Esterase-responsive pegylated conjugates containing sulfonamide groups, methods of making and using the same

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to an esterase-responsive PEGylated conjugate containing a sulfonamide group and a preparation method and application thereof. The esterase-responsive PEGylated conjugate comprises: a PEGylated hydrophilic segment; an esterase-cleavable linker arm, the linker arm comprising at least one ester bond; a copper death sensitization functional structure containing a sulfonamide group; wherein the copper death sensitization functional structure containing a sulfonamide group is covalently connected to the PEGylated hydrophilic segment through the esterase-cleavable linker arm. The esterase-responsive PEGylated conjugate containing a sulfonamide group provided by the present application can self-assemble to form nanoparticles (EsCu@NPs), and through an esterase response mechanism, L-BSO and copper ions are accurately released at a tumor site, GSH is efficiently consumed and copper death is induced, thereby triggering immunogenic cell death (ICD), promoting the maturation of dendritic cells and CD8 + T cell infiltration, and significantly enhancing the therapeutic effect of an anti-PD-1 immune checkpoint inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to esterase-responsive PEGylated conjugates containing sulfonamide groups, their preparation methods, and applications. Background Technology

[0002] Immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors) have demonstrated significant clinical efficacy in various malignant tumors, but their overall response rate is only around 20%. Most patients, especially those with a "cold tumor" microenvironment (lacking T-cell infiltration and enriched with immunosuppressive cells), cannot benefit from them. Improving the clinical response rate of immune checkpoint inhibitors and overcoming primary or acquired resistance has become a crucial scientific problem urgently needing to be solved in the field of tumor immunotherapy.

[0003] To improve the efficacy of immune checkpoint inhibitors, various combination therapy strategies have been developed, including combinations of immune checkpoint inhibitors (such as PD-1 inhibitors combined with CTLA-4 inhibitors, LAG-3 inhibitors, or TIGIT inhibitors), combination chemotherapy, combination anti-angiogenic drugs, combination radiotherapy, combination targeted therapy, engineered bispecific antibodies, antibody-drug conjugates, adoptive cell therapy, cancer vaccines, and multifunctional nanomedicines. However, the above combination strategies have the following shortcomings: (1) Although the combination of immune checkpoint inhibitors can enhance T cell activation, it significantly increases the incidence and severity of immune-related adverse events; (2) Combinations such as chemotherapy and radiotherapy lack tumor specificity, causing significant damage to normal tissues, and some combination regimens (such as KEYNOTE-867) are terminated prematurely due to failure to improve survival and increased toxicity; (3) Existing nanomedicine or prodrug designs mostly rely on microenvironmental responses such as pH and reactive oxygen species, but the tumor microenvironment is highly heterogeneous, and the response efficiency is limited; (4) There are currently no reports of drug delivery systems that can specifically induce copper death and simultaneously activate anti-tumor immune responses, thereby sensitizing immune checkpoint inhibitor therapy.

[0004] Therefore, designing an intelligent drug delivery system that can efficiently and specifically activate tumor cells at the tumor site, induce immunogenic death of tumor cells, reshape the tumor immune microenvironment, significantly enhance the therapeutic efficacy of immune checkpoint inhibitors, and has low toxicity is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] Therefore, the present invention aims to provide an esterase-responsive PEGylated conjugate (ELPM) containing a sulfonamide group, its preparation method, and its applications. The nanoparticles (EsCu@NPs) formed by assembling this esterase-responsive PEGylated conjugate containing a sulfonamide group with copper ions can significantly enhance the therapeutic efficacy of immune checkpoint inhibitors.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides an esterase-responsive PEGylated conjugate containing a sulfonamide group, the esterase-responsive PEGylated conjugate comprising: a PEGylated hydrophilic segment; an esterase-cleavable linker arm, the linker arm containing at least one ester bond; and a copper death-sensitizing functional structure containing a sulfonamide group; wherein the copper death-sensitizing functional structure containing a sulfonamide group is covalently linked to the PEGylated hydrophilic segment through the esterase-cleavable linker arm.

[0007] Preferably, in the above-mentioned esterase-responsive PEGylated conjugate, the copper death-sensitizing functional structure containing sulfonamide groups is a symmetrical bibranched structure, with each branch containing one or more sulfonamide groups at its end.

[0008] Preferably, in the above-mentioned esterase-responsive PEGylated conjugate, the copper death sensitizing functional structure containing a sulfonamide group is derived from a copper death sensitizing molecule containing a sulfonamide group, and the copper death sensitizing molecule containing a sulfonamide group is L-butyrosine sulfoxide imine.

[0009] Preferably, the structural formula of the above esterase-responsive PEGylated conjugate is shown below: , where m is 33.

