Antitumor drug nano-prodrug assembly based on dynamic covalent chemistry and preparation method and application thereof

Abstract

This invention discloses an antitumor drug nanoparticle prodrug assembly based on dynamic covalent chemistry, its preparation method, and its application, belonging to the field of pharmaceutical technology. The nanoparticle prodrug assembly is formed by the self-assembly of an antitumor drug, a traditional Chinese medicine polyphenol compound containing an ortho-diphenol structure, a natural polyamine, and formylphenylboronic acid through dynamic covalent bonds; the antitumor drug includes any one of gemcitabine, doxorubicin, and daunorubicin. This invention utilizes a one-pot reaction to form the nanoparticle prodrug assembly by the self-assembly of the antitumor drug, the traditional Chinese medicine polyphenol compound containing an ortho-diphenol structure, the natural polyamine, and formylphenylboronic acid through dynamic covalent bonds. This nanoparticle prodrug assembly solves the problems of uncontrollable drug ratios, asynchronous release, and poor targeting in existing combination therapies, achieving intelligent drug release and synergistic antitumor effects in the tumor microenvironment, thus improving the treatment efficacy of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to antitumor drug nanoprodrug assemblies based on dynamic covalent chemistry, their preparation methods, and applications. Background Technology

[0002] Gemcitabine (Gem), a nucleoside analog, is an important second-line or combination therapy option for treating recurrent or drug-resistant breast and ovarian cancer. To overcome the drawbacks of gemcitabine, such as rapid metabolism, poor stability, and weak targeting, various nanomedicine delivery systems, such as liposomes and polymer micelles, have been developed to improve tumor accumulation and efficacy. Meanwhile, active ingredients in traditional Chinese medicine (TCM) have shown synergistic potential across multiple targets and pathways in anti-tumor therapy. For example, tea polyphenols such as epigallocatechin gallate (EGCG) have been shown to exert anti-tumor effects through multiple mechanisms, including inducing apoptosis, inhibiting angiogenesis, and regulating the tumor microenvironment. Current research includes physically mixing or co-encapsulating natural polyphenols and other TCM ingredients with chemotherapeutic drugs to attempt combination therapy to enhance efficacy and reverse drug resistance.

[0003] However, existing technologies have the following problems and shortcomings: 1. Inaccurate drug synergy and uncontrollable ratios: Physical mixing or simple co-encapsulation methods cannot precisely control the dosage ratio of gemcitabine to the active ingredients of traditional Chinese medicine at the molecular level, and the final loading ratio in the delivery carrier. Inconsistent pharmacokinetic behavior of the components in vivo leads to an imbalance in the proportion of drugs reaching the tumor site, making it difficult to achieve the designed synergistic effect, and may even weaken the efficacy or increase toxicity due to the imbalance. 2. Asynchronous release, making it difficult to achieve spatiotemporal synergy: In physically mixed nanoparticles, different drugs are typically released through their respective affinity for the carrier material or simple diffusion mechanisms, resulting in varying release kinetics. This prevents gemcitabine and traditional Chinese medicine components from being released synchronously or on demand within tumor cells, making it difficult to achieve synergistic effects at critical temporal and spatial points. 3. Drug delivery systems lack intelligent responsiveness and have low targeted release efficiency: Most traditional nanocarriers are not responsive enough or specific to the tumor microenvironment (such as low pH and high reactive oxygen species). Drug release mainly depends on non-specific diffusion or carrier degradation, which leads to premature leakage during circulation and insufficient release at the tumor site, reducing the therapeutic index and increasing systemic toxicity.

[0004] The challenge in previous research lies in how to construct a gemcitabine-traditional Chinese medicine co-drug delivery system that can simultaneously achieve precise proportional loading, synchronous / sequential release, and intelligent microenvironment response without introducing complex synthetic steps or compromising drug activity. Conventional chemical coupling may result in irreversible bonds, affecting drug release; while overly complex synthetic pathways are not conducive to industrial production and clinical translation.

[0005] Based on the problems and defects of existing technologies, this paper provides a synergistic nano-prodrug assembly of gemcitabine and traditional Chinese medicine polyphenols, which has a simple construction method, precise and controllable drug ratio, and intelligent response release characteristics in the tumor microenvironment. This is of great significance for overcoming the defects of existing combined drug delivery systems, such as poor synergistic effect, asynchronous release, and insufficient targeting, thereby improving the treatment effect of ovarian cancer, especially drug-resistant ovarian cancer. Summary of the Invention

[0006] The purpose of this invention is to provide antitumor drug nanoprodrug assemblies based on dynamic covalent chemistry, their preparation methods, and applications, in order to solve the problems existing in the prior art. By using a "one-pot" reaction, gemcitabine, polyphenolic compounds, natural polyamines, and formylphenylboronic acid are self-assembled through dynamic covalent bonds to form nanoprodrug assemblies. These nanoprodrug assemblies solve the problems of uncontrollable drug ratios, asynchronous release, and poor targeting in existing combination therapies, achieving intelligent drug release and synergistic antitumor effects in the tumor microenvironment, and improving the treatment efficacy of ovarian cancer.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides an antitumor drug nanoprodrug assembly based on dynamic covalent chemistry. The nanoprodrug assembly is formed by the self-assembly of an antitumor drug, a traditional Chinese medicine polyphenol compound containing an ortho-diphenol structure, a natural polyamine, and formylphenylboronic acid through dynamic covalent bonds. The antitumor drug includes any one of gemcitabine, doxorubicin, and daunorubicin.

