Stimuli-responsive linear-dendritic block polymers and pharmaceutical uses thereof

Abstract

The application provides a kind of stimulus-responsive linear-dendritic block polymer and its pharmaceutical use, belong to the field of polymer carrier.The application prepares a kind of linear-dendritic block polymer-doxorubicin prodrug POEGMA-GFLG-(DOX)4 based on poly(ethylene glycol) methyl ether methacrylate and peptide dendrimer by reversible addition-fragmentation chain transfer polymerization.Subsequently, hydrophobic photosensitizer Ce6 is encapsulated in the nanoparticles assembled by prodrug, so as to obtain a linear-dendritic block polymer-based drug delivery system LD-DOX / Ce6 which can be used for combined treatment of cancer.LD-DOX / Ce6 shows synergistic chemophotodynamic combined antitumor efficacy (CI value is far lower than 1), which can significantly inhibit tumor growth and metastasis, and effectively reduce the toxic side effects of drugs.The linear-dendritic block polymer provided by the application has wide application prospect in the preparation of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polymeric carriers, and particularly relates to a stimuli-responsive linear-dendritic block polymer and its pharmaceutical use. BACKGROUND

[0002] Although the stimuli-responsive polymeric nanodrug delivery system (NDDS) has great potential for clinical application, its performance can still be further improved by optimizing the chemical composition, molecular structure, and molecular weight (MW), etc. Encapsulating or conjugating multiple drugs into a rationally designed multifunctional polymeric carrier can increase the solubility and stability of the drugs, and achieve tumor targeting through enhanced permeability and retention (EPR) effect, so as to minimize the toxic side effects and improve the therapeutic effect through combination therapy. Traditional polymeric structures for drug delivery and combination cancer therapy are mainly linear amphiphilic block polymers, which are relatively simple in structure and difficult to modify. In the past decade, dendritic polymers have been studied as new polymeric carriers due to their rich surface functional groups, globular conformation, and nanoscale size, etc. However, some shortcomings limit their application in cancer therapy. Generally, therapeutic drugs are mainly physically encapsulated in the internal cavity of dendritic polymers or chemically conjugated to the dendritic surface. For the physical encapsulation method, high-generation dendritic macromolecules have good encapsulation capacity, but their preparation is difficult and their biocompatibility is poor; while low-generation dendritic macromolecules are simple to prepare, but have low density and cannot retain drugs under sink conditions, thus often leading to inevitable burst release. For the chemical conjugation method, surface modification usually requires tedious synthesis steps and changes the physical and chemical properties of the carrier, such as reducing the solubility of the dendritic polymer. In addition, low-generation dendritic macromolecules usually have a small particle size (<20 nm), which makes them easily cleared from the body by glomerular filtration when used as drug carriers.

[0003] With the progress of synthetic chemistry technology, the polymeric structure has evolved from linear and dendritic to more complex branched topologies, thus further providing opportunities for improving the therapeutic effect of cancer. Linear-dendritic block polymers (LDBCs) combine the unique topological structure of branched polymers and the self-assembly characteristics of block linear polymers, and have unique advantages in the development of drug delivery systems. The dendritic / branch structure of LDBCs has multivalency, which can effectively load therapeutic drugs, and at the same time, the linear polymer segment endows it with conformational flexibility, which can form a micelle-like structure. Therefore, LDBCs can encapsulate or conjugate multiple therapeutic drugs for combination therapy of tumors.

[0004] Currently, there are some reports on the development of LDBCs as a new drug delivery system. Due to the multifunctionality and special structural features of dendrimers, LDBCs significantly improve the drug delivery efficiency and therapeutic effect compared with their linear analogues. At present, LDBCs are mainly prepared by coupling strategy, 'chain-first' strategy or 'dendron-first' strategy, and the synthesis method is highly dependent on efficient click reaction or step-by-step polymerization of macromolecules. For example, poly(ethylene glycol) (PEG) is one of the most commonly used linear polymer chains in the preparation of LDBCs, because it has good hydrophilicity and structural flexibility. However, linear mPEG or PEG can usually only be conjugated at one or two reaction sites at the end. The entanglement of high molecular weight PEG chains has high steric hindrance, which leads to low reaction efficiency of LDBCs prepared by coupling strategy, because it depends on the direct coupling of dendrimers with linear chains. Other researchers reported that the molecular weight of PEG used in LDBCs is relatively small, usually 5kDa or 10kDa. Considering the drug loading capacity, solubility and self-assembly performance, this relatively low molecular weight range of hydrophilic fragments may limit the selection of hydrophobic fragments in LDBC. In addition, for LDBCs synthesized by 'chain-first' strategy, the preparation of high-generation dendrimers requires a large number of end groups to be converted, and this step requires a very high yield reaction, otherwise it will cause defects in the molecular structure. At the same time, due to the inevitable shielding effect of polymer chains, the synthesis process is relatively complex, and the obtained product is also difficult to characterize.

[0005] In order to overcome the above problems, it is of great significance to develop a kind of LDBCs with high molecular weight, high reactivity and simple preparation method, and it is of great significance to develop a kind of new polymer carrier which can realize efficient drug delivery and therapeutic effect by regulating the composition, structure and function of LDBCs. SUMMARY

[0006] The present application aims to provide a new kind of stimuli-responsive linear-dendritic block polymer and its pharmaceutical use.

[0007] The present application provides a polymer shown in formula I:

[0008]

[0009] wherein x is selected from 20 to 60, and y is selected from 1 to 8.

[0010] R a is R b , R c , R d , R e each independently selected from an amino protecting group.

[0011] Further, x is selected from 43-53, and y is selected from 1-3.

[0012] Further, the amino protecting group is a Boc group.

[0013] Further, M n is 23.7 kgmol -1 , M w is 28.9 kgmol -1 .

[0014] The present application also provides a polymer shown in formula II:

[0015]

[0016] wherein, x is selected from 20-60, and y is selected from 1-8.

[0017] Further, x is selected from 43-53, and y is selected from 1-3.

[0018] Further, the polymer shown in formula II is obtained by deprotecting the polymer shown in formula I and reacting with doxorubicin.

[0019] The present application also provides an anti-tumor drug, which is prepared from the polymer shown in formula II and a photosensitizer.

[0020] Further, the photosensitizer is a hydrophobic photosensitizer.

[0021] Further, the hydrophobic photosensitizer is chlorin e6.

[0022] Further, the mass ratio of the polymer and the photosensitizer is 20:(1-10), preferably 20:(3-4), and more preferably 20:3.24.

[0023] Further, the solvent in the solution of the polymer is methanol, and the solvent in the solution of the photosensitizer is acetone.

[0024] The present application also provides the use of the polymer shown in formula I and the polymer shown in formula II in the preparation of an anti-tumor drug.

[0025] Further, the tumor is breast cancer.

[0026] The present application solves the challenge in the synthesis of PEG-based LDBCs, and provides a new strategy for preparing LDBC-based drug delivery systems and using them for combined cancer therapy. The novel linear-dendritic block polymers of the present application are obtained by directly polymerizing the dendritic monomers with POEGMA-CTA as macro-CTA through RAFT polymerization. The present application uses POEGMA and multifunctional peptide-based dendrimers as linear segments and dendritic parts, respectively, to design and prepare a class of cathepsin B and pH dual-responsive LDBC prodrugs. Through this innovative synthesis method, the hydrophilic / hydrophobic properties, generation, functional groups and molecular weight of the peptide-based dendrimers can be precisely controlled at the molecular level, and the chemical composition and degree of polymerization of the linear segments can also be easily adjusted. The dendrimers are covalently linked to the linear polymer backbone as hydrophobic parts through a Gly-Phe-Leu-Gly (GFLG) tetrapeptide, and DOX is connected to the periphery of the dendritic polymers through a hydrazone bond, thereby preparing a class of cathepsin B and pH dual-responsive LDBC prodrugs. The amphiphilic linear-dendritic block polymer-DOX prodrug can self-assemble into tumor microenvironment-responsive and biodegradable polymer nanoparticles (NPs).

[0027] The present application prepares a class of linear-dendritic block polymers-doxorubicin (DOX) prodrugs based on poly(ethylene glycol) methyl ether methacrylate (POEGMA) and peptide-based dendrimers: POEGMA-GFLG-(DOX)4 (named as LD-DOX) through reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequently, the hydrophobic photosensitizer chlorin e6 (Ce6) is encapsulated in the nanoparticles (NPs) assembled from the prodrugs as a model drug, thereby obtaining an LDBC-based drug delivery system for combined cancer therapy: LD-DOX / Ce6.

[0028] Due to the presence of Gly-Phe-Leu-Gly (GFLG) tetrapeptide and hydrazone bond in the structure of the polymer prodrug, LD-DOX / Ce6 can be degraded into small fragments in the tumor microenvironment, thereby specifically triggering the release of DOX and Ce6. In vivo studies of the system show that LD-DOX / Ce6 significantly improves the in vivo pharmacokinetics of the contained drugs and enhances their biodistribution at the tumor site. Under 660 nm laser irradiation, LD-DOX / Ce6 shows synergistic chemical-photodynamic combined anti-tumor efficacy (CI value far lower than 1), significantly inhibits tumor growth and metastasis, and effectively reduces the toxic side effects of the drugs.

[0029] Therefore, the present application proves that the tumor microenvironment-responsive biodegradable LDBCs have great application potential as an intelligent multifunctional drug delivery carrier for efficient combined cancer therapy in the aspect of efficient anti-tumor.

[0030] Obviously, according to the above content of the present application, other various forms of modification, replacement or change can be made according to the ordinary technical knowledge and common practice in the art without departing from the above basic technical idea of the present application.

[0031] The above content of the present application will be further explained in detail by way of specific embodiments. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 . Prodrug POEGMA-GFLG-(DOX) x and POEGMA-GFGL-(DOX) x Physicochemical property characterization and dissipative particle dynamics (DPD) simulation results of (x = 1, 2 or 4). (a) Schematic design of polymer prodrug structures used for comparison in the present application. (b) POEGMA-GFLG-(DOX) x and POEGMA-GFGL-(DOX) x DOX release profile in the presence of Cathepsin B at pH 5.4. (c) Critical micelle concentration (CMC) of POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2 and POEGMA-GFLG-(DOX)4 (from left to right) measured using pyrene as a fluorescent probe. (d) DOX plasma concentration profiles of free DOX, POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2 and POEGMA-GFLG-(DOX)4 after intravenous injection in mice. (e) Chemical structure and coarse-grained model of POEGMA-GFLG-(DOX)4. (f) Modeling process of POEGMA-GFLG-(DOX)4 forming nanoparticles after encapsulating Ce6 in water. DPD simulation was performed for 200,000 time steps with an integration time step of 0.02. (g) LD-DOX / Ce6 NPs formed at different Ce6 to DOX feed weight ratios. (h) TEM image of LD-DOX / Ce6, scale bar = 50 nm. (i) Dynamic process of LD-DOX / Ce6 drug release when both hydrazone bonds and GFLG are broken simultaneously, and 100,000 time steps were used to analyze the drug release process under protonated conditions. (j) TEM image of LD-DOX / Ce6 after incubation in deionized water at pH 5.4 for 24 h, scale bar = 200 nm.

[0033] Figure 2Fig. 1. Characterization of physicochemical properties of LD-DOX / Ce6. (a) UV-Vis absorption spectra of Ce6-loaded nanoparticles with different DOX to Ce6 feed weight ratios in PBS. (b) Drug loading content (DLC) and drug loading efficiency (DLE) of Ce6-loaded nanoparticles with different DOX to Ce6 feed weight ratios. (c) Size distribution of LD-DOX and LD-DOX / Ce6 measured by DLS. (d) Absorption spectra of LD-DOX / Ce6 NPs and controls in DMSO. (e) Photostability of LD-DOX / Ce6 and free Ce6 in DMSO or PBS after laser irradiation (660 nm, 2 mW cm -2 ) (f) Colloidal stability of LD-DOX / Ce6 in PBS at pH 7.4. (g) (h) Release profiles of DOX and Ce6 at different pH values in the presence or absence of cathepsin B. (i) Singlet oxygen generation by LD-DOX / Ce6 NPs under 660 nm laser irradiation (Ce6: 1 pg / mL, 660 nm, 5 mW cm -2 ) (f) Colloidal stability of LD-DOX / Ce6 in PBS at pH 7.4. (g) (h) Release profiles of DOX and Ce6 at different pH values in the presence or absence of cathepsin B. (i) Singlet oxygen generation by LD-DOX / Ce6 NPs under 660 nm laser irradiation (Ce6: 1 pg / mL, 660 nm, 5 mW cm

[0034] Figure 3 Fig. 6. (a) Cellular uptake of LD-DOX / Ce6 in 4T1 cells (scale bar = 50 pm). (b) CLSM images of 4T1 three-dimensional multicellular tumor spheroids (MTS) after incubation with LD-DOX / Ce6 for 6 h (scale bar = 50 pm). (c) ROS generation after treatment of 4T1 cells with LD-DOX / Ce6 or free Ce6, detected using DCFH-DA staining (scale bar = 100 pm). (d) Quantitative analysis of the mean fluorescence intensity of ROS probe DCF in 4T1 cells. (e) Expression of yH2A.X detected by immunofluorescence analysis. Nuclei were stained with DAPI (blue) (scale bar = 25 pm). (f) Quantitative analysis of the mean yH2A.X expression per nucleus. (g) Cytotoxicity of Ce6+L, DOX, LD-DOX, and LD-DOX / Ce6 on 4T1 cells with or without laser irradiation. (h, i) Flow cytometry analysis of apoptosis in 4T1 cells after various treatments with or without laser irradiation. Data are presented as mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, and **P < 0.01.

[0035] Figure 4(a) Heatmap of differentially expressed genes (DEGs) in 4T1 cells after different treatments. (b) Volcano plot of DEGs in 4T1 cells after treatment with PBS and LD-DOX / Ce6+L. (c) GO enrichment analysis and (d) KEGG enrichment analysis of DEGs in 4T1 cells after treatment with LD-DOX / Ce6+L and control groups. (e) Gene set enrichment analysis (GSEA) showing positive enrichment of cell apoptosis, DNA repair and cell cycle arrest gene signatures and negative enrichment of epithelial-mesenchymal transition, angiogenesis and TGFβ signaling gene signatures in LD-DOX / Ce6+L compared with control groups. (f) Western blot analysis showing the expression of DNA damage and apoptosis-related markers in 4T1 cells after different treatments.

