Chiral cyclic peptide coordination nanosassembly with manganese ions, preparation method and application thereof

Abstract

The application provides a coordination nanometer assembly of a chiral cyclic peptide and a manganese ion, a preparation method and application thereof, the method of the application combines a manganese superoxide dismutase protein domain with a unique tumor microenvironment of a melanoma high tyrosinase, and first designs polypeptide sequences with different chirality L‑ Y D‑ h L‑ D D‑ h、 L‑ Y L‑ H L‑ D L‑ H and D‑ y D‑ h D‑ d D‑ h, regulates polypeptide chirality, improves in-vivo circulation stability and cell entry efficiency of the nanometer assembly, coordinates self-assembly of manganese and the chiral cyclic peptide, simulates in-situ oxidation of the tumor microenvironment for photothermal therapy, and coats cell membranes. The method of the application is simple, the experimental conditions are mild, and the method is easy to operate, the cell membrane coated nanometer assembly prepared by the method not only has long circulation stability and high cell entry efficiency, but also can realize mild photothermal therapy through in-situ oxidation, release manganese ions to activate the CGAS-STING pathway for tumor immunotherapy, and has potential application value in the field of tumor combination therapy.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and more particularly to a coordination nanoassembly of a chiral cyclic peptide and manganese ions. Background Technology

[0002] Malignant tumors, characterized by rapid growth, strong metastatic ability, and high recurrence rate, have become a major killer threatening human health. Traditional clinical cancer treatments such as chemotherapy and radiotherapy often damage normal tissues, have low treatment efficiency, and easily lead to tolerance. Photothermal therapy (PTT) is favored due to its highly effective local tumor treatment, low invasiveness, and minimal side effects. Peptide materials are macromolecules composed of multiple amino acids linked by peptide bonds, possessing excellent biocompatibility, including tissue compatibility and blood compatibility; they also exhibit certain mechanical strength, appropriate physicochemical properties, ease of molding and processing, and stable drug release rates. Peptide self-assembled nanomaterials, due to their advantages such as sequence editing, regulated self-assembly, low environmental toxicity, and high biocompatibility, are widely used in chemical sensing, biomedicine, and energy catalysis. To achieve broader applications, coordination between peptides and inorganic metal materials is a research direction. Through sequence design, peptide nanomaterials can coordinate and assemble with metal ions. Combining the self-assembly 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, enhance their inherent physicochemical properties, and thus promote the deeper application of peptide nanohybrid materials.

[0003] Chiral cyclic peptides, as a unique class of bioactive molecules, exhibit significant advantages in the fields of biomedicine and materials science. Their rigid conformation, formed by covalent bond closure of their cyclic structure, effectively reduces molecular degrees of freedom, enhances structural stability, significantly resists protease degradation, and prolongs in vivo circulation time. The presence of the chiral center endows them with precise stereoselectivity, enabling them to achieve high-affinity three-dimensional spatial matching with specific biological targets (such as enzyme active sites, cell membrane receptors, or ion channels), thereby improving targeting and specificity. For example, in antitumor 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 assembly with metal ions and constructing synergistic nanocomposite systems. Their chiral characteristics can also regulate the self-assembly behavior of nanoparticles, forming well-ordered nanostructures and enhancing photothermal conversion efficiency or drug loading capacity. Compared to linear peptides, the conformational constraints of chiral cyclic peptides give them an advantage in mimicking natural protein interactions, improving transmembrane transport efficiency, and regulating immunogenicity, providing an ideal molecular framework for developing novel therapeutic platforms.

[0004] Pure L-amino acid nanoparticles face multiple limitations in biological applications, with the core issue stemming from their high compatibility with natural enzymatic mechanisms. Because proteases in vivo (such as trypsin and lysozyme) specifically recognize L-amino acids through a "lock-and-key" mechanism, these nanoparticles are easily and rapidly degraded 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 synergistic intermolecular forces, leading to low rigidity in the formed α-helical or β-sheet structures, making them prone to conformational relaxation or even disintegration under pH fluctuations or changes in ionic strength. Their high similarity to endogenous peptide chains can also trigger misrecognition by the immune system, accelerating clearance by macrophages or complement adsorption. Simultaneously, they struggle to penetrate complex barriers such as biological membranes or tumor matrix, limiting their application in oral administration or targeted therapy.

