Polyamino acid-metal coordination nano-drug as well as preparation method and application thereof

Abstract

The invention provides a polyamino acid-metal coordination nano-drug. The polyamino acid-metal coordination nano-drug comprises polyamino acid, metal ions and a polyphenol compound which are connected through a metal coordination effect, at least one ortho-position or meta-position of a phenolic hydroxyl group of the polyphenol compound is a hydroxyl group or a ketone group. The invention further provides a preparation method of the polyamino acid-metal coordination nano-drug and application of the polyamino acid-metal coordination nano-drug in preparation of tumor treatment drugs. According to the application, metal ions are taken as points, polyamino acid and polyphenol compounds are taken as organic ligands, a stable nano structure is formed through metal-ligand coordination, and the tumor space-time adaptive immune cascade coordinated regulation polyamino acid-metal ion-polyphenol coordination nano-drug is constructed, so that the complexity of constructing a nano-platform is simplified, and the tumor space-time adaptive immune cascade coordinated regulation polyamino acid-metal ion-polyphenol coordination nano-drug is suitable for large-scale popularization and application. Meanwhile, space-time adaptive dynamic continuous regulation and control of collaborative immune activation, immune cell infiltration and tumor cell recognition and killing are realized, and a new solution is provided for overcoming the problem of'cold tumor 'in tumor immunotherapy.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to a polyamino acid-metal coordination nanomedicine, its preparation method and its application. Background Technology

[0002] Surgical resection remains the cornerstone of cancer treatment, but incomplete resection can lead to tumor residue, increasing the risk of postoperative metastasis. While systemic chemotherapy and radiotherapy can effectively suppress local and metastatic recurrence after surgery, they are often accompanied by severe systemic toxicity and significant side effects, limiting their clinical application. In recent years, immunotherapy, as an emerging anti-cancer strategy, has provided a new direction for cancer treatment by activating the body's immune system to precisely eliminate tumor cells. Although immunotherapy has made some progress in cancer treatment, its inherent "cold tumor" nature results in a low response rate, with only a subset of patients benefiting. Therefore, developing innovative immunotherapy strategies to overcome the limitations of existing therapies has become an urgent need in the field of cancer treatment.

[0003] Inducing tumor immunogenic cell death (ICD) is an important strategy in tumor immunotherapy. However, traditional ICD induction strategies (such as photodynamic therapy and chemotherapy drugs) have significant limitations, including complex nanodelivery systems, low bioavailability of inducers, and inherent defects such as difficulty in precisely regulating the immune microenvironment. More importantly, the tumor immune response is essentially a multi-stage, spatiotemporally dynamic, and continuous process, encompassing key steps such as immune activation, immune cell infiltration, and tumor cell recognition and killing. Although nanotechnology has been widely used in tumor immunotherapy, current technologies can only regulate a portion of this continuous process, making it difficult to fully stimulate a robust anti-tumor immune response. Summary of the Invention

[0004] In view of this, this application provides a polyamino acid-metal coordination nanomedicine, its preparation method and its application. The polyamino acid-metal coordination nanomedicine provided by this application can be directly used for cancer immunotherapy, solving the problems of insufficient immune activation effect and difficulty in accurately regulating the immune microenvironment.

[0005] This application provides a polyamino acid-metal coordination nanomedicine, comprising polyamino acids, metal ions, and polyphenolic compounds linked through metal coordination;

[0006] The phenolic hydroxyl group of the polyphenolic compound has at least one ortho- or meta-position as a hydroxyl or ketone group.

[0007] This application utilizes metal ions as nodes and polyamino acids and polyphenolic compounds as organic ligands to form stable nanostructures through metal-ligand coordination, thereby constructing a tumor spatiotemporal adaptive immune cascade synergistic regulation polyamino acid-metal ion-polyphenol coordination nanomedicine.

[0008] The polyamino acid-metal coordination nanomedicine provided in this application includes polyamino acids. Polyamino acids possess unique structural designability, excellent functional tunability, and good biocompatibility. Furthermore, their molecular side chains are rich in active functional groups such as carboxyl, hydroxyl, and amino groups, which can coordinate with metal ions to form stable structures. This application does not impose any special limitations on the polyamino acids mentioned, including but not limited to polyglutamic acid, polylysine, polyaspartic acid, polyarginine, polyserine, and polyornithine, and may be one or more of these. When the polyamino acid is a combination of multiple substances, this application does not impose any special limitations on the content of each specific substance. In some specific implementations, the polyamino acid is preferably polyglutamic acid. In some specific implementations, the polyglutamic acid is polyethylene glycol-polyglutamic acid, wherein the molecular weight of polyethylene glycol is 500~50000 Da, preferably 1000~20000 Da, and more preferably 3000~10000 Da.

[0009] This application does not impose any special restrictions on the source of the polyamino acids; they can be purchased from the market or prepared according to methods familiar to those skilled in the art. Taking polyethylene glycol-polyglutamic acid as an example, a typical preparation process is as follows:

[0010]

[0011] L-glutamic acid-5-benzyl ester reacts with triphosgene to give glutamic acid-5-benzyl ester N-carboxyl ring anhydride;

[0012] The N-carboxyl anhydride of 5-benzyl glutamate was reacted with amino-terminated polyethylene glycol monomethyl ether, and after deprotection, polyethylene glycol-polyglutamate was obtained.

[0013] This application uses L-glutamic acid-5-benzyl ester as a raw material, which is reacted with triphosgene in a solvent. The reaction temperature is 40°C to 80°C, preferably 50°C to 70°C, and the reaction time is preferably 1 h to 5 h, preferably 1.5 h to 3 h. In some specific implementations, the solvent includes, but is not limited to, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, and acetonitrile, preferably tetrahydrofuran. In some specific implementations, the mass ratio of L-glutamic acid-5-benzyl ester to triphosgene is 10 to 30:1 to 20, preferably 15 to 25:5 to 15. After the reaction is complete, excess solution is blown away with nitrogen, then precipitated with ice-cold hexane, filtered to obtain a crude product, dissolved in ethyl acetate, washed with ice-cold saturated sodium chloride solution, dried with anhydrous magnesium sulfate at -20°C, and filtered to obtain a solid product. The solid product is recrystallized and purified to obtain the N-carboxyl anhydride of 5-glutamic acid-5-benzyl ester.