[0036] Figure 5 (a) Average plasma Ce6 concentration in BALB / c mice after intravenous injection of free Ce6 and LD-DOX / Ce6 (b) Representative in vivo fluorescence images of 4T1 orthotopic breast cancer mice after intravenous injection of LD-DOX / Ce6. (c) CLSM images of time-dependent retention and penetration of LD-DOX / Ce6 in orthotopic 4T1 tumors at different time intervals after injection (tumor tissue is within the yellow circles, scale bar = 200 μm). (d) Representative fluorescence images of major organs extracted from mice 24 h after intravenous injection of LD-DOX / Ce6 (H: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; Tu: tumor). (e) Tumor cryosections observed by CLSM at different time points after injection of LD-DOX / Ce6 (x400, scale bar = 50 μm). Green fluorescence is from anti-CD31 labeled blood vessels, red fluorescence is from Ce6, and blue fluorescence is from DAPI-stained nuclei. (f) Treatment regimen of 4T1 subcutaneous tumor-bearing BALB / c mice. For the light group, mice were administered with LD-DOX / Ce6 for 24 h before laser irradiation (660 nm, 250 mW cm -2Figure 6. (a) Schematic diagram of the experimental design. (b) Tumor volume growth curves of subcutaneous 4T1 tumors treated with various formulations with or without laser irradiation. (c) Body weight changes of 4T1 tumor-bearing mice treated with various formulations. (d) Average tumor weight of mice in each treatment group. (e) Tumor volume growth curves of orthotopic 4T1 tumors treated with various formulations. (f) Survival curves of orthotopic 4T1 tumor-bearing mice after various treatments. (g) Subcutaneous 4T1 tumor volume growth curves of various formulations with or without laser irradiation. (h) Body weight changes of 4T1 tumor-bearing mice after treatment with various formulations. (i) Average tumor weight of mice in each treatment group. (j) Orthotopic 4T1 tumor volume growth curves after treatment with various formulations. (k) Survival curves of orthotopic 4T1 tumor-bearing mice after various treatments. (l) Number of nodules of tumor metastasis in the lung at the end of treatment. (m) Lung metastasis of orthotopic 4T1 tumor-bearing mice after various treatments. From top to bottom: 3D images reconstructed by Micro CT (red, white, green and blue represent trachea, bone, lung and tumor metastasis, respectively), lung photographs and lung H&E staining sections (scale bar = 2 mm). Group 1 (G1): normal saline; Group 2 (G2): laser alone; Group 3 (G3): free Ce6 + L (8.9 mg Ce6 kg -1 ); Group 4 (G4): free DOX (4 mg DOX kg -1 ); Group 5 (G5): LD-DOX / Ce6 without laser irradiation (4 mg DOX kg -1 , 4.4 mg Ce6 kg -1 ); Group 6 (G6): LD-DOX / Ce6 without laser irradiation (8 mg DOX kg -1 , 8.9 mg Ce6 kg -1 ); Group 7 (G7): LD-DOX / Ce6 + L with laser irradiation (4 mg DOX kg -1 , 4.4 mg Ce6 kg -1 ); Group 8 (G8): LD-DOX / Ce6 + L with laser irradiation (8 mg DOX kg -1 , 8.9 mg Ce6 kg -1 ); Group 9 (G9): free DOX (8 mg DOX kg -1 ). ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05, ns means no significant difference. Figure 6 Figure 2. Schematic diagram of the synthesis route of monomers (a) MA-GFLG-NHNHBoc, (b) MA-GFLG-(NHNHBoc)2, and (c) MA-GFLG-(NHNHBoc)4. Schematic diagram of the synthesis route of monomers (a) MA-GFGL-OH, (b) MA-GFGL-NHNHBoc, (c) MA-GFGL-(NHNHBoc)2, and (d) MA-GFGL-(NHNHBoc)4.

[0037] Figure 7(a) Schematic diagram of the synthetic route of amphiphilic block polymer prodrugs POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2 and POEGMA-GFLG-(DOX)4. (b) Schematic diagram of the synthetic route of amphiphilic block polymer prodrugs POEGMA-GFGL-DOX, POEGMA-GFGL-(DOX)2 and POEGMA-GFGL-(DOX)4.

[0038] Figure 8 . Left: MALDI-TOF mass spectrum of MA-GFLG-OH, [M+H] + , m / z = 461.117. Right: H NMR chart of MA-GFLG-OH in DMSO-d6. 1

[0039] Figure 9 . Left: MALDI-TOF mass spectrum of MA-GFLG-(O t Bu)2, [M+H] + , m / z = 702.312. Right: H NMR chart of MA-GFLG-Glu-(OtBu)2 in DMSO-d6. 1

[0040] Figure 10 . Left: MALDI-TOF mass spectrum of MA-GFLG-(O t Bu)4, [M+Na] + , m / z = 1094.659. Right: H NMR chart of MA-GFLG-(OtBu)4 in DMSO-d6. t 1

[0041] Figure 11 . Left: MALDI-TOF mass spectrum of MA-GFLG-NHNHBoc, [M+Na] + , m / z = 597.163. Right: H NMR chart of MA-GFLG-NHNHBoc in DMSO-d6. 1

[0042] Figure 12 . Left: MALDI-TOF mass spectrum of MA-GFLG-(NHNHBoc)2, [M+Na] + , m / z = 840.366. Right: H NMR chart of MA-GFLG-(NHNHBoc)2 in DMSO-d6. 1

[0043] Figure 13 ​​​​​​Left image: MALDI-TOF mass spectrum of MA-GFLG-(NHNHBoc)4, [M+Na] + m / z = 1326.958. Right figure: MA-GFLG-(NHNHBoc)4 in DMSO-d6 1 H NMR spectrum.

[0044] Figure 14 Left image: MALDI-TOF mass spectrum of Boc-GF-OH, [M+H] + m / z = 323.265. Right figure: Boc-GFGL-OH in DMSO-d6 1 H NMR spectrum.

[0045] Figure 15 Left image: MALDI-TOF mass spectrum of NH2-GL-OMe, [M+H] + m / z = 202.812. Right figure: NH2·TFA-GL-OMe in DMSO-d6 1 H NMR spectrum.

[0046] Figure 16 Left image: MALDI-TOF mass spectrum of Boc-GFGL-OMe, [M+H] + m / z = 507.161. Right figure: Boc-GFGL-OMe in DMSO-d6 1 H NMR spectrum.

[0047] Figure 17 Left image: MALDI-TOF mass spectrum of MA-GFGL-OH, [M+H] + m / z = 461.173. Right figure: MA-GFGL-OH in DMSO-d6 1 H NMR spectrum.

[0048] Figure 18 Left image: MA-GFGL-(O) t MALDI-TOF mass spectrum of Bu)2, [M+H + m / z = 702.325. Right figure: MA-GFGL-(O t Bu)2 in DMSO-d6 1 H NMR spectrum.

[0049] Figure 19 Left image: MA-GFGL-(O t MALDI-TOF mass spectrum of Bu)4, [M+H +, m / z = 1072.414. Right: MALDI-TOF mass spectrum of MA-GFGL-(0 t Bu)4in DMSO-d6 1 H NMR plot.

[0050] Figure 20 . Left: MALDI-TOF mass spectrum of MA-GFGL-NHNHBoc, [M+Na] + , m / z = 597.184. Right: H NMR plot of MA-GFGL-NHNHBoc in DMSO-d6 1 H NMR plot.

[0051] Figure 21 . Left: MALDI-TOF mass spectrum of MA-GFGL-(NHNHBoc)2, [M+Na] + , m / z = 840.360. Right: H NMR plot of MA-GFGL-(NHNHBoc)2in DMSO-d6 1 H NMR plot.

[0052] Figure 22 . Left: MALDI-TOF mass spectrum of MA-GFGL-(NHNHBoc)4, [M+Na] + , m / z = 1326.542. Right: H NMR plot of MA-GFGL-(NHNHBoc)4in DMSO-d6 1 H NMR plot.

[0053] Figure 23 . Left: MALDI-TOF mass spectrum of POEGMA-CTA, POEGMA-GFLG-(NHNHBoc) x and POEGMA-GFGL-(NHNHBoc) x (x = 1, 2 or 4) in DMSO-d6.

[0054] Figure 24 . Left: H NMR plot of POEGMA-GFLG-(NHNHBoc) x and POEGMA-GFGL-(NHNHBoc) x (x = 1, 2 or 4) in DMSO-d6. 1 H NMR plot. Right: 1 POEGMA-GFLG-(DOX) x and POEGMA-GFGL-(DOX) x (x = 1, 2 or 4) in DMSO-d6. 1 H NMR plot.

[0055] Figure 25 (a) POEGMA-CTA, (b) POEGMA-GFLG-(NHNHBoc)4, and (c) POEGMA-GFLG-(DOX)4(LD-DOX) in DMSO-d6. 1 H NMR spectra. (d) UV-Visible absorption spectra of free DOX and LD-DOX in DMSO. (e) HPLC spectra of each polymeric prodrug dissolved in methanol. DOX concentration was 10 pg / mL, free DOX as control. Elution from 5% to 95% buffer B in 30 min; flow rate: 1.0 mL / min; wavelength: 480 nm; buffer A: deionized water with 0.1% TFA; buffer B: methanol. (f) DOX release profile of POEGMA-GFLG-(DOX) x and POEGMA-GFGL-(DOX) x of DOX release profile of POEGMA-GFLG-(DOX)

[0056] Figure 26 (a) Coarse-grained model of POEGMA-GFLG-(DOX)4. (b) Modeling process of POEGMA-GFLG-(DOX)4forming nanoparticles (NPs) in neutral solvents. DPD simulation was performed for 200,000 time steps with an integration time step of 0.02.

[0057] Figure 27 Particle size distribution of polymeric prodrug LD-DOX (a) and Ce6-loaded nanoparticles LD-DOX / Ce6 (b) detected by DLS. Figure 28 Dynamic drug release process of LD-DOX / Ce6 upon cleavage of (a) hydrazone bond (acidic environment, pH 5.4) or (b) GFLG (in the presence of cathepsin B). DPD simulation was performed for 100,000 time steps to analyze the drug release process under protonated conditions. (c) Drug release profile of DOX and (d) Ce6 under different conditions obtained from DPD simulation. The drug release profile was obtained by calculating the number of drug beads in the nanoparticles at different times.

[0058] Figure 29 Encapsulation of PTX into LD-DOX as a multifunctional nanoplatform. Size distribution of LD-DOX before (a) and after (b) encapsulation of PTX measured by dynamic light scattering (DLS). (c) Drug loading capacity (DLC) and encapsulation efficiency (DLE) of LD-DOX / PTX nanoparticles at different DOX to PTX feed weight ratios. (d) Release profile of PTX under different incubation conditions.

[0059] Figure 30UV-Vis absorption spectra of LD-DOX / Ce6 in DMSO at different DOX:Ce6 feed ratios.

[0060] Figure 31 Stability of LD-DOX / Ce6 in 10% FBS (a) and cell culture medium (b) (incubation at 37°C on a shaker); (c) Stability of LD-DOX / Ce6 under storage at 4°C; (d) Photographs of LD-DOX and LD-DOX / Ce6 prepared by the film method before and after re-dispersion in water after lyophilization (LD-DOX concentration; 3 mg / mL).

[0061] Figure 32 (a) HPLC chromatogram of LD-DOX / Ce6 after incubation at pH 5.4 for 24 h in the presence of cathepsin B. Free DOX as a control. (b) MALDI-TOF mass spectrum of the eluted sample at 21.608 min in the HPLC chromatogram after incubation of LD-DOX / Ce6. 544.212 is the characteristic peak of DOX ([M+H] + ) and 597.154 is the characteristic peak of Ce6 ([M+H] + ).(c) MALDI-TOF mass spectrum of the degradation products of LD-DOX / Ce6 nanoparticles after incubation at pH 5.4 in the presence of cathepsin B. The peak at m / z = 566.117 is the characteristic peak of DOX ([M+Na] + ) and the peak at m / z = 597.154 is the characteristic peak of Ce6 ([M+H]

[0062] Figure 33 Changes in the fluorescence emission spectra of SOSG in free Ce6 (a) without laser irradiation, LD-DOX / Ce6 (b) without laser irradiation, free Ce6 (c) with laser irradiation, free DOX (d) with laser irradiation and LD-DOX (e) with laser irradiation.

[0063] Figure 34 (a) Cellular uptake of LD-DOX / Ce6 after incubation with 4T1 cells for 4 h. Average fluorescence intensity of DOX (b) and Ce6 (c) in 4T1 cells after incubation with free DOX, free Ce6, LD-DOX and LD-DOX / Ce6 for 1, 2 and 4 h, respectively. Fluorescence intensity quantification was analyzed by flow cytometry.

[0064] Figure 35 CLSM images of 4T1 tumor spheroids after treatment with LD-DOX / Ce6 for 2 h (a) and 4 h (b), respectively (scale bar, 50 μm).

[0065] Figure 36(a) Cytotoxicity of free Ce6 on 4T1 cells at different laser intensities and (b) IC 50 values of (c) free Ce6 (3 pg mL -1 ) on 4T1 cells at the same laser intensity but different light exposure times.

[0066] Figure 37 (a) POEGMA-CTA and (b) MA-GFLG-(NHNH2)4 cytotoxicity on 4T1 cells after 24h incubation at different concentrations. (c) IC 50 values of different formulations on 4T1 cells without laser irradiation (DOX, LD-DOX and LD-DOX / Ce6) and with laser irradiation (LD-DOX / Ce6+L, Ce6+L).

[0067] Figure 38 Combination index (CI) of LD-DOX prodrug and laser-irradiated Ce6 on 4T1 cells.

[0068] Figure 39 Gene set enrichment analysis (GSEA) showing the enriched pathways between different treatment groups: (a) LD-DOX / Ce6+L vs Ce6+L, (b) LD-DOX / Ce6+L vs DOX (b), (c) DOX vs Control and (d) Ce6+L vs Control.

[0069] Figure 40 Western blot semi-quantification of cleaved Caspase 3, Caspase 3, yH2A.X, Bax, cleaved PARP and PARP protein expression levels in each experimental group after 4T1 cells were treated with PBS, Ce6+L, DOX or LD-DOX / Ce6+L.

[0070] Figure 41 Body weight changes of normal mice after treatment with normal saline, free DOX, free Ce6, LD-DOX and LD-DOX / Ce6. In the DOX dosing groups, one mouse died on day 13 at a DOX dose of 10 mg kg -1 ; two mice died on day 9 and two mice died on day 11 at a DOX dose of 15 mg kg -1 . No deaths occurred in the mice of the other groups during the experiment.

[0071] Figure 42H&E-stained tissue sections of major organs (heart, liver, spleen, lung, and kidney) from normal mice treated with physiological saline, free DOX (10 mg kg⁻¹), free Ce₆ (10 mg kg⁻¹), and LD-DOX. The DOX-treated group showed disordered cardiomyocyte structure and inflammatory cell infiltration in the liver. No significant abnormalities were observed in other experimental groups.

[0072] Figure 43 Use free DOX (10 mg / kg) -1 ), free Ce6 (10 mg kg) -1 LD-DOX (equivalent to DOX 10mg / kg) -1 ) and LD-DOX / Ce6 (equivalent to 10 mg kg of DOX) -1 Ce6 11.1 mg kg -1 Biocompatibility assessment in mice treated with the treatment. Mice treated with saline were used as controls. Complete blood count analysis: (a) WBC, (b) RBC, (c) HGB, (d) HCT, (e) MCV, (f) MCH, (g) CHC, (h) RDW, and (i) PLT (mean ± SD, n = 4, *P < 0.05).

[0073] Figure 44 Use free DOX (10 mg / kg) -1 ), free Ce6 (10 mg kg) -1 LD-DOX (equivalent to DOX 10mg / kg) -1 ) and LD-DOX / Ce6 (equivalent to 10 mg kg of DOX) -1 Ce6 11.1 mg kg -1 Biocompatibility assessment in mice treated with the treatment. Mice treated with saline were used as controls. Blood biochemistry analysis: (a) ALP, (b) ALT, (c) AST, (d) URE, and (e) CRE (mean ± SD, n = 5, *P < 0.05).

[0074] Figure 45 Representative phototoxicity photographs and H&E-stained sections of skin from mice after different treatments following dorsal shaving. Red circles indicate laser-irradiated areas, and blue arrows indicate necrotic areas in the skin tissue. (Scale bar, 200 μm).