[0010] In another aspect, the present invention provides a method for preparing the above-mentioned esterase-responsive PEGylated conjugate, the method comprising: (1) linking a mercapto alcohol compound to a polymer precursor containing an alkyne group via a thiol-alkyne click reaction to obtain a hydroxyl-containing polymer; (2) linking a copper death sensitizer molecule containing a sulfonamide group to the hydroxyl-containing polymer via an esterification reaction to obtain the above-mentioned esterase-responsive PEGylated conjugate containing a sulfonamide group.

[0011] In another aspect, the present invention provides a nanoparticle formed by self-assembly of the above-mentioned esterase-responsive PEGylated conjugate containing sulfonamide groups and copper ions.

[0012] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described esterase-responsive PEGylated conjugate containing a sulfonamide group or the above-described nanoparticles, and an immune checkpoint inhibitor.

[0013] Preferably, in the above-mentioned pharmaceutical composition, the immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0014] In another aspect, the present invention provides a medicament comprising the above-described pharmaceutical composition.

[0015] In another aspect, the present invention provides the use of the above-mentioned esterase-responsive PEGylated conjugate containing sulfonamide groups or the above-mentioned nanoparticles in the preparation of a medicament for enhancing the sensitivity of tumors to immune checkpoint inhibitor therapy, wherein the tumor is selected from one or more of breast cancer, lung cancer, liver cancer, melanoma, colorectal cancer, pancreatic cancer, kidney cancer, cervical cancer or soft tissue sarcoma.

[0016] The beneficial effects of this invention include at least the following: The esterase-responsive PEGylated conjugate containing sulfonamide groups provided by this invention can self-assemble into nanoparticles (EsCu@NPs), precisely releasing L-BSO and copper ions at the tumor site through an esterase-responsive mechanism, efficiently consuming GSH and inducing copper death, thereby triggering immunogenic cell death (ICD), promoting dendritic cell maturation and CD8+. + T-cell infiltration transforms "cold tumors" into "hot tumors," significantly enhancing the therapeutic efficacy of anti-PD-1 immune checkpoint inhibitors. It also exhibits good biosafety and tumor targeting, providing a novel technical solution for overcoming drug resistance in tumor immunotherapy. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of ELPM prepared in Example 1; where, label 1 represents the PEGylated hydrophilic segment (purple), label 2 represents the esterase-cleavable linker arm (yellow), and label 3 represents the copper death-sensitizing functional structure containing a sulfonamide group (blue). Figure 2 This is a synthesis route diagram for ELPM in this invention.

[0018] Figure 3 The 1H NMR spectrum of mPEG-NCA prepared in DMSO for Example 1 ( 1 HNMR, 400MHz).

[0019] Figure 4 The 1H NMR spectrum of ELPM prepared in DMSO for Example 1 ( 1 HNMR, 400MHz).

[0020] Figure 5 Characterization, esterase response, and release profiles of EsCu@NPs prepared in Example 1; wherein, Figure 5 A is a transmission electron microscope (TEM) image of EsCu@NPs; Figure 5 B is the dynamic light scattering (DLS) particle size distribution of EsCu@NPs; Figure 5 C represents the Zeta potential diagram of EsCu@NPs; Figure 5 D is a TEM image of EsCu@NPs after esterase treatment; Figure 5E is a comparison of the DLS particle size of EsCu@NPs before and after esterase treatment. Figure 5 F and Figure 5 G represents the copper ion release curves of EsCu@NPs under different conditions (with or without esterase); Figure 6 Figure 1 shows the flow cytometry results of ICG@NPs uptake by 4T1 cells. Figure 7 Figure 1 shows the results of copper content detection in 4T1 cells after treatment with different preparations. Figure 8 Figure 1 shows the results of glutathione (GSH) content detection in 4T1 cells after treatment with different preparations. Figure 9 Figure 1 shows the Annexin V-FITC / PI double staining flow cytometry results of apoptosis rate of 4T1 cells after treatment with different agents. Figure 10 The figure shows the experimental results of EsCu@NPs inducing immunogenic cell death (ICD) and remodeling the tumor immune microenvironment; among them, Figure 10 A is a schematic diagram of the detection process for ICD and tumor microenvironment reprogramming; Figure 10 B is a graph showing the results of detecting the release of lactate dehydrogenase (LDH) from cells after different treatments; Figure 10 C shows the results of extracellular ATP level detection after different treatments; Figure 10 D represents mature dendritic cells (mDCs, CD80) within the tumor after different treatments. + CD86 + The results of flow cytometry analysis at a ratio of 1 / 2. Figure 10 E represents intratumoral CD8 after different treatments. + Figure showing the flow cytometry results of the T cell ratio; Figure 10 F represents intratumor CD4 levels after different treatments. + Figure showing the flow cytometry results of the T cell ratio; Figure 10 G represents the ELISA results of intratumoral IFN-γ content after different treatments; Figure 10 H represents the ELISA results of intratumoral IL-4 levels after different treatments; Figure 10 Figure I shows the ELISA results of intratumoral TNF-α content after different treatments; Figure 11 This is a fluorescence imaging image of ICG@NPs enriched in tumor-bearing mice. Figure 12 The figure shows the results of quantitative analysis of tumor enrichment of ICG@NPs in tumor-bearing mice.