[0014] After obtaining the N-carboxyl anhydride of 5-benzyl glutamic acid, it is reacted with terminal amino polyethylene glycol monomethyl ether in a reaction solvent at room temperature for 2 to 4 days. In some specific implementations, the reaction solvent includes, but is not limited to, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, dioxane, toluene, etc., preferably N,N-dimethylformamide. In some specific implementations, the mass ratio of the N-carboxyl anhydride of 5-benzyl glutamic acid to terminal amino polyethylene glycol monomethyl ether is 1~10:0.1~5, preferably 2~5:0.5~1. In some specific implementations, the terminal amino polyethylene glycol monomethyl ether is preferably pretreated with dehydration, specifically by azeotropically removing water with a dehydrating agent. In some specific implementations, the dehydrating agent includes, but is not limited to, toluene, xylene, etc., preferably toluene. In some specific implementations, the azeotropic dehydration temperature is 100°C to 150°C, and the time is 1 h to 3 h.

[0015] After the reaction of the N-carboxyl anhydride of 5-benzyl glutamate with the terminal amino group of polyethylene glycol monomethyl ether is complete, the mixture is precipitated with ice-cold diethyl ether and dried under vacuum to obtain benzyl-protected polyethylene glycol-polyglutamic acid. Deprotection of this product yields polyethylene glycol-polyglutamic acid. This application does not impose any particular limitation on the deprotection method; acid pyrolysis can be used. For example, benzyl-protected polyethylene glycol-polyglutamic acid can be dissolved in trifluoroacetic acid, and a hydrobromic acid / acetic acid solution can be added to initiate the reaction. After the reaction is complete, the mixture is precipitated with ice-cold diethyl ether. The resulting white solid is dissolved, dialyzed, and freeze-dried to obtain polyethylene glycol-polyglutamic acid.

[0016] The polyamino acid-metal coordination nanomedicine provided in this application includes metal ions, such as copper ions, manganese ions, and iron ions. These metal ions can trigger a Fenton-like reaction to convert excess low-toxicity hydrogen peroxide (H2O2) in the tumor microenvironment into more cytotoxic hydroxyl radicals (•OH), killing tumor cells, inducing ICD, and significantly enhancing tumor immunogenicity, thereby achieving effective tumor suppression. In some specific implementations, the metal ions include, but are not limited to, copper ions, manganese ions, iron ions, or zinc ions, preferably Mn. 2+ or Cu 2+ .

[0017] The polyamino acid-metal coordination nanomedicine provided in this application includes polyphenolic compounds. Polyphenols (such as quercetin, epigallocatechin gallate, etc.) have high antioxidant capacity and have been widely studied and confirmed to have anticancer activity. For example, quercetin can reduce α-SMA. + The expression of collagen and the remodeling of the tumor extracellular matrix (ECM) enhance T cell infiltration in tumor tissues. Simultaneously, by downregulating the expression of tumor cell programmed death ligand 1 (PD-L1), it inhibits immune escape and enhances the T cell recognition and killing effect on tumor cells. In this application, at least one ortho- or meta-position of the phenolic hydroxyl group of the polyphenolic compound is a hydroxyl or ketone group. This ketone or hydroxyl group coordinates with the phenolic hydroxyl group and metal ions, enhancing the stability of the nanomedicine. In some specific implementations, the polyphenolic compound includes, but is not limited to, epigallocatechin gallate, quercetin, catechin, proanthocyanidins, chlorogenic acid, kaempferol, and resveratrol, and may be one or more of these. When the polyphenolic compound is a combination of multiple substances, this application does not have a specific limitation on their specific proportions. In some specific implementations, the polyphenolic compound is preferably epigallocatechin gallate and quercetin.

[0018] In some specific implementations, the mass ratio of the polyamino acid, metal ions and polyphenolic compounds is 30~50:5~15:1~10, preferably 32~48:8~14:1~8, more preferably 36~44:9~13:2~6, and most preferably 38~42:10~12:3~5.

[0019] In some specific implementations, the particle size of the polyamino acid-metal coordination nanomedicine is 30 nm to 500 nm, preferably 40 nm to 250 nm, and more preferably 50 nm to 200 nm.

[0020] This application also provides a method for preparing the polyamino acid-metal coordination nanomedicine described in the above technical solution, comprising the following steps:

[0021] Polyamino acids, metal salts, and polyphenolic compounds were mixed and dialyzed to obtain polyamino acid-metal coordination nanomedicines.

[0022] Specifically, this application first dissolves polyamino acids to form a polyamino acid solution, then adjusts the pH of the polyamino acid solution to 8-10, preferably 8.5-9, and then mixes it with an aqueous solution of metal salts. After stirring for 5 min-20 min, the pH is adjusted again to 8-10, preferably 8.5-9, and then mixed with a polyphenol compound solution. After stirring for 5 h-20 h, the mixture is dialyzed to obtain nanomedicine.

[0023] This application also provides the application of the polyamino acid-metal coordination nanomedicine described above in the preparation of immunotherapeutic drugs for treating tumors. In some specific implementations, the tumor is breast cancer, colon cancer, etc.