[0075] Figure 46. Semi-quantitative average fluorescence signal intensity of free Ce6 administration group (a) and LD-DOX / Ce6 administration group (b) in major organs and tumor sites. (c) Semi-quantitative average fluorescence signal intensity of free Ce6 administration group and LD-DOX / Ce6 administration group in tumor sites. (d) Fluorescence imaging pictures of major organs and tumors at different time points after intravenous injection of saline (first row), free Ce6 (second row) or LD-DOX / Ce6 (third row) in 4T1 tumor-bearing mice (H: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; Tu: tumor).

[0076] Figure 47 . The frozen sections of tumor tissues at different time points after free Ce6 administration were taken by CLSM (x400, scale bar = 50 μm). Red fluorescence came from Ce6, green fluorescence came from anti-CD31 labeled tumor vessels, and blue fluorescence came from DAPI stained nuclei.

[0077] Figure 48 (a) Photographs of tumors of mice in each experimental group after treatment; (b) Tumor inhibition rates of mice in each experimental group. Group 1: saline; Group 2: laser irradiation only; Group 3: free Ce6 plus laser irradiation (8.9 mg Ce6 / kg); Group 4: free DOX (4 mg DOX / kg); Group 5: LD-DOX / Ce6 NPs without laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 6: LD-DOX / Ce6 without laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg); Group 7: LD-DOX / Ce6 plus laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 8: LD-DOX / Ce6 plus laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg).

[0078] Figure 49Tumor H&E stained sections of each group (red arrow points to tumor necrotic area, scale bar = 100 pm) and histological analysis of tumor sections after different treatments, respectively stained with CD31, Ki-67 and tunel (x400, scale bar = 50 pm). Group 1: normal saline; Group 2: laser irradiation only; Group 3: free Ce6 plus laser irradiation (8.9 mg Ce6 / kg); Group 4: free DOX (4 mg DOX / kg); Group 5: LD-DOX / Ce6 NPs without laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 6: LD-DOX / Ce6 without laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg); Group 7: LD-DOX / Ce6 plus laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 8: LD-DOX / Ce6 plus laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg).

[0079] Figure 50 . H&E stained sections of main organs (heart, liver, spleen, lung, kidney) of 4T1 tumor-bearing mice after 21 days of treatment by different administration methods (red loops in the lung represent tumor metastatic lesions, scale bar = 100 pm). Group 1: normal saline; Group 2: laser irradiation only; Group 3: free Ce6 plus laser irradiation (8.9 mg Ce6 / kg); Group 4: free DOX (4 mg DOX / kg); Group 5: LD-DOX / Ce6 NPs without laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 6: LD-DOX / Ce6 without laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg); Group 7: LD-DOX / Ce6 plus laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group 8: LD-DOX / Ce6 plus laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg). DETAILED DESCRIPTION

[0080] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0081] Among them, 4-cyanopentanoic acid dithiobenzoate (CTA), poly(ethylene glycol) monomethyl ether methacrylate (OEGMA, Mw=500 Da), 4-methoxyphenol, 2,2'-[azobis(1-methylethylidene)]bis[4,5-dihydro-1H-imidazole] dihydrochloride (VA044), methacryloyl chloride, Cathepsin B, doxorubicin hydrochloride (DOX-HCl) and chlorin e6 (Ce6) were purchased from Sigma-Aldrich (USA). Boc-glycine (Boc-Gly-OH), L-phenylalanine methyl ester hydrochloride (H-Phe-OMe-HCl), Boc-L-leucine (Boc-Leu-OH-H2O), glycine methyl ester hydrochloride (H-Gly-OMe-HCl), L-glutamic acid di-tert-butyl ester hydrochloride (H-Glu(OtBu)-OtBu-HCl), Boc-hydrazide (NH2NHBoc), 1-hydroxybenzotriazole hydrate (HOBt), N,N-diisopropylethylamine (DIPEA), N,N,N',N'-tetramethyl-(1H-benzotriazol-1-yl)phosphonium hexafluorophosphate (HBTU), 2-(7-Aza-1Hbenzotriazole-1-yl)-1,1,3,3-hexafluorophosphate tetramethyluronium (HATU), N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine (EDC) and trifluoroacetic acid (TFA) were purchased from GL Biochem (Shanghai, China). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher, USA). CCK-8 detection kit was purchased from Dojindo Molecular Technologies (Japan). Singlet oxygen sensor green (SOSG) was purchased from Life Technologies (NY, USA). Reactive oxygen species (ROS) detection kit, Annexin V-FITC apoptosis detection kit and Hoechst 33342 were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies for western blotting included Bax (2772S), Caspase-3 (D3R6Y) (14220S), PARP (9542S) and γ-H2A.X (7631S) purchased from Cell Signaling Technology (CST); Cleaved caspase-3 (ab2302) purchased from Abeam; GAPDH (7E4) (200306-7E4) purchased from ZEN BIO (Chengdu, China). HRP-labeled secondary antibodies were purchased from Jackson Lab (115-035-003, 111-035-003).

[0082] Example 1: Synthesis of linear-dendritic block polymeric prodrug POEGMA-GFLG-(DOX)4 (named as LD-DOX)

[0083] 1. Synthesis of methacrylate monomer

[0084] 1.1 Synthesis of compound MA-GFLG-Glu-(OtBu)2

[0085] H-Glu(OtBu)-OtBu-HCl (2.46 g, 8.3 mmol), MA-GFLG-OH (S1 and S2) (3.188 g, 6.9 mmol), EDC (1.86 g, 9.7 mmol) and HOBt (1.31 g, 9.7 mmol) were dissolved in 40 mL of chromatographically pure DMF under N2protection. DIEA (6.8 mL, 41.1 mmol) was added slowly under ice-bath stirring. Subsequently, the reaction bottle was placed at 25 °C for further reaction, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed (36 h), most of the solvent was removed by distillation under reduced pressure, and the remaining solution was poured into 400 mL of ethyl acetate, which was washed with saturated NaHC03solution, dilute hydrochloric acid (0.1 M) and saturated NaCl solution, respectively. The washed organic phase was dried over anhydrous MgS04, and the filtrate was collected by filtration. After the solvent was removed by distillation under reduced pressure and dried in vacuum, the target product MA-GFLG-Glu-(OtBu)2was obtained (white solid, yield 92%). The molecular structure was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Autoflex III, Bruker) (left panel) and proton nuclear magnetic resonance (1H NMR, right panel). Figure 9 1 H NMR, Figure 9

[0086] 1.2 Synthesis of MA-GFLG-(OtBu)4 t Bu)4

[0087] MA-GFLG-Glu-(OtBu)2(3.9 g, 5.6 mmol) was dissolved in 8.6 mL of chromatographically pure DCM under ice-bath stirring, and an equal volume of trifluoroacetic acid (8.6 mL, 112 mmol) was added. Subsequently, the reaction was continued at room temperature for 10 h, and the reaction progress was detected by TLC. After the reaction was completed, the DCM was removed by distillation under reduced pressure, and washed with anhydrous ether, and a large amount of white powdery solid appeared. After standing, the supernatant was removed (repeated twice). After the residual ether was removed by distillation under reduced pressure, MA-GFLG-Glu-(OH)2was obtained.

[0088] ​​MA-GFLG-Glu-(OH)2 (3.3 g, 5.6 mmol), H-Glu(OtBu)-OtBu-HCl (3.61 g, 12.2 mmol), HOBt (1.93 g, 14.3 mmol), EDC (2.74 g, 14.3 mmol) were dissolved in 40 mL of chromatographically pure DMF. DIEA (10.1 mL, 61.2 mmol) was added slowly under ice-bath stirring. The reaction flask was then placed in a 25 °C water bath and the reaction was continued. The progress of the reaction was monitored by TLC. After the reaction was completed (48 h), most of the solvent was removed by distillation under reduced pressure, and the remaining solution was poured into 300 mL of ethyl acetate, which was washed with saturated NaHCO3solution, dilute HCl (0.1 M), and saturated NaCl solution, successively. The washed ethyl acetate layer was dried over anhydrous MgSO4, and the filtrate was collected by filtration. The solvent was removed by distillation under reduced pressure and dried in vacuum to give the target product MA-GFLG-Glu-(OtBu)4as a white solid (90% yield). The molecular structure was confirmed by MALDI-TOF MS (left) and1H NMR spectra (right). Figure 10 1 Figure 10

[0089] 1.3 Synthesis of MA-GFLG-(NHNHBoc)4

[0090] MA-GFLG-G2(Glu)-(OtBu)4was deprotected according to the above procedure. MA-GFLG-G2(Glu)-(OH)4(1.50 g, 1.8 mmol), NH2NHBoc (1.88 g, 14.2 mmol), HATU (5.40 g, 14.2 mmol) were dissolved in 50 mL of chromatographically pure DMF, and DIEA (4.8 mL, 28.8 mmol) was added slowly under ice-bath stirring. The reaction was continued at 25 °C, and the progress of the reaction was monitored by TLC. After the reaction was completed (48 h), the reaction solution was poured into 400 mL of ethyl acetate, which was washed with saturated NaHCO3solution, dilute HCl (0.1 M), and saturated NaCl solution, successively. White solid was precipitated from the organic layer after washing with saturated NaCl solution. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography to give the product MA-GFLG-(NHNHBoc)4with a yield of 84%. The molecular structure was confirmed by MALDI-TOF MS (left) and1H NMR spectra (right). Figure 11 1 Figure 11 Figure 12 to Figure 22

[0091] 2. Synthesis of linear-dendritic block copolymer prodrug​​​​​​​

[0092] 2.1 Synthesis of macromolecular chain transfer agent POEGMA-CTA

[0093] OEGMA (4.125 g, 8.25 mmol), CTA (23.1 mg, 82.5 μmol) and VA044 initiator (8.8 mg, 27.5 μmol) were dissolved in 18.7 mL of mixed solvent (H2O / CH3OH = 4:1, v / v). The mixture was purged with argon for 30 min in an ice bath and the polymerization was carried out at 44 °C in the dark for 12 h under a sealed condition. The resulting solution was dialyzed (MWCO, 3.5 kDa) against deionized water at 4 °C for 1.5 days and then lyophilized to give the product POEGMA-CTA with a yield of 58% (2.42 g). The number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) of the polymer were determined by GPC to be 2.42 kDa, 2.48 kDa and 1.02, respectively. n w The GPC analysis was performed on a Shimadzu prominence HPLC system (columns: Shodex Asahipak GF-1 G 7B and Shodex Asahipak GF-510 HQ, detector: Shimadzu RID-20A refractive index (RI) detector, flow rate: 0.5 mL / min, mobile phase: 0.2 M LiCl / DMF = 35:65, v / v, column temperature: 45 °C, injection volume: 50 μL).

[0094] 2.2 Synthesis of POEGMA-GFLG-(NHNHBoc)4

[0095] POEGMA-CTA (1.73 g), MA-GFLG-(NHNHBoc)4(593.6 mg, 44.4 mmol) and VA044 (6.2 mg, 19.4 μmol) were dissolved in 10.5 mL of mixed solvent (H2O / CH3OH = 1:4, v / v). The mixture was purged with argon for 30 min at 0 °C and the polymerization was carried out at 45 °C in the dark for 17 h under a sealed condition. The resulting solution was dialyzed (MWCO, 8 kDa) against deionized water at 4 °C for 2 days and then lyophilized to give 1.69 g of product POEGMA-GFLG-(NHNHBoc)4. The number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) of the polymer were determined by GPC to be 2.42 kDa, 2.48 kDa and 1.02, respectively. n w Determined by GPC on a Shimadzu prominence HPLC system as above. Table 1 lists the Mn, Mw and PDI of each copolymer. n w Determined by GPC on a Shimadzu prominence HPLC system as above. Table 1 lists the Mn, Mw and PDI of each copolymer.​​​​​

[0096] 2.3 Synthesis of amphiphilic linear-dendritic block polymer prodrug POEGMA-GFLG-(DOX)4

[0097] POEGMA-GFLG-(NHNHBoc)4(850.0 mg, 0.27 mmol) was dissolved in a mixed solution of DCM (10 mL) and TFA (10 mL) and the solution was stirred in an ice bath. Then, the reaction was allowed to proceed at room temperature for 20 hours. The solvent was removed by rotary evaporation. The residue was dissolved in deionized water. After dialysis (MWCO, 2 kDa) for 2 days, the solution was lyophilized to give the product POEGMA-GFLG-(NHNH2·TFA)4.

[0098] The deprotected conjugate (500 mg) and DOX·HCl (200 mg) were dissolved in 20 mL of NH4OAc buffer (0.1 M, pH 5.7). The solution was then stirred in the dark at room temperature for 48 hours. Subsequently, the solution was dialyzed (MWCO, 2000) at 4 °C to remove excess free DOX. Finally, the solution was lyophilized to give 530 mg of the final product POEGMA-GFLG-(DOX)4(named as LD-DOX).

[0099] Example 2: Preparation of LD-DOX / Ce6 nanoparticles

[0100] The preparation method of LD-DOX / Ce6 nanoparticles (NPs) was as follows: POEGMA-GFLG-(DOX)4(20 mg) was dissolved in 20 mL of methanol, and 6.48 mL of an acetone solution containing Ce6 (0.5 mg mL -1 ) was added dropwise to it under vigorous stirring. After the mixture was further stirred at room temperature for 2 hours, the solvent was removed by distillation under reduced pressure to form a uniform thin film on the wall of the reaction flask. The residual organic solvent was then completely removed by vacuum drying overnight. Subsequently, deionized water was added dropwise to hydrate the Ce6-loaded thin film. After stirring at 4 °C for 4 hours, the resulting mixed solution was filtered to remove the unloaded Ce6. The filtered solution was freeze-dried to give POEGMA-GFLG-(DOX)4 / Ce6 nanoparticles (named as LD-DOX / Ce6 nanoparticles). It was stored at 4 °C and used for subsequent characterization. The whole experimental process was carried out in the dark.

[0101] The following is the preparation method of the control sample.

[0102] Example 1: Preparation of control amphiphilic linear-dendritic block polymer prodrug

[0103] The following control amphiphilic linear-dendritic block polymeric prodrugs were synthesized according to the method of Example 1 : POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2, POEGMA-GFGL-DOX, POEGMA-GFGL-(DOX)2, POEGMA-GFGL-(DOX)4.

[0104] Control Example 2: Preparation of control nanoparticles

[0105] POEGMA-GFLG-(DOX)4 / Ce6 nanoparticles with different ratios of DOX and Ce6 were prepared according to the method of Example 2.

[0106] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0107] I. Experimental Methods

[0108] 1. Physicochemical parameter characterization

[0109] Monomer and polymer 1 H NMR was characterized by a Bruker Avance II NMR spectrometer (Germany) at 400 MHz. Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a MALDI-7090 TOF-TOF mass spectrometer (Shimadzu, Japan). Weight average (Mw) and number average (Mn) molecular weights of polymers were determined by gel permeation chromatography (GPC) and a GPC column of Shodex Asahipak GF-510HQ (7.5 mm ID x 300 mm L). The sample (2 mg / mL) was injected at 50 μL. The mobile phase was a 0.2 M lithium chloride solution (H2O:DMF = 35:65, v / v) at a flow rate of 0.5 mL / min (45 °C). Mw, Mn, and dispersity (D) were calculated using a calibration curve of narrow polystyrene standards. Measurements were performed using pullulan as a standard. The zeta potential and hydrodynamic diameter of the nanoparticles were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, UK) and the results were analyzed using DTS software version 3.32. The morphology of the resulting nanoparticles and their degradation products were observed by transmission electron microscopy (TEM, Tecnai GF20S-TWIN, USA). The drug loading of DOX and Ce6 in the polymer conjugates and nanoparticles were determined by UV-visible absorption spectroscopy (Perkin-Elmer, USA) and fluorescence spectrophotometer (Hitachi F-7000, Japan). High performance liquid chromatography (HPLC) analysis was performed on a Shimadzu prominence HPLC system (column: Shim-pack GIST C18, pore size: 5 μm, size: 4.6 x 250 mm).