[0021] Figure 13This is a schematic diagram illustrating the evaluation of the antitumor efficacy of EsCu@NPs combined with aPD-1 in a 4T1 tumor-bearing mouse model.

[0022] Figure 14 Tumor growth curves for each group of mice.

[0023] Figure 15 This is a comparative chart showing the tumor weight of mice in each group at the end of treatment.

[0024] Figure 16 Gross photographs of tumors in each group of mice at the end of treatment; Figure 17 The graph shows the changes in body weight of mice in each group. Detailed Implementation

[0025] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0027] In a first aspect, embodiments of the present invention provide an esterase-responsive PEGylated conjugate containing a sulfonamide group, the esterase-responsive PEGylated conjugate comprising: a PEGylated hydrophilic segment; an esterase-cleavable linker arm, the linker arm containing at least one ester bond; and a copper death-sensitizing functional structure containing a sulfonamide group; wherein the copper death-sensitizing functional structure containing a sulfonamide group is covalently linked to the PEGylated hydrophilic segment via the esterase-cleavable linker arm.

[0028] It should be noted that this invention covalently integrates three functional structures—a hydrophilic PEG segment, an esterase-sensitive ester bond linker, and a bibranched sulfonamide-type copper death sensitizing core—into a single conjugate, distinguishing it from existing single-function prodrugs or small-molecule copper death formulations without enzyme-controlled release. The PEG chain forms a hydrated cryptic layer in vivo, enhancing molecular water solubility and enriching solid tumors through the EPR effect, reducing hepatic and renal clearance and non-specific adsorption. The intermediate ester bond serves as a specific cleavage site for tumor intracellular esterases; normal tissues have low esterase levels, and the conjugate is stable and does not release the active ingredient, significantly reducing systemic toxicity. The terminal sulfonamide functional fragment targets and inhibits glutathione synthesis, reducing intracellular copper chelation capacity by depleting GSH, increasing free copper concentration, and inducing tumor copper death, achieving an integrated design of "long-circulating enrichment + tumor-targeted drug release + copper death sensitization."

[0029] In some specific examples, the copper death sensitizing functional structure containing sulfonamide groups in the above-mentioned esterase-responsive PEGylated conjugates is a symmetrical bibranched structure, with each branch containing one or more sulfonamide groups at its end.

[0030] It should be noted that the copper death sensitizing structure adopts a symmetrical bibranched configuration, with each branch terminal carrying a sulfonamide group, forming a multivalent GSH-targeting molecular structure. Compared with monosulfonamide-modified molecules, the dual-site synergistic effect can significantly improve the inhibitory efficiency against γ-glutamylcysteine ​​synthase, more strongly deplete intracellular GSH, amplify copper accumulation and copper death effects, and effectively enhance tumor sensitizing activity.

[0031] In some specific examples, in the above-mentioned esterase-responsive PEGylated conjugates, the copper death sensitizing functional structure containing a sulfonamide group is derived from a copper death sensitizing molecule containing a sulfonamide group, which is L-butyrosine sulfoxide imine.

[0032] It should be noted that L-butyrosine sulfoxide imine (L-BSO) was chosen as the functional core. This compound has a sulfonamide pharmacophore group and is a recognized key enzyme inhibitor in GSH synthesis. Its pharmacological mechanism is clear and commercial raw materials are readily available. The molecule has a carboxyl group, which can be efficiently bonded to hydroxylated PEG intermediates through esterification reaction. This facilitates the precise construction of esterase-cleavable ester bonds at the linker arm site, which is suitable for the synthetic process design of this conjugate.

[0033] In some specific examples, the structural formulas of the above esterase-responsive PEGylated conjugates are shown below: , where m is 33.

[0034] It should be noted that the above structural formula is the preferred molecular configuration of this invention, precisely constraining the PEG chain length, ester bond connection sites, and the arrangement of the bibranched sulfonamides. The ester bonds are fixed in the linker arm segment, ensuring that they break only in the tumor hyperesterase microenvironment, and releasing the sulfonamide-containing BSO active fragment completely after cleavage. The structure is controllable, with strong batch stability, and is the optimal configuration after synthesis and cell screening. Specifically, as shown... Figure 1 As shown, label 1 represents the PEGylated hydrophilic segment (purple), label 2 represents the esterase-cleavable linker arm (yellow), and label 3 represents the copper death-sensitizing functional structure containing a sulfonamide group (blue).

[0035] In a second aspect, embodiments of the present invention provide a method for preparing the above-mentioned esterase-responsive PEGylated conjugate, the method comprising: (1) linking a mercaptool compound to a polymer precursor containing an alkyne group via a thiol-alkyne click reaction to obtain a hydroxyl-containing polymer; (2) linking a copper death sensitizer molecule containing a sulfonamide group to the hydroxyl-containing polymer via an esterification reaction to obtain the above-mentioned esterase-responsive PEGylated conjugate containing a sulfonamide group.

