Mannose functionalized supramolecular nanoparticles as well as preparation method and application thereof

Abstract

The invention provides mannose functionalized supramolecular nanoparticles and a preparation method and application thereof, the nanoparticles are formed by supermolecule self-assembly of a water-soluble amphiphilic compound MaCa in a water phase, and the nanoparticles are used for targeted therapy and early diagnosis of triple negative breast cancer (TNBC). A surface mannose unit of the nanoparticle is specifically combined with a TNBC marker alpha-mannosidase 2C1, and a hydrophobic core loaded drug (such as paclitaxel) or an imaging reagent (such as DiR and MRI contrast agents). Experiments show that the enrichment amount of the particles at the tumor part is 9.3 times of that of a control group, the tumor volume disappears after the particles are treated for 7 days, and the particles have no obvious toxicity. The process is simple and convenient, and an innovative solution is provided for TNBC diagnosis and treatment integration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mannose functionalized supramolecular nanoparticles, particularly to a kind of mannose functionalized supramolecular nanoparticles and its preparation method and application in preparing drug for treating triple negative breast cancer. BACKGROUND

[0002] Triple negative breast cancer (TNBC) is a highly invasive subtype of breast cancer, accounting for about 10-20% of all breast cancer cases. Its main feature is the lack of expression of estrogen receptor, progesterone receptor and HER2 receptor, which makes traditional hormone therapy and HER2 targeted therapy ineffective. Currently, the treatment of TNBC mainly relies on chemotherapy, such as paclitaxel drugs. However, chemotherapy drugs usually lack selectivity, killing cancer cells while also damaging normal cells, causing serious side effects such as nausea, hair loss and immunosuppression. In addition, TNBC has a high recurrence rate and poor prognosis, and the five-year survival rate of patients is significantly lower than that of other breast cancer subtypes. This makes it a research focus and urgent need to develop new treatment methods and early diagnosis techniques.

[0003] The existing technology for triple negative breast cancer (TNBC) has significant limitations, and the present application aims to solve the following key technical problems to achieve efficient targeted treatment and early diagnosis of TNBC.

[0004] 1. Limitations of existing treatment methods TNBC lacks estrogen receptor, progesterone receptor and HER2 receptor, making traditional hormone therapy and targeted therapy ineffective. Clinically, it mainly relies on chemotherapy. However, chemotherapy drugs (such as paclitaxel) lack selectivity, causing serious damage to normal cells while killing tumor cells, resulting in side effects such as nausea, hair loss and immunosuppression in patients, significantly reducing their quality of life. In addition, TNBC has a high recurrence rate and poor prognosis, with a five-year survival rate lower than other breast cancer subtypes. To address this issue, the present application develops a nano-delivery system specifically targeting TNBC, using the overexpression of α-mannosidase 2C1 in TNBC cells as a target, and achieving efficient targeted drug delivery through MaCa nanoparticles, reducing damage to normal cells and improving treatment effectiveness.

[0005] 2. Difficulty in early diagnosis Traditional imaging techniques (such as CT, X-ray and ultrasound) rely on physical changes to detect tumors, making it difficult to detect abnormalities in the early stages of TNBC (when the tumor is small), resulting in late-stage diagnosis and missing the best treatment window. Molecular imaging can achieve early diagnosis through tumor markers, but TNBC lacks traditional biomarkers, limiting its application. The present application designs MaCa nanoparticles to load imaging reagents such as fluorescent dyes or MRI contrast agents, which specifically recognizeα - Mannosidase 2C1, enabling high sensitivity early imaging of TNBC.

[0006] 3. Deficiency of targeted delivery and imaging probes Although existing nanotechnology can achieve passive targeting through the EPR effect, the efficiency varies due to individual differences, and there are few active targeting systems for TNBC, which are complex to prepare and have poor stability. The development of imaging probes is also time-consuming and laborious due to the need to adapt to different imaging methods. The present application uses supramolecular self-assembly technology to construct MaCa nanoparticles that are simple to prepare, have high stability and good biocompatibility, integrate targeted delivery, drug loading and imaging functions, simplify the process and improve the potential for clinical translation.

[0007] 4. The need for integrated diagnosis and treatment Existing technologies cannot simultaneously achieve early diagnosis and precise treatment of TNBC. The present application constructs an integrated diagnosis and treatment platform by loading hydrophobic drugs and imaging reagents into MaCa nanoparticles, thereby improving treatment accuracy and effectiveness.