[0024] This application utilizes a one-step metal-ligand coordination method to construct polyamino acid-metal ion-polyphenol coordination nanomedicines. This not only simplifies the complexity of nanoplatform construction but also achieves spatiotemporal adaptive dynamic and continuous regulation of immune activation, immune cell infiltration, and tumor cell recognition and killing, providing a new solution to overcome the "cold tumor" problem in tumor immunotherapy. Experimental results show that the polyamino acid-metal coordination nanomedicines provided in this application can be efficiently taken up by tumor cells, exhibit dose-dependent toxicity to tumor cells, effectively induce ROS production in tumor cells, effectively induce ICD in tumor cells, and possess tumor targeting capabilities. They effectively inhibit tumor growth, promote dendritic cell maturation, enhance cytotoxic T lymphocyte activation, significantly downregulate PD-L1 expression, and reduce α-SMA expression. + Fibroblast and collagen deposition effectively remodel the immunosuppressive matrix, thereby promoting CD8. + T cell infiltration in tumor tissue. Attached Figure Description

[0025] Figure 1 The 1H NMR spectrum of the L-Glu NCA monomer prepared in Example 1;

[0026] Figure 2 The carbon NMR spectrum of the L-Glu NCA monomer prepared in Example 1;

[0027] Figure 3 The mass spectrum of the L-Glu NCA monomer prepared in Example 1;

[0028] Figure 4 The 1H NMR spectrum of the polymer prepared before deprotection in Example 1 of this application;

[0029] Figure 5 mPEG prepared in Example 1 of this application5k - The proton NMR spectrum of PGA;

[0030] Figure 6 This is a schematic diagram of nanomedicine preparation and specific coordination structure provided in Example 1 of this application;

[0031] Figure 7 This is a particle size distribution diagram of the nanomedicine prepared in Example 1 of this application;

[0032] Figure 8 This is a schematic diagram of the potential of the nanomedicine prepared in Example 1 of this application;

[0033] Figure 9 Transmission electron microscopy (TEM) image of the nanomedicine prepared in Example 1 of this application;

[0034] Figure 10 The infrared spectrum of the nanomedicine prepared in Example 1 of this application;

[0035] Figure 11 The ultraviolet absorption spectrum of the nanomedicine prepared in Example 1 of this application;

[0036] Figure 12 X-ray photoelectron spectroscopy of the nanomedicine prepared in Example 1 of this application;

[0037] Figure 13 The results of cell endocytosis characterization provided in Example 1 of this application;

[0038] Figure 14 The cytotoxicity results of the nanomedicine provided in Example 1 of this application;

[0039] Figure 15 Characterization of intracellular reactive oxygen species generation of the nanomedicine provided in Example 1 of this application;

[0040] Figure 16 Characterization of the in vitro induced ICD effect of the nanomedicine provided in Example 1 of this application;

[0041] Figure 17 This is an in vivo bioimaging characterization of nanomedicines provided in Example 1 of this application;

[0042] Figure 18 Characterization of the in vitro antitumor effect of the nanomedicine provided in Example 1 of this application;

[0043] Figure 19 Characterization of the in vivo immune activation effect of the nanomedicine provided in Example 1 of this application;

[0044] Figure 20 Characterization of the in vivo microenvironment for reversing immunosuppression of the nanomedicine provided in Example 1 of this application;

[0045] Figure 21 CD8 provided for Embodiment 1 of this application + Characterization of T-cell tumor tissue infiltration;

[0046] Figure 22 This is a schematic diagram of nanomedicine preparation and specific coordination structure provided in Example 2 of this application;

[0047] Figure 23 The characterization results of the nanomedicine provided in Example 2 of this application;

[0048] Figure 24 The ultraviolet absorption spectrum of the nanomedicine provided in Example 2 of this application;

[0049] Figure 25 Infrared spectrum of the nanomedicine provided in Example 2 of this application;

[0050] Figure 26 X-ray photoelectron spectroscopy of the nanomedicine provided in Example 2 of this application;

[0051] Figure 27 Cellular endocytosis characterization of the nanomedicine provided in Example 2 of this application;

[0052] Figure 28 Cytotoxicity characterization of the nanoparticles provided in Example 2 of this application;

[0053] Figure 29 Cell viability staining results for the nanomedicine provided in Example 2 of this application;

[0054] Figure 30 The results of intracellular reactive oxygen species generation for the nanomedicine provided in Example 2 of this application;

[0055] Figure 31 Characterization of the in vitro induced ICD effect of the nanomedicine provided in Example 2 of this application;

[0056] Figure 32 In vivo bioimaging characterization of the nanomedicine provided in Example 2 of this application;

[0057] Figure 33 Characterization of the in vitro antitumor effect of the nanomedicine provided in Example 2 of this application;

[0058] Figure 34 Characterization of the in vivo immune activation effect of the nanomedicine provided in Example 2 of this application. Detailed Implementation

[0059] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0060] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0061] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0062] This application provides a polyamino acid-metal coordination nanomedicine, comprising polyamino acids, metal ions, and polyphenolic compounds linked through metal coordination;

[0063] The phenolic hydroxyl group of the polyphenolic compound has at least one ortho- or meta-position as a hydroxyl or ketone group.

[0064] This application utilizes metal ions as nodes and polyamino acids and polyphenols as organic ligands to form stable nanostructures through metal-ligand coordination, constructing a spatiotemporally adaptive immune cascade synergistic regulation polyamino acid-metal ion-polyphenol coordination nanodrug. This not only simplifies the complexity of nanoplatform construction but also achieves spatiotemporally adaptive dynamic continuous regulation of immune activation, immune cell infiltration, and tumor cell recognition and killing, providing a new solution to overcome the "cold tumor" problem in tumor immunotherapy. Experimental results show that the polyamino acid-metal coordination nanodrug provided in this application can be efficiently taken up by tumor cells, exhibits dose-dependent toxicity to tumor cells, effectively induces ROS production in tumor cells, effectively induces ICD in tumor cells, and has tumor targeting ability. It effectively inhibits tumor growth, effectively promotes dendritic cell maturation, enhances cytotoxic T lymphocyte activation, significantly downregulates PD-L1 expression, and reduces α-SMA. + Fibroblast and collagen deposition effectively remodel the immunosuppressive matrix, thereby promoting CD8. + T cell infiltration in tumor tissue.

[0065] The present application is further illustrated below with reference to embodiments. The scope of protection of the present application is not limited to the following embodiments.

