Chiral cyclic peptide and manganese ion coordination nano assembly and preparation method and application thereof

Abstract

The invention provides a coordination nano assembly of chiral cyclopeptide and manganese ions as well as a preparation method and application of the coordination nano assembly. According to the method, a manganese superoxide dismutase protein structural domain and a unique tumor microenvironment of melanoma high tyrosinase are combined, and polypeptide sequences L-YD-hL-DD-h, L-YL-HL-DL-H and D-yD-hD-dD-h with different chirality are designed for the first time; the chirality of the polypeptide is regulated and controlled, the in-vivo circulation stability and the cell entry efficiency of the nano assembly are improved, the coordination self-assembly of manganese and different chiral cyclopeptides is realized, and the in-situ oxidation of an in-vivo tumor microenvironment is simulated for photo-thermal therapy and cell membrane coating. The method disclosed by the invention is simple, mild in experimental condition and easy to operate, the prepared cell membrane coated nano assembly not only has long circulation stability and high cell entry efficiency, but also can realize mild photo-thermal therapy through in-situ oxidation, releases manganese ions to activate a CGAS-STING pathway for immunotherapy of tumors, and has a wide application prospect. The potential application value is realized in the field of combined treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a coordination nanoassembly of a chiral cyclic peptide and a manganese ion. Background Art

[0002] Malignant tumors, characterized by rapid growth, strong metastatic potential, and high recurrence rates, have become a major threat to human health. Traditional clinical cancer treatments, such as chemotherapy and radiotherapy, often damage normal tissues, have low therapeutic efficacy, and are prone to developing tolerance. Photothermal therapy (PTT) has gained popularity due to its highly effective local tumor treatment, minimal invasiveness, and minimal side effects. Peptides, macromolecules composed of multiple amino acids linked by peptide bonds, exhibit excellent biocompatibility, including tissue and blood compatibility; possess mechanical strength, suitable physical and chemical properties, ease of molding, and stable drug release rates. Peptide self-assembling nanomaterials, due to their advantages such as sequence editability, regulated self-assembly, low environmental toxicity, and high biocompatibility, are widely used in chemical sensing, biomedicine, and energy catalysis. To achieve broader applications, the coordination of peptides with inorganic metal materials is a research direction. Through sequence design, peptide nanomaterials can be coordinated and assembled with metal ions. Combining the structural and functional advantages of both, multifunctional peptide hybrid nanomaterials with supramolecular structures can be constructed. This method can improve the dispersibility, stability, and biocompatibility of inorganic materials and enhance the inherent physical and chemical properties of the materials, thereby promoting deeper applications of polypeptide nanohybrid materials.

[0003] Chiral cyclic peptides, a unique class of bioactive molecules, have demonstrated significant advantages in biomedicine and materials science. Their cyclic structures, formed by covalent closure and forming a rigid conformation, effectively reduce molecular freedom, enhance structural stability, significantly resist protease degradation, and prolong in vivo circulation. The presence of chiral centers imparts precise stereoselectivity, enabling high-affinity, three-dimensional binding to specific biological targets (such as enzyme active sites, cell membrane receptors, or ion channels), thereby enhancing targeting and specificity. For example, in anti-tumor drug design, chiral cyclic peptides can precisely recognize tumor-associated antigens through stereocomplementarity, reducing off-target toxicity to normal tissues. Furthermore, the cyclic topology can form hydrophobic cavities or expose specific functional groups, promoting coordination and assembly with metal ions and constructing nanocomplex systems with synergistic functions. Their chirality can also modulate the self-assembly behavior of nanoparticles, forming well-organized nanostructures and enhancing photothermal conversion efficiency or drug loading capacity. Compared with linear peptides, the conformational constraints of chiral cyclic peptides give them more advantages in simulating natural protein interactions, transmembrane transport efficiency and immunogenicity regulation, providing an ideal molecular skeleton for the development of new integrated diagnosis and treatment platforms.