[0008] Therefore, in view of the lack of biomarkers for TNBC, the large side effects of treatment and the difficulty of early diagnosis, it is necessary to develop a supramolecular nanoparticle for targeted delivery and early diagnosis of triple-negative breast cancer to achieve the dual goals of efficient treatment and early diagnosis. SUMMARY

[0009] The purpose of the present application is to provide a mannose-functionalized supramolecular nanoparticle and its preparation method and application in the preparation of a drug for treating triple-negative breast cancer. The mannose-functionalized supramolecular nanoparticle not only can load drugs to achieve precise treatment, but also can achieve early detection of TNBC through multi-modal imaging, which has important scientific significance and potential clinical application value.

[0010] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In the first aspect of the present application, a mannose-functionalized supramolecular nanoparticle is provided, which is formed by supramolecular self-assembly of a water-soluble amphiphilic compound MaCa in an aqueous phase, wherein the compound MaCa is a mannose-carbazole conjugate represented by formula (I):

[0011] Formula I.

[0012] Further, the mannose unit in the mannose-carbazole conjugate is connected to the triethylene glycol through a β 1,4 glycosidic bond, and the terminal of the triethylene glycol is combined with the carbazole unit through an ether bond.

[0013] Further, the concentration of the compound MaCa in the mannose functionalized supramolecular nanoparticle is 5-10 mg / mL.

[0014] Further, the mannose functionalized supramolecular nanoparticle further comprises a hydrophobic substance selected from at least one of paclitaxel, docetaxel, DiR fluorescent dye or Mn-BnO-TyEDTA contrast agent; the hydrophobic inner core of the compound MaCa embeds the hydrophobic substance through π-π stacking.

[0015] Further, the encapsulation efficiency of the nanoparticle is ≥ 90% and the drug loading is 5-20% (w / w).

[0016] In the second aspect of the present application, a preparation method of the mannose functionalized supramolecular nanoparticle is provided, Step S1, mannose is connected with triethylene glycol through glycosylation reaction to generate intermediate A; the intermediate A is connected with a carbazole unit through etherification reaction to generate intermediate B; the acetyl protecting group in the intermediate B is removed to obtain the compound of formula (I); Step S2, the compound of formula (I) is dissolved in water with a concentration of 5-10 mg / mL, and ultrasonic treatment is performed to form nanoparticles with a particle size of 300-400 nm.

[0017] The step S1 specifically comprises: (1) synthesis of intermediate A: mannose is connected with triethylene glycol through glycosylation reaction to generate intermediate A; (2) synthesis of intermediate B: intermediate A is connected with a carbazole unit through etherification reaction to generate intermediate B; (3) deprotection reaction: the acetyl protecting group in the intermediate B is removed to obtain the compound of formula (I); In step (1), the glycosylation reaction conditions are: using fully acetylated mannose as raw material, boron trifluoride etherate (BF3·Et2O) as catalyst; the reaction solvent is anhydrous dichloromethane (DCM), the reaction temperature is 0°C to room temperature, and the reaction time is 12-24 hours.

[0018] In step (2), the etherification reaction conditions are: using 1,5-dibromo-2,4-dimethoxybenzene as crosslinking agent, potassium carbonate (K2CO3) as base, and cuprous iodide (CuI) as catalyst; the reaction solvent is o-dichlorobenzene, the reaction temperature is 160-180°C, and the reaction time is 36-48 hours. boron tribromide (BBr3) is used as deprotection reagent; The reaction solvent is dichloromethane (DCM), the reaction temperature is 0°C to room temperature, and the reaction time is 24-48 hours.

[0019] Furthermore, in step S2, the conditions for the ultrasonic treatment include: power of 80-120 W, frequency of 40 kHz, and time of 5-10 minutes.

[0020] Furthermore, the method further includes: co-dissolving the hydrophobic substance and the nanoparticles of the compound of formula (I) in an organic solvent, removing the solvent by dialysis, wherein the hydrophobic substance is embedded in the core of the nanoparticles through hydrophobic interaction.

[0021] Furthermore, the organic solvent includes dimethyl sulfoxide, N , N At least one of dimethylformamide, wherein the hydrophobic substance is in a mass ratio of 1:5 to 1:20 with respect to the compound of formula (I).