[0110] 2. Measurement of DOX release properties of different block polymer prodrugs

[0111] The DOX release profiles of the pH or pH / enzyme dual-responsive polymer prodrugs were investigated by a dialysis method and POEGMA-GFLG-(DOX)4was taken as an example to illustrate the process. Briefly, 3 mL of POEGMA-GFLG-(DOX)4at a concentration of 1 mg mL -1 was placed in a dialysis bag (MWCO, 1 kDa) and immersed in 40 mL of release medium (pH 5.4, containing 2.8 μΜ cathepsin B) at 37 °C. At predetermined time points, 5 mL of solution was collected and an equal volume of fresh medium was added. The concentration of DOX was measured by fluorescence spectrophotometer (Hitachi F-7000, Japan).

[0112] 3. Measurement of critical micelle concentration of different block polymer prodrugs

[0113] The critical micelle concentration (CMC) of the prodrugs was determined with pyrene as a fluorescence probe and POEGMA-GFLG-(DOX)4was taken as an example to illustrate the process. Briefly, 200 μL of pyrene in acetone (1.2 x 10 -5 M) was added to a 10 mL sample vial. After the complete evaporation of acetone, 2 mL of LD-DOX aqueous solution at different concentrations (0.01-1000 μg / mL) was added (n = 3), respectively. The solution was then incubated at 37 °C for 12 h. The fluorescence spectrum was detected and recorded by fluorescence spectrophotometer (λex= 300-380 nm, λem= 390 nm). The ratio of the fluorescence intensity at 339 nm and 334 nm was calculated. The CMC value of the polymer prodrug LD-DOX was determined by the inflection point of the ratio.

[0114] 4. Pharmacokinetic evaluation of dual-sensitive responsive block copolymer prodrugs

[0115] For pharmacokinetic studies, healthy female BALB / c mice (body weight: 20 ± 2 g, n = 5) were administered with 5 mg DOX kg -1 Doses of mice were injected via tail vein with POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2, POEGMA-GFLG-(DOX)4 and free DOX-HCl. 20 μL blood was extracted from the orbital sinus at different time points from 1 min to 12 h and stored at -20 °C. After thawing at room temperature, 200 μL dimethyl sulfoxide (DMSO) was added, vortexed for 5 min and left at 4 °C overnight. Subsequently, the mixture was vortexed for 1 min and then centrifuged at 14,000 rpm for 5 min. Finally, 100 μL supernatant was transferred into a black 96-well plate and DOX fluorescence intensity (480 nm excitation and 590 nm emission) was measured using a Varioskan Flash (Thermo Scientific, MA, USA). The content of DOX in each blood sample was calculated by a standard curve of free DOX. Blood pharmacokinetic parameters were analyzed by non-compartmental model using PK Solver software.

[0116] 5. Nanoparticle formation, drug loading and drug release simulation

[0117] The present invention uses dissipative particle dynamics (DPD) to examine the formation of nanoparticles and the loading and release of drugs by the nanoparticles. DPD is a coarse-grained simulation technique that employs momentum-conserving thermostats and soft repulsive interactions between beads representing various molecules. This allows physical phenomena occurring on relatively large time and spatial scales to be simulated. Currently, DPD has been successfully used to simulate the self-assembly of polymers and nanoparticles dispersed in Newtonian fluids. In the simulation of the present invention, the present invention sets the time step Δt = 0.04, p = 1, m = 1, r c = 1, a ii = 25, kBT = 1 and the solvent number density p = 3. For all bead interactions, the present invention sets γ = 4.5. Unless otherwise stated, all dimension values are given in DPD units. The coarse-grained model of POEGMA-GFLG-(DOX)4 is shown in Figure 26 a. Each polymer chain has a hydrophilic block (blue) and a hydrophobic dendritic block connected by a harmonic spring potential U bond = K b ((r - b) / r c ) 2 , where K b = 64k B T and b = 0.5r crespectively, bond constant and equilibrium bond length. The coarse-grained model of the polymer was designed according to the branched structure used in the experiment, each coarse-grained bead contains almost the same volume and molecular weight. DOX molecules (red) are linked to glutamic acid dendrons (brown) through pH-sensitive bonds, and Ce6 molecules (green) are modeled by three beads connected in a triangle. In all simulations, the volume fraction of the polymer is set to 10%, while the fraction of Ce6 molecules varies. A 30 x 30 x 30 r c 3 Rectangular simulation box. The DPD simulation was performed for 200,000 time steps with an integration time step of 0.02 to achieve thermodynamic equilibrium and obtain drug-loaded nanoparticles under neutral conditions. Then, in order to describe the response behavior of the polymer chain under different environments, i.e., under lower pH conditions or in the presence of cathepsin B in the system, the present invention breaks some specific chemical bonds (hydrazone bonds or GFLG tetrapeptides), resulting in the separation of DOX or dendrimers from the polymer, respectively. After that, another 100,000 time steps were used to analyze the drug release process under protonated conditions.

[0118] 6. Drug loading and drug loading efficiency characterization

[0119] The hydrodynamic diameter and zeta potential of the resulting nanoparticles were measured by dynamic light scattering (DLS, Malvern Zetasizer NANO ZS, UK) and the results were analyzed with DTS software version 3.32. Transmission electron microscopy (TEM, Tecnai GF20S-TWIN, USA) was used to detect the morphology of the nanoparticles and their degradation products. The drug loading of DOX and Ce6 at 280 nm was determined by ultraviolet-visible absorption spectroscopy (Perkin-Elmer, USA). Drug loading (DLC) and drug loading efficiency (DLE) were calculated using the following formulas:

[0120] DLC (wt%) = (drug loading weight / total weight of polymer and drug) x 100

[0121] DLE (wt%) = (drug loading weight / drug delivery weight) x 100

[0122] The characterization results of POEGMA-GFLG-(DOX)4 / Ce6 NPs at different feed ratios are summarized in Table 4. PTX was used as another model drug to evaluate the ability of LD-DOX as a nanoplatform to encapsulate hydrophobic therapeutic agents. PTX was encapsulated using the same method as Ce6. The characterization results of POEGMA-GFLG-(DOX)4 / PTX NPs at different feed ratios are summarized in Table 5. The content of PTX was analyzed by an HPLC system (column: Shim-pack GIST C18, pore size: 5 μm, size: 4.6 x 250 mm).

[0123] 7. Stability of NPs and drug release detection

[0124] POEGMA-GFLG-(DOX)4 / Ce6 NPs (named as LD-DOX / Ce6) (1.0 mg mL -1 ) were incubated in three different buffer solutions at 37 °C: PBS (pH 7.4), PBS containing 10% FBS (pH 7.4) and cell culture medium without FBS. At predetermined time points, the particle size and PDI of NPs were detected by DLS. In addition, the long-term storage stability of LD-DOX / Ce6 was also evaluated by storing at 4 °C. The photo-stability of LD-DOX / Ce6 in different media (PBS or DMSO) was evaluated by UV-vis spectra. Sample solutions were prepared at a Ce6 concentration of 2 μΜ, and irradiated with a 2 mW cm -2 at 660 nm. The UV-vis absorbance of samples at 664 nm was detected at predetermined time points. Free Ce6 was used as a control.

[0125] The in vitro drug release behavior of LD-DOX / Ce6 for DOX and Ce6 was detected by dialysis method, by simulating normal physiological conditions and tumor tissue microenvironment. Briefly, 3 mL of LD-DOX / Ce6 (1.0 mg / mL) was placed in a dialysis bag (MWCO 1 kDa) and immersed in 40 mL of different release media (McIlvaine's buffer solution at pH 5.4, McIlvaine's buffer solution at pH 7.4 and McIlvaine's buffer solution at pH 5.4 containing 2.8 μΜ cathepsin B). The solution was incubated in a 37 °C shaker (100 rpm). At different time intervals, 5 mL of buffer solution was removed and an equal volume of fresh buffer was added. The concentrations of DOX and Ce6 in the solution were measured by a fluorescence spectrophotometer. Meanwhile, another sample of LD-DOX / Ce6 (0.5 mg mL -1 ) was incubated in acidic aqueous solution (pH 5.4) for 24 h, and the morphological changes of LD-DOX / Ce6 were observed under TEM. The drug release characteristics of LD-DOX-encapsulated PTX were determined according to the above method. The concentration of PTX was measured by HPLC (SHIMADZU, Japan).

[0126] 8. In vitro 1 O2 detection

[0127] To evaluate the photochemical activity of Ce6 in LD-DOX / Ce6, the present invention detected the singlet oxygen generated by photosensitizer Ce6 after laser irradiation using singlet oxygen fluorescent probe SOSG. Briefly, 0.5 mL of LD-DOX / Ce6 (0.5 mg mL 1O2 generation was investigated. Free Ce6 and LD-DOX / Ce6 NPs (Ce6 concentration: 1.0 μg / mL) were dissolved in SOSG (2.5 μM) solution, respectively, and then irradiated with a 660 nm laser (power density: 5.0 mW / cm²). 2 Duration: 4 min. At predetermined time points (0 s, 30 s, 60 s, 120 s, 180 s, 240 s), the SOSG fluorescence intensity of each sample at 525 nm was recorded using a fluorescence spectrophotometer (excitation wavelength: 490 nm). Samples not irradiated by laser served as a control group.

[0128] 9. Cell culture and animal models

[0129] The 4T1 cell line (mouse-derived breast cancer cells) used in this experiment was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The 4T1 cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Throughout the experiment, the cells were cultured in a humidified incubator containing 5% CO2 / 95% air. For animal experiments, 6-8 week old BALB / c mice (approximately 20±2g) were purchased from Chengdu Dashuo Biotechnology Co., Ltd. The purchased mice were randomly assigned and fed in a sterile room with constant temperature and a regular simulated day-night cycle. All animals were acclimatized to the environment for one week before the experiment. All animal experiments used in this study were conducted strictly in accordance with relevant national policies and regulations and the relevant regulations of the Animal Ethics Committee. 1.5×10⁻⁶ cells were suspended in 70μL PBS. 5 A subcutaneous 4T1 tumor model was established by subcutaneously injecting 4T1 cells into the lower right back of female BALB / c mice. An orthotopic 4T1 tumor model was established by suspending 1.5 × 10⁻⁶ cells in 70 μL of PBS. 5 4T1 cells were injected into the right side of the fourth fat pad of female BALB / c mice. All animal procedures were performed in accordance with the Laboratory Animal Care and Use Guidelines of West China Hospital, Sichuan University, and approved by the Chinese Animal Ethics Committee. Tumor volume was calculated using the following formula: Tumor volume V (mm²) 3 = 1 / 2 × length (mm) × width (mm) × width (mm).

[0130] 10. Cellular uptake and drug penetration in three-dimensional multicellular tumor spheroids (MTS)

[0131] To study cellular uptake of LD-DOX / Ce6, 4T1 cells were seeded in glass-bottomed culture dishes (1×10⁶ cells / year). 4cells / mL) and incubated for 24 h. Subsequently, the original medium was replaced with fresh medium containing free DOX, free Ce6, LD-DOX or LD-DOX / Ce6 (equivalent to 5.0 μg mL"1DOX or 5.5 μg mL"1Ce6), respectively. After 4 h incubation, the cells were washed with cold PBS for 3 times and the nuclei were stained with Hoechst 33342 according to the standard protocol provided by the supplier. The intracellular drug distribution of LD-DOX / Ce6 in 4T1 cells was observed under confocal laser scanning microscope (CLSM). Further, 4T1 MTSs were prepared to reveal the uptake and penetration of nanoparticles in tumor in vitro. 2 mL of molten 2% agarose solution was added into a 5 cm diameter petri dish. After the agarose solution solidified, 4 mL of 4T1 cell suspension was added on top of the agarose, 5 x 10 6 cells / mL and fresh RMPI 1640 medium. The MTSs were formed after 5 days of incubation with a diameter of about 200 μm for incubation with materials.

[0132] 11. Intracellular ROS detection

[0133] To detect the intracellular ROS level, 4T1 cells were seeded in 6-well plates (1 x 10 6 cells / well) and incubated for 24 h. Subsequently, the original medium was replaced with fresh medium containing free Ce6 or LD-DOX / Ce6 at a Ce6 concentration of 1.0 μg / mL, respectively. After 6 h incubation, the medium was discarded and the 4T1 cells were incubated with 1 μM of 2',7'-dichlorofluorescin diacetate (DCFH-DA, a ROS-sensitive probe) for 20 min. Subsequently, fresh medium was added and the cells were irradiated with 660 nm laser (10 mW / cm 2 ) for 30 s. The fluorescence signal of intracellular DCF was detected under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). In addition, the intracellular ROS level after treatment with different Ce6 formulations was quantitatively detected by flow cytometry. After the 4T1 cells were treated with laser irradiation as described above, the cells in each group were collected and the DCF fluorescence intensity was quantitatively analyzed by flow cytometry (FITC channel). Untreated and unirradiated cells were used as control.

[0134] 12. DNA damage analysis

[0135] γH2A.X staining was used to evaluate the DNA double-strand breakage of 4T1 cells. 4T1 cells were seeded in 6-well plates (5 x 10 5 cells / mL) and incubated for 24 h. Subsequently, the cells were treated with free DOX, free Ce6, LD-DOX, LD-DOX / Ce6 (equivalent to 0.3 μg mL -1DOX or 0.33 μg mL -1 Ce6) treatment and incubation for 12 h. Subsequently, fresh medium was replaced. The cells in experimental groups were irradiated with 660 nm laser for 30 s (10 mW cm -2 ) for 30 s (10 mW cm-2). Twelve hours after laser irradiation, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Trition X-100. After blocking in 5% BSA for 1 h, the cells were incubated with anti-histone γH2A.X rabbit monoclonal antibody at 4°C overnight, and then with fluorescently labeled goat anti-rabbit secondary antibody in 1% BSA for 1 h. After that, the cells were stained with DAPI to locate the nuclei and observed with CLSM. Quantitative analysis of γH2AX foci density (foci per nucleus) was counted using ImageJ software.