[0036] It should be noted that this preparation method consists of two core reactions, relying on thiol-alkyne click chemistry and esterification to achieve controllable synthesis. In the first step, alkynyl-modified PEG and mercaptool undergo a thiol-alkyne click reaction initiated by UV light. This reaction site is specific, produces few byproducts, and precisely introduces hydroxyl groups into the polymer side chain. In the second step, the hydroxyl group esterifies with the L-BSO carboxyl group, generating an esterase-sensitive ester bond in situ, thus completing the construction of the responsive linker in one step. The overall synthesis conditions are mild, purification is simple, and reproducibility is good, avoiding the cumbersome multi-step protection and deprotection processes of existing methods, facilitating the large-scale preparation of ELPM conjugates.

[0037] Thirdly, embodiments of the present invention provide a nanoparticle (EsCu@NPs nanoparticle), which is formed by the self-assembly of the above-mentioned esterase-responsive PEGylated conjugate containing sulfonamide groups and copper ions.

[0038] It should be noted that the ELPM conjugate side-chain sulfonamide can react with Cu 2+ Through coordination, EsCu@NPs nanoparticles are formed by self-assembly via solvent displacement. The nanoparticle size is controllable and the dispersibility is excellent. They passively target and enrich tumor tissues by relying on the EPR effect of solid tumors. After entering tumor cells, the ester bonds expressed intracellularly are hydrolyzed and broken. The disintegration of the nanoparticles releases free BSO and coordinated copper ions simultaneously. The two work together to deplete GSH, increase intracellular free copper levels, and induce copper death. This solves the shortcomings of existing formulations such as poor water solubility of free BS, short in vivo half-life, lack of tumor targeting, and high systemic toxicity.

[0039] Fourthly, embodiments of the present invention provide a pharmaceutical composition comprising the above-described esterase-responsive PEGylated conjugate containing a sulfonamide group or the above-described nanoparticles, and an immune checkpoint inhibitor.

[0040] It should be noted that this pharmaceutical composition is a combination of the conjugate / self-assembled nanoparticles of this invention and an immune checkpoint inhibitor, overcoming the industry pain point that the current clinical objective response rate of ICI monotherapy is only 20%. Existing ICIs are often combined with chemotherapy, targeted drugs, dual antibody regimens, etc., which generally have the problem of increased side effects from combination therapy; the nano-formulation in this composition can induce tumor immunogenic cell death (ICD) through the copper death pathway, releasing tumor-associated antigens, promoting dendritic cell maturation, and increasing intratumoral CD8. + T lymphocyte infiltration transforms immunosuppressive cold tumors into immunosensitive hot tumors, breaking down ICI resistance at the tumor microenvironment level. It complements the action pathways of PD-1 / PD-L1 inhibitors, synergistically enhancing anti-tumor efficacy, and its combination therapy offers superior safety.

[0041] In some specific examples, the immune checkpoint inhibitor in the above-mentioned pharmaceutical composition is a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0042] It should be noted that PD-1 inhibitors and PD-L1 inhibitors are clinically approved immune checkpoint drugs. Their mechanism of action is to block the PD-1 / PD-L1 immune escape pathway and restore the anti-tumor activity of the body's T cells. When used in combination with the EsCu@NPs of this invention, the tumor microenvironment remodeled by copper death can significantly improve the tumor invasion efficiency of ICI and greatly improve the primary drug resistance problem of various solid tumors.

[0043] Fifthly, embodiments of the present invention provide a medicament comprising the above-described pharmaceutical composition.

[0044] It should be noted that, using the above composition as the core active ingredient, and combined with commonly used pharmaceutical excipients such as lyophilized excipients and injection solvents, commonly used clinical dosage forms such as lyophilized powder injections and sterile injection solutions can be prepared, which are suitable for intravenous infusion administration and meet the clinical needs of combined drug use for various solid tumors such as breast cancer and lung cancer.

[0045] In a sixth aspect, embodiments of the present invention provide the use of the above-mentioned esterase-responsive PEGylated conjugate containing sulfonamide groups or the above-mentioned nanoparticles in the preparation of a medicament for enhancing the sensitivity of tumors to immune checkpoint inhibitor therapy.

[0046] It should be noted that this conjugate or self-assembled nanoparticle can be used to prepare immune checkpoint inhibitor sensitizing drugs with a clear mechanism of action: the nanoparticles target and enrich the tumor via EPR → tumor esterase triggers carrier dissociation, simultaneously releasing BSO and copper ions → BSO depletes intracellular GSH, free copper overload induces tumor copper death → copper death induces ICD, releases damage-related molecular patterns and tumor antigens, and remodels the anti-tumor immune microenvironment → improves the tumor's drug sensitivity to ICI, significantly improving the objective response rate of immunotherapy.

[0047] In some specific examples, in the above applications, the tumor is selected from one or more of breast cancer, lung cancer, liver cancer, melanoma, colorectal cancer, pancreatic cancer, kidney cancer, cervical cancer, or soft tissue sarcoma.