[0008] Preferably, the traditional Chinese medicine polyphenolic compound containing the ortho-diphenol structure includes any one of epigallocatechin gallate, ellagic acid, tanshinone, myricetin, quercetin, and tannic acid; The natural polyamines include any one of spermidine, polyethyleneimine, diethylenetriamine, 3,3'-diaminodipropylamine, putrescine, and spermine; The formylphenylboronic acid includes any one of 2-formylphenylboronic acid, 3-formylphenylboronic acid, and 4-formylphenylboronic acid.

[0009] More preferably, the traditional Chinese medicine polyphenol compound containing the ortho-diphenol structure is epigallocatechin gallate, the natural polyamine is spermidine, and the formylphenylboronic acid is 2-formylphenylboronic acid.

[0010] This invention also provides a method for preparing the aforementioned nanoprodrug assembly, comprising the following steps: Gemcitabine, epigallocatechin gallate, spermidine, and 2-formylphenylboronic acid are self-assembled via dynamic covalent imine-boronic acid ester bonds.

[0011] Preferably, the method for forming by dynamic covalent bond self-assembly includes: The traditional Chinese medicine polyphenolic compound containing the ortho-diol structure is dissolved in water or an organic solvent to prepare a polyphenol solution; Natural polyamines are dissolved in water to prepare polyamine solutions; Gemcitabine and formylphenylboronic acid were dissolved in organic solvents to prepare gemcitabine solution and formylphenylboronic acid solution, respectively. The polyamine solution, gemcitabine solution, and formylphenylboronic acid solution are mixed to obtain a premix; under stirring conditions, the polyphenol solution is added dropwise to the premix, and stirring is continued until micelles are completely formed, thus obtaining the nano-prodrug assembly.

[0012] Preferably, in the nano-prodrug assembly, the molar ratio of gemcitabine, polyamine, formylphenylboronic acid and traditional Chinese medicine polyphenol compound containing an ortho-diol structure is 2:(0.5-2):(3-6):(0.5-4).

[0013] Preferably, the organic solvent includes dimethyl sulfoxide.

[0014] The present invention also provides the use of the aforementioned nanoprodrug assembly in the preparation of medicaments for treating breast cancer, ovarian cancer and / or cervical cancer.

[0015] The present invention also provides the application of the aforementioned nanoprodrug assembly in the preparation of intelligent drug delivery systems with pH and / or ROS-responsive release properties.

[0016] The present invention also provides a medicament for treating breast cancer, ovarian cancer and / or cervical cancer, comprising the aforementioned nanoprodrug assembly.

[0017] The present invention discloses the following technical effects: (1) Structure and release synergy The drug ratio is precisely controllable, avoiding the problem of imbalance caused by physical mixing; The nanoprodrug assembly has a stable structure and exhibits dual pH and ROS responsiveness, enabling intelligent triggering release in the tumor microenvironment, thereby increasing local drug concentration in the tumor and reducing systemic toxicity.

[0018] (2) Promotes synergistic effects between drug endocytosis and pharmacokinetics Compared to drug-to-cell administration alone, nano-prodrug assemblies can enhance the ability of drugs to enter cells and simultaneously release drugs in the low pH and high ROS environment of tumor cell lysosomes, ensuring the combined effect of drugs.

[0019] (3) Pharmacodynamic synergy Compared to physically mixed drugs, nanoprodrug assemblies constructed using dynamic covalent chemical reactions can induce a higher proportion of apoptosis, promote ferroptosis in tumor cells, and enhance the autophagy capacity of tumor cells.