[0136] 13. In vitro cytotoxicity and apoptosis

[0137] The present application first analyzed the photodynamic therapy cytotoxicity of free Ce6 on 4T1 cells with CCK-8 kit. 4T1 cells were incubated in 96-well plates (5 x 103cells / well) for 24 h to adhere. Subsequently, the original culture medium was removed, fresh culture medium containing different concentrations of Ce6 (0.0005-90 μg / mL) was added and incubated for 12 h. Subsequently, the cells were washed twice with PBS, fresh culture medium was added and the cells were irradiated with 660 nm laser at a power of 10 mW / cm 3 2 The cells after laser irradiation were incubated in the cell incubator for 36 h and then detected for cell survival rate under different light conditions by CCK-8. In addition, the present application also studied the cytotoxicity of 4T1 cells under different laser energies when the concentration of Ce6 was 3 μg mL -1

[0138] To analyze the cytotoxicity of the combined chemotherapy-photodynamic therapy, four experimental groups were set, i.e. free DOX group, LD-DOX NPs group, LD-DOX / Ce6 (without laser irradiation) group and LD-DOX / Ce6 (with laser irradiation) group. 4T1 cells were incubated in 96-well plates (5 x 103cells / well) for 24 h to adhere. Subsequently, the original culture medium was removed, and the corresponding treatment reagents were added to the four experimental groups, respectively, and the concentration of DOX ranged from 0.0005 to 90 μg / mL. After 12 h of incubation, the cell culture medium of the LD-DOX / Ce6 NPs (with laser irradiation) group was discarded and replaced with fresh culture medium, and then 660 nm laser was used at a power of 10 mW / cm 2 ​​The cells were then incubated at 37 °C for another 36 h, and the survival rate of each group was detected by CCK-8 kit. The combination index (CI) of LD-DOX and Ce6+L was calculated by CompuSyn software.

[0139] In the apoptosis analysis, 4T1 cells were seeded in 6-well plates (1 x 10 6 cells / well) and cultured for 24 h to adhere. Then, the cells were treated with various drug formulations (equivalent to 0.3 pg / mL of DOX or 0.33 pg / mL of Ce6). After 12 h of continuous incubation, the original culture medium was replaced with fresh medium. The cells of the experimental groups were irradiated with a 660 nm laser for 30 s (10 mW / cm 2 After 24 h of irradiation treatment, each group of cells was treated with an Annexin V-FITC apoptosis detection kit according to the standard protocol provided by the supplier, and finally the apoptosis was quantitatively analyzed by flow cytometry.

[0140] 14. Transcriptomic data processing and analysis

[0141] 14.1. Construction and sequencing of mRNA library

[0142] TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate and purify the total DNA of each sample. According to the manufacturer's recommendations, Dynabeads Oligo(dT) 25-61005 (Thermo Fisher, CA, USA) was used to extract Poly(A) RNA.

[0143] 14.2. Alignment of transcriptomic data

[0144] Fastp removed the adapters and low-quality reads in the original fasta file using the default parameters. And the processed fatsa file was used for subsequent analysis, including alignment, quantification and differential identification. In addition, STAR was used to generate the alignment file with the mouse genome reference mm10.

[0145] 14.3. Identification of differentially expressed genes

[0146] Rsamtools and GenomicAlignments were used to quantify the expression level of each gene in different samples, singleEnd = FALSE, fragments = FALSE. DESeq2 was used to normalize the counts by removing the effect of gene length and library size for each sample. All the visualizations and pathway enrichment were implemented on these processed data. In addition, DESeq2 was also used to identify the significantly expressed genes with p.adj < 0.05 and |log2-foldchange| > 0.5.

[0147] 14.4. Gene set enrichment analysis

[0148] Gene ontology database (GO) and KEGG enrichment analysis were implemented by clusterprofiler, pvalueCutoff = 0.01, pAdjustMethod = BH, qvalueCutoff = 0.05. Log transformation has been used for the expression data after DESeq2 processing. Gene set enrichment analysis was performed by GSEA (java version) software version 7 gene set.

[0149] 14.5. Visualization of heat map and volcano plot

[0150] Volcano plot and heat map were used to show the expression pattern of differentially expressed genes. In volcano plot, -log10(p-value) was re-ranked as y-axis, log2-foldchange value was re-ranked as x-axis. The highlighted genes in volcano plot were from the significantly enriched 7thversion gene set in GSEA. In heat map, genes were differentially expressed genes, ordered by their log2-foldchange. The labeled genes in heat map were also from the 7thversion gene set in GSEA. In addition, the visualization of volcano plot was performed by ggplot2.

[0151] 15. Western blot analysis

[0152] For western blot analysis, 4T1 cells were seeded in 6-well plates (5 x 10 5 cells / mL) and incubated for 24 hours. Subsequently, cells were treated with PBS, free DOX, Ce6+L or LD-DOX / Ce6+L (equivalent to 0.3 pg mL -1 DOX or 0.33 pg mL -1 Ce6), respectively, and incubated for 12 hours. Subsequently, fresh medium was replaced. The irradiated experimental group cells were irradiated with 660 nm laser for 30 s (10 mW cm -2). After 24 h of irradiation, cells of each group were collected and immediately prepared into cell lysates. Electrophoresis was performed using 10% SDS-polyacrylamide gels and transferred to Immobilon-P (Millipore) membranes. Primary antibodies were diluted 1:500-1:2000 in 5% BSA and incubated overnight at 4°C. HRP-conjugated secondary antibodies were applied at a dilution of 1:10000. Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, MILIPORE) was used for visualization of proteins.

[0153] 16. Acute toxicity experiment

[0154] 130 healthy BALB / c mice (half male and half female) were randomly divided into 13 groups (n=10, 5 male and 5 female): saline group, 3 DOX groups (doses of 5 mg kg -1 , 10 mg kg -1 , 15 mg kg -1 ), 3 Ce6 groups (doses of 10 mg kg-1, 20 mg kg-1, 30 mg kg-1), 3 LD-DOX groups (10 mg kg -1 , 20 mg kg -1 , 30 mg kg -1 at equivalent DOX doses), and 3 LD-DOX / Ce6 groups (10 mg kg -1 / 11.1 mg kg -1 , 20 mg kg -1 / 22.2 mg kg -1 , 30 mg kg -1 / 33.3 mg kg -1 at equivalent DOX / Ce6 doses). The mice were fasted for 12 hours before being administered the above preparations via the tail vein. On the day of administration, the mice were continuously monitored for 4 hours, and then their body weight, general behavior changes, and deaths were recorded every other day for a total of 14 days. At the end of the experiment, the mice in each group were sacrificed. Blood routine and biochemical analyses were performed on the saline group, the DOX group (10 mg kg -1 ), the Ce6 group (10 mg kg -1 ), the LD-DOX group (10 mg kg -1 ), and the LD-DOX / Ce6 mouse group (equivalent DOX / Ce6 dose of 10 mg kg -1 / 11.1 mg kg -1 ), and hematoxylin-eosin staining (H&E) was performed on the main organs.

[0155] 17. Evaluation of skin photosensitivity

[0156] Healthy BALB / c female mice were depilated 3 days before the light exposure to ensure that all skin lesions had recovered. Shaved mice were randomly divided into four groups (n = 4): (a) injection of normal saline without laser irradiation; (b) laser irradiation; (c) free Ce6 + laser irradiation (Ce6 dose of 2 mg kg -1 ); (d) LD-DOX / Ce6 + laser irradiation (equivalent Ce6 dose of 2 mg kg -1 ). After administration of the above preparations, the anesthetized mice were immediately irradiated with a 660 nm laser (200 mW cm -2 , 4 min), and the mouse back with an area of about 1 cm 2 was exposed to laser irradiation. The back skin of these groups was photographed on days 1, 2, 3, 4 and 5 after irradiation, respectively. Finally, the skin of each group of mice was peeled off and analyzed by H&E staining.

[0157] 18. In vivo pharmacokinetics and fluorescence imaging

[0158] In the pharmacokinetic study, normal female BALB / c mice (20-22 g) were randomly divided into 2 groups (3 mice in each group). Free Ce6 and LD-DOX / Ce6 NPs were injected through the tail vein (Ce6 dose equivalent to 5 mg / kg mouse), respectively. At the pre-set time points (1 min to 24 h), 20 μL of blood was taken from the orbital plexus and collected in a heparin tube, and stored in a -20 °C refrigerator. After all the blood samples at different time points were taken, the blood samples were thawed at room temperature, diluted with 100 μL of acetonitrile, vortexed and then left to stand overnight at 4 °C. After standing, the samples were vortexed for 1 min and then centrifuged (14000 rpm) using a high-speed centrifuge. 100 μL of supernatant was taken and placed in a black 96-well plate, and the fluorescence intensity of Ce6 in the supernatant was detected using a multifunctional enzyme label instrument (excitation wavelength: 400 nm, emission wavelength: 660 nm). The relevant pharmacokinetic parameters were calculated by fitting the Ce6 concentration in the plasma with a non-compartment model using PKsolver 2.0 software.

[0159] The present application first investigated the tumor targeting ability of LD-DOX / Ce6 in 4T1-bearing orthotopic breast cancer mice. After administration of the drug to the mice, the biodistribution of LD-DOX / Ce6 in the tumor was evaluated by fluorescence imaging. Mice with a tumor volume of about 150 mm 3 were injected with LD-DOX / Ce6 through the tail vein (n = 3), and the Ce6 dose administered to each mouse was 5 mg kg -1 . Subsequently, fluorescence imaging was performed at 1, 6, 24 and 48 hours after administration. In addition, the present application also investigated the penetration of nanoparticles from tumor blood vessels into tumors using CLSM. When the volume of the orthotopic 4T1 tumor reached 150 mm3 At the same time, after intravenous administration of LD-DOX / Ce6 (5 mg Ce6 / kg mouse), the mice were anesthetized with isoflurane and the skin covering the tumor surface was removed, and the fluorescence changes in the tumor site were immediately observed using a fluorescence microscope.

[0160] The present application studied the in vivo distribution of LD-DOX / Ce6 NPs by 4T1 subcutaneous xenograft tumor model. When the tumor volume reached about 100 mm 3 At the same time, after intravenous administration of LD-DOX / Ce6 (5 mg Ce6 / kg mouse), the mice were anesthetized with isoflurane and the skin covering the tumor surface was removed, and the fluorescence changes in the tumor site were immediately observed using a fluorescence microscope.

[0161] 19. Evaluation of in vivo anti-tumor effect of chemo-photodynamic therapy

[0162] The subcutaneous 4T1 tumor model and orthotopic 4T1 tumor model were established as described above. For the subcutaneous tumor model, when the tumor volume of female BALB / c mice reached about 70 mm 3 At the same time, after intravenous administration of LD-DOX / Ce6 (5 mg Ce6 / kg mouse), the mice were anesthetized with isoflurane and the skin covering the tumor surface was removed, and the fluorescence changes in the tumor site were immediately observed using a fluorescence microscope. 2Mice were irradiated for 20 minutes. Throughout the experiment, mouse weight and tumor volume were recorded every two days. On day 21, all mice were euthanized, and major organs and tumors were dissected. Tumors in each group were weighed, and the tumor growth inhibition rate (TGI) was calculated using the following formula: TGI = (1 - W1 / W2) × 100%, where W1 and W2 represent the average tumor weight of the treatment and control groups, respectively. Organs and tumors in each experimental group were stained with H&E, and their morphology and pathological characteristics were observed. Tumor tissues from each experimental group were further used for immunohistochemical analysis.

[0163] For the in situ 4T1 tumor model, when the 4T1 tumor volume reaches approximately 70 mm... 3 Mice were divided into 6 groups: a saline group, a free Ce6+ light-treated group (8.9 mg / kg), and a 24-month-old mouse group. -1 Mice), free DOX (4 mg kg) -1 ), LD-DOX (DOX = 8 mg / kg) -1 ), LD-DOX / Ce6 without laser irradiation (DOX = 8 mg kg-1, Ce6 = 8.9 mg kg-1) and LD-DOX / Ce6 + laser irradiation group (DOX = 8 mg kg-1). -1 Ce6 = 8.9 mg / kg -1 Mice in each experimental group were administered the drug via tail vein injection on days 1, 5, 9, and 13. For the laser irradiation group, a 660nm laser (250mW / cm²) was applied 24 hours after each drug administration. 2 Mice were irradiated for 20 min. Throughout the experiment, mouse body weight and tumor volume were recorded every two days. On day 21, tumor metastases in the lungs of each group of mice were detected using a Quantum GX Micro-CT imaging system (Quantum GX, PerkinElmer, USA). Micro-CT scan parameters were as follows: voltage, 80 kV; current, 100 μA; pixel size, 150 μm. 3D images were reconstructed using Analysis 12.0 software (PerkinElmer, USA) to generate a visual representation of the results. After euthanasia, the lungs were harvested and fixed with 4% paraformaldehyde. H&E staining analysis of mouse lung sections and calculation of metastatic nodules on the lung surface were performed.

[0164] For the in situ 4T1 tumor model, when the 4T1 tumor volume reaches approximately 70 mm... 3The mice were divided into 6 groups and were injected with saline, free Ce6+L, free DOX, LD-DOX, LD-DOX / Ce6 and LD-DOX / Ce6+L, respectively, with equivalent DOX and Ce6 (equivalent to 4 mg DOX / kg mouse and 4.4 mg Ce6 / kg mouse) via tail vein injection on days 1, 5, 9 and 13, respectively. For the laser irradiation group, the mice were irradiated after each administration, and 660 nm laser (250 mW cm-2) was used for 20 minutes at 24 hours after injection. The body weight and tumor volume were monitored as described above. The survival rate was recorded for 30 days (when the mouse weight decreased by more than 20% or the longest diameter of the tumor was greater than 12 mm, the mouse was considered dead). On day 21, the tumor metastasis in the lungs of mice in each group was detected by Quantum GX Micro-CT imaging system (Quantum GX, PerkinElmer, USA). The micro-CT scan parameters were as follows: voltage, 80 kV; current, 100 μΑ; pixel size, 150 μιη. The 3D images of tumor metastasis in the lungs of mice were reconstructed using Analyze 12.0 software (PerkinElmer, USA). At the end of the experiment, the mice were sacrificed and the lung tissue was stripped and fixed with 4% paraformaldehyde. The tumor nodules on the surface of the mouse lung were calculated, and the mouse lung lobe sections were analyzed by H&E staining.

[0165] II. Experimental results

[0166] Table 1. Molecular weight distribution (MWD) statistical parameters of POEGMA-CTA, POEGMA-GFLG-(NHNHBoc) x and POEGMA-GFGL-(NHNHBoc) x Molecular weight distribution (MWD) statistical parameters of POEGMA-GFLG-(DOX)x and POEGMA-GFGL-(DOX)x (x = 1, 2 or 4)

[0167]

[0168] Table 2. Drug loading capacity (DLC), critical micelle concentration (CMC) and self-assembly particle size of POEGMA-GFLG-(DOX)x and POEGMA-GFGL-(DOX)x (x = 1, 2 or 4)

[0169]

[0170]

[0171] Table 3. Survival rate of mice treated with 5 mg kg -1Pharmacokinetic parameters of female Balb / c mice after intravenous injection of equivalent DOX doses of free DOX, POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2, and POEGMA-GFLG-(DOX)4 (data obtained by fitting a non-compartmental model using PKSolver 2.0 software).

[0172]

[0173] Table 4. Characterization of Ce6-loaded nanoparticles obtained at different therapeutic agent loading ratios (DLC represents drug loading, DLE represents drug encapsulation efficiency).

[0174]

[0175] Table 5. Characterization of PTX-loaded nanoparticles obtained at different therapeutic agent loading ratios (DLC represents drug loading, DLE represents drug encapsulation efficiency).

[0176]

[0177] Table 6. 5 mg / kg -1 Pharmacokinetic parameters of female Balb / c mice after intravenous administration of equivalent Ce6 doses of Ce6 and LD-DOX / Ce6 (data obtained by fitting a non-compartmental model using PKSolver 2.0 software).