[0005] In recent years, tumor immunotherapy has developed rapidly, primarily exerting its effects by enhancing certain active components in the immune system or relieving immunosuppression. It has become another major pillar in cancer treatment following surgery, radiotherapy, and chemotherapy. Among these advancements, significant clinical progress has been made in regulating the anti-tumor immune system by activating the CGAS-STING pathway, establishing it as a novel anti-cancer weapon. Summary of the Invention

[0006] The purpose of this invention is to propose a chiral cyclic peptide-manganese ion coordination nanoassembly with high in vivo long-term circulation stability and high cell entry efficiency, as well as its preparation method and application.

[0007] To achieve the above objectives, this invention proposes a chiral cyclic peptide-manganese ion coordination nanoassembly, characterized in that the nanoassembly is Mn@ L- Y D- h L- D D- h, via chiral cyclic peptide L- Y D- h L- D D- h is obtained by coordination with manganese ions; among which, chiral cyclic peptides are obtained. L- Y D- h L- D D- h selects the optimal chiral peptides based on their characteristics, thereby regulating the chirality of cyclic peptides.

[0008] Further characteristics include internal circulation stability, cell entry efficiency, and immune activation capacity.

[0009] This invention also proposes a method for preparing a coordination nanoassembly of a chiral cyclic peptide and manganese ions, comprising the following steps:

[0010] S1: Different chiral cyclic peptides were dissolved in ultrapure water, and manganese chloride solution was added dropwise. The mixture was then stirred, centrifuged, washed, and dried to obtain the nano-assemblies 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 screen for chiral nanoassemblies with the best in vitro cell entry efficiency, strongest immune activation ability, and strongest in vivo circulation stability, Mn@ L- Y D- h L- D D- h-peptide was used as a subsequent material, in which the ratio of chiral cyclic peptide to manganese chloride solution was 10-12 mg : 0.1-0.15 mL;

[0011] S2: Mn@ L- Y D- h L- D D- h was dissolved in PBS, tyrosinase was added, the mixture was stirred, centrifuged, washed, and dried to obtain oxidized nanoassemblies Mn@m L- Y D- h L- D D- h, of which the amount of tyrosinase used is 0.1-0.15 mL;

[0012] S3: Transfer Mn@m from 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. After centrifugation, washing, and drying, cell membrane-coated oxidized nanoassemblies Mn@m were obtained. L- Y D- h L- D D- h@CM, where the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2 to 1:1.8.

[0013] Furthermore, the concentration of manganese chloride in S1 was 1 mg / mL, and the reaction time was 8-10 h.

[0014] Furthermore, the concentration of tyrosinase in S2 is 1 mg / mL.

[0015] Furthermore, the S2 temperature was 37°C, and the stirring time was 22-24 h.

[0016] Furthermore, the solvent in S3 is PBS buffer, and the cell membrane concentration is 1 mg / mL.

[0017] This invention also proposes the application of a chiral cyclic peptide-manganese ion coordination nanoassembly for the preparation of tumor therapeutic drugs.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] The nanoassemblies of this invention utilize a breakthrough strategy of introducing D-type amino acids through chiral engineering: mixing D / L-type amino acids can interfere with protease recognition and extend the half-life by more than 10 times; the non-natural hydrogen bond network enhances hydrophobic stacking, forming a dense and stable nanotopological structure; providing a new direction for nano-drug delivery systems.

[0020] 2. The present invention provides a novel nano-assembly of tumor cell membrane-coated chiral cyclic peptides and ion-coordinated nanoassemblies. Through chiral regulation, it effectively solves the problems of poor in vivo long-term circulation stability and low cell entry efficiency of nanoassemblies. At the same time, through in-situ oxidation in the tumor microenvironment, it can be used for photothermal therapy and immunotherapy, and has potential application value in the field of tumor treatment.

[0021] (2) The nano-assemblies prepared by the present invention have good water solubility and biocompatibility.

[0022] (3) The nano-assembly preparation method of the present invention has mild experimental conditions and is easy to operate. Attached Figure Description

[0023] Figure 1 Mn@m in this invention L- Y D- h L- D D- Schematic diagram of the synthesis process of the h@CM complex;

[0024] Figure 2 Mn@ with different chiralities in Embodiment 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- Circular dichroism chromatograms of h and its free polypeptide;

[0025] Figure 3 Mn@ with different chiralities in Embodiment 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- Circulatory stability of h in animals;

[0026] Figure 4 Mn@ with different chiralities in Embodiment 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 entry efficiency diagram;

[0027] Figure 5 Mn@ with different chiralities in Embodiment 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- Graph showing the in vitro activation of the immune system by h;

[0028] Figure 6 Mn@ in Embodiment 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 microscope image of the h@CM preparation process;

[0029] Figure 7 Mn@ in Embodiment 7 of the present inventionL- 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 microscope images of h@CM;