[0048] It should be noted that the listed solid tumors, such as breast cancer, lung cancer, liver cancer, and melanoma, are generally typical cold tumors, and they generally present clinical challenges such as immunosuppression of the tumor microenvironment and high incidence of primary ICI resistance. These are the core cancer types for which this sensitizing agent is applicable, and the drug has a wide clinical application coverage and outstanding translational value.

[0049] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0050] Preparation Example

[0051] Example 1: Preparation of esterase-responsive PEGylated conjugates (ELPM) containing sulfonamide groups

[0052] ELPM synthesis route as follows Figure 2 As shown, the specific preparation method is as follows.

[0053] (1) Synthesis of the cyclic monomer DL-PGNCA

[0054] DL-propargylglycine (5.0 g, 44.2 mmol, 1 equivalent) was dissolved in 60 mL of anhydrous tetrahydrofuran under argon protection. α-pinene (32 mL, 4.6 equivalent) was added, and the mixture was heated to 50 °C. Triphosgene (10.98 g, 37.1 mmol, 0.84 equivalent) was added dropwise in 15 mL of tetrahydrofuran over 1 hour. The mixture was then stirred at 50 °C for 2.5 hours until the solution was clear. The solvent was removed by vacuum distillation, the residue was washed with diethyl ether, and the solution was stored overnight in 20 mL of dry diethyl ether at -20 °C. The precipitate was redissolved in 20 mL of tetrahydrofuran, filtered, and recrystallized from diethyl ether. This recrystallization was repeated three times to obtain the N-carboxylic anhydride monomer.

[0055] (2) Preparation of alkynyl polymer (mPEG-NCA) by ring-opening polymerization

[0056] The block copolymer mPEG-2K-NH2 (163 mg, 0.815 mmol, 1 equivalent) and the N-carboxylic anhydride monomer (800 mg, 5.705 mmol, 7 equivalents) were dissolved separately in anhydrous dimethyl sulfoxide (DMSO) to obtain two different solutions, which were then dried by toluene azeotropic distillation under a nitrogen atmosphere. After drying, the two solutions were mixed and reacted at 37 °C for 5 days. The product was purified by water dialysis for 2 days and then freeze-dried to obtain the peptide product mPEG-NCA.

[0057] (3) Photo-initiated thiol-alkyne click reaction to prepare hydroxyl-containing polymer (mPEG-NCA-OH)

[0058] Under a nitrogen atmosphere, mPEG-NCA (300 mg, 0.0058 mmol, 1 equivalent) was dissolved in DMSO (30 mL). Then, 2-mercaptoethanol (374 mg, 0.04785 mmol, 82.5 equivalents) and photoinitiator DMPA (80 mg) were added. The reaction mixture was irradiated with ultraviolet light (365 nm) for 4 hours with stirring. The reaction product was purified by dialysis using a dialysis membrane (molecular weight cutoff 1 kDa), first dialyzing with buffer for 24 hours, then with deionized water for 24 hours. Lyophilization yielded a white solid modified copolymer, mPEG-NCA-OH.

[0059] (4) Preparation of ELPM by L-BSO coupling (esterification reaction)

[0060] L-Butylthionine sulfoxide imine (L-BSO, 205 mg, 0.924 mmol, 66 equivalents) was co-activated for 10 minutes with HOBT (1-hydroxybenzotriazole) (125 mg, 0.924 mmol, 66 equivalents), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (350 mg, 0.924 mmol, 66 equivalents), and DIPEA (N,N-diisopropylethylamine) (119 mg, 0.924 mmol, 66 equivalents). This solution was then added to mPEG-NCA-OH (150 mg, 0.014 mmol, 1 equivalent) dissolved in DMSO. The crude product was purified by dialysis with a 1 kDa molecular weight cutoff membrane using 50% ethanol / water solution, and lyophilized to obtain the solid-state conjugate ELPM (an esterase-responsive PEGylated conjugate containing a sulfonamide group).

[0061] Example 2 Assembly of copper death-sensitizing nanoparticles (EsCu@NPs)

[0062] (1) Preparation of ELPM solution: Dissolve the ELPM prepared in Example 1 in DMSO to prepare a 10 mg / mL ELPM solution.

[0063] (2) EsCu solution: Ilismo (Es) and metal salt CuCl2 are mixed in a stoichiometric ratio of 1:1 and dissolved in DMSO to obtain a clear solution, namely EsCu solution (Es concentration is 100 μg / mL).

[0064] (3) Nanoprecipitation and self-assembly: Take 100 μL of LEP M solution (10 mg / mL) and slowly disperse it in 1 mL of deionized water to obtain mixed solution A; add 10 μL of LESCu solution (100 μg / mL) to the above mixed solution A to obtain mixed solution B; treat mixed solution B with ultrasound (200 W, 40 kHz) for 5 minutes to promote the uniform formation of nanoparticles, reduce the particle size and ensure stability; obtain EsCu@NPs aqueous dispersion.