[0066] Example 1

[0067] 1.1 Synthesis of mPEG-PGA

[0068] Synthesis of L-glutamic acid NCA: A clean, anhydrous 500 mL three-necked round-bottom flask was prepared. Under nitrogen protection, 100 mL of tetrahydrofuran (THF) was added. Subsequently, 20 g of L-glutamic acid-5-benzyl ester and 9 g of trichloromethyl carbonate (BTC) were added sequentially, and the mixture was stirred at 60°C for 2 h. After the reaction was complete, excess solution was purged with nitrogen to a final volume of 50 mL. The solution was then precipitated with ice-cold hexane and filtered through a Buchner funnel to obtain the crude product. The crude product was dissolved in an appropriate amount of ice-cold ethyl acetate, washed with ice-cold saturated sodium chloride solution, and the organic phase was collected. After drying with anhydrous magnesium sulfate at -20°C overnight, the solution was filtered, and the filtrate was connected to a cold trap and the solvent was removed by a vacuum pump to obtain the solid product. After recrystallization and purification, pure L-glutamic acid-5-benzyl ester N-carboxyl anhydride (L-Glu NCA) was obtained. The specific synthetic route is as follows:

[0069]

[0070] The L-Glu NCA was characterized, and the results are shown in [reference needed]. Figure 1 , Figure 2 and Figure 3 , Figure 1 The above is the 1H NMR spectrum of the L-GluNCA monomer prepared in Example 1. Figure 2 The carbon NMR spectrum of the L-Glu NCA monomer prepared in Example 1 is shown below. Figure 3 The mass spectrum of the L-Glu NCA monomer prepared in Example 1 is shown.

[0071] Synthesis of mPEG-PGA: 1 g of mPEG-NH2 was added to 200 mL of dry toluene and azeotropically dehydrated at 125°C for 2 h. Subsequently, the toluene was dried under vacuum, and 35 mL of dry anhydrous N,N-dimethylformamide (DMF) and 2.5 g of L-Glu NCA were added sequentially. The reaction was carried out at room temperature for 3 days. After the reaction was complete, the mixture was precipitated with ice-cold diethyl ether, and the white solid was collected and dried under vacuum to obtain the intermediate product. The intermediate product was dissolved in 10 times its mass volume of trifluoroacetic acid (TFA), and then 3 times its mass volume of hydrobromic acid / acetic acid solution was added. After reacting for 1 h, the mixture was precipitated with ice-cold diethyl ether, and the white solid was collected. The white solid was dissolved in an appropriate amount of DMF, placed in a 3500 Da dialysis bag, dialyzed in deionized water for 3 days, and then freeze-dried to obtain the mPEG-PGA polymer. The reaction process is as follows:

[0072]

[0073] The product before deprotection and mPEG-PGA were characterized by NMR. The results are shown in [link to NMR diagram]. Figure 4 and Figure 5 , Figure 4 The 1H NMR spectrum of the polymer prepared before deprotection in the embodiments of this application is shown. Figure 5 The 1H NMR spectrum of mPEG-PGA prepared for the embodiments of this application is shown. The experimental results indicate that mPEG-PGA was successfully synthesized.

[0074] 1.2 Preparation and Characterization of Nanomedicines

[0075] Taking the preparation of polyamino acid-manganese ion-quercetin coordination nanomedicine as an example: mPEG-PGA was prepared into a 10 mg / mL solution, and the pH was adjusted to 8.8 with 1 M sodium hydroxide solution. Then, 150 μL of 5 mg / mL manganese chloride aqueous solution was added, and the mixture was stirred for 10 min. Next, a certain amount of Tris HCl solution at pH 8.8 was added, and the mixture was stirred for 5 min. Finally, 125 μL of 20 mg / mL DMSO solution of quercetin was added, and the mixture was stirred overnight. After dialysis using a 7000 Da dialysis bag for 24 h, the PGA-Mn-Quer nanomedicine was obtained. A schematic diagram of the nanomedicine preparation and the specific coordination structure are shown below. Figure 6 As shown, Figure 6 The diagram shows the preparation of nanomedicines and their specific coordination structures as provided in the embodiments of this application.

[0076] The prepared nanomedicine was diluted to a solution of 0.5 mg / mL, and the particle size of the nanomedicine was measured using a dynamic light scattering instrument. The results are shown in [reference needed]. Figure 7 , Figure 8 and Figure 9 , Figure 7 This is a particle size distribution diagram of the nanomedicine prepared in the embodiments of this application. Figure 8 This is a schematic diagram of the potential of the nanomedicine prepared in the embodiments of this application. Figure 9 Transmission electron microscopy images of the nanomedicines prepared for the embodiments of this application, by Figures 7-9 It is known that the nanoparticles prepared in this application have a particle size of about 100 nm, a negatively charged surface, and a spherical and relatively regular morphology.

[0077] Quer drug loading assay: After lyophilizing the prepared sample, the lyophilized sample was accurately weighed and dissolved in DMSO, then the pH was adjusted to 2 with 1 M hydrochloric acid solution. The absorbance at 365 nm was measured using a microplate reader. The DLC and DLE of Quer in the nanomedicine were calculated using the following equations:

[0078] DLC = Drug content in micelles / Total mass of drug-loaded micelles × 100%;

[0079] DLE = Drug amount in micelles / Total drug amount × 100%;

[0080] The results showed that its drug loading was 4.4%.

[0081] Mn 2+ Content testing: Take a certain amount of lyophilized sample, dissolve it in water, and directly send it for ICP testing to detect Mn. 2+ The content was determined, and the results showed that its content was 0.7%.

[0082] The formation mechanism of nanomedicines was characterized using multispectral techniques including ultraviolet absorption spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The results are as follows: Figure 10 , Figure 11 and 12 As shown, Figure 10 The infrared spectrum of the nanomedicine prepared in the embodiments of this application is shown below. Figure 11 The image shows the ultraviolet absorption spectrum of the nanomedicine prepared in the embodiments of this application. Figure 12 X-ray photoelectron spectroscopy of the nanomedicine prepared for the embodiments of this application. Figures 10-12 It is known that the nanoparticles prepared in this application contain mPEG-PGA, manganese ions, and polyphenol quercetin.