[0178]

[0179]

[0180] To obtain structurally optimized LDBCs, this invention first synthesized a series of multifunctional polymer prodrugs with different structures and evaluated their potential as drug carriers. For example... Figure 1As shown in FIG. 1, in these polymer prodrugs, the chemotherapeutic drug DOX is attached to the periphery of the dendrimer through a hydrazone bond, while the dendrimer is covalently linked to the polymer backbone through a tetrapeptide (GFLG or GFGL). The GFLG linker can be cleaved in the presence of cathepsin B, which is overexpressed in a variety of tumor cells including breast cancer. As a control, the present application prepared monomers modified with a cathepsin B less sensitive tetrapeptide sequence GFGL to compare the differences in their physicochemical properties (Schemes S2 and S4). In addition, the present application also prepared dendrimers of different generations to investigate the influence of dendrimers on the assembly characteristics of LDBCs (Schemes S3-S4). These polymer-DOX prodrugs were synthesized through a series of steps: first, macromolecular chain transfer agents based on OEGMA (POEGMA-CTA, Scheme S3) were prepared by controlled RAFT polymerization, and GFLG or GFGL functionalized dendrimers were prepared by a solution synthesis method. Subsequently, macromolecular chain transfer agents POEGMA-CTA were subjected to RAFT polymerization with MA-GFLG-NHNHBoc, MA-GFLG-(NHNHBoc)2, MA-GFLG-(NHNHBoc)4, MA-GFGL-NHNHBoc, MA-GFGL-(NHNHBoc)2, and MA-GFGL-(NHNHBoc)4, respectively, to prepare polymer intermediates with different structures. After de-Boc treatment, the antitumor drug DOX was covalently attached to the periphery of the dendrimer through a pH-sensitive hydrazone bond, thereby preparing a series of amphiphilic linear-dendritic block polymer prodrugs (POEGMA-GFLG / GFGL-(DOX) x )(where x = 1, 2, or 4).

[0181] The present application first prepared cathepsin B sensitive monomers MA-GFLG-NHNHBoc, MA-GFLG-(NHNHBoc)2, MA-GFLG-(NHNHBoc)4, and cathepsin B less sensitive monomers MA-GFGL-NHNHBoc, MA-GFGL-(NHNHBoc)2, and MA-GFGL-(NHNHBoc)4, and their chemical structures were confirmed by proton nuclear magnetic resonance ( 1 H NMR) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) ( Figure 8 to Figure 22 ). Polymer intermediates with different structures were also characterized by 1 H NMR and gel permeation chromatography (GPC) ( Figure 23 to Figure 24(See Table 1). As shown in Table 1, the obtained polymer intermediates had high molecular weight (MW > 28 kDa) and narrow molecular weight distribution (polydispersity index < 1.3). After deprotection treatment, the DOX content covalently linked to the polymer side chains via hydrazone bonds was determined by UV-Vis spectroscopy. As shown in Table 2, POEGMA-GFLG-(DOX)4 and POEGMA-GFGL-(DOX)4 showed the highest DOX loadings compared to their analogues, at 10.8% and 11.3%, respectively.

[0182] Because polymer intermediates and products have similar structures, they have similar properties. Figure 24 ) 1 1H NMR spectrum. Specifically, taking POEGMA-GFLG-(DOX)4 as an example, compared with POEGMA-CTA, characteristic peaks of dendritic monomers were observed at 7.0-10.0 and 1.3 ppm, while the characteristic peaks of olefin bonds in functionalized monomers disappeared. Figure 25 (a and 25b) indicate that LDBC was successfully synthesized via RAFT polymerization. After the carbonyl reaction with DOX, the characteristic peak of the -Boc group at 1.3 ppm in POEGMA-GFLG-(NHNHBoc)4 disappeared, while characteristic peaks of DOX were observed at 7.5-8.5 ppm and 5.3 ppm. Figure 25 c). Furthermore, a significant UV-vis absorption peak of DOX in LD-DOX was observed at 480 nm. Figure 25 d) The purity results by HPLC showed that the polymer prodrug did not contain free DOX. Figure 25 e) indicates that DOX was successfully linked to the polymer via hydrazone bonds. These results demonstrate that the present invention successfully prepared a linear-dendritic polymer prodrug based on POEGMA and peptide dendritic molecules.

[0183] The GFLG linker between dendrites and the polymer backbone can be cleaved by cathepsin B, while the hydrazone bond between DOX and dendritic molecules can be cleaved in a weakly acidic environment. Therefore, in the microenvironment of tumor cells, dual-responsive polymer prodrugs hold promise for rapid degradation and release of their contained DOX. To investigate the DOX release behavior of polymer prodrugs co-triggered by cathepsin B and pH, different samples were incubated in a simulated tumor cell microenvironment. Figure 1b, the dual-responsive polymer prodrugs POEGMA-GFLG-DOX, POEGMA-GFLG-(DOX)2 and POEGMA-GFLG-(DOX)4 showed fast drug release properties, with the cumulative release of DOX close to 80% within 8 h, which was significantly higher than the cathepsin B low-sensitive polymer prodrugs modified with GFGL (less than 50%) and the polymer prodrugs modified with GFLG in the presence of cathepsin B at pH 7.4 (less than 30%) Figure 25 f). This fast release property can allow the drug to rapidly reach therapeutic concentrations in tumor cells, thereby reducing the likelihood of drug resistance. In addition, the CMC of the dual-responsive polymer prodrugs was measured to evaluate their ability to self-assemble into micelle-like structures of nanoparticles. As shown in Figure 1 c, POEGMA-GFLG-(DOX)4 had the lowest CMC value (2.22 μg mL -1 ), which indicated that high-generation dendrimers were expected to improve the stability and drug loading capacity of linear-dendritic polymers. Subsequently, the present application studied the pharmacokinetic properties of different dual-sensitive polymer prodrugs in mice. Figure 1 d shows the change of DOX concentration in plasma over time after intravenous injection of three prodrugs and free DOX into mice. The pharmacokinetic parameters (Table 3) show that the POEGMA-based prodrugs significantly prolonged the half-life of DOX. Among them, the blood half-life of POEGMA-GFLG-(DOX)4 was the longest, which was 1.78 times and 1.41 times that of POEGMA-GFLG-DOX and POEGMA-GFLG-(DOX)2, respectively. Based on the above analysis, POEGMA-GFLG-(DOX)4 (named LD-DOX, i.e. the LD-DOX prepared in Example 1) was selected as the best carrier for further study.

[0184] Notably, LD-DOX has several significant advantages compared to previously reported linear dendritic polymers. First, the high molecular weight chain transfer agent POEGMA-CTA has a higher polymerization activity, which enables it to effectively polymerize with the methacrylated dendrimers. The present application first designed and prepared this new type of LDBC that can be obtained by one-step RAFT polymerization. The dendritic part of the LDBCs prepared by this method can covalently polymerize multiple dendrimers, thus effectively avoiding the problem of fewer reaction sites in traditional linear polymers. Second, the peptide-based dendrimers that make up the dendritic part of the LDBCs have higher biocompatibility, and their appropriate generation number and lower molecular weight can reduce steric hindrance during the reaction process and improve polymerization efficiency. At the same time, the peptide-based dendrimers are prepared by step-by-step organic synthesis, so their structure can be precisely controlled, and it is also easy to realize multifunctionalization, such as endowing them with cathepsin B and pH responsiveness. In addition, low-generation dendrimers also effectively avoid complex synthesis steps and structural defects that may exist in high-generation dendrimers. Finally, the structure and properties of the linear polymer chain can be easily adjusted by RAFT polymerization, and the resulting POEGMA linear segment has a controlled molecular weight and low polydispersity. The molecular weight of the linear polymer chain of LD-DOX is much higher than that of the PEG-based linear-dendritic polymers reported so far, and it has a higher reaction activity, which provides more possibilities for selecting dendritic parts for polymerization. In addition, a high proportion of hydrophilic segments is also more conducive to the assembly of LD-DOX into stable spherical micelle-like structures, thereby improving its stability and drug loading capacity. Therefore, by combining the advantages of dendritic structures and the advantages of block copolymers, LD-DOX is considered to be a promising candidate for constructing drug delivery systems.

[0185] The present application simulates the self-assembly of LD-DOX and its ability to encapsulate drugs by dissipative particle dynamics (DPD) calculation. The polymer structure and molecular model of LD-DOX are shown in FIG. 1. Figure 1 Figure 26 The process of dynamic formation of nanoparticles of the prodrug LD-DOX in an aqueous solution is depicted. Due to the presence of hydrophilic linear POEGMA and hydrophobic dendritic parts, LD-DOX first forms smaller incomplete aggregates through hydrophilic and hydrophobic interactions, and then gradually forms larger complete micelle-like nanoparticles. This result shows that the prodrug based on amphiphilic LDBCs has good self-assembly ability. Further DPD simulation was performed using the photosensitizer Ce6 to demonstrate the ability of the polymer prodrug nanoparticles to encapsulate hydrophobic drugs Figure 1 ​f) Initially, the hydrophobic domains of the polymer interact with the hydrophobic Ce6 molecules to minimize the volume fraction of Ce6. Then, smaller, incomplete micelle-like aggregates form. Finally, larger, complete micelles appear with Ce6 encapsulated in their hydrophobic core. In addition, by adjusting the concentration of Ce6, the present application compares the nanoparticle formation of the polymer prodrug at different Ce6:DOX weight ratios. Figure 1 g shows snapshots of the equilibrium structures of these polymers self-assembled at different Ce6:DOX feed ratios. It can be noted that when the weight ratio of Ce6 to DOX does not exceed 1, the polymer prodrug and Ce6 form complete nanoparticles. Further increasing the Ce6 concentration (DOX:Ce6 = 1:1.5, wt / wt) will lead to saturation of the number of Ce6 molecules in the micellar hydrophobic core. Under this condition, the excess Ce6 molecules exist in the form of colloids or precipitates in the solvent.

[0186] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to detect the size and morphology of the polymer prodrug-based nanoparticles. As shown in Figure 27 a, DLS measurements confirmed that the LD-DOX prodrug self-assembled into nanoparticles with a hydrodynamic size of about 46.8 nm in aqueous solution. Notably, the Ce6-loaded nanoparticles LD-DOX / Ce6 (Ce6:DOX = 1.5, wt / wt) showed a larger hydrated particle size (about 67.7 nm, Figure 27 b), and TEM detection results showed that the nanoparticles had a regular spherical morphology Figure 1 h). In addition, the present application used 100,000 time steps to simulate the drug release process of the nanoparticles in the presence of cathepsin B at pH 5.4. As shown in Figure 1 i and Figure 47 The presence of cathepsin B and acidic pH value will cause the cleavage of GFLG tetrapeptide and hydrazone bond, respectively, leading to the disintegration of the nanostructure and drug release. After incubation at pH 5.4 for 24 hours, TEM detection showed that the nanoparticles LD-DOX / Ce6 presented a loose morphology Figure 1 j), indicating that the structure of the nanoparticles was decomposed under acidic conditions. The above results show that the nanoparticles can specifically degrade and release the loaded therapeutic agents in the tumor microenvironment. The DPD simulation release curve shows that the drug release rate of the nanoparticles under the dual response of pH and enzyme is significantly accelerated compared to the single response Figure 28c and 28d). These simulations provide a reasonable explanation of how the LDBC-based prodrug forms nanoparticles, how Ce6 is loaded into these particles, and the release mechanism of DOX and Ce6. The results show that hydrophobic interactions are the main driving force for nanoparticle formation and drug loading, and the disassembly of the nanoparticle assembly structure caused by the breaking of specific chemical bonds (GFLG peptide and hydrazone bond) is the main driving force for drug release.

[0187] Subsequently, the present application evaluated the potential of LDBC-based LD-DOX prodrug to co-deliver hydrophobic therapeutic agents to achieve combination anticancer therapy. Table 4 and Table 5 summarize the results of encapsulating PTX or Ce6 into LD-DOX by the thin film hydration method. Figure 29 The results show that LD-DOX can effectively encapsulate and control the release of PTX. Therefore, LD-DOX can be a multifunctional, efficient nanodrug delivery platform. The present application next selected Ce6 as a dye-based photosensitizer to encapsulate into LD-DOX for preparing a model nanodrug for chemical-photodynamic combination antitumor therapy. UV absorption spectra show that the characteristic absorption peak of Ce6 (660 nm) is observed in PBS( Figure 2 a) or DMSO( Figure 30 ) solution, indicating that it is successfully loaded into the nanoparticles. With the increase of Ce6 feeding amount, the drug loading of Ce6 in LD-DOX NPs gradually increases, and when the DOX:Ce6 ratio is 1:1.5 (wt / wt), the encapsulation efficiency of Ce6 reaches a maximum value (74.3%) ( Figure 2 b and Table 4). Therefore, the present application selected the nanoparticles obtained at this ratio (Ce6 drug loading of 10.0 wt%) for further study and named them as LD-DOX / Ce6 (i.e. LD-DOX / Ce6 prepared in Example 2).

[0188] It is worth noting that the particle size of LD-DOX / Ce6 increases( Figure 2 c) compared with LD-DOX, which can be due to the entry of Ce6 into the hydrophobic core of the nanoparticles, causing the swelling of the nanoparticle core. Further, the present application compared the UV-Vis absorption spectra of LD-DOX / Ce6, free DOX, and free Ce6. As shown in Figure 2 d, the characteristic peak corresponding to Ce6 is observed in LD-DOX / Ce6, but not in LD-DOX, which further indicates the successful encapsulation of Ce6 into the nanoparticles. Figure 2 e shows that LD-DOX / Ce6 has similar photo-stability as free Ce6 and can well resist photobleaching under long-time laser irradiation, indicating that it can be used as a good photosensitizer candidate for photodynamic therapy. The colloidal stability of the NPs solution has an important influence on its distribution in vivo and accumulation at the tumor site. As shown in Figure 2f, the changes in the particle size and polydispersity index (PDI) of LD-DOX / Ce6 were negligible after incubation in PBS for up to 48 h. Similar results were detected when it was incubated in PBS containing 10% fetal bovine serum (FBS) or cell culture medium without FBS Figure 31 a and 31b). In addition, LD-DOX showed good stability under storage condition at 4 °C and was well re-dispersed in aqueous solution after lyophilization Figure 31 c and 31d).

[0189] The drug release profile of LD-DOX / Ce6 NPs was analyzed by dialysis method. As shown in Figure 2 g and 2h, at pH 7.4, in the absence of cathepsin B, only about 12% of DOX and 25% of Ce6 were released within 48 h. As a control, at pH 5.4, the release of DOX and Ce6 reached 82% and 66% within 48 h, respectively. Notably, in the presence of cathepsin B, the release rate of DOX and Ce6 was significantly accelerated, with 78% of DOX and 63% of Ce6 released after 12 h of incubation. In the structure of LD-DOX / Ce6 NPs, DOX is covalently linked to the polymer through a pH-sensitive hydrazone bond, thus DOX can be rapidly released from the carrier under acidic conditions depending on the cleavage of the hydrazone bond. After the release of DOX, the balance between hydrophilicity and lipophilicity in the nanoparticle structure is destroyed, and the π-π interaction is weakened, thus the nanoparticle structure disintegrates and releases the encapsulated Ce6. Meanwhile, as the drug-loaded dendrimer is linked to the polymer backbone through GFLG short peptides, GFLG is cleaved under the action of high concentration of cathepsin B, which further promotes the disintegration of the nanostructure and accelerates the release of DOX and Ce6.