[0065] (4) Post-processing and purification: The EsCu@NPs aqueous dispersion was dialyzed to remove unreacted free Es, CuCl2 and residual DMSO; it was replaced multiple times with deionized water and low-concentration buffer until no free copper ions and drugs were detected in the external aqueous phase, thus obtaining EsCu@NPs nanoparticles.

[0066] Characterization test

[0067] The 1H NMR spectrum (¹H NMR, 400 MHz, DMSO) of the alkynyl polymer (mPEG-NCA) prepared in step (2) of Example 1 is as follows: Figure 3 As shown, the alkynyl polymer mPEG-NCA was successfully synthesized.

[0068] The 1H NMR spectrum (¹H NMR, 400 MHz, DMSO) of the esterase-responsive PEGylated conjugate (ELPM) containing sulfonamide groups prepared in step (4) of Example 1 is as follows: Figure 4 As shown, this confirms that L-BSO was successfully linked to the polymer via ester bonds.

[0069] The physicochemical properties of the EsCu@NPs prepared in Example 2 were characterized, and the results are as follows: Figure 5 A to Figure 5 As shown in Figure C. Transmission electron microscopy (TEM) observation revealed that EsCu@NPs exhibit a spherical morphology with an average particle size of 32±5 nm. Figure 5 A). Dynamic light scattering (DLS) measured the hydrated particle size to be approximately 197 nm. Figure 5 B), the Zeta potential is +19.6mV ( Figure 5 C) indicates that the nanoparticles have good colloidal stability.

[0070] The EsCu@NPs prepared in Example 2 were placed in DMEM medium containing esterase (15 U / mL) and incubated at 37°C for 24 hours. TEM imaging showed that the nanoparticle structure underwent significant degradation after esterase treatment. Figure 5 D). DLS analysis showed that, in the presence of esterase, the hydrated particle size increased sharply from 95.0 nm to 255.5 nm (D). Figure 5 E). Copper ion release experiment ( Figure 5 F and 5G showed that copper release was significantly increased in esterase-containing media.

[0071] The above results demonstrate that the ester bonds in EsCu@NPs can be specifically cleaved by esterases, triggering the dissociation of nanoparticles and the release of loaded EsCu and L-BSO, thus proving their rationale as an intelligent nanocarrier system for enzyme-responsive drug release.

[0072] In vitro cell experiments

[0073] (a) Cellular uptake experiment

[0074] EsCu@NPs prepared in Example 2 were coated with the fluorescent dye ICG to obtain ICG@NPs. 4T1 mouse breast cancer cells were seeded at a rate of 200,000 per well in six-well plates and cultured overnight. After incubation, the cells were co-incubated with ICG@NPs for different times (1 hour, 10 hours, and 24 hours), and the intensity of intracellular red fluorescence was detected by flow cytometry (FCM).

[0075] The results are as follows Figure 6 As shown, the fluorescence intensity of cells treated for 24 hours was significantly higher than that treated for 1 hour and 10 hours, indicating that the uptake of ICG@NPs by cells is time-dependent and continuously efficient.

[0076] (II) Detection of intracellular copper accumulation

[0077] 4T1 cells were divided into 7 groups, including a blank control group (Control) and experimental groups. The blank control group received no treatment. The experimental groups were treated with Es, CuCl2, EsCu, Free@NPs (prepared in Example 2, using PBS instead of EsCu, otherwise the same as in Example 2), Es@NPs (prepared in Example 2, using Es instead of EsCu, otherwise the same as in Example 2), and EsCu@NPs (prepared in Example 2), respectively. Specifically, the above different reagents were added to make the final DMEM culture medium contain either ES or CuCl2. 2+ The concentration of the active ingredient was 4 μmol / L, and the cells were cultured overnight (12 hours). Intracellular copper content was then quantitatively detected using inductively coupled plasma (ICP) technology.

[0078] The results are as follows Figure 7As shown, the EsCu@NPs group had the highest intracellular copper level, indicating a significantly enhanced copper delivery capacity.

[0079] (III) Detection of intracellular glutathione (GSH) levels

[0080] After treating 4T1 cells according to the grouping method described in (II) above, the intracellular GSH level was detected (Beyotime, BC1175).

[0081] The results are as follows Figure 8 As shown, all formulations containing L-BSO prodrugs (Free@NPs, Es@NPs, and EsCu@NPs) significantly reduced intracellular GSH levels, with the EsCu@NPs group exhibiting the most significant GSH depletion, exhibiting significantly lower GSH levels than the other groups. This indicates that EsCu@NPs can achieve efficient copper delivery and GSH depletion, predicting enhanced copper-dependent cytotoxicity.

[0082] (iv) Apoptosis detection

[0083] After treating 4T1 cells according to the grouping method described in (II) above, the apoptosis status was detected by Annexin V-FITC / PI double staining.