[0022] In a third aspect of the invention, the use of the aforementioned mannose-functionalized supramolecular nanoparticles in the preparation of targeted delivery carriers or drugs for the treatment of triple-negative breast cancer is provided.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention discloses for the first time a novel mannose-functionalized supramolecular nanoparticle for treating triple-negative breast cancer, and develops a novel water-soluble amphiphilic compound, MaCa. This compound consists of mannose units and carbazole units, and can self-assemble into nanoparticles in water. The mannose unit, acting as a hydrophilic head, not only endows the nanoparticles with excellent water solubility but also serves as a targeting group, specifically recognizing the overexpressed [specific gene] in TNBC cells. α - Mannosidase 2C1; the carbazole unit forms a hydrophobic core, providing space for loading hydrophobic drugs or imaging reagents. This supramolecular self-assembled nanoparticle combines active targeting and loading functions, providing a new platform for the integrated diagnosis and treatment of TNBC. The following details its effects and advantages from multiple dimensions, including yield, quality, efficiency, energy consumption, ease of operation, and environmental friendliness, and provides supporting evidence through experimental data and comparisons with background technologies.

[0024] (1) Highly efficient targeted delivery, improving quality and efficiency, and reducing side effects. Effects and benefits: MaCa nanoparticles specifically recognize overexpressed [a specific gene] in TNBC cells through surface mannose units. α - Mannosidase 2C1 enables highly efficient targeted delivery. Drugs (such as PTX) are primarily concentrated at the tumor site, significantly reducing damage to normal cells, improving treatment quality and efficiency, and significantly reducing side effects.

[0025] Experimental data: In vivo fluorescence imaging shows that the enrichment amount of DiR@MaCa at the tumor site is 9.3 times that of the control group DiR@F127 (test conditions: mouse model, fluorescence imaging). In the treatment experiment, PTX@MaCa almost disappeared the tumor volume within 7 days, while the tumor volume of the control group increased by 5 times (test conditions: TNBC mouse model).

[0026] Comparison with prior art: Traditional chemotherapy drugs lack selectivity and have large side effects (such as nausea, hair loss, etc.); the present application improves drug bioavailability and reduces systemic toxicity through targeted design, which is significantly better than the prior art.

[0027] (2) Strong early diagnosis ability, improving diagnosis accuracy and efficiency Effect and advantage: After MaCa nanoparticles load imaging reagents, the signal can be detected on the first day of tumor implantation, greatly improving the diagnosis accuracy and early detection efficiency, and gaining time for the best treatment opportunity.

[0028] Experimental data: DiR&Mn@MaCa detected tumor signals through fluorescence and MRI imaging on the first day, with an 85% increase in MRI signal intensity (test conditions: mouse model, fluorescence and MRI dual-mode imaging).

[0029] Comparison with prior art: Traditional CT, X-ray and other technologies can only be detected after the tumor grows to a certain size, with low diagnosis accuracy and poor efficiency; the present application significantly improves the early diagnosis ability by using molecular level recognition.

[0030] (3) Integration of diagnosis and treatment, improving ease of use and efficiency Effect and advantage: MaCa nanoparticles can simultaneously load drugs and imaging reagents, realizing simultaneous diagnosis and treatment, simplifying the clinical operation process and improving efficiency.

[0031] Experimental data: DiR&Mn@MaCa shows excellent in vivo and in vitro imaging effects, and PTX@MaCa significantly inhibits tumor growth in treatment.

[0032] Comparison with prior art: In the prior art, diagnosis and treatment are usually performed independently, and the operation is complex; the present application integrates diagnosis and treatment functions, making the operation more simple and efficient.

[0033] (4) Simple preparation process, improving yield and saving process Effect and advantage: MaCa nanoparticles are prepared by supramolecular self-assembly, without complex chemical synthesis, with simple process, high yield, easy to scale up production, and saving processing procedures.

[0034] Experimental data: MaCa synthesis yield up to 85%, preparation only needs 5 minutes of ultrasound and 24 hours of dialysis (test conditions: laboratory standard conditions).

[0035] Compared with the prior art: traditional nanomaterial preparation involves multi-step reaction and complex purification, low yield, and multiple processes; the present application simplifies the process, improves the yield, and reduces the production cost.

[0036] (5) Good biocompatibility, high quality, and reduced toxicity Effects and advantages: MaCa nanoparticles have excellent biocompatibility and no significant toxicity, ensuring product quality and reducing health risks during treatment.

[0037] Experimental data: CCK-8 experiment showed that MaCa had no significant toxicity to MCF-10a and MDA-MB-231 cells; in animal experiments, the body weight of mice was stable after PTX@MaCa treatment, there was no pathological damage to major organs, and hematological indexes were normal.

[0038] Compared with the prior art: traditional chemotherapy drugs have high toxicity and affect the quality of life of patients; the present application has better quality and significantly reduced toxicity through targeted delivery and biocompatibility design.

[0039] (6) Multifunctionality and adjustability to meet diverse needs Effects and advantages: MaCa nanoparticles can load various hydrophobic substances (such as drugs, dyes, and contrast agents), and the assembly behavior is adjustable, which adapts to different diagnosis and treatment needs and improves the flexibility of use.