[0004] Purely L-amino acid nanoparticles face multiple limitations in biological applications, a core issue stemming from their high compatibility with natural enzymatic degradation mechanisms. Because proteases in the body (such as trypsin and lysozyme) specifically recognize L-amino acids through a "lock-and-key" mechanism, these nanoparticles are susceptible to rapid degradation in blood or tissue fluids. For example, particles containing L-phenylalanine have a half-life of only a few hours in serum. Furthermore, the single L-chiral configuration results in insufficient intermolecular cooperativity, resulting in low rigidity of the α-helical or β-sheet structures, which are susceptible to conformational relaxation or even disintegration when exposed to pH fluctuations or changes in ionic strength. Their high similarity to endogenous peptide chains can also trigger misidentification by the immune system, accelerating clearance by macrophages or complement adsorption. Furthermore, they struggle to penetrate complex barriers such as biological membranes and tumor stroma, limiting their application in oral administration or targeted therapy.

[0005] In recent years, tumor immunotherapy has rapidly developed. It primarily works by boosting certain active components of the immune system or relieving immunosuppression. It has become a major pillar of cancer treatment following surgery, radiotherapy, and chemotherapy. In particular, modulating the anti-tumor immune system by activating the CGAs-STING pathway has achieved significant clinical progress, becoming a novel anti-cancer weapon. Summary of the Invention

[0006] The purpose of the present invention is to provide a chiral cyclic peptide and manganese ion coordination nanoassembly with high in vivo long-term circulation stability and high cellular efficiency, as well as a preparation method and application thereof.

[0007] To achieve the above object, the present invention proposes a chiral cyclic peptide and manganese ion coordination nanoassembly, characterized in that the nanoassembly is Mn@ L- Y D- h L- D D- h, through chiral cyclic peptides L- Y D- h L- D D- h is coordinated with manganese ions; among them, chiral cyclic peptide L- Y D- h L- D D- h Based on the characteristics of different chiral cyclic peptides, the chirality of the cyclic peptides is regulated.

[0008] Furthermore, the characteristics include endocytic stability, intracellular efficiency and immune activation ability.

[0009] The present invention also provides a method for preparing a coordination nanoassembly of a chiral cyclic peptide and a manganese ion, comprising the following steps: S1: Dissolve different chiral cyclic peptides in ultrapure water, add manganese chloride solution dropwise, then stir to react, centrifuge, wash, and dry to obtain nanoassembly Mn@ L- Y D- h L- D D- h、Mn@ L- Y L- H L- D L- H and Mn@ D- y D- h D- d D- h, screen the chiral nanoassemblies with the best in vitro cell entry efficiency, the strongest immune activation ability, and the strongest in vivo circulation stability, and select Mn@ L- Y D- h L- D D- h polypeptide as the subsequent material, wherein the ratio of chiral cyclic peptide to manganese chloride solution is 10-12 mg: 0.1-0.15 mL; S2: Mn@ L- Y D- h L- D D- h was dissolved in PBS, tyrosinase was added, stirred for reaction, centrifuged for washing, and dried to obtain the oxidized nanoassembly Mn@m L- Y D- h L- D D- h, wherein the amount of tyrosinase used is 0.1-0.15 mL; S3: Change Mn@m in S2 L- Y D- h L- D D- h was dissolved in PBS, and the prepared B16 cell membrane was added. After ultrasonic dispersion, the membrane was repeatedly pushed and pulled 30 times using a membrane coating device, centrifuged, washed, and dried to obtain the cell membrane-coated oxidized nanoassembly Mn@m L- Y D- h L- D D- h@CM, in which the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2-1:1.8.

[0010] Furthermore, the concentration of manganese chloride in S1 was 1 mg / mL, and the reaction time was 8-10 h.

[0011] Furthermore, the tyrosinase concentration in S2 was 1 mg / mL.