[0020] In summary, this invention achieves orderly synergy between gemcitabine and active ingredients of traditional Chinese medicine at multiple levels, including carrier construction, drug metabolism, and pharmacodynamic synergy, through a dynamic covalent chemical strategy. It has shown therapeutic potential in killing breast cancer and ovarian cancer cells, providing a new method for constructing novel nanomedicine systems with high synergy and clinical translational potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the process of constructing a nanoprodrug assembly (DCNA) and its entry into tumor cells to exert its effects is shown, with the structure of the A1B1C1D1 nanoprodrug assembly (A1, spermidine; B1, 2-formylphenylboronic acid; C1, epigallocatechin gallate; D1, gemcitabine) as an example. Figure 2 Here are the compound structural formulas for modules A, B, and C used in the preparation of DCNA; Figure 3 To screen the particle size of DCNAs formed by different combinations (A1-A3 combined with other modules); Figure 4 To screen the particle size of DCNAs formed by different combinations (A4-A6 combined with other modules); Figure 5The dynamic covalent bond formation and structural properties of DCNA formed from epigallocatechin gallate (EGCG) and spermidine (Spd) were characterized as an example; preparation and characterization of DCNA; (A) photographs of the Tyndall effect observed in the prepared Mix (physical mixture of drugs without 2-FPBA) and DCNA; (B) particle size distribution of DCNA (n=3); (C) Zeta potential of DCNA (n=3); (D) NMR of Gem, EGCG, Spd, and 2-FPBA. 1 H NMR, (E)2-FPBA 11 B. NMR spectroscopy; (F) Transmission electron microscopy observation of DCNA structure; (G) Infrared spectral analysis of DCNA; (H) Ultraviolet-Vis absorption spectrum of DCNA; (I) Changes in particle size of nano-prodrug assemblies under pH and / or ROS conditions; Figure 6 To determine the survival rates of OVCAR-3 (A), HeLa (B), and 4T1 (C) cells after treatment with different concentrations of DCNA, Mix, and Gem using the CCK-8 assay (n=3). Figure 7 To study the endocytosis efficiency of nanomedicines using DCNA as an example, OVCAR-3 cells were used. (A) Flow cytometry was used to detect the endocytosis rate of DCNA and Mix at 1, 2, and 6 h. The figure shows the efficiency of cell uptake of the two nanoparticles (DCNA and Mix) at different time points, reflecting their endocytosis kinetics. (B) Statistical analysis of endocytosis rate (n=3). (C) Confocal microscopy images of OVCAR-3 cells after incubation with PBS, Mix, and DCNA for 6 h. Nile red fluorescent labeling was used to locate the distribution of nanoparticles in the cells, visually presenting the intracellular uptake and distribution characteristics of DCNA and Mix. (D) Fluorescence images showing the co-localization of nanoparticle prodrug assemblies and lysosomes at different time points. (E) Statistical analysis of data in Figure D. (F) Co-localization of nanoparticle prodrug assemblies and lysosomes at 2 h. (G) Co-localization of nanoparticle prodrug assemblies and lysosomes at 4 h. (H) Co-localization of nanoparticle prodrug assemblies and lysosomes at 8 h. Figure 8Taking DCNA as an example, this study used OVCAR-3 cells to observe the differences in gene levels among different groups after different drug treatments, and transcriptome sequencing revealed these differences. (A) Principal component analysis (PCA) of the transcriptome of OVCAR-3 cells under different drug treatments showed the biological reproducibility and inter-group differences in the overall expression profiles of the PBS, GEM, EGCG, and DCNA treatment groups. (BD) Volcano plot analysis of differentially expressed genes (DEGs) among the DCNA vs PBS, EGCG vs DCNA, and DCNA vs GEM treatment groups, respectively; red dots represent significantly upregulated genes, and green dots represent significantly downregulated genes (screening criteria: |log2FC|>1 and p<0.05). (E) Heatmap analysis of the expression levels of core differentially expressed genes related to ferroptosis among the groups, showing the Top 5 pathways that were significantly downregulated, involving cell proliferation and migration regulation. (FG) WikiPathways differential pathway enrichment analysis between the DCNA group and the PBS group; significantly upregulated Top 5 pathways. Four pathways were identified, with a focus on enrichment in ferroptosis and stress response networks; bubble size represents the number of enriched genes (Count), and color intensity represents the significance level (-log10_Qvalue). Figure 9 This study investigated the level of ferroptosis induced by DCNA at the cellular level; (AB) Assessment of DCNA-induced intracellular reactive oxygen species (ROS) accumulation in OVCAR-3 cells, with representative ROS fluorescence images (A) and quantitative analysis (B) obtained by CLSM (scale bar = 50 μm); (C, D) Histograms of ROS fluorescence distribution (C) and statistical analysis of average fluorescence intensity (D) obtained by flow cytometry; (EF) Flow cytometry detection of Fe2+ labeled with FerroOrange probe in each group. 2+ Fluorescence distribution histogram (E) and quantitative statistics (F); (G) CLSM observation of intracellular ferrous ion fluorescence imaging in each group (red: Fe) 2+ (Blue: cell nucleus) and (H) quantitative analysis (scale bar = 50 μm); (IL) qRT-PCR detection of the relative mRNA expression levels of ferroptosis-related genes NCOA4, ACSL4, GPX4 and HMOX1 in OVCAR-3 cells after treatment in each group; data are expressed as Mean ± SD. p<0.05, p<0.01, p<0.001, p<0.0001; Figure 10Taking DCNA as an example, a mouse tumor model was constructed using OVCAR-3 cells to detect the inhibitory effect of nanomedicine on tumors; (A) Anatomical image of the dissected tumor; (B) Dynamically monitored tumor growth curve; (C) Trend of tumor volume change; (D) Detailed diagram of tumor growth; (E) Weight of tumors in different groups; (F) Ki67 immunohistochemical staining results; (G) TUNEL immunofluorescence staining results; (HK) respectively NCOA4 , ACSL4, GPX4, HMOX1 The expression situation; Figure 11 Results of in vivo hemolysis and biosafety studies; (A) Hemolysis of the nano-prodrug assembly at concentrations of 0-250 µg / mL; (B) Hemolysis rate of the nano-prodrug assembly at concentrations of 0-250 µg / mL; (C) Blood parameters after treatment with the nano-prodrug assembly. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides a nano-prodrug assembly of gemcitabine and traditional Chinese medicine components based on dynamic covalent chemistry, its preparation method, and its application in anti-ovarian cancer (see flowchart). Figure 1 This nano-prodrug assembly comprises four modules, which are self-assembled into the following components: Module A (Polyamine Functional Regulatory Components): Selected from spermidine (A1), polyethyleneimine (PEI, molecular weight 800, A2), diethylenetriamine (DETA, A3), 3,3'-diaminodipropylamine (DPTA, A4), putrescine (A5), and spermine (A6). Its main functions are to regulate the surface charge of the assembly, promote cellular uptake, and may play an adjunctive antitumor role.