[0083] 1.3 Cellular uptake

[0084] The cellular uptake of PGA-Mn-Quer nanoparticles was quantitatively detected by flow cytometry (FCM). 4T1 cells were cultured at 4 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured for 24 h. Cells were then treated with Cy5-labeled PGA-Mn-Quer nanoparticles for 2, 4, 6, and 8 h, with untreated cells serving as a negative control. After treatment, cells were collected, washed with PBS, and intracellular fluorescence intensity was measured by flow cytometry. Flow cytometry results of cell uptake are shown below. Figure 13 As shown, Figure 13 The cell endocytosis characterization results provided in the embodiments of this application are shown in the left figure, which is a flow cytometry spectrum, and the right figure is a statistical bar chart of quantitative cell endocytosis. Figure 13 It is known that the nanoparticles provided in this application can be efficiently taken up by tumor cells.

[0085] 1.4 Tumor Cytotoxicity of Nanomedicines

[0086] Logarithmically growing 4T1 cells were digested, resuspended, and diluted before counting to determine cell density (cells / mL). The cell suspension was diluted and seeded at a density of 5000 cells / well in 96-well plates. PGA-Mn-Quer nanomedicine and Mn+Quer mixed experimental groups were set up. After cell attachment, different drug concentrations were added (at least three biological replicates were set up for each concentration, including blank and negative control wells), and the cells were incubated at 37°C for 24 h. Then, 10 μL of prepared MTT assay reagent (5 mg / mL) was added to each well, and the cells were incubated at 37°C for another 4 h. The supernatant was discarded, and DMSO (100 μL / well) was added. After shaking for 10 min, the OD value was measured at 490 nm using a microplate reader to calculate cell viability. Cytotoxicity results are as follows: Figure 14 As shown, Figure 14 The cytotoxicity results of the nanomedicine provided in the embodiments of this application show that tumor cells exhibit dose-dependent toxicity to the nanoparticles.

[0087] 1.5 Detection of intracellular reactive oxygen species (ROS)

[0088] Press 1×10 4 At a cell / well density of 4 T1 cells, 24-well plates were seeded and incubated overnight. Experimental groups included a control group, a PGA-Mn-Quer nanomedicine group, and a Mn+Quer group. Based on IC50... 50 After adding the drug at the specified concentration and incubating for 24 h, the culture medium was discarded, the cells were washed three times with PBS, and fresh culture medium was added along with the ROS detection probe. The cells were then incubated for 30 min. Subsequently, the culture medium was discarded, the cells were washed three times with PBS, and fresh culture medium was added. Intracellular ROS levels were then detected using laser confocal microscopy. The ROS experimental results are as follows: Figure 15 As shown, Figure 15 The intracellular reactive oxygen species (ROS) generation of the nanomedicine provided in the embodiments of this application was characterized, and the results showed that the nanoparticles could effectively induce tumor cells to produce ROS.

[0089] 1.6 Nanoparticle-induced ICD

[0090] To investigate whether nanomedicines could induce intracellular drug reaction (ICD) in tumor cells in vitro, flow cytometry (FCM) was used to detect the expression of calreticulin (CRT) exposed on the cell membrane surface, ELISA kits were used to detect the secretion level of high-mobility group box 1 (HMGB1), and ATP release levels were detected using chemiluminescence immunoassay. Experimental groups included a PBS group, a Mn+Quer mixed group, and a PGA-Mn-Quer nanomedicine group.

[0091] ①FCM assay for CRT: 4T1 cells were seeded at a density of 400,000 per well in 6-well plates, with 2.0 mL of culture medium per well. The cells were cultured overnight until fully adhered, then the culture medium was discarded. 2.0 mL of a nanomedicine-based IC50 assay was then added. 50 After incubation in concentrated culture medium for 24 h, remove the medium and wash three times with pre-chilled PBS. Add 0.5 mL of trypsin-EDTA digestion solution to each well and incubate at 37°C for 2 min. Gently pipette the cells and resuspend them evenly in 1 mL of pre-chilled PBS. Centrifuge at 800 rpm for 3 min, discard the supernatant, and resuspend the cells in 0.1 mL of pre-chilled PBS. Add 1.0 μL of MIAlexa Fluor 647-CRT antibody and stain at room temperature in the dark for 30 min. After staining, resuspend the cells in 0.5 mL of pre-chilled PBS, centrifuge at 1200 rpm for 5 min, discard the supernatant, and repeat the process. Resuspend the cells in 0.5 mL of pre-chilled PBS again and centrifuge at 1200 rpm for 5 min, discarding the supernatant. Finally, resuspend the cells in 0.5 mL of pre-chilled PBS and analyze them using flow cytometry.

[0092] ② HMGB1 and ATP assay: 4T1 cells were seeded in 6-well plates (500,000 cells / well), and 2.0 mL of culture medium was added to each well. The plates were incubated overnight at 37°C. The culture medium was discarded, and 2.0 mL of fresh nanomedicine-based IC50 assay was added. 50 Concentrated culture medium. After culturing for 24 h, collect the cell culture supernatant. Perform the procedure according to the operating instructions of the mouse HMGB1 ELISA kit, and detect the experimental results on a microplate reader. Perform the procedure according to the operating instructions of the enhanced ATP assay kit.

[0093] Results of CRT expression, HMGB1 and ATP extracellular release were as follows: Figure 16 As shown, Figure 16 The in vitro induction of ICD effect by nanomedicine provided in the embodiments of this application is characterized. The left figure shows the ATP release result, the middle figure shows the HMGB1 release result, and the right figure shows the CRT expression result. The results show that nanoparticles can induce CRT expression level, increase the extracellular release levels of HMGB1 and ATP, and thus effectively induce ICD in tumor cells.

[0094] 1.7 Biodistribution within the body

[0095] By 1×10 6A BALB / c mouse model of 4T1 tumors was established by subcutaneous injection of 100 cells into the right axilla. On day 7 post-inoculation, Cy5-labeled nanoparticles were injected via the tail vein. Mice were sacrificed at 0, 6, and 12 hours post-inoculation, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for in vitro fluorescence imaging. Data were analyzed using Living Image software. Experimental results are as follows: Figure 17 As shown, Figure 17 The in vivo bioimaging characterization of nanomedicines provided in this application embodiment shows images from left to right at 0 h, 6 h, 12 h, and 24 h. Figure 17 It is evident that PGA-Mn-Quer nanoparticles possess excellent tumor-targeting capabilities, supporting their potential application in the treatment of solid tumors.