[0012] Furthermore, the S2 temperature is 37° and the stirring time is 22-24 h.

[0013] Furthermore, the solvent in S3 was PBS buffer, and the cell membrane concentration was 1 mg / mL.

[0014] The present invention also proposes an application of a coordination nanoassembly of a chiral cyclic peptide and a manganese ion for preparing a tumor therapeutic drug.

[0015] Compared with the prior art, the advantages of the present invention are: The nanoassembly of the present invention introduces D-amino acids through chiral engineering, which is a breakthrough strategy: the mixed D / L type can interfere with protease recognition and extend the half-life by more than 10 times; the non-natural hydrogen bond network enhances hydrophobic stacking to form a dense and stable nanotopological structure; providing a new direction for nano drug delivery systems.

[0016] 2. The chiral cyclic peptide coated with tumor cell membrane and ion-coordinated nanoassembly of the present invention effectively solves the problems of poor long-term circulation stability and low cell entry efficiency of the nanoassembly through chirality regulation for the first time. At the same time, it can be used for photothermal therapy and immunotherapy through in situ oxidation of the tumor microenvironment, and has potential application value in the field of tumor treatment.

[0017] (2) The nanoassembly prepared by the present invention has good water solubility and biocompatibility.

[0018] (3) The nanoassembly preparation method of the present invention has mild experimental conditions and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Mn@m in the present invention L- Y D- h L- D D- Schematic diagram of the synthesis process of h@CM complex; Figure 2 is the Mn@ of different chirality in Example 1 of the present invention L- Y D- h L- D D- h,Mn@ L- Y L- H L- D L- H, Mn@ D- y D- h D- d D- h and circular dichroism spectra of its free peptide; Figure 3 is the Mn@ of different chirality in Example 1 of the present invention L- Y D- hL- D D- h,Mn@ L- Y L- H L- D L- H, Mn@ D- y D- h D- d D- h Circulation stability diagram in animals; Figure 4 is the Mn@ of different chirality in Example 1 of the present invention L- Y D- h L- D D- h,Mn@ L- Y L- H L- D L- H, Mn@ D- y D- h D- d D- h Cell invasion efficiency diagram; Figure 5 is the Mn@ of different chirality in Example 1 of the present invention L- Y D- h L- D D- h,Mn@ L- Y L- H L- D L- H, Mn@ D- y D- h D- d D- h Analysis of immune system activation in vitro; Figure 6 Mn@ in Example 6 of the present invention L- Y D- h L- D D- h,Mn@m L- Y D- h L- D D- h,Mn@m L- Y D- h L- D D- Transmission electron microscopy images of the h@CM preparation process; Figure 7 Mn@ in Example 7 of the present invention L- Y D- h L- D D- h,Mn@m L- Y D- h L- D D-h,Mn@m L- Y D- h L- D D- Scanning electron microscopy images of h@CM; Figure 8 Mn@m in Example 8 of the present invention L- Y D- h L- D D- Curves showing the changes in the hydrodynamic diameter of h@CM in water, PBS solution, and culture medium over time. Figure 9 is Mn@m in Example 9 of the present invention L- Y D- h L- D D- The heating curves of h@CM at different concentrations (808 nm, 5 min, 1.0 W / cm 2 ); Figure 10 is Mn@m in Example 10 of the present invention L- Y D- h L- D D- Stability curve of h@CM under cyclic illumination (808 nm, 5 min, 1.0 W / cm 2 ); Figure 11 Mn@m in Example 11 of the present invention L- Y D- h L- D D- Release kinetics of manganese ions from h@CM under different conditions; Figure 12 Mn@m in Example 12 of the present invention L- Y D- h L- D D- Cell viability of h@CM and B16-F10 cells with and without laser irradiation after 24 hours of incubation; Figure 13 is Mn@m in Example 13 of the present invention L- Y D- h L- D D- Schematic diagram of h@CM's resistance to nonspecific protein adsorption; Figure 14 Mn@ in Example 14 of the present invention L- Y D- h L- D D- h Schematic diagram of the simulated in vivo oxidation process; Figure 15Mn@m in Example 15 of the present invention L- Y D- h L- D D- Detection of extracellular ATP content in h@CM after laser irradiation and incubation of B16-F10 cells for different time periods; Figure 16 Mn@m in Example 16 of the present invention L- Y D- h L- D D- Flow cytometry analysis of h@CM maturation of DC cells; Figure 17 Mn@m in Example 16 of the present invention L- Y D- h L- D D- h@CM detects and analyses cytokines after DC cell maturation; Figure 18 In Example 17 of the present invention, PBS, manganese chloride, Mn@ L- Y D- h L- D D- h@CM,Mn@m L- Y D- h L- D D- Flow cytometric analysis of CD4+T cells and CD8+T cells in tumor tissues on day 14 after h@CM irradiation; Figure 19 This is a graph showing the fluorescence intensity analysis of CD4+ T cells in tumor tissue in Example 17 of the present invention; Figure 20 This is a graph analyzing the fluorescence intensity of CD8+ T cells in tumor tissue in Example 17 of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0021] Unless otherwise noted, all chemical reagents were commercially available and used without further purification. B16-F10 cells (a murine melanoma cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI-1640 medium (1640 medium, GIBCO, Invitrogen, Carlsbad, CA), fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (HyClone, Thermo Scientific, Logan, UT), and trypsin 0.25% solution (HyClone) were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou, China). Approximately 4–6 weeks old black mice were purchased from the Shanghai Slac Laboratory Animal Center (Shanghai, China). Water used in all experiments with a resistivity greater than 18.2 MΩ·cm was purified using a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0022] For ease of presentation, the Figure 1-20 middle, L- Y D- h L- D D- h is represented by YhDh, L- Y L- H L- D L- H represents YHDH; D- y D- h D- d D- h is represented as yhdh.