[0029] 2. Module B (Dynamically Covalently Linked and Responsive Component): Selected from bifunctional molecules containing both aldehyde and phenylboronic acid groups, preferably formylphenylboronic acid, including 2-formylphenylboronic acid (2-FPBA, B1), 3-formylphenylboronic acid (3-FPBA, B2), 4-formylphenylboronic acid (4-FPBA, B3), or their structural analogs. This module acts as a molecular bridge; its aldehyde group can form an imine bond with the primary amine group of the A module's structural domain, and its phenylboronic acid group can form a borate ester bond with the ortho-dihydroxyl group of the C module's structural domain, thereby dynamically linking different modules and endowing the entire assembly with responsiveness to the tumor microenvironment (low pH, high ROS).

[0030] 3. Module C (Functional Components of Traditional Chinese Medicine): Selected from active ingredients of traditional Chinese medicine containing ortho- and tho-dihydroxy groups, such as epigallocatechin gallate (EGCG, C1), ellagic acid (C2), salvianolic acid (C3), myricetin (C4), quercetin (C5), and tannic acid (C6). These components act as nano-scaffold components and exert multiple functions, including synergistic anti-tumor activity and reversal of drug resistance.

[0031] 4. Module D (Core Chemotherapy Drugs): Gemcitabine (Gem, D1), Doxorubicin Hydrochloride (DOX, D2), and Daunorubicin (Dua, D3). As the main effector molecules in antitumor therapy, their primary amine groups participate in the formation of dynamic covalent bonds.

[0032] The four modules mentioned above are interconnected through reversible dynamic covalent bonds, encompassing a wide range of possible combinations. This allows for the rapid construction of a series of nano-prodrug assemblies with different functions, forming a platform-based technology. The following example uses the A1B1C1D1 nano-prodrug assembly (A1, spermidine; B1, 2-formylphenylboronic acid; C1, epigallocatechin gallate; D1, gemcitabine; specific chemical structures can be found in [link to chemical structure]). Figure 2 Taking [example 1] as an example, the preparation of all other combined nanoprodrug assemblies is similar to the steps described below. The particle size of the nanoparticles formed by dynamic covalent bonds from different modules is as follows: Figure 3 and Figure 4 As shown, this method confirms that nanomedicines with particle sizes in the range of 100-800 nm can be formed.

[0033] Example 1: A method for preparing a nano-prodrug assembly of gemcitabine and traditional Chinese medicine components based on dynamic covalent chemistry 1. Experimental Materials Taking the preparation of A1B1C1D1 nanoprodrug assemblies as an example, gemcitabine (Gem), epigallocatechin gallate (EGCG), spermidine (Spd), 2-formylphenylboronic acid (2-FPBA), and phosphate buffered saline (PBS, pH 7.4) can all be purchased through conventional commercial channels.

[0034] 2. Take appropriate amounts of Gem, Spd, 2-FPBA, and Gem respectively, and prepare each stock solution as follows: EGCG: Dissolve in ultrapure water to prepare a 10 mg / mL EGCG solution; Spd: Dissolve in ultrapure water to prepare a 20 mg / mL Spd solution; 2-FPBA: Dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mg / mL 2-FPBA solution; Gem: Dissolved in dimethyl sulfoxide to prepare a 10 mg / mL Gem solution.

[0035] Subsequently, a premix was prepared by mixing gemcitabine, spermidine, and 2-formylphenylboronic acid in a molar ratio of 1:1:3. Under stirring conditions at room temperature and 300 rpm, EGCG solution was slowly added dropwise to the premix at a rate of 1 drop every 10 seconds, with a molar ratio of EGCG to Gem of 1:1. Stirring was continued for 0.5 h, and a colloidal solution spontaneously formed. Figure 5 (A) is used to obtain the nano-prodrug assembly.