[0030] Figure 8 Mn@m in Embodiment 8 of the present invention L- Y D- h L- D D- The curves showing the hydrodynamic diameter of h@CM over time in water, PBS solution, and culture medium;

[0031] Figure 9 Mn@m in Embodiment 9 of the present invention L- Y D- h L- D D- Temperature rise curves of h@CM at different concentrations under illumination (808 nm, 5 min, 1.0 W / cm²) 2 );

[0032] Figure 10 Mn@m in Embodiment 10 of the present invention L- Y D- h L- D D- Stability curves of h@CM under cyclic illumination (808 nm, 5 min, 1.0 W / cm²) 2 );

[0033] Figure 11 Mn@m in Embodiment 11 of the present invention L- Y D- h L- D D- Release kinetics curves of manganese ions under different conditions for h@CM;

[0034] Figure 12 Mn@m in Embodiment 12 of the present invention L- Y D- h L- D D- Cell viability graphs of h@CM and B16-F10 cells after 24 hours of incubation with and without laser irradiation;

[0035] Figure 13 Mn@m in Embodiment 13 of the present invention L- YD- h L- D D- Schematic diagram of h@CM's resistance to nonspecific protein adsorption;

[0036] Figure 14 Mn@ in Embodiment 14 of the present invention L- Y D- h L- D D- h Schematic diagram simulating the in vivo oxidation process;

[0037] Figure 15 Mn@m in Embodiment 15 of the present invention L- Y D- h L- D D- Figure 1. Detection of extracellular ATP content in h@CM cells after laser irradiation and B16-F10 cells after incubation for different times;

[0038] Figure 16 Mn@m in Embodiment 16 of the present invention L- Y D- h L- D D- h@CM flow cytometry analysis of DC cell maturation;

[0039] Figure 17 Mn@m in Embodiment 16 of the present invention L- Y D- h L- D D- h@CM analysis of cytokines in matured DC cells;

[0040] Figure 18 In Example 17 of this invention, PBS and manganese chloride were injected via the tail vein. L- Y D- h L- D D- h@CM,Mn@m L- Y D- h L- D D- Flow cytometry analysis of CD4+ T cells and CD8+ T cells in tumor tissue on day 14 after h@CM light exposure;

[0041] Figure 19 This is a fluorescence intensity analysis diagram of CD4+ T cells in tumor tissue in Example 17 of the present invention;

[0042] Figure 20 This is a fluorescence intensity analysis diagram of CD8+ T cells in tumor tissue in Example 17 of the present invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0044] Unless otherwise specified, all chemical reagents were commercially available and ready for use without further purification. B16-F10 cells (mouse 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 0.25% trypsin 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). All water with a resistivity higher than 18.2 MΩ·cm used in the experiments was purified using a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0045] For ease of explanation, see appendix Figure 1-20 middle, L- Y D- h L- D D- h is represented as YhDh. L- Y L- H L- D L- H is represented as YHDH; D- y D- h D- d D- h is represented as yhdh.

[0046] Example 1

[0047] (1) Cyclic peptides of different chirities were dissolved in ultrapure water, and manganese chloride solution was added dropwise. The mixture was then stirred, centrifuged, washed, and dried to obtain nanoassemblies of different chirities, 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, and in vitro immune activation efficiency of different chiral assemblies, and to screen them. L- Y D- hL- D D- Further studies on chiral peptides (screening steps as described in Examples 2-5) were conducted, in which the ratio of chiral cyclic peptide to manganese chloride solution was 10-12 mg : 0.1-0.15 mL.

[0048] (2) Mn@ L- Y D- h L- D D- h was dissolved in PBS, tyrosinase was added, the reaction was carried out with open stirring, centrifuged, washed, and dried to obtain oxidized nanoassemblies (Mn@m L- Y D- h L- D D- (h), of which the amount of tyrosinase used is 0.1-0.15 mL.

[0049] (3) Take Mn@m from 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. After centrifugation, washing, and drying, cell membrane-coated oxidized nanoassemblies (Mn@m) were obtained. L- Y D- h L- D D- h@CM), where the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2 to 1:1.8.

[0050] 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 sequences of h are the same, both being: YHDH (tyrosine-histidine-lysine-histidine).