[0023] Example 1 (1) Dissolve the cyclic peptides of different chirality in ultrapure water, add manganese chloride solution dropwise, then stir the reaction, centrifuge and wash, and dry to obtain nanoassemblies of different chirality, Mn@ L- Y D- h L- D D- h,Mn@ L- Y L- H L- D L- H and Mn@ D- y D- h D- d D- h, to investigate the in vivo circulation stability, in vitro cell entry efficiency, in vitro immune activation efficiency, and screening of different chiral assemblies L- Y D- h L- D D-h chiral peptides were further studied (screening steps were as described in Examples 2-5), wherein the ratio of chiral cyclic peptide to manganese chloride solution was 10-12 mg: 0.1-0.15 mL.

[0024] (2) Mn@ L- Y D- h L- D D- h was dissolved in PBS, tyrosinase was added, the reaction was stirred in an open container, centrifuged and washed, and dried to obtain the oxidized nanoassembly (Mn@m L- Y D- h L- D D- h), wherein the amount of tyrosinase used is 0.1-0.15 mL.

[0025] (3) Replace Mn@m in step (2) L- Y D- h L- D D- h was dissolved in PBS, and the prepared B16 cell membrane was added. After ultrasonic dispersion, the membrane was repeatedly pushed and pulled 30 times using a membrane coating device, centrifuged, washed, and dried to obtain the cell membrane-coated oxidized nanoassembly (Mn@m L- Y D- h L- D D- h@CM), in which the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2-1:1.8.

[0026] Among them, chiral cyclic peptides L- Y D- h L- D D- h, L- Y L- H L- D L- H and D- y D- h D- d D- The amino acid sequence of h is the same: YHDH (tyrosine-histidine-lysine-histidine).