[0036] Example 2: Physicochemical characterization of nanoprodrug assemblies 1. Particle size and potential analysis Experimental group sample dilution: Take 100 μL of the nano-prodrug assembly prepared in Example 1, add 900 μL of PBS buffer (pH 7.4), and gently invert to mix 3-5 times. Control group samples (Mix group): Treat the Mix prepared in the physical mixing group (see the preparation method of Mix in Example 2) in the same way as the experimental group. Set instrument parameters: temperature 25.0 ± 0.1℃, equilibration time 120 s, and measure each sample 3 times consecutively. Record the hydrodynamic diameter (Z-Average, d.nm) as follows. Figure 3 As shown, different reaction modules A, B, C, and D can all react and assemble in an aqueous system. Their particle size is between 100 and 600 nm, PDI is between 0.1 and 0.5, and the Zeta potential is mostly negative.

[0037] like Figure 5 As shown in Figure B, the results indicate that the Z-Average of the nano-produce A1B1C1D1 of this invention is 126.73 ± 3.1 nm, and the PDI is 0.234 ± 0.04. The Z-Average of the Mix group sample is 2036 ± 110.1 nm, and the PDI is 0.26 ± 0.031. This demonstrates that the method of this invention can enable the four components to interact and form nanoparticles.

[0038] 1.3 Zeta potential test Inject the same diluted sample into a dedicated folded potential cell (Malvern DTS1070) to ensure that there are no air bubbles in the cell.

[0039] Parameter settings: temperature 25.0℃, equilibration time 60s, 5 consecutive measurements per sample. Select “ZetaPotential - M3-PALS” mode, and the instrument will automatically record the zeta potential (mV) and its distribution.

[0040] like Figure 5 As shown in Figure C, the results show that the Zeta potential of the nano-prodrug assembly A1B1C1D1 of the present invention is -15 mV, which is significantly higher than that of the Mix group, indicating that the nano-prodrug assembly prepared by the present invention has a completely different surface charge situation compared with the physical mixing group.

[0041] 2. Spectroscopic characterization of drugs: infrared spectroscopy, nuclear magnetic resonance. 1 H NMR, 11 Characterization by B NMR, transmission electron microscopy (TEM), and ultraviolet-visible spectroscopy.

[0042] The nano-prodrug assembly A1B1C1D1 was freeze-dried (-50 °C, 48 h) to obtain a dry, loose solid powder. Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy were used to measure the infrared and nuclear magnetic resonance spectra of each individual active pharmaceutical ingredient (Gem, EGCG, Spd, 2-FPBA) under the same conditions. 1 H NMR, 11 B NMR spectrum.

[0043] like Figure 5 As shown in D, 1 ¹H NMR showed that the characteristic hydrogen peak of the aldehyde group in 2-FPBA decreased significantly after the reaction. 11 B NMR spectroscopy showed that sp in 2-FPBA 2 After the boric acid structure is formed into nanoparticles, it transforms into sp. 3 The structure of the borate ester structure ( Figure 5 Transmission electron microscopy revealed that the nanoparticles exhibited a regular spherical structure, with elements such as C, N, O, F, and B uniformly distributed in low-level distributions within the nanoparticles. Figure 5 (Middle F). In infrared spectroscopy analysis, it appears at ~1000 cm⁻¹. -1 The nearby absorption peaks are attributed to characteristic vibrations of the B–O–C structure, further confirming the successful construction of boronic ester bonds in the system. Meanwhile, at ~1650 cm⁻¹... -1 The absorption peak observed at this point corresponds to the stretching vibration mode of the C=N bond, proving the formation of the imine bond. Figure 5 The UV-Vis absorption spectrum of the nano-prodrug assembly shows a characteristic absorption at 257 nm, which can be considered as a superposition and fusion of the characteristic absorptions of 2-FPBA (254 nm) and Gem (268 nm). This indicates that both components are successfully loaded in the nano-assembly, and its electronic absorption environment is adjusted due to the formation of dynamic covalent bonds and nanoscale stacking. Figure 5 (H). Furthermore, the particle size of this nanoprodrug assembly changes under low pH and high ROS conditions in tumor tissue. Figure 5 (I) This change is due to the pH and ROS responsiveness of the formed imine-boronate bonds.

[0044] Preparation of assembled drug samples: Take an appropriate amount of the nanoprodrug assembly prepared in Example 1 and dilute it appropriately with buffer (PBS, pH 7.4) to ensure that the absorbance of the sample to be tested is within the linear range of the instrument (usually controlled between 0.1-1.0 Abs). Use a UV-Vis spectrophotometer, set the spectral scanning range to 200–800 nm, and the scanning speed to medium speed (e.g., 200 nm / min).

[0045] control sample ① The steps for synthesizing physical mixtures: Take appropriate amounts of epigallocatechin gallate, spermidine, and gemcitabine respectively, and prepare stock solutions as follows: EGCG: Dissolve in ultrapure water to prepare a 10 mg / mL EGCG solution; Spd: Dissolve in ultrapure water to prepare a 20 mg / mL Spd solution; Gem: Dissolved in dimethyl sulfoxide to prepare a 10 mg / mL Gem solution.

[0046] The above-mentioned stock solutions were mixed at the same drug molar ratio as those used for preparing the nano-prodrug assemblies, with the 2-FPBA solution replaced by an equal volume of ultrapure water. The mixed solutions were then gently agitated to obtain a physically mixed control group sample, labeled Mix.