[0084] The results are as follows Figure 9 As shown, the apoptosis rate of the EsCu@NPs group was significantly higher than that of the EsCu group or the Es@NPs group, proving that the copper accumulation induced by EsCu@NPs effectively triggered cell death.

[0085] (v) Detection of immunogenic cell death (ICD) markers

[0086] After treating 4T1 cells according to the grouping in (3) above, typical ICD markers were detected: extracellular high mobility group 1 (HMGB1), ATP release and lactate dehydrogenase (LDH) leakage.

[0087] The results are as follows Figure 10 A, Figure 10 B and Figure 10 As shown in C, the EsCu@NPs group significantly increased LDH release and extracellular ATP levels ( Figure 10 B and 10C), indicating that it can effectively induce ICD.

[0088] In vivo anti-tumor and immune microenvironment remodeling experiments

[0089] (I) Analysis of immune cells in the tumor microenvironment

[0090] (1) Mouse model construction

[0091] To establish a 4T1 subcutaneous tumor model, 4T1 cells (1×10⁻⁶) were suspended in 100 μL of RPMI-1640 medium.6 (Number) was injected into the right inguinal region of BALB / c mice (5-6 weeks old, weighing 25-30g).

[0092] (2) Grouping and administration

[0093] 4T1 subcutaneous tumor-bearing mice were divided into 7 groups of 3 mice each. The conditions and treatments of each group are as follows: Control group: Injected with normal saline (volume 100uL, same as other experimental groups) via tail vein. Es group: Es solution (solvent is physiological saline, volume is 100uL, Es dosage is 6mg / kg) was injected into the tail vein. CuCl2 group: CuCl2 solution (solvent is physiological saline, volume is 100uL, CuCl2 dosage is 1.8mg / kg) was injected via tail vein. EsCu group: EsCu solution (solvent is physiological saline, volume is 100uL, Es dosage is 6mg / kg) was injected into the tail vein. Free@NPs group: Free@NPs solution (solvent is physiological saline, volume is 100uL, Es dosage is 6mg / kg) was injected via tail vein. The preparation of Free@NPs was the same as in Example 2, except that PBS was used instead of EsCu.

[0094] Es@NPs group: Free@NPs solution (solvent is physiological saline, volume is 100uL, Es dosage is 6mg / kg) was injected via tail vein. The preparation of Free@NPs was the same as in Example 2, except that Es was used instead of EsCu.

[0095] EsCu@NPs group: Free@NPs solution (solvent is physiological saline, volume is 100uL, Es dosage is 6mg / kg) was injected via tail vein. Free@NPs was prepared in Example 2.

[0096] (3) Tests and Results

[0097] Tumors were collected 9 days after drug administration, and immune cell subsets were analyzed by flow cytometry. Results are as follows: Figure 10 D、 Figure 10 E and Figure 10 As shown in Figure F: the EsCu@NPs group significantly increased mature dendritic cells (mDCs, CD80) + CD86 + The proportion of ) Figure 10 D); Intratumoral CD8 + T cell infiltration significantly increased ( Figure 10 E), CD4 + T cell infiltration was significantly reduced ( Figure 10F). Simultaneously, the levels of IFN-γ, IL-4, and TNF-α within the tumor were significantly increased (F). Figure 10 G, Figure 10 H and Figure 10 I). The above results indicate that EsCu@NPs can induce ICD, promote DC maturation, and reprogram the immunosuppressive tumor microenvironment.

[0098] (ii) Tumor enrichment experiment

[0099] (1) Mouse model construction

[0100] The mouse model was constructed using the same method as described in "(I) Analysis of Tumor Microenvironment Immune Cells".

[0101] (2) Experiments and Results

[0102] The EsCu@NPs prepared in Example 2 were encapsulated with the fluorescent dye ICG to obtain ICG@NPs; 4T1 subcutaneous tumor-bearing mice were divided into two groups: the ICG group was injected with free ICG via the tail vein, and the ICG@NPs group was injected with ICG@NPs (100µL, 0.5mg / mL) via the tail vein. In vivo fluorescence imaging was then performed at different time points.

[0103] like Figure 11 and Figure 12 As shown, the tumor fluorescence of free ICG decreased rapidly within 1 hour after injection, while ICG@NPs remained relatively stable within 12 hours and were still significantly enriched at the tumor site after 48 hours, indicating that the nanocarrier achieved effective tumor-targeted delivery.

[0104] (III) Antitumor efficacy of EsCu@NPs combined with aPD-1

[0105] (1) Mouse model construction

[0106] The mouse model was constructed using the same method as described in "(I) Analysis of Tumor Microenvironment Immune Cells".