[0027] Example 2: The different chiral assemblies obtained in Example 1 were dissolved in ultrapure water to prepare solutions with a concentration of 1 mg / mL. To measure circular dichroism (CD) spectra, the peptide nanoparticles (Mn-YhDh, Mn-YHDH, Mn-yhdh) and their corresponding free peptides (YhDh, YHDH, yhdh) were prepared into 1 mg / mL aqueous solutions. 5% trifluoroacetic acid (TFA) was added to the free peptide samples to inhibit assembly. All samples were measured using a JASCO spectrometer at a constant temperature of 25°C ( Figure 2 The results indicate that the chirality of the free peptides and the assembled peptides varies, with a pair of opposite peaks at 208 nm exhibiting an effect related to the nanoparticle's α-helical structure. Mn-YhDh also exhibits a positive peak at 208 nm. In contrast, Mn-YHDH exhibits a mirror-image spectrum relative to Mn-yhkh, indicating a complete reversal of the molecular chirality.

[0028] Example 3 To measure the in vivo circulatory stability of different chiral assemblies, the assemblies obtained in Example 1 (Mn-YhDh, Mn-YHDH, and Mn-yhdh) were dissolved in PBS to prepare a 1 mg / mL solution. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital before the experiment. Following intravenous injection of Mn-YhDh, Mn-YhDh, or Mn-YhDh, serum was collected at different time points and digested with aqua regia. Mn was analyzed by ICP-OES. 2+ The results showed that ( Figure 3 ) The pure D-type and DL-type chiral assemblies have good stability, reaching 42 and 29 μg / mL respectively after 8 h.

[0029] Example 4 To measure the cellular entry efficiency of different chiral assemblies, the assemblies obtained in Example 1 (Mn-YhDh, Mn-YHDH, and Mn-yhdh) were dissolved in culture medium to prepare solutions with concentrations of 0.1 and 0.25 mg / mL. Melanoma cells were incubated with the assemblies for 24 h and then harvested. After trypsinization and counting, the supernatant was centrifuged, digested with aqua regia (1 mL), and diluted to 5 mL with deionized water. Intracellular Mn was determined by ICP-OES. 2+ The results showed that ( Figure 4 ) Pure L-type and DL-type have the highest cell entry efficiency, reaching 17 and 11 picograms per cell at the highest concentration.

[0030] Example 5 To measure the in vitro immune activation abilities of different chiral assemblies, the assemblies obtained in Example 1 were dissolved in culture medium to prepare solutions at a concentration of 0.1 mg / mL (1: PBS blank control, 2: Mn-YHDH, 3: Mn-yhdh, and 4: Mn-YhDh). Dendritic cells were co-cultured with the different chiral assemblies for 24 hours, and IL-6 cytokine concentrations in the cell culture fluid were measured by ELISA. The results showed that the DL-type peptide assembly had the most potent in vitro immune system activation ability, reaching 120 picograms per milliliter.

[0031] Example 6 The assembly obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. The solution was then dropped onto the surface of a copper mesh with a carbon film and dried at room temperature to obtain a sample. The sample was examined using a transmission electron microscope at 200 kV ( Figure 6 Transmission electron microscopy showed that the particle size of the prepared nanoassembly was 150 ± 20 nm.

[0032] Example 7 The assembly obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. The solution was dropped onto the surface of a silicon wafer and dried at room temperature to prepare a sample. The sample was examined using a scanning electron microscope at 10 kV ( Figure 7 Transmission electron microscopy showed that the particle size of the prepared nanoassembly was 150 ± 20 nm.

[0033] Example 8 The Mn@mYhDh@CM prepared in Example 1 was dissolved in water, PBS solution and cell culture medium respectively, and the hydration kinetic diameter was measured. Figure 8 As shown in the figure, the hydration kinetic diameter of Mn@mYhDh@CM can remain unchanged for a long time in the above solutions. The sizes in PBS solution in water and cell culture medium on the first day are approximately 120 ± 3 nm, 123 ± 2 nm and 131 ± 2 nm, respectively. On the fifth day, they are 123 ± 3 nm, 125 ± 3 nm and 140 ± 2 nm, respectively, demonstrating that Mn@mYhDh@CM has good colloidal stability.