[0040] Experimental data: Successfully loaded Nile Red, DiR, Mn-BnO-TyEDTA, and PTX, showing versatility.

[0041] Compared with the prior art: traditional delivery systems are designed for specific substances and have a narrow range of applications; the present application has strong versatility and obvious advantages.

[0042] (7) Environmentally friendly, low energy consumption, and reduced labor intensity Effects and advantages: The preparation process does not use toxic reagents, the process is mild, the energy consumption is low, it meets the green chemistry concept, the operation is simple, and the labor intensity is reduced.

[0043] Experimental data: Preparation only needs ultrasound and dialysis, without high temperature and pressure or special equipment (test conditions: standard laboratory environment).

[0044] Compared with the prior art: traditional nanomaterial preparation often involves organic solvents or high energy consumption conditions, with heavy environmental burden; the present application is environmentally friendly, with significantly reduced energy consumption and labor intensity. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0046] Figure 1 For the preparation of molecular assembly (MA) and the activation process of nanoparticle probe targeting. Figure 2 For the dynamic light scattering (DLS) results of MaCa nanoparticles in Example 2.

[0047] Figure 3 For the atomic force microscope (AFM) results of MaCa nanoparticles in Example 2.

[0048] Figure 4 For the CLSM images of MaCa taken up by different cells in Example 4.

[0049] Figure 5 For the target connection rate of MaCa detected by flow cytometry analysis in Example 4.

[0050] Figure 6 For the fluorescence imaging observation of the fluorescence intensity of tumors and main organs in nude mice after injection of DiR@MaCa and DiR@F127 in Example 5.

[0051] Figure 7 For the active tumor targeting effect in vivo at different time points after injection of DiR@MaCa in Example 6. Figure 8 For the magnetic resonance imaging at different time points before and after injection of DiR@MaCa in Example 6.

[0052] Figure 9 For the tumor volume growth curve during treatment in Example 7.

[0053] Figure 10 For the body weight change of nude mice during treatment in Example 7.

[0054] Figure 11 For the tissue staining results of healthy nude mice before and after injection of MaCa in Example 8. DETAILED DESCRIPTION

[0055] The advantages and various effects of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, but not to limit the present application.

[0056] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood in the manner in which they are commonly used in the art. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification prevails.

[0057] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present invention can be purchased on the market or obtained by existing methods.

[0058] The general idea of the present invention is as follows: According to a typical embodiment of the present invention, a mannose functionalized supramolecular nanoparticle is provided: The MaCa nanoparticle, for short, is formed by supramolecular self-assembly of a water-soluble amphiphilic compound MaCa, and the surface of the water-soluble amphiphilic compound MaCa is modified with mannose units, which can specifically recognize the overexpressed α - Mannosidase 2C1. The hydrophobic core can load drugs (such as paclitaxel PTX) or imaging agents (such as DiR and Mn-BnO-TyEDTA), realizing diagnosis and treatment integration.

[0059] 1. Product structure, position and mutual relationship of each component The MaCa nanoparticle is composed of the following core components: Mannose unit (hydrophilic head): located on the surface of the nanoparticle, serving as a targeting group and being connected to the core through a covalent bond.

[0060] Carbazole unit (hydrophobic tail): constituting the aromatic hydrophobic core of the nanoparticle, located inside the particle and providing a loading space.

[0061] Loaded substances: such as drugs (PTX) or imaging agents (DiR, Mn), which are uniformly distributed in the hydrophobic core and combined with the carbazole unit through hydrophobic interaction.

[0062] Mutual relationship: the mannose unit forms a hydrophilic shell on the outer layer, protecting the core and realizing targeting; the carbazole unit self-assembles to form a hydrophobic core on the inner layer, wrapping the loaded substances; the loaded substances stably exist through physical embedding and depend on the hydrophobic environment of the core.

[0063] 2. Function, shape, position and connection relationship of each component (1) Mannose unit: Function: targeted recognition of α - Mannosidase 2C1 in TNBC cells, enhancing particle selectivity; providing water solubility.

[0064] Shape: monosaccharide molecule, arranged in a spherical shape on the surface of the particle.

[0065] Location: evenly distributed on the outer layer of the nanoparticle.

[0066] Connection: covalently linked to the carbazole unit via ether or amide bond.

[0067] (2) Carbazole unit: Function: drive self-assembly, form a hydrophobic core, and load hydrophobic substances.

[0068] Shape: planar aromatic structure, stacked to form the core.

[0069] Location: located inside the nanoparticle.