[0047] ② Single-drug group tests were conducted under the same conditions: Gem, 2-FPBA, Spd, and EGCG.

[0048] Example 3: Evaluation of in vitro antitumor effects 1. Cell lines: OVCAR-3 (human ovarian cancer cells), HeLa cells (cervical cancer cells), 4T1 (breast cancer cells).

[0049] 2. Grouping DCNA group: Cells were treated with the nanoprodrug assembly A1B1C1D1 prepared in Example 1; Mix group: Cells were treated with Mix solution prepared using a physical mixing method; Gem monotherapy group: Cells were treated with Gem; PBS control group: Cells were treated with PBS.

[0050] 3. CCK-8 Experiment (1) When the tumor cells reached a cell density of 70–80%, a single-cell suspension was prepared by trypsin digestion. 8000 cells were seeded into each well of a 96-well plate, with 100 μL of the single-cell suspension added to each well. The seeded plates were incubated in a cell culture incubator for 4 h. After confirming cell adhesion under a microscope, the supernatant was aspirated, and different concentrations of Gem, nano-prodrug assembly A1B1C1D1, or Mix were added to the wells. The concentration of Gem was kept consistent across all components, with final drug concentrations of 0, 20, 40, and 80 μg / mL, respectively. The plates were incubated in the cell culture incubator for another 36 h. After incubation, the supernatant was aspirated under dark conditions, and 100 μL of 1640 medium containing 10% FBS and 10 μL of CCK-8 solution were added to each well. The plates were returned to the incubator for further incubation until the absorbance (OD value) of the control group reached between 0.8 and 1.2. The culture plate was wrapped with aluminum foil to protect it from light, and the absorbance of each well was measured at 450 nm using a microplate reader. A cell growth curve was plotted with drug concentration on the x-axis and absorbance on the y-axis, and the drug inhibition rate was calculated using the following formula: Inhibition rate = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%.

[0051] like Figure 6 As shown in Figure A, after 36 h of treatment with OVCAR-3 ovarian cancer cells in the Gem, DCNA, and Mix groups, the IC50 concentration was significantly reduced. 50 The values ​​were 29.58 μg / mL, 7.95 μg / mL, and 18.4 μg / mL, respectively. Based on the CCK-8 assay results, a drug concentration range of 15–30 μg / mL was selected for subsequent cell cycle and apoptosis experiments of the nanoprodrug assembly system.

[0052] like Figure 6 As shown in Figure B, after 36 h of treatment with HeLa cervical cancer cells in the Gem, DCNA, and Mix groups, the IC50 concentration was significantly higher than that of the target cells. 50 The values ​​were 26.96 μg / mL, 13.37 μg / mL and 19.96 μg / mL, respectively.

[0053] like Figure 6 As shown in Figure C, after 36 h of treatment with 4T1 breast cancer cells from the Gem, DCNA, and Mix groups, the IC50 concentration was significantly higher. 50 The values ​​were 10.67 μg / mL, 4.71 μg / mL and 10.49 μg / mL, respectively.

[0054] The above results confirm that the nano-prodrug assembly can significantly improve the killing ability of a broad spectrum of tumor cells, including OVCAR-3 ovarian cancer cells, HeLa cervical cancer cells, and 4T1 breast cancer cells, compared with Gem monotherapy or physical combination therapy of different drugs.

[0055] Meanwhile, this invention also tested the killing effect of nanoassemblies or physical mixtures prepared by replacing Gem with other drugs on OVCAR-3 ovarian cancer cells, and the results are shown in Table 1.

[0056] Table 1 IC 50 Test Results 4. Cell endocytosis experiment and lysosomal escape experiment In the endocytosis assay, OVCAR-3 cells were used as the representative cell line. When the cell density reached 70-80%, trypsin digestion was performed. After terminating digestion with complete culture medium, 2 × 10⁶ cells were added to each well. 5 Cells were seeded into 12-well plates at a density of 1000 cells / well and cultured in an incubator for 4 h. 1 mg of Nilered was dissolved in 1 mL of DMSO to prepare a 1 mg / mL stock solution. Nanoparticle preparation: 528 µL of PBS (pH 7.4) buffer, 100 µL of gemcitabine solution, 85.5 μL of 2-FPBA solution, 10 μL of Nilered stock solution, 27.5 μL of spermidine solution, and 174 μL of EGCG solution were added sequentially to the sample vials, and the mixture was stirred using a magnetic stirrer. After synthesis, the solution was added to an ultrafiltration tube and centrifuged at 8000 rpm for 10 min. The lower filtrate was discarded, and the upper residue was dissolved in PBS (pH 7.4) buffer and mixed thoroughly. The Nilered concentration was determined using a UV-Vis spectrophotometer to ensure that the Mix group and the A1B1C1D1 group had the same Nilered concentration. After cell adhesion, a portion of the cells were collected and incubated with PBS, A1B1C1D1, and a mixture, respectively. After incubation in the dark for 1 h, 6 h, and 12 h, the cells were washed with pre-cooled PBS to terminate endocytosis. The cells were then digested from the wells and collected as single-cell suspensions. The mean intracellular fluorescence intensity was detected using flow cytometry and laser confocal microscopy.