[0034] Example 9 The Mn@mYhDh@CM aqueous solution was prepared in Example 1, and 200 μL was placed in an EP tube. Ultrapure water was used as a control group and the solution was purified by laser (808 nm, 1.0 W / cm 2 ) irradiate each of the above solutions for 120 s, and use a thermocouple to monitor and record the temperature of the solution. Figure 9As shown in the figure, the solution temperature rises more with time, reaching 42°. This indicates that the prepared Mn@mYhDh@CM has good photothermal conversion efficiency and can be used for photothermal therapy of tumors.

[0035] Example 10 The dried Mn@mYhDh@CM nanomaterials prepared in Example 1 were prepared into a 2 mg / mL aqueous solution and added to an EP tube. The solution was then illuminated by a laser (808 nm, 1.0 W / cm 2 ) irradiated an EP tube containing a solution with a concentration of 2 mg / mL and performed five heating-cooling cycles. In each cycle, the solution was first exposed to the laser and heated for 120 s, then the laser was turned off and cooled to room temperature. The temperature of the solution was monitored and recorded using a thermocouple. Figure 10 As shown, after four cycles of heating and cooling, the maximum temperature and the minimum temperature after cooling of the Mn@mYhDh@CM solution remained nearly constant. These results demonstrate the excellent photothermal stability of the Mn@mYhDh@CM nanoassembly, suggesting its potential for photothermal therapy of tumors.

[0036] Example 11 Prepare buffer solutions of pH = 7.4 and pH = 5.0 respectively, dissolve the Mn@mYhDh@CM prepared in Example 1 with 1 mL of the above different buffer solutions to a 1 mg / mL solution and place it in a dialysis bag, place the dialysis bag in a container containing 9 mL of the above different buffer solutions, and shake it in a constant temperature shaker at 37 ° C. At different time points, draw 1 mL from the liquid outside the dialysis bag, and then add 1 mL of the corresponding buffer solution to the container, and measure the manganese content by inductively coupled plasma atomic emission spectrometry. After the sustained release is completed, the drug release curve of Mn@mYhDh@CM under different conditions is plotted. Figure 11 As shown, Mn@mYhDh@CM releases manganese slowly in a pH 7.4 buffer solution, with a release rate of 19.48%. At pH 5.0, the release rate is 78.46%, significantly higher than the former. This is because, under acidic conditions, hydrogen ions dissociate the manganese chelate coordination between the manganese and the chiral cyclic peptide. This indicates that the release of manganese from Mn@mYhDh@CM is significantly pH-responsive.

[0037] Example 12 B16-F10 cells in the logarithmic growth phase were collected and 1×10 4The cells were seeded at a density of 100 cells / mL in a 96-well plate, placed in 5% CO2, and incubated at 37°C for 12 h. The original culture medium was discarded, and different concentrations of Mn@mYhDh@CM were added to each well plate for illumination experiments, and no illumination was set as a control. Co-cultured with cells at 5% CO2 and 37°C for 24 h. After that, the well plate was removed, the original culture medium was discarded, washed three times with PBS, and fresh culture medium containing 10% (v / v) CCD-8 was added, and the cells were incubated in the incubator for another 3 h. Finally, the absorbance of each well was tested at a wavelength of 450 nm using a multifunctional enzyme reader. The cells treated with PBS were used as blank controls, and the cell viability was recorded as 100%. The results are shown in the figure. Figure 12 As shown, the cell viability of the light-irradiated group decreased with the increase of material concentration, while there was almost no cell casualties in the control group, indicating that Mn@mYhDh@CM has good biocompatibility and phototoxicity.

