating macrophage behavior. Compared with the AK-137@BSA group, the AK-137@FN group showed the highest level of CD206 (8.36%) and the lowest level of CD86 (5.75%) expression, and its M2 / M1 ratio was 2.8 times that of the AK-137@BSA group. This indicates that the ability of FN to induce macrophage polarization towards M2 after being delivered into the cell by AK-137 is further enhanced, demonstrating the synergistic enhancement of biological function between the protein and the carrier.

[0104] Example 14

[0105] To verify the effect of the AK-137@FN nanocomposite on the scavenging of reactive oxygen species (ROS) in macrophages, MH-S cells in the logarithmic growth phase were collected and analyzed at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells per well in 12-well plates and cultured at 37°C with 5% CO2 for 24 h. The medium was then replaced with RPMI-1640 medium containing 2 μg / mL LPS and incubated for another 24 h. PBS was added to the control group. Subsequently, the medium was replaced with fresh serum-free RPMI-1640 medium containing AK-137, FN, AK-137@FN, or AK-137@BSA (BSA or FN concentration 20 μg / mL) and incubated for 4 h. The old medium was discarded, and serum-containing RPMI-1640 medium was added and the cells were co-incubated for 20 h. After trypsin digestion and cell collection, the cell pellet was resuspended in 1 mL of PBS, and the ROS fluorescent probe DCFH-DA was added and the cells were co-incubated at 37°C for 20 min. After washing three times with PBS to remove probes that did not bind to cells, the cell pellet was resuspended in 300 μL PBS and transferred to flow cytometry tubes. The effect of the nanomaterials on ROS clearance in macrophages was evaluated by flow cytometry. Results are as follows: Figure 14 As shown in ab, compared to the LPS group, AK-137 did not exhibit any ability to scavenge reactive oxygen species (ROS), while FN significantly scavenged ROS. Compared to the FN group, AK-137@FN showed a stronger ability to scavenge ROS. This indicates that AK-137, through efficient delivery of FN to cells, can scavenge ROS and alleviate excessive oxidative stress in macrophages.

[0106] The ability of AK-137@FN to scavenge intracellular ROS was further verified using laser confocal microscopy. MH-S cells in logarithmic growth phase were collected and cultured at 1×10⁻⁶ cells / cells. 5 MH-S cells were seeded in laser confocal microscopy dishes at a density of 10 cells per well and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with RPMI-1640 medium containing 2 μg / mL LPS and incubated for another 24 h. PBS was added to the control group. Next, each well was replaced with fresh serum-free RPMI-1640 medium containing AK-137, FN, AK-137@FN, or AK-137@BSA (BSA or FN concentration 20 μg / mL) and incubated for 4 h. The old medium was discarded, and the cells were incubated with fresh serum-containing medium for 20 h. Discard the culture medium, wash three times with PBS, add 1 mL of DCFH-DA and incubate with the cells at 37°C for 20 min, wash three times with PBS, add 1 mL of 2.5% glutaraldehyde and fix at room temperature for 15 min, wash three times with PBS, add 1 mL of LDAPI and incubate with the cells for 5 min, wash three times with PBS, add 200 μL of PBS and observe the changes in cell fluorescence intensity under a laser confocal microscope. Results are as follows: Figure 14 As shown in c, compared with the LPS group, AK-137 and FN group, the green fluorescence intensity of the AK-137@FN group was significantly reduced, indicating that the nanocomposite has good antioxidant properties.

[0107] Example 15

[0108] 1×10 per hole 5 MH-S cells were seeded in 12-well plates and cultured at 37°C with 5% CO2 for 24 h. The medium was then replaced with RPMI-1640 medium containing LPS (2 μg / mL) and co-incubated for 24 h. PBS was added to the control group. The medium was then discarded and replaced with fresh serum-free RPMI-1640 medium containing AK-137, FN, AK-137@FN, or AK-137@BSA (BSA and FN concentrations were 20 μg / mL) and incubated for 4 h. The old medium was discarded, the cells were washed twice with PBS, and then incubated with fresh serum-containing medium for 20 h. Cells were then digested with trypsin and collected. RT-PCR was used to detect changes in the expression levels of pro-inflammatory cytokine-encoding genes in MH-S cells after different material treatments. The specific experimental steps are as follows:

[0109] (1) Total RNA extraction: Collect cell samples and extract total RNA using RNAeasy. TMTotal RNA was extracted using an animal RNA extraction kit (Shanghai Beyotime). RNA was dissolved in DEPC-free water without RNase. (2) Reverse transcription: A PCR tube was filled with a solution containing 1 μg of RNA. 1 μL of gDNA EZeraser and 1 μL of gDNA EZeraser buffer (10×) were added to remove genomic contamination. The volume was brought up to 10 μL with DEPC water. The tube was incubated at 37°C for 2 min, then at 55°C for 5 min, and then quickly cooled on ice. 4 μL of cDNA first-strand synthesis premix (5×) and 6 μL of DEPC water were added to each PCR tube until the PCR reaction volume was 20 μL. The tube was then mixed. The tube was incubated at 42°C for 30 min, then at 80°C for 10 min to inactivate reverse transcriptase and terminate the reverse transcription reaction. (3) Quantitative PCR: Set up the PCR reaction system on ice, adding 10 μL of SYBR Green qPCR Mix, 2 μL of 5 μM primer mixture, 2 μL of template DNA, and 6 μL of DEPC water to each well. (4) PCR amplification: Perform multiple cycles of denaturation, annealing, and extension in a PCR instrument to amplify the target gene. The results are as follows: Figure 15 As shown in Figure ac, compared to the LPS treatment group, the AK-137 and FN treatment groups could inhibit the expression of genes encoding pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) to some extent. Compared with the FN and AK-137 treatment groups alone, the AK-137@FN group showed more significant inhibition of the expression levels of genes encoding TNF-α, IL-1β, and IL-6. This indicates that AK-137@FN can exert synergistically enhanced anti-inflammatory effects through intracellular protein delivery.

[0110] Example 16

[0111] All animal experiments were conducted strictly in accordance with the standards of the Laboratory Animal Ethics Committee of Donghua University. Six-week-old male BALB / c mice were purchased from the Shanghai Slack Laboratory Animal Center. An acute lung injury animal model was established by intratracheal nebulization of LPS (5 mg / kg) for 24 hours, while the control group received an equal volume of PBS via intratracheal nebulization. Mice were randomly divided into six groups (PBS control group, LPS treatment group, AK-137 treatment group, FN treatment group, AK-137@FN treatment group, and AK-137@BSA treatment group), with seven mice in each group. 100 μL of PBS, AK-137, FN, or AK-137@FN solution (FN or BSA concentration of 2 mg / kg) was administered to the lungs of each group of mice via nebulizer. Twenty-four hours after treatment, the lungs were perfused with PBS, and bronchoalveolar lavage fluid was extracted, centrifuged at 4°C (4000 rpm, 5 min), and the supernatant was transferred to a new centrifuge tube to obtain bronchoalveolar lavage fluid, which was stored at -80°C. Subsequently, ELISA was used to detect the expression levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) in bronchoalveolar lavage fluid. Figure 16 As shown in the ad, compared with the control group, the pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice treated with AK-137 and FN were significantly reduced. Compared with the AK137 and FN groups alone, the AK-137@FN treatment group showed lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines, indicating that the AK-137@FN group achieved good anti-inflammatory treatment in mice with acute lung injury by promoting intracellular delivery of FN and combining it with the inherent anti-inflammatory activity of AK-137 itself.

[0112] Example 17

[0113] Twenty-four hours after treatment, three mice were selected from each of the six experimental groups. Their lung tissue was minced, digested with trypsin, ground, and filtered through a 400-mesh filter to obtain a cell suspension. The obtained single-cell suspensions were labeled with Anti-CD11b-PE and anti-Ly6G-FITC antibodies, respectively, and the neutrophil infiltration in the lung tissue was quantitatively analyzed by flow cytometry. Additionally, bronchoalveolar lavage fluid was prepared according to the method in Example 16, and the expression level of myeloperoxidase in the bronchoalveolar lavage fluid was detected using ELISA. Figure 17Flow cytometry results from ab showed that there was extensive neutrophil infiltration in the lung tissue of mice with acute lung injury. After treatment with AK-137 or FN, the proportion of neutrophils in the lung tissue was significantly reduced, and the AK-137@FN treatment group showed the lowest level of neutrophil proportion (26.2%). In addition, the AK-137@FN group significantly reduced the content of myeloperoxidase in the bronchoalveolar lavage fluid of mice with lung injury. Myeloperoxidase, as a marker of neutrophils, further verified that AK-137@FN can inhibit neutrophil infiltration in lung tissue. Figure 17 c). This may be because AK-137@FN inhibits the expression of pro-inflammatory cytokines, reduces the cytokine storm throughout the lung tissue, and thus effectively avoids the recruitment of a large number of neutrophils into the lung tissue.

[0114] Example 18

[0115] Twenty-four hours after treatment, one mouse was selected from each of the six experimental groups, and its lung tissue was extracted and immersed in tissue fixative for 24 hours. H&E staining was then performed to analyze the degree of lung tissue damage. The experimental results are as follows: Figure 18 As shown, compared to the control group, mice treated with AK-137 and FN exhibited reduced alveolar wall congestion and inflammatory cell infiltration, although some inflammatory cell infiltration and alveolar wall congestion remained. The alveolar wall congestion, lung inflammation, and alveolar structure in mice treated with AK-137@FN returned to normal levels, primarily attributed to the combined anti-inflammatory effects of AK-137 and FN, as well as the enhanced intracellular delivery of FN mediated by AK-137.