[0070] Connection: chemically bonded to the mannose unit and interacted through π-π stacking inside the core.

[0071] (3) Loaded substances: Function: achieve therapeutic (PTX) or diagnostic (DiR, Mn) functions.

[0072] Shape: determined by the characteristics of the molecule (e.g., PTX is a small molecule, and DiR is a dye).

[0073] Location: dispersed in the hydrophobic core.

[0074] Connection: combined with the carbazole unit through hydrophobic interaction without chemical bonds.

[0075] 3. How the components work together to achieve the advantages of the invention High efficiency targeting: specific binding of the mannose unit α - Mannosidase 2C1, ensuring that the nanoparticles are preferentially enriched in TNBC cells; the carbazole unit maintains particle stability, ensuring that the targeting process does not disintegrate.

[0076] Integrated diagnosis and treatment: the hydrophobic core loads drugs and imaging agents, and the mannose unit guides to the tumor site, achieving simultaneous diagnosis and treatment.

[0077] Stability and biocompatibility: the hydrophilicity of the mannose unit prevents particle aggregation, and the hydrophobicity of the carbazole unit ensures that the loaded substances do not leak, and the synergistic effect of the two reduces toxicity.

[0078] According to another typical embodiment of the present invention, a preparation method of a mannose functionalized supramolecular nanoparticle is provided, which comprises: Step S1, MaCa synthesis: The mannose is connected to the carbazole unit through multi-step organic reactions (such as etherification and amidation), and the yield is optimized to more than 85%.

[0079] Conditions: reaction temperature 50°C, solvent DMSO, catalyst EDC / NHS, reaction time 12 hours.

[0080] Step S2, nanoparticle preparation: MaCa was dissolved in water (concentration 5 mg / mL), ultrasonic treatment (power 100 W, frequency 40 kHz, 10 minutes), forming self-assembled nanoparticles.

[0081] Optimization parameter: ultrasonic time is shortened to 5 minutes to avoid excessive dispersion of particles.

[0082] Step S3, embedding of loaded substances: MaCa and loaded substances (such as PTX, mass ratio 1:0.2) were dissolved in DMSO, and organic solvents were removed by dialysis (dialysis bag MWCO 3500, 48 hours).

[0083] Optimization: dialysis time is shortened to 24 hours, and embedding efficiency is improved to 90%.

[0084] Configuration and function Ultrasonic process: ensure uniform self-assembly of MaCa, and control particle size at 300-400 nm.

[0085] Dialysis method: remove organic solvents to ensure biocompatibility and improve loading efficiency.

[0086] Parameter optimization: shorten process time, improve production efficiency, and reduce cost.

[0087] It should be noted that the components can be replaced as follows: (1) Targeting group: replace: mannose can be replaced by polysaccharides (such as dextran) or small molecule inhibitors.

[0088] Principle: still targeting α Mannosidase 2C1 as the target, the recognition mechanism remains unchanged.

[0089] (2) Hydrophobic unit: replace: carbazole can be replaced by fluorene or naphthalene.

[0090] Principle: maintain hydrophobicity and π-π stacking ability, and optimize core stability.

[0091] (2) Loaded substances: replace: PTX can be replaced by docetaxel, and DiR can be replaced by ICG.

[0092] Principle: hydrophobic drugs or dyes can be embedded in the core, and have similar functions.

[0093] In summary, the technical solution of the present application has the following advantages: Targeting: in vivo experiments show that the enrichment amount of DiR@MaCa in TNBC tumor is 9 times that of the control group.

[0094] Early diagnosis: DiR & Mn@MaCa can detect signals on the first day of tumor implantation.

[0095] Therapeutic effect: PTX@MaCa makes the tumor volume disappear within 7 days, with low toxicity.

[0096] Simple process: Self-assembly preparation does not require complex equipment and is easy to scale up production.

[0097] The technical solution realizes the efficiency and practicality of TNBC diagnosis and treatment through structure optimization and process improvement, laying a foundation for clinical transformation.

[0098] The present application will be described in detail below in conjunction with examples and experimental data.

[0099] Example 1: Synthesis of MaCa compound 1. Synthesis steps of MaCa compound

[0100] Step 1: Dissolve triethylene glycol (29.82 g, 0.20 mol) in 20 mL THF, add NaOH solution (1.59 g, 39.60 mmol, dissolved in 15 mL H2O) R1. Cool the mixture with ice water to 0°C, then add TsCl (4.74 g, 248.00 mmol) dissolved in THF (100 mL) dropwise into the mixture while starting the reaction. Continue stirring for 1 h, evaporate the solvent. Dissolve the residue in DCM and wash with brine. Dry with MgSO4, filter and concentrate under reduced pressure to obtain the target compound (3.74 g) as a yellow oil, yield: 50%.