[0038] Example 13 Serum albumin (BSA) was incubated with Mn@mYhDh@CM and Mn@mYhDh at 37°C for 2 hours. The mixture was then centrifuged at 10,000 rpm at 4°C for 20 minutes, and the supernatant was collected. The absorbance of the BSA solution before incubation and the supernatant after incubation was measured at 278 nm, and the difference in absorbance was calculated. The results showed that within the studied concentration range, the absorbance change of Mn@mYhDh@CM was significantly lower than that of Mn@mYhDh (Figure 13), indicating that coating with tumor cell membranes imparts excellent protein resistance to Mn@mYhDh@CM.

[0039] Example 14 The dried Mn@YhDh nanomaterials in Example 1 were prepared into 1 mg / mL, 100 μL of tyrosine oxidase was added, and the mixture was placed in a 37° oven. The color change was observed at regular intervals. Figure 14 As shown, the solution turned light gray after 1 h of oxidation and completely turned black after 8 h, indicating that Mn@YhDh was successfully oxidized and converted into a melanin-like structure, which can be used for subsequent photothermal therapy.

[0040] Example 15 B16-F10 cells in the logarithmic growth phase were collected and 1×10 4 The cells were seeded in a 96-well plate at a density of 100 cells / well and incubated at 37°C with 5% CO2 for 12 h. The original culture medium was discarded, and PBS and Mn@mYhDh@CM were added to the plate. After irradiation for 2 min, the cells were further incubated (808 nm, 1.0 W / cm 2) The non-illumination group served as the control. The culture medium from the top layer of cells in each group was aspirated. 100 μL of ATP assay working solution was added to the 96-well plate. After 3-5 minutes, 20 μL of culture medium sample was added to the wells. After mixing, the RLU value was measured using a multifunctional microplate reader to detect extracellular ATP. The results are shown in Figure 2. Figure 15 As shown in the figure, the amount of ATP released by the Mn@mYhDh@CM light-exposed group was significantly higher than that of the other three groups. The released ATP will help promote the phagocytosis of apoptotic tumor cells by dendritic cells and enhance the anti-tumor immune response.

[0041] Example 16 B16-F10 cells in the logarithmic growth phase were collected and 1×10 5 The cells were seeded at a density of 100 cells / mL in a Transwell plate and incubated at 5% CO2 and 37°C for 12 h. The original culture medium was discarded, and PBS and Mn@mYhDh@CM were added to the plate, respectively. After irradiation for 2 minutes, the cells were further incubated (808 nm, 1.0 W / cm 2 ) The non-illumination group was used as the control. DC cells in the logarithmic growth phase were collected and 1×10 5 The cells were cultured in the lower chamber of the Transwell at a density of 100 cells / mL. After 24 hours, DC cells were collected and the expression of CD80, CD86, and MCH-II was detected by flow cytometry. At the same time, 100 μL of DC cell culture supernatant was collected and IL6 and TNFα were detected by ELISA reaction. Figure 16 and 17 As shown in the data, after co-culture of tumor cells treated with Mn@mYhDh@CM light group and DCs, the levels of CD80, CD86 and MCH-II increased to 53%, 55% and 73%, respectively. The related maturation cytokines were also much higher than those in the other three groups, indicating that the immunogenic cell death caused by photothermal reaction can effectively mature DC cells and activate the body's anti-tumor immune response.