[0057] Subsequently, in parallel experiments, lysosomal staining experiments were performed on cells at different times to monitor the co-localization of nanomedicine and lysosomes and to analyze its lysosomal escape ability.

[0058] Experimental results: such as Figure 7 As shown in the AC diagram, both confocal microscopy and flow cytometry results indicate that the cell uptake efficiency of the DCNA group was higher than that of the Mix group. Figure 7The DH assay showed that the nano-prodrug assembly and lysosomes had strong co-localization ability at 2 h, but this ability weakened at 8 h. This demonstrates that the nano-prodrug assembly can achieve lysosomal escape into the cytoplasm to exert its cytotoxic effect.

[0059] 7. Transcriptome sequencing OVCAR-3 cells were used as the representative cell line for the endocytosis experiment. Cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded into 12-well plates at a density of 100 cells / mL and stimulated with PBS, Gem, EGCG, and A1B1C1D1 (20 μg / mL), respectively. Transcriptome sequencing was then performed.

[0060] Experimental results: such as Figure 8 As shown, the enrichment of key genes, KEGG pathway, and GSEA indicates that treatment with the nanoprodrug assembly can significantly enhance the ferroptosis pathway.

[0061] Example 4: Study on nanomedicines promoting ferroptosis pathway in tumor cells The endocytosis experiment used OVCAR-3 cells as the representative cell line to study the effects of different drug components on ROS elevation and intracellular Fe in the OVCAR-3 ferroptosis pathway. 2+ The effects of enrichment.

[0062] Experimental results: such as Figure 9 As shown in the middle AE, compared with the Mix group, the DCNA group showed a significant increase in intracellular ROS (manifested as a significant increase in the green fluorescence intensity of the reactive oxygen species dye DCFH-DA), while Fe... 2+ The content of [unclear] increased significantly (manifested as intracellular Fe [unclear]). 2+ The red fluorescence intensity of the dye Ferro-orange increased significantly. This demonstrates that nanomedicine therapy can significantly promote the ferroptosis pathway and kill tumor cells.

[0063] Example 5: In vivo anti-tumor effect study 1. Animal model: BALB / c nude mice were selected. After disinfecting the right axillary region of the mice with 75% ethanol cotton balls, 100 μL of cell suspension (containing 5×10⁻⁶ cells) was drawn using a 1 mL sterile syringe. 7 One OVCAR-3 cell was slowly injected subcutaneously into the right axilla of a nude mouse. The mice were observed daily after inoculation, and the long diameter (L) and short diameter (W) of the tumor were measured with calipers every two days.

[0064] 2. After tumor formation, different drugs were injected subcutaneously into the tumor using an insulin injector: PBS group, Gem monotherapy group, Mix group, DCNA group (the equivalent dose of Gem in the nanoprodrug assembly A1B1C1D1 was 7.5 mg / kg) for treatment, and changes in tumor volume and body weight were monitored.

[0065] The results showed that the DCNA group significantly inhibited tumor growth in vivo. (See the anatomical image of the dissected tumor.) Figure 10 In the DCNA group (Category A), the tumor volume was significantly smaller than that in the PBS treatment group, GEM group, and Mix group. Dynamically monitored tumor growth curves (Category A) Figure 10 The trend was clearly demonstrated in the study (Case B): from day 12 of treatment, the tumor volume growth in the DCNA group began to slow significantly; by day 32, the endpoint of the experiment, its average tumor volume was only about 40% of that in the other groups, and its growth curve was the flattest. Figure 10 (C) Detailed tumor growth diagram for each mouse ( Figure 10 (D) further confirmed that the tumor-suppressing effect of the DCNA group showed good consistency and reproducibility among samples. The weighing results of the dissected tumor ( Figure 10 The results (E) also showed that the DCNA group had the lightest average tumor weight, which was highly consistent with the trend of volume change. To further explore the cellular mechanism by which the nano-prodrug assembly inhibits tumor growth, this invention performed proliferation and apoptosis-related tests on tumor tissue sections. Ki67 immunohistochemical staining results showed ( Figure 10 In the DCNA group, the proportion of Ki67-positive proliferating cells (brown nuclei) in the tumor tissue was significantly lower than that in the control group, indicating that the proliferative activity of the tumor tissue was significantly inhibited. Simultaneously, TUNEL immunofluorescence staining results ( Figure 10 The results showed that the number of apoptotic cells (green fluorescence) in the DCNA group tumor tissue was significantly increased, suggesting that the nano-prodrug assembly can also effectively induce programmed cell death in ovarian cancer cells in vivo. Based on the previous in vitro experiments, the mRNA expression levels of core regulatory genes of ferroptosis in tumor tissue were further detected by qRT-PCR. The results confirmed that the nano-prodrug assembly successfully remodeled the iron metabolism homeostasis of tumor cells and activated the ferroptosis pathway in vivo. Compared with the PBS group, the nano-prodrug assembly treatment significantly upregulated proferroptosis-related genes. NCOA4 , ACSL4 and HMOX1 The expression, particularly crucially, shows that the nano-prodrug assembly significantly downregulated key negative regulators of ferroptosis. GPX4 mRNA expression ( Figure 10(HK). This change in gene expression profile indicates that the nano-prodrug assembly successfully achieved precise inhibition of the tumor suppressor defense system in vivo. Combined with the aforementioned proliferation inhibition and apoptosis induction effects, these results collectively reveal the molecular mechanism by which the nano-prodrug assembly exerts its powerful in vivo antitumor activity through the synergistic effect of apoptosis and ferroptosis.