[0096] 1.8 Nanoparticles and their antitumor effects

[0097] Take 1×10 6 Four T1 cells were inoculated into the right axilla of BALB / c mice on day 0. Tumor formation was observed one week later, and the mice were randomly divided into three groups: a saline group, a Mn+Quer mixed group, and a PGA-Mn-Quer nanomedicine group. Treatment was administered via tail vein injection, once every three days for a total of four doses. 2+ The dosage was 1.5 mg / kg, and the Quer dosage was 5 mg / kg. Mouse body weight and tumor volume were recorded concurrently with administration. Three days after the last administration, mice were sacrificed, and their tumors were harvested, weighed, and photographed. Experimental results are as follows: Figure 18 As shown, Figure 18 The in vitro antitumor effect characterization of the nanomedicine provided in the embodiments of this application is shown in the left figure, which is the tumor volume change curve, and the right figure is a tumor photograph. Figure 18 It is known that PGA-Mn-Quer nanoparticles can effectively inhibit tumor growth.

[0098] 1.9 Detection of in vivo immune activation effect

[0099] 4T1 tumor-bearing mice were euthanized, and tumor tissue was obtained. The tissue was gently ground and filtered through a 300-mesh nylon sieve to obtain a single-cell suspension. The single-cell suspension was then washed with FACS buffer, and individual cells were stained with FITC-CD80 and PE-CD86 monoclonal antibodies. Additionally, immune cells were stained with APC-CD3, FITC-CD4, and PECD8a. The cells were then fixed with 4% paraformaldehyde. Flow cytometry analysis was performed. The results were analyzed using FlowJo.X software. Experimental results are shown below. Figure 19 As shown, Figure 19 The in vivo immune activation effect of the nanomedicine provided in the embodiments of this application is characterized, wherein the left figure shows the results of DC cells, and the middle figure shows the results of CD4.+ T cell results, right figure shows CD8 + T cell results, by Figure 19 It is known that PGA-Mn-Quer nanoparticles can effectively promote the maturation of dendritic cells and enhance the activation of cytotoxic T lymphocytes.

[0100] 1.10 Detection of In vivo immune microenvironment regulation

[0101] Tumor tissue was isolated from 4T1 tumor-bearing mice, washed three times in PBS, fixed in 4% (w / v) paraformaldehyde, dehydrated with ethanol, embedded in paraffin, and sectioned to obtain paraffin sections with a thickness of approximately 5 μm. The sections were baked in a 68°C incubator for 2 h, then cooled to room temperature. The sections were then dewaxed by soaking in xylene for 10 min, followed by another 10 min soak in fresh xylene. Subsequently, they were hydrated by soaking in 100% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 10 min each, and then gently washed with diluted water for 1 min. The sections were then placed in citrate buffer and heated in an autoclave until boiling for 150 seconds, then cooled to room temperature. The sections were placed in a humidified chamber, blocked with goat serum, and incubated at 37°C for 20 min. The serum was discarded, and the sections were then subjected to collagen, PD-L1, and α-SMA assays. + Fibroblast staining, immunohistochemical staining, and immunofluorescence staining analysis. CD8+ was analyzed using immunofluorescence staining. + T cell infiltration in tumor tissue. Results as follows: Figure 20 and Figure 21 As shown, Figure 20 Characterization of the in vivo immunosuppression reversal microenvironment for nanomedicines provided in the embodiments of this application. Figure 21 CD8 provided for embodiments of this application + Characterized by T-cell tumor tissue infiltration. (By...) Figure 20 and Figure 21 It is evident that the nanoplatform provided in this application can significantly downregulate PD-L1 expression, reduce α-SMA⁺ fibroblast and collagen deposition, effectively remodel the immunosuppressive matrix, and thereby promote CD8. + T cell infiltration in tumor tissue.

[0102] Example 2

[0103] 2.1 Synthesis of mPEG-PGA

[0104] The preparation process is the same as in Example 1.

[0105] 2.2 Preparation and Characterization of PGA-Cu-EGCG Nanomaterials

[0106] mPEG-PGA was dissolved in deionized water (10 mg / mL), and the pH of the solution was adjusted to 8.8 using 1 M NaOH. Then, copper chloride solution (5 mg / mL, 150 μL) was slowly added dropwise to the solution, and the mixture was magnetically stirred for 10 min. Tris-HCl buffer (pH 8.8) was then added, and stirring was continued for another 5 min. Epigallocatechin gallate (EGCG, 20 mg / mL, 125 μL) dissolved in DMSO was then added, and the reaction was carried out overnight under continuous stirring. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 7000 Da and dialyzed against deionized water for 24 h to obtain PGA-Cu-EGCG nanoparticles. See [link to relevant documentation] Figure 22 , Figure 22 This is a schematic diagram of the preparation and specific coordination structure of the nanomedicine provided in Example 2 of this application.

[0107] The obtained nanoparticles were diluted to 0.5 mg / mL, and their particle size and morphology were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results are as follows: Figure 23 As shown, Figure 23 The characterization results of the nanomedicine provided in Example 2 of this application are shown in the figure. The left figure is the particle size distribution map, the middle figure is the potential map, and the right figure is the transmission electron microscopy (TEM) photograph. Figure 23 It is known that the nanoparticles prepared in Example 2 of this application have a particle size of about 90 nm and a negative potential; transmission electron microscopy shows that their morphology is spherical and relatively regular.

[0108] EGCG drug loading test: After lyophilizing the prepared sample, the lyophilized sample was accurately weighed and dissolved in DMSO, then the pH was adjusted to 2 with 1 M hydrochloric acid solution. The UV absorbance at 365 nm was measured using a microplate reader. The DLC and DLE of EGCG in the nanomedicine were calculated using the following equations:

[0109] DLC = Drug content in micelles / Total mass of drug-loaded micelles × 100%;

[0110] DLE = Drug amount in micelles / Total drug amount × 100%.

[0111] The results showed that its drug loading was 43.78%.