[0042] Example 17 Mice with tumors were divided into 4 groups and injected with PBS, manganese chloride solution, Mn@mYhDh@CM, and Mn@mYhDh@CM plus light treatment through the tail vein. The injection volume of all solutions was 100 μL, and the injection time was on the 1st, 4th, and 8th day. The laser group was irradiated with laser for 2 minutes on the 1st, 4th, and 8th day. After 14 days, the mice were killed and their tumor tissues were removed under sterile conditions, chopped and ground, and filtered through a 400-mesh filter to obtain a cell suspension. Lymphocytes were then separated using various animal tumor-infiltrating tissue lymphocyte separation kits. Finally, T lymphocyte suspensions were obtained using nylon wool columns. The T cells obtained above were labeled with CD4 / CD8 antibodies, and CD4+T cells and CD8+T cells in tumor tissues were quantitatively analyzed by flow cytometry. The results are shown in Figure 2. Figure 18 、 19 As shown in Figures 20, the highest expression of CD4 / CD8 was found in the Mn@mYhDh@CM plus light irradiation group. This is because the immunogenic death of tumor cells caused by photothermal therapy and the activation of the CGAS-STING pathway jointly stimulated the maturation of dendritic cells, enhanced the ability of dendritic cells to present antigens, thereby enhancing the activation of T cells and promoting the typing of cytotoxic T cells.

[0043] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A chiral cyclic peptide and manganese ion coordination nanoassembly, characterized in that: The nanoassembly is Mn@ L- Y D- h L- D D- h, through chiral cyclic peptides L- Y D- h L- D D- h is coordinated with manganese ions; wherein the chiral cyclic peptide L- Y D- h L- D D- h Based on the characteristics of different chiral cyclic peptides, the chirality of the cyclic peptides is regulated.

2. The chiral cyclic peptide and manganese ion coordination nanoassembly according to claim 1, characterized in that: The characteristics include endocytic stability, cytosolic efficiency and immune activation ability.

3. A method for preparing a coordination nanoassembly of a chiral cyclic peptide and a manganese ion, comprising preparing the coordination nanoassembly as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: S1: Dissolve different chiral cyclic peptides in ultrapure water, add manganese chloride solution dropwise, then stir to react, centrifuge, wash, and dry to obtain nanoassembly Mn@ L- Y D- h L- D D- h、Mn@ L- Y L- H L- D L- H and Mn@ D- y D- h D- d D- h, screen the chiral nanoassemblies with the best in vitro cell entry efficiency, the strongest immune activation ability, and the strongest in vivo circulation stability, and select Mn@ L- Y D- h L- D D- h polypeptide as the subsequent material, wherein the ratio of chiral cyclic peptide to manganese chloride solution is 10-12 mg: 0.1-0.15 mL; S2: Mn@ L- Y D- h L- D D- h was dissolved in PBS, tyrosinase was added, stirred for reaction, centrifuged for washing, and dried to obtain the oxidized nanoassembly Mn@m L- Y D- h L- D D- h, wherein the amount of tyrosinase used is 0.1-0.15 mL; S3: Change Mn@m in S2 L- Y D- h L- D D- h was dissolved in PBS, and the prepared B16 cell membrane was added. After ultrasonic dispersion, the membrane was repeatedly pushed and pulled 30 times using a membrane coating device, centrifuged, washed, and dried to obtain the cell membrane-coated oxidized nanoassembly Mn@m L- Y D- h L- D D- h@CM, in which the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2-1:1.

8.

4. The method for preparing the coordination nanoassembly of a chiral cyclic peptide and a manganese ion according to claim 3, characterized in that: The concentration of manganese chloride in S1 was 1 mg / mL, and the reaction time was 8-10 h.

5. The method for preparing the coordination nanoassembly of a chiral cyclic peptide and a manganese ion according to claim 3, characterized in that: The tyrosinase concentration in S2 was 1 mg / mL.

6. The method for preparing the coordination nanoassembly of a chiral cyclic peptide and a manganese ion according to claim 3, characterized in that: The S2 temperature is 37° and the stirring time is 22-24 h.

7. The method for preparing the coordination nanoassembly of a chiral cyclic peptide and a manganese ion according to claim 3, characterized in that: The solvent in S3 is PBS buffer, and the cell membrane concentration is 1 mg / mL.

8. An application of a coordination nanoassembly of a chiral cyclic peptide and a manganese ion, using the coordination nanoassembly as described in claim 1, characterized in that: Used to prepare tumor treatment drugs.

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