[0101] Step 2: Dissolve peracetylated mannose (2.00 g, 5.12 mmol) in anhydrous dichloromethane (50 mL), stir, add boron trifluoride etherate (3.2 mL, 25.61 mmol) at 0°C, then add compound 1 (3.74 g, 12.29 mmol). Stir the resulting reaction mixture at room temperature overnight. Monitor the reaction progress using thin layer chromatography (TLC). After the reaction is complete, dilute with DCM, and wash the organic layer with sodium bicarbonate solution, water and brine in turn. Subsequently, the organic layer is dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to obtain crude compound 2. Purify the pure component by flash column chromatography using ethyl acetate and hexane as eluent, with an eluent of 40% ethyl acetate and hexane mixture. Spin evaporate the product to obtain a viscous oil, with a yield of 65% (2.12 g).

[0102] Step 3: 1,3-Dimethoxybenzene (5.30 g, 38.36 mmol) and THF (50 mL) were added to a 100 mL glass flask. Subsequently, a solution of 1,3-dibromo-5,5-dimethylhydantoin (DBH, 12.06 g, 42.19 mmol) in THF (50 mL) was added to the flask at 0ºC and the resulting mixture was stirred at room temperature for 1 day. After that, the mixture was poured into water and the crude product was extracted with chloroform. The combined organic phase was dried over MgS04, filtered, and concentrated under reduced pressure. The crude product was then washed with cold methanol and hexane to obtain white solid 3 (10.25 g, yield 90%).

[0103] Step 4: Carbazole (6.12 g, 36.60 mmol), 1,5-dibromo-2,4-dimethoxybenzene (3.55 g, 12.00 mmol), 18-crown-6 (0.99 g, 3.73 mmol), cuprous iodide (0.68 g, 3.58 mmol), potassium carbonate (20.10 g, 0.15 mol), and dry o-dichlorobenzene (80 mL) were mixed. The resulting mixture was stirred at 180ºC for 48 hours. After filtration and concentration under reduced pressure, the crude product was washed with water, hot methanol, and hexane in sequence to obtain white solid 4 (2.11 g, yield 38%).

[0104] Step 5: Compound 4 (1.00 g, 2.13 mmol) was added to a 100 mL two-necked glass flask and purged with N2. Anhydrous DCM (30 mL) was added to the flask. At the same time, 20 mL of BBr3 (1.85 mL, 19.20 mmol) in DCM was carefully added at 0ºC R4. The resulting reaction mixture was stirred and allowed to warm to room temperature for 48 hours. To quench the reaction, H2O (150 mL) was added. The two layers were separated and the aqueous layer was extracted with DCM (3 x 100 mL). Next, the combined organic layers were dried over Na2S04, filtered, and concentrated under reduced pressure. The resulting crude product was washed with hexane to obtain 2 (0.80 g, 85%) as a gray solid.

[0105] Step 6: In a two-necked glass flask purged with N2, 5 (0.05 g, 0.11 mmol), Cs2C03(0.14 g, 0.44 mmol), 2 (0.29 g, 0.45 mmol) and anhydrous DMF (5 mL) were combined to give R5. The reaction mixture was stirred at room temperature for 3 hours. Subsequently, the resulting mixture was concentrated in vacuo. To the resulting oil was added water, which was then extracted with CHC13. The combined organic phase was dried over Na2S04, filtered, and concentrated in vacuo. Purification by flash column chromatography with MeOH and DCM as eluents gave pure components eluted in 2% mixture to give colorless oil containing 6 and its partially deprotected derivatives (92 mg, 60% yield).

[0106] Step 7: Compound 6 and its partially deprotected derivatives (93 mg, 68 pmol) were mixed with Amberlyst A26 hydroxide (0.20 g) and methanol (10 mL) in a one-necked glass flask R5. The reaction mixture was stirred at room temperature for 5 days. After the resulting solid was filtered and the solvent was evaporated, the resulting yellow solid was washed with hexane and acetone to give MA as a pink oil (60 mg, 86% yield).

[0107] Characterization data: 1H NMR (400 MHz, DMSO-d6): d 7.82 (d, J = 8.0 Hz, 2H, carbazole-H), 7.45 (t, J = 7.2 Hz, 2H), 5.21 (s, 1H, mannose-H), 4.10 (m, 4H, triethylene glycol-OCH2), 3.55 (m, 4H, triethylene glycol-CH2). ESI-MS: m / z 689.2 [M+H]+. Example 2, Preparation of MaCa nanoparticles (without loading) 1, Step: MaCa compound (10 mg) was dissolved in 1 mL DMSO, which was added dropwise into 9 mL deionized water pre-cooled to 4 °C in an ice bath. Ultrasonic treatment (power 100 W, frequency 40 kHz, time 5 minutes) and dialysis (MWCO 3500, 24 hours) were used to remove DMSO to obtain a MaCa nanoparticle suspension.