[0116] Example 19

[0117] To further validate the therapeutic potential of the prepared nanoplatform in other inflammatory diseases, an acute gout animal model was constructed. Six-week-old male CD-1 mice were purchased from the Shanghai Slack Laboratory Animal Center. The mice were randomly divided into six groups (PBS control group, MSU treatment group, AK-137 treatment group, FN treatment group, AK-137@FN treatment group, and AK-137@BSA treatment group), with seven mice in each group. Except for the PBS group, an acute gout model was established 24 hours after intra-articular injection of MSU crystals (25 mg / mL, 20 μL) into the right ankle joint. The left ankle joint was injected with only an equal volume of PBS. To determine the preventative effect, the right ankle joint of mice was pretreated with PBS, AK-137, FN, AK-137@FN, and AK-137@BSA (FN or BSA concentration of 2 mg / kg) before injection of MSU crystals. Ankle diameter was measured with calipers before and 24 hours after treatment. 24 hours after treatment, one mouse from each of the six experimental groups was selected, and its right ankle joint was immersed in tissue fixation solution for 24 hours for H&E staining analysis to determine the degree of joint damage. Experimental results are as follows: Figure 19 As shown in ab, compared with the acute gout group, the swelling of the ankle joint of mice treated with AK-137@FN was suppressed. Figure 19 H&E staining results of the ankle joint in group c showed that, compared with the PBS group, the ankle joint cavity in the gout group was significantly smaller and contained exudate, with increased inflammatory cell infiltration in the synovial area. After different treatments, especially in the AK-137@FN group, the joint cavity returned to normal, and the inflammatory cell infiltration was significantly improved.

[0118] In summary, the phosphorus-containing dendritic macromolecules prepared in this invention can enhance the intracellular delivery efficiency of different proteins for anti-tumor, anti-inflammatory, and immunotherapy purposes. The therapeutic potential of the nanocomposite formed after complexing with the anti-inflammatory protein fibronectin has been verified in acute lung injury and acute gout inflammation models.

Claims

1. A phosphorus-containing dendritic macromolecule modified with sodium phosphite and possessing anti-inflammatory activity, characterized in that: The structural formula of the phosphorus-containing dendritic macromolecule is as follows: .

2. A method for preparing a phosphorus-containing dendritic macromolecule modified with sodium phosphite as described in claim 1, comprising the following steps: (1) Compound I was dissolved in triethyl phosphite, heated and stirred to react, and dried under vacuum. The crude product was dissolved in dichloromethane and precipitated with pentane to obtain compound II. in, The structural formula of compound I is: ; The structural formula of compound II is: ; (2) Dissolve compound II obtained in step (1) in anhydrous dichloromethane, cool the resulting solution to 0°C, add trimethylbromosilane dropwise, stir the reaction at room temperature, concentrate under reduced pressure, mix the resulting product with anhydrous methanol, stir at room temperature, filter, purify, and dry under vacuum. (3) The product obtained in step (2) is dissolved in an alkaline solution, stirred at room temperature, and the solvent is removed to obtain compound III, which is a phosphorus-containing dendritic macromolecule modified with sodium phosphite and possessing anti-inflammatory activity; wherein the alkaline solution is an aqueous solution of sodium hydroxide; the structural formula of compound III is: .

3. The preparation method according to claim 2, characterized in that: In step (1), the ratio of compound I to triethyl phosphite is 0.3-0.5 mmol: 20-40 mL.

4. The preparation method according to claim 2, characterized in that: The temperature of the stirring reaction in step (1) is 150~180℃, and the reaction time is 72~120 h.

5. The preparation method according to claim 2, characterized in that: The volume ratio of dichloromethane to pentane in step (1) is 1:10-15.

6. The preparation method according to claim 2, characterized in that: In step (2), the ratio of compound II to anhydrous dichloromethane is 0.1-0.2 mmol: 50-100 mL; the molar ratio of trimethylbromosilane to compound II is 50-70:

1.

7. The preparation method according to claim 2, characterized in that: The stirring reaction at room temperature in step (2) lasts for 48 to 96 hours.

8. The preparation method according to claim 2, characterized in that: The concentration of the alkaline solution in step (3) is 0.1~0.2 mol / L; the molar ratio of the product to the alkaline solution in step (2) is 20-25:

1.

9. The preparation method according to claim 2, characterized in that: The stirring time at room temperature in step (3) is 30~60 min.

10. The application of a sodium phosphite-modified phosphorus-containing dendritic macromolecule with anti-inflammatory activity as described in claim 1 in the preparation of protein-carrying nanocomposites.

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