[0112] Cu 2+ Content testing: Take a certain amount of lyophilized sample, dissolve it in water, and then directly send it for ICP testing to detect Cu. 2+ The content, the results showed, its Cu 2+ The content is 5.34%.

[0113] The formation mechanism of nanomedicines was characterized using multispectral techniques including ultraviolet absorption spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The results are as follows: Figure 24 , Figure 25 and Figure 26 As shown, Figure 24 The ultraviolet absorption spectrum of the nanomedicine provided in Example 2 of this application is shown. Figure 25 The infrared spectrum of the nanomedicine provided in Example 2 of this application. Figure 26 The X-ray photoelectron spectroscopy of the nanomedicine provided in Example 2 of this application is obtained by... Figures 24-26 It is known that the nanoparticles prepared in this application contain mPEG-PGA, copper ions, and polyphenol epigallocatechin gallate.

[0114] 2.3 Flow cytometry analysis of cell uptake

[0115] CT26 cells were loaded at 4 × 10⁻⁶ 4 Cells were seeded at a density of 100 cells / well in 24-well plates and cultured overnight. Subsequently, cells were co-incubated with Cy5-labeled PGA-Cu-EGCG nanoparticles for 2, 4, 6, or 8 h, with untreated cells serving as a control group. After incubation, cells were collected, washed with PBS, and resuspended. Flow cytometry (FCM) was used to quantify intracellular uptake of the nanoparticles at different time points. The flow cytometry results of cell uptake are shown below. Figure 27 As shown, Figure 27 The cellular endocytosis characterization of the nanomedicine provided in Example 2 of this application is shown in the left figure as a flow cytometry spectrum and the right figure as a quantitative statistical bar chart of cellular endocytosis. The experimental results show that the nanoparticles can be efficiently taken up by tumor cells.

[0116] 2.4 In vitro cytotoxicity assay

[0117] The cytotoxicity of PGA-Cu-EGCG nanoparticles was assessed using the MTT assay and compared with that of free Cu+EGCG. Briefly, CT26 cells in logarithmic growth phase were digested, resuspended, and counted, then analyzed at 5 × 10⁻⁶. 3 Cells were seeded at a density of 10 cells / well in 96-well plates and allowed to adhere overnight. Subsequently, cells were treated with different concentrations of PGA-Cu-EGCG or Cu+EGCG (at least three biological replicates for each concentration, including blank and negative control wells) and incubated at 37°C for 24 h. After treatment, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued at 37°C for 4 h. The supernatant was carefully discarded, and 100 μL of DMSO was added to each well to dissolve the generated formazan crystals. After shaking for 10 min, the absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated using the untreated control group as a reference. Cytotoxicity results are as follows: Figure 28As shown, Figure 28 The cytotoxicity characterization of the nanoparticles provided in Example 2 of this application showed that tumor cells exhibited dose-dependent toxicity to the nanoparticles.

[0118] 2.5 Live / Dead Cell Staining Assay

[0119] After digesting, resuspending, and counting CT26 cells in the logarithmic growth phase, they were divided into 5 × 10⁻⁶ cells. 3 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated overnight. Subsequently, they were seeded separately using their respective IC50 values. 50 Cells were treated with PGA-Cu-EGCG or free Cu+EGCG at a concentration of [concentration not specified] for 24 h, with at least three replicates per group. After treatment, Calcein-AM / PI working solution was prepared using the Calcein / PI Cell Viability and Cytotoxicity Assay Kit (Beyotime, China), with 100 μL added to each well and incubated at 37°C for 30 min in the dark. After staining, images were captured using a fluorescence microscope to assess cell viability and death. Cell viability and death staining results are shown below. Figure 29 As shown, Figure 29 The results of cell viability and mortality staining for the nanomedicine provided in Example 2 of this application further demonstrate that the nanoparticles effectively induce tumor cell death.

[0120] 2.6 Intracellular ROS Detection

[0121] CT26 cells were loaded at 1 × 10⁻⁶ 4 Cells were seeded at a density of 100 cells / well in confocal culture dishes and incubated overnight. The experiment was divided into a control group, a PGA-Cu-EGCG group, and a Cu+EGCG group. All groups were incubated using IC50. 50 Cells were treated with the concentration for 24 h. After treatment, the culture medium was discarded, and the cells were washed three times with PBS. Fresh culture medium containing 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added, and the cells were incubated for 30 min. The cells were then washed three more times with PBS, fresh culture medium was added, and intracellular ROS levels were observed using a confocal laser scanning microscope. The ROS experiment results are as follows: Figure 30 As shown, Figure 30 The results of intracellular reactive oxygen species generation of the nanomedicine provided in Example 2 of this application show that the nanoparticles can effectively induce tumor cells to produce ROS.

[0122] 2.7 Nanomedicine-induced ICD

[0123] In vitro CRT expression analysis: CT26 cells were cultured at 4 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in 6-well plates, with 2 mL of culture medium added to each well. The plates were incubated overnight until fully adherent. Subsequently, they were seeded using IC50 assay. 50Cells were treated with PGA-Cu-EGCG or Cu+EGCG at various concentrations for 24 h. After treatment, the culture medium was discarded, and the cells were washed three times with ice-cold PBS. 0.5 mL of trypsin-EDTA was added to each well, and the cells were digested at 37°C for 2 min. After gentle resuspending, the cells were collected in 1 mL of ice-cold PBS. After centrifugation at 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 0.1 mL of ice-cold PBS. 1 μL of Alexa Fluor 647-labeled anti-CRT antibody was added, and the cells were incubated at room temperature in the dark for 30 min. After staining, the cells were washed twice by centrifugation at 1200 rpm for 5 min, and finally resuspended in 0.5 mL of PBS. The exposure level of CRT on the cell surface was quantitatively analyzed by flow cytometry.

[0124] In vitro HMGB1 and ATP release assay: CT26 cells were cultured at 5 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in 6-well plates, with 2 mL of culture medium per well, and incubated overnight at 37°C. Subsequently, they were seeded using IC50. 50 Cells were treated with a concentration of PGA-Cu-EGCG for 24 h. After treatment, the culture supernatant was collected. Following the kit instructions, the HMGB1 content in the supernatant was quantitatively detected using a mouse HMGB1 ELISA kit, and the absorbance was measured using a microplate reader. Extracellular ATP content was measured using a commercially available ATP assay kit.