[0108] 2, Characterization results: Dynamic light scattering (DLS): average particle size 320 nm, PDI 0.18.

[0109] Atomic force microscopy (AFM): spherical particles, particle size 300-350 nm Figure 2 .

[0110] Zeta potential: -25 mV.

[0111] Example 3: Preparation of paclitaxel-loaded (PTX@MaCa) 1. Raw materials: MaCa (10 mg), paclitaxel (PTX, 2 mg), DMSO (1 mL).

[0112] 2. Process steps: (1) Nile Red (NR) loading: NR (0.2 mg) and MaCa (10 mg) were co-dissolved in 1 mL DMSO, added dropwise to 9 mL water, ultrasonicated for 5 minutes, and dialyzed (MWCO 3500) for 24 hours to remove DMSO, obtaining NR@MaCa.

[0113] (2) Paclitaxel (PTX) loading: PTX (2 mg) and MaCa (10 mg) were co-dissolved in 1 mL DMSO, added dropwise to 9 mL water, ultrasonicated for 5 minutes, and dialyzed for 24 hours, obtaining PTX@MaCa.

[0114] (3) DiR and Mn-BnO-TyEDTA (Mn) loading: DiR (0.2 mg), Mn (1 mg), and MaCa (10 mg) were co-dissolved in 1 mL DMSO, added dropwise to 9 mL water, ultrasonicated for 5 minutes, and dialyzed for 24 hours, obtaining DiR&Mn@MaCa.

[0115] 3. Quality control: Drug loading: 15.2% (w / w) as determined by HPLC.

[0116] Encapsulation efficiency: 92.5% (calculation formula: encapsulation efficiency = actual drug loading / theoretical drug loading x 100%).

[0117] Example 4: In vitro targeting performance test 1. Cell uptake experiment Cell lines: MDA-MB-231 (TNBC cells), SK-BR-3 (breast cancer cells), MCF-10a (normal breast epithelial cells).

[0118] Method: NR@MaCa (NR concentration 10 μM) was co-incubated with cells for 1 hour, and the uptake was observed under a fluorescence microscope.

[0119] The results are shown in Figure 4 MDA-MB-231 cells had the highest uptake of NR@MaCa, followed by SK-BR-3, and MCF-10a had the lowest uptake.

[0120] 2. Flow cytometry analysis Method: Cells were co-incubated with NR@MaCa for 1 hour, and the fluorescence intensity was detected by flow cytometry.

[0121] Results are shown in Figure 5 Figure 2. The average fluorescence intensity of MDA-MB-231 cells was 2000, that of SK-BR-3 was 1100, and that of MCF-10a was 720.

[0122] Example 5: Test of in vivo targeting performance of MaCa nanoparticles Establishment of a TNBC model: MDA-MB-231 cells (1 x 10 6 ) were injected subcutaneously into BALB / c nude mice.

[0123] Imaging experiment: DiR@MaCa (DiR dose 0.5 mg / kg) was injected via the tail vein, and fluorescence imaging was performed 24 hours later.

[0124] Results are shown in Figure 6 Figure 4. The fluorescence intensity of DiR@MaCa at the tumor site was 9.3 times that of the control DiR@F127.

[0125] Example 6: Early diagnosis ability of MaCa nanoparticles 1. Animal model: BALB / c nude mice were inoculated subcutaneously with MDA-MB-231 cells (tumor volume about 50 mm 3 ).

[0126] 2. Imaging procedure: Fluorescence imaging: DiR@MaCa (DiR dose 0.5 mg / kg) was injected via the tail vein.

[0127] Results are shown in Figure 7 Figure 6. The fluorescence intensity at the tumor site was 9.3 times that of the control 24 hours after injection.

[0128] MRI imaging: Mn@MaCa (Mn dose 1 mg / kg) was injected.

[0129] Results are shown in Figure 8 Figure 7. The T1-weighted signal intensity was increased by 85%, and the tumor boundary was clearly visible.

[0130] Example 7: Evaluation of in vivo anti-tumor efficacy 1. Experimental design: Grouping: PTX@MaCa group (5 mg / kg PTX), free PTX group (5 mg / kg), and blank control group (PBS).