[0190] Photodynamic therapy (PDT) is a method of killing tumor cells by irradiating a photosensitizer with a specific wavelength of laser light, thereby generating reactive oxygen species (ROS) with cytotoxicity. In this work, the present invention used singlet oxygen green fluorescent probe (SOSG) to study the singlet oxygen (SO) generated by LD-DOX / Ce6 NPs and free Ce6 under 660 nm laser irradiation. As shown in Figure 33 a-e, the SOSG fluorescence intensity of the control groups (free Ce6, LD-DOX / Ce6 NPs without laser irradiation, DOX with laser irradiation or LD-DOX with laser irradiation) remained at baseline levels, while an irradiation time-dependent enhancement of SOSG fluorescence intensity was observed in LD-DOX / Ce6 solution after irradiation Figure 2 i), which was similar to free Ce6 under the same irradiation conditions, indicating that LD-DOX / Ce6 NPs effectively generated ROS at an equivalent level to free Ce6 after irradiation.

[0191] The present application studies the cellular uptake of LD-DOX / Ce6 NPs by detecting the fluorescence signals of Ce6 and DOX. The fluorescence intensities of free Ce6, free DOX and LD-DOX / Ce6 NPs are observed by confocal laser scanning microscopy (CLSM). As shown in FIG. 3a, in the LD-DOX / Ce6 NPs treatment group, the strong coexistence of DOX and Ce6 fluorescence in the cells is observed. In addition, the fluorescence of free DOX is distributed in the nucleus, while under the same conditions, the fluorescence of DOX in LD-DOX / Ce6 NPs mainly exists in the cytoplasm, further indicating that the intracellular distribution of DOX may be affected by the LD-DOX / Ce6 delivery system. Figure 3 Figure 34 ) As shown in FIG. 4a, the fluorescence intensities of DOX and Ce6 in the MCS increase with the increase of incubation time. After 6 hours of incubation, significant fluorescence distribution is observed in the MCS at a depth of 60 μm (FIG. 4b), indicating that LD-DOX / Ce6 NPs have a good in vitro three-dimensional (3D) model simulating 4T1 tumor tissue. Figure 35 Figure 3

[0192] In order to determine the level of reactive oxygen species produced after 4T1 cells uptake free Ce6 or LD-DOX / Ce6, the present application incubates the two preparations with 4T1 cells respectively, and detects the intracellular reactive oxygen species with the fluorescent probe DCFH-DA. In the cell, DCFH-DA can be hydrolyzed by esterase to DCFH which cannot penetrate the cell membrane, so as to be loaded into the cell. The non-fluorescent DCFH can be oxidized to fluorescent DCF by intracellular reactive oxygen species, so the level of intracellular reactive oxygen species can be evaluated by detecting the fluorescence of DCF. As shown in FIG. 5c, without laser irradiation, the fluorescence intensities of free Ce6 and LD-DOX / Ce6 NPs treatment groups are very weak, and there is no obvious difference compared with the PBS group. In contrast, after laser irradiation, the fluorescence intensity of the control group does not change significantly, while the fluorescence intensities of LD-DOX / Ce6 NPs and free Ce6 treatment groups are significantly enhanced. As shown in FIG. 5d, the fluorescence intensity of the control group is not significantly different from that of the PBS group, while the fluorescence intensity of the LD-DOX / Ce6 NPs treatment group is significantly higher than that of the free Ce6 treatment group, indicating that the LD-DOX / Ce6 NPs can produce more reactive oxygen species than free Ce6. Figure 3 Figure 3 ​​​​d, flow cytometry quantitative analysis results showed that after laser irradiation, the DCF fluorescence intensity in Ce6 or LD-DOX / Ce6 NPs treated cells was significantly higher than that in PBS treated group, and the DCF fluorescence intensity level in LD-DOX / Ce6 NPs treated cells was close to that in Ce6 treated cells, which was consistent with the results observed by fluorescence microscope. The above results showed that under the condition of 660 nm laser irradiation, Ce6 could significantly promote the generation of reactive oxygen species in tumor cells, thereby playing its role in photodynamic therapy. γH2A.X is a marker of double-strand DNA break and is widely used to detect early DNA damage. Previous studies have shown that both DOX and PDT can cause significant and irreversible damage to DNA. As shown in Figs. Figure 3 e and 3f, compared with the control group, each experimental group caused significant cell DNA damage after treatment, indicating that chemotherapy and photodynamic therapy can cause damage to cancer cells through DNA damage.

[0193] The present application used CCK-8 detection kit to detect the in vitro cytotoxicity of different free therapeutic agents and LD-DOX / Ce6 NPs on 4T1 cells. As shown in Fig. Figure 36 a, under light shielding conditions, free Ce6 had no obvious toxicity to 4T1 cells, even when the Ce6 concentration was as high as 30 μg mL -1 , the cell survival rate was still above 95%. In contrast, after laser irradiation (660 nm, 10 mW cm -2 ) for 30 s, obvious concentration-dependent cytotoxicity was observed. When the Ce6 concentration was greater than 10 μg mL -1 , it showed great cytotoxicity to 4T1 cells under light irradiation. When the light irradiation time was 0.5 min, 1 min and 2 min, respectively, the IC 50 values of 4T1 cells gradually decreased, which were 6.9 μg mL -1 , 4.1 μg mL -1 and 1.6 μg mL -1 ( Figure 36 b), respectively. In addition, when the laser energy increased from 0 to 4.511 J cm -2 at the Ce6 concentration of 3 μg mL -1 , the survival rate of 4T1 cells gradually decreased from 100% to 5% ( Figure 36 c), indicating that the laser irradiation induced cytotoxicity was also related to the energy. The above results confirmed that the photodynamic therapy agent Ce6 could produce obvious cytotoxicity to 4T1 cells under the condition of specific wavelength laser irradiation.

[0194] Next, the present application studies the in vitro anti-tumor efficacy of LD-DOX / Ce6 on 4T1 cells. POEGMA and low-generation glutamic acid dendrimers do not produce cytotoxicity on 4T1 cells even at high concentrations Figure 37 a and 37b). As shown in Fig. Figure 3 g, five different formulations or treatment methods all produce concentration-dependent cytotoxicity on 4T1 cells. Among them, the nanoparticle administration group irradiated by laser (LD-DOX / Ce6 + L) shows the strongest cytotoxicity. Quantitative analysis results show that the IC 50 values of LD-DOX / Ce6 NPs + L, LD-DOX / Ce6 NPs, LD-DOX NPs and Ce6 + L on 4T1 cells are 0.141, 1.03, 2.01 μg / mL and 6.94 μg / mL, respectively Figure 37 c). This result shows that compared with single chemotherapy, the combined treatment method combining chemotherapy and photodynamic therapy can more effectively inhibit the growth of tumor cells, and has a very significant synergistic anti-tumor effect. It is worth noting that free DOX usually has better cytotoxicity than its prodrug because it can quickly enter cells through free diffusion. In this study, LD-DOX / Ce6 can achieve combined treatment of chemotherapy and photodynamic therapy after laser irradiation, and its therapeutic effect is significantly improved, reaching the level of cytotoxicity on 4T1 cells comparable to free DOX (the IC 50 value of free DOX on 4T1 cells is 0.14 μg / mL). In addition, the present application also analyzes the combination index (CI) of LD-DOX NPs and Ce6, and the result shows that the CI value is much lower than 1, which further indicates that LD-DOX NPs and Ce6 synergistically inhibit the proliferation of 4T1 cells Figure 38 .

[0195] The present application quantitatively analyzes the apoptosis level of 4T1 cells by flow cytometry to further evaluate the effectiveness of the combined chemotherapy-photodynamic therapy on 4T1 cells. Figure 3 i and 3h show the apoptosis of 4T1 cells after treatment with different formulations, in which Q1, Q2, Q3 and Q4 represent dead cells, late apoptotic cells, early apoptotic cells and living cells, respectively. As shown in the figures, after 660 nm laser irradiation (10 mW / cm 2) After 30s continuous irradiation, there was no significant difference in apoptosis between 4T1 cells receiving laser irradiation alone and the control group, indicating that laser irradiation had little effect on cell viability. In contrast, cells in other administration groups showed varying degrees of increased apoptosis. Notably, the combined administration group (LD-DOX / Ce6+L) showed the highest level of apoptosis, with a total apoptosis rate of up to 81.9%, indicating significant cytotoxicity. The flow cytometry results were consistent with the cytotoxicity results determined by CCK-8, confirming that LD-DOX / Ce6, which has a combined chemotherapy-photodynamic therapy function, has a better in vitro anti-tumor effect than single chemotherapy or photodynamic therapy after light treatment.

[0196] To further understand the anti-tumor molecular mechanisms of DOX and Ce6 combination therapy, the present application compares the transcriptomic changes in tumor cell samples after different treatments by RNA sequencing. Figure 4 a The heatmap shows the differentially expressed genes (DEG) after PBS, free DOX, Ce6+L, and LD-DOX / Ce6+L treatment of 4T1 cells. These DEGs are mainly related to DNA damage repair (Brca1, Ercc1 Uvssa, and Rpa2), apoptosis (CD44), epithelial-mesenchymal transition (MMP3, MEST, TGFB1, FOXC2, BMP1, Hmox1, and Hspb1), cell cycle (Ccne2, Erbb3, Ccnb2, and Ccnd1), and proliferation pathways (Isg15 and Btg2). The volcano plot also shows that the LD-DOX / Ce6+L treatment group has upregulated genes related to DNA repair (Eid3, Prdm9, and Fancg) and cell cycle arrest (Ccne2), while downregulated genes related to tumor growth (Dach1 and App), TGFβ pathway (Tgfb1), and epithelial-mesenchymal transition (Mmp9 and Stc1) compared to the control group Figure 4 b) Gene ontology (GO) term enrichment analysis and KEGG enrichment analysis show that, compared to the control group, the upregulated genes after LD-DOX / Ce6+L treatment mainly include DNA repair and DNA replication pathways Figure 4 c and 4d).

[0197] In addition, gene set enrichment analysis (GSEA) of LD-DOX / Ce6+L treated cells showed positive enrichment of genes characteristic of apoptosis (NES = 1.76, FDR = 0.04), DNA repair (NES = 1.75, FDR = 0.04), and cell cycle arrest (NES = 1.79, FDR = 0.03) compared to control groups. DNA repair is a corrective response of cells to DNA damage and alterations. Therefore, LD-DOX / Ce6+L treatment can directly induce cell death by causing DNA damage, blocking cell cycle, and inducing apoptosis. As shown in FIGS. 16a-16c, LD-DOX / Ce6+L treated cells showed positive enrichment of genes characteristic of apoptosis (NES = 1.76, FDR = 0.04), DNA repair (NES = 1.75, FDR = 0.04), and cell cycle arrest (NES = 1.79, FDR = 0.03) compared to other control groups. Figure 4 As shown in FIGS. 17a-17c, LD-DOX / Ce6+L treated cells showed negative enrichment of genes characteristic of epithelial-mesenchymal transition (EMT, NES = -2.02, FDR = 0.0), angiogenesis (NES = -1.85, FDR = 5.79E-4), and TGF signaling (NES = -2.07, FDR = 0.0) compared to other control groups. Previous reports have shown that EMT and TGF pathway are closely related to the migration and invasion of tumor cells, thus these data suggest that LD-DOX / Ce6+L combination therapy significantly inhibited the metastatic ability of cancer cells. Importantly, the combination treatment further inhibited the EMT pathway, TGF pathway, angiogenesis, and hypoxia pathway (FIGS. 17a-17c) compared to cells treated with Ce6+L or DOX alone. Figure 39 a and 39b). Hypoxia-inducible factor 1 (HIF1) has been reported to be an important factor in promoting cancer development. The HIF1A pathway has been shown to be closely related to tumor metastasis, angiogenesis, poor prognosis of patients, and drug resistance of tumors to chemotherapy. The above data suggest that the combination therapy significantly inhibited tumor metastasis compared to DOX or Ce6+L alone. At the same time, the combination therapy upregulated the DNA repair pathway (NES = 1.53, FDR = 0.01 and NES = 1.82, FDR = 0.04 compared to free DXO and Ce6+L), which indicates that the combination therapy induced more severe DNA damage (FIG. 18a). Figure 39 a). In addition, the combination treatment directly induced higher levels of apoptosis (NES = 2.03, FDR = 0.001) (FIG. 18b) compared to those treated with Ce6+L. Figure 39 a). Therefore, the combination therapy can not only inhibit tumor growth, but also significantly reduce tumor metastasis compared to DOX or Ce6+L therapy.

[0198] DOX treatment induced apoptosis (NES = 1.49, FDR = 0.03) and inhibited Myc target pathway (NES = -2.62, FDR = 0.0) and HIF1A pathway (NES = -1.68, FDR = 0.12) compared to control groups (FIGS. 19a-19c). Figure 39c), which indicates that DOX treatment promotes cancer cell death and inhibits tumor growth and metastasis. Compared with the control group, Ce6+L group not only inhibits the cell proliferation pathway (NES = -1.95, FDR = 0.008), but also inhibits the HIF1A pathway (NES = -1.82, FDR = 0.02) and the NF-κB pathway (NES = -2.36, FDR = 0.0) Figure 39 d). The NFκB pathway is widely involved in the occurrence and progression of cancer, which mediates tumor cell proliferation, survival and angiogenesis by regulating the expression of TNFA, IL6, BCL2 and VEGF. Therefore, treatment with DOX or Ce6+L can inhibit tumor growth and metastasis.

[0199] Consistent with bioinformatics analysis, Western blot analysis results show that after treatment with free DOX, Ce6+L or LD-DOX / Ce6+L, apoptosis-related proteins, including Bax, cleaved caspase-3 and cleaved PARP, are significantly up-regulated Figure 4 f and Figure 40 ) In addition, the expression of DNA damage-related marker γH2A.X in cells is also significantly enhanced Figure 4 f and Figure 40 ) after treatment with free DOX, Ce6+L or LD-DOX / Ce6+L.

[0200] Encouraged by the excellent anti-tumor effect of LD-DOX / Ce6 in vitro, the present application further evaluates its biocompatibility through acute toxicity test. As shown in Figure 41 , free DOX shows obvious dose-dependent toxicity, and injection of high-dose DOX into mice (10 mg kg -1 and 15 mg kg -1 ) leads to severe weight loss and even death of mice. In contrast, mice in LD-DOX and LD-DOX / Ce6 treatment groups survive at a dose equivalent to 30 mg DOX kg -1 . In addition, the results of H&E staining of mice treated with 10 mg kg -1 DOX show that the liver and heart exhibit obvious pathological changes Figure 42 , which is consistent with the results of their severe weight loss. In addition, the results of blood routine and biochemical tests show that the ALT, AST, RBC and PLT of the DOX treatment group have significant differences with the control group Figure 43 and 44 , further indicating that DOX has serious side effects. However, these blood test indicators of LD-DOX- and LD-DOX / Ce6-treated groups have no significant difference with the control group at the equivalent dose of DOX Figure 43 and 44, indicating that the systemic toxicity of DOX was significantly reduced and both LD-DOX and LD-DOX / Ce6 had good biocompatibility. In addition, the skin photosensitivity of Ce6 and LD-DOX / Ce6 was also evaluated. As shown in Figure 45 Fig. 6b, the skin of mice treated with Ce6 was severely damaged after exposure to 660 nm laser. H&E sections showed severe necrosis of the skin. In contrast, mice treated with LD-DOX / Ce6 only showed mild edema after laser irradiation, and there was no significant difference in H&E sections between the LD-DOX / Ce6 treatment group and the control group after 5 days. Overall, these results strongly support the in vivo biocompatibility and biosafety of LD-DOX / Ce6, laying a solid foundation for its application in vivo as a chemo-photodynamic combination therapy.