[0107] (2) Grouping and administration (administration regimen such as...) Figure 13 (As shown)

[0108] 4T1 subcutaneous tumor-bearing mice were divided into 4 groups of 6 mice each. The conditions and treatments of each group are as follows: Control group: 4T1 subcutaneous tumor-bearing mice were intravenously injected with an equal volume of physiological saline; aPD-1 group: 4T1 subcutaneous tumor-bearing mice were intravenously injected with aPD-1 (Hengrui Medicine, HRP00262-023) (solvent was physiological saline, the same below), the injection dose was 10mg / kg; EsCu@NPs group: 4T1 subcutaneous tumor-bearing mice were intravenously injected with EsCu@NPs at a dose of 6 mg / kg (solvent was physiological saline, the same below). EsCu@NPs+aPD-1 group: 4T1 subcutaneous tumor-bearing mice were intravenously injected with aPD-1 at a dose of 10 mg / kg (injection timing according to...). Figure 13 In addition, EsCu@NPs were administered intravenously at a dose of 6 mg / kg.

[0109] Each group of mice was given the drug a total of 4 times, with the timing of administration according to... Figure 13 The procedure involved injecting EsCu@NPs on days 0, 3, 6, and 9 after successful mouse modeling, and on days 1, 4, 7, and 10 after successful mouse modeling.

[0110] The changes in tumor volume in each group of mice are as follows: Figure 14 As shown, the results indicate that the control group (1398mm) 3 ±191mm 3 ) and aPD-1 group (1143mm) 3 ±260mm 3 The tumor grew rapidly; the EsCu@NPs group (295mm) 3 ±104mm 3 Tumor progression was moderately slowed, while the EsCu@NPs+aPD-1 group (62mm) showed a more moderate slowdown. 3 ±61mm 3 It significantly inhibited tumor growth; in addition, at the end of treatment (day 12 after successful mouse modeling), the tumor weight of each group of mice was as follows: Figure 15 As shown, the treatment amounts were: control group (1.42g ± 0.22g), aPD-1 group (1.19g ± 0.15g), EsCu@NPs group (0.30g ± 0.09g), and EsCu@NPs+aPD-1 group (0.05g ± 0.04g). Additionally, tumor images were taken at the end of treatment. Figure 16 This further confirms the superior antitumor efficacy of the combined treatment group. Furthermore, the mice's body weight remained stable during treatment. Figure 17 This indicates that the treatment regimen was well tolerated.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An esterase-responsive PEGylated conjugate containing a sulfonamide group, characterized in that, Esterase-responsive PEGylated conjugates include: PEGylated hydrophilic segments; The esterase-cleavable linker arm contains at least one ester bond; A copper death-sensitizing functional structure containing a sulfonamide group; Among them, the copper death-sensitizing functional structure containing sulfonamide groups is covalently linked to the PEGylated hydrophilic segment through an esterase-cleavable linker arm.

2. The esterase-responsive PEGylated conjugate according to claim 1, characterized in that, The copper death sensitizer structure containing sulfonamide groups is a symmetrical bibranched structure, with each branch containing one or more sulfonamide groups at its end.

3. The esterase-responsive PEGylated conjugate containing a sulfonamide group according to claim 2, characterized in that, The copper death sensitizing functional structure containing a sulfonamide group is derived from a copper death sensitizing molecule containing a sulfonamide group, namely L-butyrosine sulfoxide imine.

4. The esterase-responsive PEGylated conjugate containing a sulfonamide group according to claim 1, characterized in that, The structural formula of the esterase-responsive PEGylated conjugate containing a sulfonamide group is shown below: , where m is 33.

5. The method for preparing the esterase-responsive PEGylated conjugate according to any one of claims 1 to 4, characterized in that, Preparation methods include: (1) A mercaptool compound is attached to a polymer precursor containing an alkyne group via a thiol-alkyne click reaction to obtain a polymer containing a hydroxyl group; (2) The copper death sensitizer containing sulfonamide groups is linked to a hydroxyl-containing polymer via esterification to obtain the esterase-responsive PEGylated conjugate containing sulfonamide groups as described in any one of claims 1 to 4.

6. A nanoparticle, characterized in that, The nanoparticles are formed by self-assembly of the esterase-responsive PEGylated conjugate containing sulfonamide groups as described in any one of claims 1 to 4 and copper ions.

7. A pharmaceutical composition, characterized in that, Includes the esterase-responsive PEGylated conjugate containing a sulfonamide group as described in any one of claims 1 to 4, or the nanoparticles as described in claim 6, and immune checkpoint inhibitors.

8. The pharmaceutical composition according to claim 7, characterized in that, Immune checkpoint inhibitors are PD-1 inhibitors and / or PD-L1 inhibitors.

9. A drug, characterized in that, The drug comprises the pharmaceutical composition of claim 7 or 8.

10. The use of the esterase-responsive PEGylated conjugate containing a sulfonamide group as described in any one of claims 1 to 4 or the nanoparticles as described in claim 6 in the preparation of a medicament for enhancing the sensitivity of tumors to immune checkpoint inhibitor therapy, wherein the tumor is selected from one or more of breast cancer, lung cancer, liver cancer, melanoma, colorectal cancer, pancreatic cancer, kidney cancer, cervical cancer, or soft tissue sarcoma.

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