[0066] Example 6: In vivo hemolysis and biosafety study One mL of fresh mouse blood was diluted with 10 mL of phosphate buffer and thoroughly mixed. The mixture was then centrifuged at 4000 rpm for 5 minutes. The supernatant was discarded, and the precipitated red blood cells were collected. The cells were washed five times with 10 mL of phosphate buffer to obtain a purified red blood cell pellet. The red blood cells were then resuspended in the same buffer to prepare a 4% (v / v) red blood cell suspension. An equal volume of the red blood cell suspension was mixed with different concentrations of the test material solutions and incubated at 37°C for 1 hour. After incubation, the mixture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected. The absorbance at 545 nm was measured using a microplate reader. Red blood cell systems treated with Triton X-100 solution and physiological saline were used as positive and negative controls, respectively.

[0067] Hemolysis rate is calculated using the following formula: Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100% In the formula, OD sample, OD negative control and OD positive control represent the absorbance measurements at 545 nm of the test sample group, negative control group and positive control group, respectively.

[0068] Results analysis: A hemolysis rate exceeding 5% was considered mild hemolysis, 10% to 50% was moderate hemolysis, and above 50% was considered significant hemolysis. Figure 11 As shown in Figure AB, the hemolysis rate of the nano-prodrug assembly was less than 5% at concentrations ranging from 0 to 250 µg / mL, demonstrating its good biocompatibility.

[0069] Furthermore, different treatments were administered to mice, and their blood parameters were monitored. The results showed that the blood parameters after treatment with the nano-prodrug assembly were all within the healthy range. Figure 11 (C) proves that it has good in vivo biocompatibility.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nanoprodrug nanoassembly for antitumor drugs based on dynamic covalent chemistry, characterized in that, The nano-prodrug assembly is formed by the dynamic covalent self-assembly of an antitumor drug, a traditional Chinese medicine polyphenol compound containing an ortho-diphenol structure, a natural polyamine, and formylphenylboronic acid; the antitumor drug includes any one of gemcitabine, doxorubicin, and daunorubicin.

2. The nanoprodrug assembly as described in claim 1, characterized in that, The traditional Chinese medicine polyphenolic compounds containing the ortho-diphenol structure include any one of epigallocatechin gallate, ellagic acid, tanshinone, myricetin, quercetin, and tannic acid; The natural polyamines include any one of spermidine, polyethyleneimine, diethylenetriamine, 3,3'-diaminodipropylamine, putrescine, and spermine; The formylphenylboronic acid includes any one of 2-formylphenylboronic acid, 3-formylphenylboronic acid, and 4-formylphenylboronic acid.

3. The method for preparing the nanoprodrug assembly as described in claim 1 or 2, characterized in that, Includes the following steps: Gemcitabine, traditional Chinese medicine polyphenols containing ortho-diol structures, natural polyamines, and formylphenylboronic acid are formed through dynamic covalent self-assembly.

4. The preparation method according to claim 3, characterized in that, The method of forming via dynamic covalent bond self-assembly includes: The traditional Chinese medicine polyphenolic compound containing the ortho-diol structure is dissolved in water or an organic solvent to prepare a polyphenol solution; Natural polyamines are dissolved in water to prepare polyamine solutions; Gemcitabine and formylphenylboronic acid were dissolved in organic solvents to prepare gemcitabine solution and formylphenylboronic acid solution, respectively. The polyamine solution, gemcitabine solution, and formylphenylboronic acid solution are mixed to obtain a premix; under stirring conditions, the polyphenol solution is added dropwise to the premix, and stirring is continued until micelles are completely formed, thus obtaining the nano-prodrug assembly.

5. The preparation method according to claim 4, characterized in that, In the nano-prodrug assembly, the molar ratio of gemcitabine, polyamine, formylphenylboronic acid, and traditional Chinese medicine polyphenols containing an ortho-diol structure is 2:(0.5-2):(3-6):(0.5-4).

6. The preparation method according to claim 4, characterized in that, The organic solvent includes dimethyl sulfoxide.

7. The use of the nanoprodrug assembly as described in claim 1 or 2 in the preparation of medicaments for treating breast cancer, ovarian cancer and / or cervical cancer.

8. The use of the nanoprodrug assembly as described in claim 1 or 2 in the preparation of a smart drug delivery system with pH and / or ROS-responsive release properties.

9. A drug for treating cervical cancer, breast cancer, and / or ovarian cancer, characterized in that, It contains the nanoprodrug assembly as described in claim 1 or 2.

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