[0131] Dosing regimen: Injection via the tail vein on days 1, 3, and 5.

[0132] 2. Results: (1) Change in tumor volume: After 7 days, the tumor volume of the PTX@MaCa group almost disappeared, and the tumor volume of the control group increased 5 times. Figure 9 and Figure 10 ).

[0133] (2) Toxicity evaluation: Methods: The body weight of mice was monitored during treatment, and HE staining of major organs and hematological tests were performed 28 days after treatment. Results: The body weight of mice was stable, there was no pathological damage to organs, and hematological indexes were normal.

[0134] Methods: CCK-8 method was used to detect the toxicity of MaCa to MCF-10a and MDA-MB-231 cells.

[0135] The results of tissue staining of healthy nude mice before and after injection of MaCa are shown in Figure 11 After 48 hours, the cell survival rate was >90%.

[0136] Example 8: Comparative experiment (with traditional nanoparticles) 1. Comparison object: F127 nanoparticles (Pluronic surfactant). 2. Experimental content: (1) Targeting comparison: The fluorescence intensity of DiR@F127 at the tumor site was only 1 / 9 of that of DiR@MaCa.

[0137] (2) Stability comparison: MaCa nanoparticles: 4°C storage for 30 days, change in particle size <5%.

[0138] F127 nanoparticles: aggregation occurred after 7 days (particle size increased to 600 nm).

[0139] Finally, it should be noted that the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusions, such that processes, methods, articles, or devices that comprise a list of elements are not limited to those elements, but can also include other elements not expressly listed, or inherent to such processes, methods, articles, or devices. Although preferred embodiments of the present application have been described, those skilled in the art, once armed with the basic inventive concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application. The present application also intends to include these modifications and variations, provided that they fall within the scope of the claims of the present application and their equivalents.

Claims

1. A mannose-functionalized supramolecular nanoparticle, characterized in that, The nanoparticles are formed by supramolecular self-assembly of the water-soluble amphiphilic compound MaCa in an aqueous phase. The compound MaCa is of formula (I). The mannose-carbazole conjugate shown: Formula I.

2. The mannose-functionalized supramolecular nanoparticles according to claim 1, characterized in that, In the mannose-carbazole conjugate, the mannose unit is linked to triethylene glycol via a β-1,4 glycosidic bond, and the triethylene glycol terminal is linked to the carbazole unit via an ether bond.

3. The mannose-functionalized supramolecular nanoparticles according to claim 1, characterized in that, In the mannose-functionalized supramolecular nanoparticles, the concentration of the compound MaCa is 5-10 mg / mL.

4. The mannose-functionalized supramolecular nanoparticles according to claim 1, characterized in that, The mannose-functionalized supramolecular nanoparticles also include hydrophobic substances selected from at least one of paclitaxel, docetaxel, DiR fluorescent dye, or Mn-BnO-TyEDTA contrast agent; the hydrophobic core of the compound MaCa encapsulates the hydrophobic substances through π-π stacking and hydrophilic-hydrophobic interactions.

5. The mannose-functionalized supramolecular nanoparticles according to claim 4, characterized in that, The nanoparticles have an encapsulation efficiency of ≥90%, a particle size of 300-400 nm, and a drug loading of 5-20% (w / w).

6. A method for preparing mannose-functionalized supramolecular nanoparticles according to any one of claims 1-5, characterized in that, The method includes: Step S1: Connect mannose to triethylene glycol via glycosylation to generate intermediate A; connect intermediate A to carbazole unit via etherification to generate intermediate B; remove the acetyl protecting group from intermediate B to obtain compound (I); Step S2: Dissolve the compound shown in formula (I) in water to a concentration of 5-10 mg / mL, and then sonicate to form nanoparticles.

7. The preparation method according to claim 6, characterized in that, The conditions for the ultrasonic treatment include: power of 80-120 W, frequency of 40 kHz, and time of 5-10 minutes.

8. The preparation method according to claim 6, characterized in that, The method further includes: co-dissolving the hydrophobic substance and the nanoparticles of the compound of formula (I) in an organic solvent, dialyzing to remove the organic solvent, wherein the hydrophobic substance is embedded in the core of the nanoparticles through hydrophobic interaction.

9. The preparation method according to claim 8, characterized in that, The organic solvent includes dimethyl sulfoxide, N , N At least one of dimethylformamide, wherein the hydrophobic substance is in a mass ratio of 1:5 to 1:20 with respect to the compound of formula (I).

10. The use of the mannose-functionalized supramolecular nanoparticles according to any one of claims 1-5 in the preparation of a targeted delivery carrier or drug for the treatment of triple-negative breast cancer.

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