[0201] PEGylated nanomedicine delivery systems are expected to significantly improve the pharmacokinetic properties of small molecule drugs, thereby enhancing their anti-tumor therapeutic effect. Therefore, the inventors investigated the in vivo pharmacokinetic properties of LD-DOX / Ce6. Female BALB / c mice were injected intravenously (iv) with free Ce6 and LD-DOX / Ce6 at an equivalent Ce6 dose of 5.0 mg / kg, respectively, and blood samples were collected at different time points after injection, and the concentration in the blood was determined by measuring the Ce6 fluorescence. As shown in Figure 5 Fig. 7a, no Ce6 fluorescence signal was detected in the free Ce6 administration group 8 hours after injection, indicating that free Ce6 had been eliminated from the blood circulation system. In contrast, the retention time of Ce6 in the blood was significantly prolonged after administration of LD-DOX / Ce6, and the fluorescence signal of Ce6 was still detectable in the blood 12 hours after injection (1.17 μg mL -1 ). The pharmacokinetic parameters by non-compartment analysis are summarized in Table 6. As shown in the data in the table, the elimination half-life (t1 / 2) of LD-DOX / Ce6 NPs was 13.6 h, which was 8.5 times that of free Ce6 (1.6 h). This result indicates that Ce6 can significantly prolong its in vivo circulation time after being encapsulated by NPs.

[0202] The prolonged blood circulation time of LD-DOX / Ce6 prompted the inventors to further investigate the targeting ability of NPs in the orthotopic 4T1 breast tumor model of BALB / c mice. As shown in Figure 5 Fig. 7b, LD-DOX / Ce6 showed excellent targeting in the orthotopic 4T1 breast tumor model, and accumulated a large amount of LD-DOX / Ce6 in the primary tumor after intravenous administration. In addition, the results of the window model also confirmed that the fluorescence intensity at the orthotopic 4T1 breast tumor gradually increased within 6 hours after administration, indicating that NPs can effectively enrich and spread at the tumor site Figure 5 c).

[0203] This invention further analyzed the biodistribution of LD-DOX / Ce6 in tumor-bearing mice by detecting the fluorescence of Ce6. Figure 5 As shown in Figure d, in vitro fluorescence imaging results indicated that 1 hour after administration, both free Ce6 and LD-DOX / Ce6 were widely distributed in the heart, liver, spleen, lungs, kidneys, and tumors of mice. The difference was that free Ce6 exhibited very high fluorescence intensity in the liver, indicating significant accumulation in the liver after in vivo. Within 12 hours after administration, the fluorescence intensity of Ce6 in various organs of mice treated with free Ce6 gradually decreased over time, becoming very weak after 12 hours. Conversely, the fluorescence intensity in various organs of mice treated with LD-DOX / Ce6 remained at a high level, with a gradually increasing trend in the tumor site. 24 hours after administration, almost no Ce6 fluorescence signal was observed in any organs of the free Ce6 group, while significant Ce6 fluorescence was still detectable in the tumor site of the LD-DOX / Ce6 NPs treatment group.

[0204] To further analyze the distribution of LD-DOX / Ce6 NPs in vivo, this invention performed a semi-quantitative analysis of fluorescence intensity in tumors and major organs. For example... Figure 46 As shown in a and 46b, compared with the free Ce6 group, the LD-DOX / Ce6 group showed stronger fluorescence signals of Ce6 in various organs and tissues. Notably, compared to the slight fluorescence signal changes of free Ce6 at the tumor site, the fluorescence signal in the LD-DOX / Ce6 group at the tumor site showed a gradually increasing trend, peaking at 24 hours after administration and remaining at a relatively high level for 72 hours. Figure 46 c). These results are consistent with pharmacokinetic data, indicating that LD-DOX / Ce6 NPs effectively prolong the circulation time of Ce6 in vivo, improve its biodistribution, and increase its accumulation at tumor sites.

[0205] Figure 5 e represents frozen sections of tumor tissues from tumor-bearing mice in the free Ce6 group and the LD-DOX / Ce6 group at different administration time points, obtained by CLSM. 24 hours after administration, significant Ce6 fluorescence signals were observed in the tumor tissues of the LD-DOX / Ce6 NPs group, while the Ce6 signal in the tumor sites of the free Ce6 group was very weak at all time points. Figure 47 These results are consistent with those obtained from in vitro fluorescence imaging, further demonstrating that LD-DOX / Ce6 NPs can effectively deliver Ce6 to the tumor site via the EPR effect.

[0206] The in vivo anti-tumor efficacy of LD-DOX / Ce6 NPs was evaluated by establishing a xenograft 4T1 tumor model on BALB / c mice Figure 5 f) When the tumor volume reached about 70 mm 3 (n=7) were randomly divided into nine groups and treated according to the following administration methods: Group I: normal saline; Group II: laser irradiation alone; Group III: free Ce6 plus laser irradiation (8.9 mg Ce6 / kg); Group IV: free DOX (4 mg DOX / kg); Group V: LD-DOX / Ce6 NPs without laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group VI: LD-DOX / Ce6 NPs without laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg); Group VII: LD-DOX / Ce6 NPs plus laser irradiation (4 mg DOX / kg, 4.4 mg Ce6 / kg); Group VIII: LD-DOX / Ce6 NPs plus laser irradiation (8 mg DOX / kg, 8.9 mg Ce6 / kg); Group IX: free DOX (8 mg DOX / kg). The body weight and tumor volume of mice in each group were measured and recorded at predetermined time points, and the changes in tumor volume of mice in different treatment groups are shown in Figure g. Figure 5 g.

[0207] During the entire treatment period, the tumor of mice treated by laser alone grew rapidly, with a growth trend close to that of the normal saline group, indicating that laser irradiation had no inhibitory effect on tumor growth. Compared with single free formulation treatment (free DOX at a dose of 4 mg / kg or free Ce6 plus laser irradiation at a dose of 8.9 mg / kg), the LD-DOX / Ce6 administration group without laser irradiation (corresponding to a DOX administration dose of 4 mg / kg or 8 mg / kg) showed a moderate tumor inhibition effect. After laser irradiation, the tumor inhibition effect of the LD-DOX / Ce6 NPs administration group was significantly enhanced, and the tumor growth inhibition was most obvious, indicating that the combined chemotherapy-photodynamic therapy could significantly improve the anti-tumor efficacy of small molecule therapeutic agents. Notably, as the administration dose increased, the anti-tumor effect of various formulations also correspondingly increased. However, although free DOX at a dose of 8 mg / kg had a significant inhibitory effect on tumor growth, the body weight of these mice decreased to less than 80% of the initial body weight after 13 days of administration, and they died before the end of the experiment, indicating that they had severe systemic toxicity. In contrast, when LD-DOX / Ce6 NPs were administered at a DOX dose of 8 mg / kg, this administration method had the best inhibitory effect on the tumor of mice, and no significant change in the body weight of the mice was observed Figure 5h). The above results indicate that LD-DOX / Ce6 NPs significantly reduced the toxic side effects of free DOX, which may be related to the fact that the nano-drug delivery system can significantly improve the in vivo distribution of DOX and increase its accumulation at the tumor site. After the experiment, the tumors of each group of mice were dissected and photographed (h). Figure 48 a) The tumor weight of mice in each experimental group was weighed and the growth inhibition rate was calculated. Figure 5 i). like Figure 48 As shown in a and b, compared with the saline group (Group I), each experimental group showed different degrees of antitumor effect. Compared with the saline group, the tumor inhibition rates of Groups II to VIII were 3.3%, 15.9%, 29.3%, 35.2%, 39.7%, 63.2%, and 76.2%, respectively. Among them, Group VIII (LD-DOX / Ce6 NPs irradiated by laser, with a dose of 8 mg DOX / kg mice and 8.9 mg Ce6 / kg mice) had the lightest mean tumor weight (253 ± 47 mg) and the highest TGI (76.2%), which is consistent with the trend of the tumor volume change curve.

[0208] Subsequently, the present invention performed H&E staining analysis on the tumor tissue and major organs. For example... Figure 49 As shown, compared with the control group, varying degrees of structural damage were observed in the tumor sections of all other treatment groups. Among them, the most significant tumor tissue necrosis was observed in the tumor sections of group VIII, manifested as extensive incomplete cell morphology, nuclear shrinkage, and increased intercellular spaces. To elucidate the antitumor mechanism of LD-DOX / Ce6NPs, this invention performed IHC analysis on the tumors of each group of mice. Figure 49 As shown, tumor microvascular staining revealed that the vascular density in the LD-DOX / Ce6 NPs group (8 mg DOX / kg, 8.9 mg Ce6 / kg) after laser irradiation was significantly lower than that in other treatment groups, indicating that the combined chemo-photodynamic therapy effectively inhibited tumor angiogenesis. Tumor Ki-67 staining showed a significant reduction in Ki-67-positive cells in group VIII, indicating that tumor cell proliferation was significantly inhibited after chemo-photodynamic therapy. Furthermore, TUNEL staining results showed that the administration route in group VIII induced the most significant tumor cell apoptosis, indicating that the combined chemo-photodynamic therapy effectively induced tumor cell apoptosis, a result consistent with H&E staining results of tumor tissue. These in vivo data collectively demonstrate that LD-DOX / Ce6 NPs based on chemo-photodynamic therapy exhibits better therapeutic effects than chemotherapy or PDT alone, exerting its anti-tumor effect by reducing angiogenesis, inhibiting tumor cell proliferation, and inducing apoptosis.

[0209] To investigate the systemic toxicity of different administration routes to tumor-bearing mice, this invention performed H&E staining analysis on the major organs of mice in each group after treatment. For example... Figure 50 As shown, in tissue sections of major organs (including heart, liver, spleen, kidney, and lung), significant tumor metastasis was observed in the lungs of mice in the saline group, the laser-only irradiation group, and the Ce6 plus laser irradiation group. Simultaneously, significant lesions and tumor metastases were also observed in the livers of these three groups. In contrast, no significant pathological changes or tumor metastasis were observed in the other treatment groups. These results indicate that the LD-DOX / Ce6 NPs prepared in this invention can effectively inhibit the metastasis of 4T1 tumors, while also exhibiting good in vivo biocompatibility.

[0210] Furthermore, this invention also evaluated the therapeutic effect of LD-DOX / Ce6 on an orthotopic breast cancer model. As expected, the combination therapy group (LD-DOX / Ce6 NPs+L) showed the best antitumor therapeutic effect compared to monotherapy. Figure 5 j), and the survival time of mice was significantly prolonged ( Figure 5 k). It is noteworthy that breast cancer, especially in situ breast cancer, readily metastasizes to the lungs. Therefore, this invention detected lung tumor metastasis in mice of each treatment group using CT imaging and H&E histological analysis. Figure 5 l and 5m). Figure 5 The 3D lung model shown in m indicates that numerous tumor metastases were detected in the lungs of mice treated with saline and Ce6+L 21 days after treatment. In contrast, single chemotherapy groups (DOX, LD-DOX, and LD-DOX / Ce6) only partially alleviated tumor metastasis. Notably, no visible tumor metastases were found in mice treated with LD-DOX / Ce6 NPs+L. At the endpoints of various treatments, H&E staining and counting of metastatic tumor nodules in mouse lung lobe sections further confirmed severe metastasis of in situ breast cancer in the lungs. Figure 5 (l and 5m). Obvious metastatic tumor nodules were observed in the saline and Ce6+L groups (indicated by yellow arrows), while the LD-DOX / Ce6+L treatment group exhibited a normal appearance similar to normal lung tissue. This result is consistent with the significant prolongation of survival in mice treated with LD-DOX / Ce6+L. These results indicate that chemotherapy combined with PDT not only inhibits tumor growth but also significantly prolongs survival time in mice by suppressing lung metastasis.

[0211] To sum up, the application firstly prepares a linear-dendritic polymer-based amphiphilic prodrug loaded with DOX through two-step RAFT polymerization, and then encapsulates photosensitizer Ce6 to prepare a combined chemical-photodynamic therapy polymer nanodrug delivery system: LD-DOX / Ce6 NPs. In vivo experiments prove that LD-DOX / Ce6 NPs can significantly prolong the in-vivo circulation time of free small molecule therapeutic agents, and enhance their accumulation at the tumor site through the EPR effect. In the intracellular microenvironment of tumor cells with overexpressed cathepsin B and weakly acidic conditions, the nanostructure of LD-DOX / Ce6 NPs can be rapidly degraded and simultaneously release DOX and Ce6. After Ce6 is irradiated by 660 nm laser, it can promote the generation of intracellular ROS, thereby producing a significantly improved synergistic therapeutic effect on tumors together with the chemotherapeutic drug DOX. Antitumor mechanism research shows that LD-DOX / Ce6 mainly exerts synergistic therapeutic effect by promoting apoptosis, inhibiting DNA repair and causing cell cycle arrest, and inhibits tumor metastasis by inhibiting EMT pathways, TGFbeta pathways, angiogenesis and hypoxia pathways. In-vivo antitumor experiments show that LD-DOX / Ce6 NPs have excellent in-vivo antitumor effect and good biological safety. The research results of the application show that the linear-dendritic polymer prodrug can be explored as a new multifunctional drug carrier, and can be used for cancer treatment by loading various therapeutic agents for combination of various treatment methods.

Claims

1. A polymer of formula I: ###0001### I wherein: x is selected from 20 to 60, and y is selected from 1 to 8. wherein x is selected from 43 to 53, and y is selected from 1 to 3; and the amino protecting group is a Boc group. R a For R b , R c , R d , R e are each independently selected from amino protecting groups.

2. The polymer of claim 1, wherein 4. A polymer of formula II: ###0002### II wherein: x is selected from 20 to 60, and y is selected from 1 to 8.

3. The polymer of claim 2, wherein M of the polymer is 23.7 kgmol n M of the polymer is 23.7 kgmol -1 M of the polymer is 23.7 kgmol w M of the polymer is 23.7 kgmol -1 M of the polymer is 23.7 kgmol x is selected from 43 to 53, and y is selected from 1 to 3. wherein It is prepared from the polymer of any one of claims 4-5 and a photosensitizer.

5. The polymer of claim 4, wherein The photosensitizer is a hydrophobic photosensitizer.

6. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The hydrophobic photosensitizer is chlorin e6.

7. The antitumor drug according to claim 6, wherein The mass ratio of the polymer to the photosensitizer is 20:(1-10).

8. The antitumor drug according to claim 7, wherein The mass ratio of the polymer to the photosensitizer is 20:(3-4).

9. The antitumor medicament according to any one of claims 6 to 8, characterized by, The mass ratio of the polymer to the photosensitizer is 20:3.

24.

10. The antitumor drug according to claim 9, wherein The method comprises the following steps: adding dropwise a solution of the photosensitizer into a solution of the polymer, continuing to stir for 1-3 hours after the dropwise addition is completed, then removing the solvent to form a film, adding water, stirring for 2-6 hours, filtering, and retaining the liquid.

11. The antitumor drug according to claim 10, wherein In the solution of the polymer, the solvent is methanol; and in the solution of the photosensitizer, the solvent is acetone.

12. A method for preparing the antitumor agent according to any one of claims 6 to 11, characterized by:

14. Use of the polymer of any one of claims 1-3, or the polymer of any one of claims 4-5, in the preparation of a medicament for the treatment of a tumor.

13. The method of claim 12, wherein, The tumor is breast cancer. ​ 15. Use according to claim 14, characterized in that, ​

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