[0125] Results of CRT expression, HMGB1 and ATP extracellular release were as follows: Figure 31 As shown, Figure 31 The in vitro induction of ICD by the nanomedicine provided in Example 1 of this application is characterized, wherein the left figure shows the ATP release results, the middle figure shows the HMGB1 release results, and the right figure shows the CRT expression results. The results show that the nanoparticles can induce CRT expression levels and increase the extracellular release levels of HMGB1 and ATP, thereby effectively inducing ICD in tumor cells.

[0126] 2.8 Biodistribution within the body

[0127] 1 × 10 6 CT26 cells were subcutaneously inoculated into the right axilla of BALB / c mice. On day 7 post-inoculation, Cy5-labeled PGA-Cu-EGCG nanoparticles were injected via the tail vein. Mice were sacrificed at 0, 6, and 12 h post-inoculation, and tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested. In vitro fluorescence imaging was performed to assess the in vivo distribution of the nanoparticles, and all images were quantitatively analyzed using Living Image software. Experimental results are as follows: Figure 32 As shown, Figure 32The in vivo bioimaging characterization of the nanomedicine provided in Example 2 of this application shows, from left to right, 0 h imaging, 6 h imaging, 12 h imaging and statistical graph. These results indicate that PGA-Cu-EGCG nanoparticles have excellent tumor targeting ability, supporting their potential application in the treatment of solid tumors.

[0128] 2.9 In vivo antitumor therapy experiment

[0129] 1 × 10 6 CT26 cells were subcutaneously inoculated into the right axilla of BALB / c mice, and tumor growth was monitored. Nine days later, the mice were randomly divided into three groups: a saline group (control), a Cu+EGCG mixture group, and a PGA-Cu-EGCG nanoparticle group. All groups received the drug via tail vein injection, once every three days for a total of four administrations. 2+ The dosage was 1.5 mg / kg, and the EGCG dosage was 5 mg / kg. Tumor volume and mouse weight were recorded regularly during treatment. Mice were sacrificed 3 days after the last administration, and the tumors were dissected, weighed, and photographed. Experimental results are as follows: Figure 33 As shown, Figure 33 The in vitro antitumor effect of the nanomedicine provided in Example 2 of this application is characterized. The left figure is a graph of tumor volume change, and the right figure is a tumor photograph. These findings indicate that PGA-Cu-Quer nanoparticles can effectively inhibit tumor growth.

[0130] 2.10 Detection of in vivo immune response

[0131] After euthanizing CT26 tumor-bearing mice, tumor tissue was removed, gently minced, and filtered through a 300-mesh nylon sieve to prepare a single-cell suspension. Cells were washed with FACS buffer and then subjected to immunocellular phenotypic analysis using fluorescently labeled antibodies: CD4⁺ T cells (FITC anti-mouse CD3, PE anti-mouse CD4), CD8⁺ T cells (FITC anti-mouse CD3, PE anti-mouse CD8a), and dendritic cells (DCs; FITC anti-mouse CD11c, PE anti-mouse CD80, APC anti-mouse CD86). For a more comprehensive T-cell analysis, APC-CD3, FITC-CD4, and PE-CD8a staining were performed separately. After staining, cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry. Data were analyzed using FlowJoX software to determine the proportions of various immune cells in the tumor microenvironment. Experimental results are as follows: Figure 34 As shown, Figure 34The in vivo immune activation effect of the nanomedicine provided in Example 2 of this application is characterized, with the results from left to right showing the detection of dendritic cells, CD4⁺ T cells, and CD8⁺ T cells. These findings indicate that PGA-Cu-Quer nanoparticles can effectively promote dendritic cell maturation and enhance cytotoxic T lymphocyte activation.

[0132] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and application concept of this application, should be included within the scope of protection of this application.

Claims

1. A polyamino acid-metal coordination nanodrug, comprising a polyamino acid, a metal ion and a polyphenol compound connected by metal coordination. At least one of the ortho- or meta-positions of the phenolic hydroxyl group of the polyphenol compound is a hydroxyl group or a ketone group.

2. The polyamino acid-metal coordination nanodrug of claim 1, wherein, The polyphenol compound is one or more of epigallocatechin gallate, quercetin, catechin, proanthocyanidin, chlorogenic acid, kaempferol and resveratrol.

3. The polyamino acid-metal coordination nanodrug of claim 1, wherein, The polyamino acid is one or more of polyglutamic acid, polylysine, polyaspartic acid, polyarginine, polyseryl acid and polyornithine. The metal ion is a copper ion, a manganese ion, an iron ion or a zinc ion.

4. The polyamino acid-metal coordination nanomedicine according to any one of claims 1 to 3, characterized in that, The mass ratio of the polyamino acid, the metal ion and the polyphenol compound is 30-50:5-15:1-10.

5. The polyamino acid-metal coordination nanomedicine of claim 4, wherein, The polyamino acid is polyethylene glycol-polyglutamic acid.

6. The polyamino acid-metal coordination nanomedicine of claim 5, wherein, The molecular weight of the polyethylene glycol is 1000 Da-50000 Da.

7. The polyamino acid-metal coordination nanomedicine according to any one of claims 1 to 6, characterized in that, The particle size is 50 nm-200 nm. 8.A method for preparing the polyamino acid-metal coordination nanodrug of any one of claims 1-7, comprising the following steps: Mixing the polyamino acid, the metal salt and the polyphenol compound, and obtaining the polyamino acid-metal coordination nanodrug after dialysis.

9. The production method according to claim 8, characterized by, Specifically comprising: Adjusting the pH value of the polyamino acid solution to 8-10, mixing with the aqueous metal salt solution, adjusting the pH value to 8-10, mixing with the polyphenol compound solution, stirring for 5 h-20 h, and then dialysis. 10.The use of the polyamino acid-metal coordination nanodrug of any one of claims 1-7 in the preparation of a drug for immunotherapy of tumors.

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