[0051] Example 2:

[0052] Different chiral assemblies obtained in Example 1 were dissolved in ultrapure water to prepare solutions with a concentration of 1 mg / mL. For circular dichroism (CD) spectroscopy measurements, peptide nanoparticles (Mn-YhDh, Mn-YHDH, Mn-yhdh) and their corresponding free peptides (YhDh, YHDH, yhdh) were prepared as 1 mg / mL aqueous solutions. The free peptide samples were treated with 5% trifluoroacetic acid (TFA) to inhibit assembly. All samples were measured using a JASCO spectrometer under isothermal conditions at 25°C. Figure 2 The results showed that different free peptides and assemblies possessed different chiralities, exhibiting a pair of opposite peaks at 208 nm, which was related to the α-helical structure of the nanoparticles. Mn-YhDh also showed a positive peak at 208 nm. In contrast, Mn-YHDH exhibited a mirror spectrum relative to Mn-yhkh, indicating a complete reversal of molecular chirality.

[0053] Example 3

[0054] To measure the in vivo circulation stability of different chiral assemblies, the assemblies obtained in Example 1 (Mn-YhDh, Mn-YHDH, Mn-yhdh) were dissolved in PBS to prepare solutions with a concentration of 1 mg / mL. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital before the experiment. After intravenous injection of Mn-YhDh, Mn-YhDh, or Mn-YhDh, serum was collected at different time points and analyzed by ICP-OES after digestion with aqua regia. 2+ The content of . The results showed that ( Figure 3 The pure D-type and DL-type chiral assemblies exhibit good stability, with energy reaching 42 and 29 micrograms per milliliter, respectively, after 8 hours.

[0055] Example 4

[0056] To measure the cell entry efficiency of different chiral assemblies, the assemblies obtained in Example 1 (Mn-YhDh, Mn-YHDH, Mn-yhdh) were dissolved in culture medium to prepare solutions with concentrations of 0.1 and 0.25 mg / mL. Melanoma cells were cultured with the assemblies for 24 h, and then the cells were collected. After trypsinization and counting, the supernatant was collected by centrifugation, digested with aqua regia (1 mL), diluted with deionized water to 5 mL, and intracellular Mn was measured by ICP-OES. 2+ Content. 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 concentrations, respectively.

[0057] Example 5

[0058] To measure the in vitro immune activation capacity of different chiral assemblies, the assemblies obtained in Example 1 were dissolved in culture medium to prepare solutions with a concentration of 0.1 mg / mL (1 is PBS blank control, 2 is Mn-YHDH, 3 is Mn-yhdh, and 4 is Mn-YhDh). Dendritic cells were co-cultured with different chiral assemblies for 24 h, and the concentration of IL-6 cytokine in the cell culture medium was detected by ELISA. The results showed that the DL-type polypeptide assemblies had the most efficient in vitro immune system activation capacity, reaching 120 pg / mL.

[0059] Example 6

[0060] The assembly obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. This solution was then dropped onto the surface of a copper mesh with a carbon film and dried at room temperature to obtain the 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 nanoassemblies was 150 ± 20 nm.

[0061] Example 7

[0062] The assembly obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. This solution was then dropped onto the surface of a silicon wafer and dried at room temperature to obtain the 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 nanoassemblies was 150 ± 20 nm.

[0063] Example 8

[0064] 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, the hydration kinetic diameter of Mn@mYhDh@CM remained unchanged for a long time in the above solutions. On the first day, the size in water, PBS solution, and cell culture medium was approximately 120 ± 3 nm, 123 ± 2 nm, and 131 ± 2 nm, respectively. On the fifth day, the size was 123 ± 3 nm, 125 ± 3 nm, and 140 ± 2 nm, respectively, demonstrating that Mn@mYhDh@CM has good colloidal stability.

[0065] Example 9

[0066] In Example 1, an aqueous solution of Mn@mYhDh@CM was prepared. 200 μL of this solution was placed in an EP tube, with ultrapure water used as a control group. The sample was analyzed using a laser (808 nm, 1.0 W / cm²). 2Irradiate each of the above solutions for 120 seconds, and monitor and record the temperature of the solutions using thermocouples. Figure 9 As shown, the solution temperature increases significantly with time, reaching up to 42°C. This indicates that the prepared Mn@mYhDh@CM has good photothermal conversion efficiency and can be used for photothermal therapy of tumors.

[0067] Example 10

[0068] The dried Mn@mYhDh@CM nanomaterials from Example 1 were prepared into a 2 mg / mL aqueous solution and added to an EP tube. The solution was then analyzed using a laser (808 nm, 1.0 W / cm²). 2 The solution was irradiated with an EP tube containing a 2 mg / mL solution, and five cycles of heating and cooling were performed. In each cycle, the solution was first exposed to the laser for 120 seconds to raise the temperature, then the laser was turned off and the solution was cooled to room temperature. The temperature of the solution was monitored and recorded using thermocouples. Figure 10 As shown, after four cycles of heating and cooling, the highest temperature and the lowest temperature after cooling of the Mn@mYhDh@CM solution remained almost unchanged. The results indicate that the Mn@mYhDh@CM nanoassemblies exhibit excellent photothermal stability and can be applied to photothermal therapy of tumors.

[0069] Example 11

[0070] Buffer solutions with pH = 7.4 and pH = 5.0 were prepared separately. Mn@mYhDh@CM prepared in Example 1 was dissolved in 1 mL of each of the above buffer solutions to obtain a 1 mg / mL solution, which was then placed in a dialysis bag. The dialysis bag was placed in a container containing 9 mL of each of the above buffer solutions and shaken in a 37 °C constant-temperature shaker. At different time points, 1 mL of the solution was drawn from the outside of the dialysis bag, and 1 mL of the corresponding buffer solution was added to the container. The manganese content was measured by inductively coupled plasma atomic emission spectrometry. After the sustained-release phase, drug release curves of Mn@mYhDh@CM under different conditions were plotted. Figure 11 As shown, the release of manganese from Mn@mYhDh@CM is slow in a buffer solution at pH 7.4, with a release rate of 19.48%, while the release rate is 78.46% at pH 5.0, the latter being significantly higher than the former. This is because, under acidic conditions, hydrogen ions cause the manganese to dissociate from its chelate with the chiral cyclic peptide. This indicates that the release of manganese from Mn@mYhDh@CM exhibits a significant pH responsiveness.

[0071] Example 12

[0072] Collect B16-F10 cells in the logarithmic growth phase, at a ratio of 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37 °C for 12 h in 5% CO2. The original culture medium was discarded, and each well was treated with different concentrations of Mn@mYhDh@CM for illumination experiments. No light was used as a control. The cells were co-cultured with the plates at 37 °C in 5% CO2 for 24 h. Afterward, the plates were removed, the original culture medium was discarded, and the plates were washed three times with PBS. Fresh culture medium containing 10% (v / v) CCD-8 was added, and the plates were incubated for another 3 h. Finally, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader. Cells treated with PBS served as a blank control, and cell viability was recorded as 100%. Results are as follows: Figure 12 As shown, cell viability decreased in the light-illuminated group with increasing material concentration, while the control group showed almost no cell loss, indicating that Mn@mYhDh@CM has good biocompatibility and phototoxicity.

[0073] Example 13

[0074] Serum albumin (BSA) was incubated with Mn@mYhDh@CM and Mn@mYhDh at 37 °C for 2 h. The supernatant was then collected by centrifugation at 10,000 rpm, 4 °C for 20 min. 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 the coating of tumor cell membranes endows Mn@mYhDh@CM with good anti-protein adsorption ability.

[0075] Example 14

[0076] The dried Mn@YhDh nanomaterials from Example 1 were prepared to a concentration of 1 mg / mL, and 100 μL of tyrosine oxidase was added. The mixture was then placed in a 37°C oven, and color changes were observed at regular intervals. Figure 14 As shown, the solution turned light gray after 1 hour of oxidation and turned completely black after 8 hours, indicating that Mn@YhDh was successfully oxidized and transformed into a melanin-like structure, which can be used for subsequent photothermal therapy.

[0077] Example 15

[0078] Collect B16-F10 cells in the logarithmic growth phase, at a ratio of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates 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 wells. After 2 minutes of illumination, the cells were incubated again at 808 nm, 1.0 W / cm². 2The non-illuminated group served as a control. The upper layer of cell culture medium from each group was aspirated. 100 μL of ATP detection working solution was added to each well of a 96-well plate. After incubation for 3-5 minutes, 20 μL of culture medium sample was added to each well, mixed well, and the RLU value was measured using a multi-functional microplate reader to detect extracellular ATP. Results are as follows: Figure 15 As shown, the ATP release in the Mn@mYhDh@CM light-exposed group was significantly higher than that in the other three groups. The released ATP will promote the phagocytosis of apoptotic tumor cells by dendritic cells and enhance the anti-tumor immune response.

[0079] Example 16

[0080] Collect B16-F10 cells in the logarithmic growth phase, at a ratio of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in Transwell plates 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 wells. After 2 minutes of illumination, the cells were incubated again at 808 nm (1.0 W / cm²). 2 The non-light-controlled group served as the control. Dendritic cells (DCs) in the logarithmic growth phase were collected and divided into groups of 1 × 10⁻⁶ cells per well. 5 The cells were seeded at a density of [number] and cultured in the lower chamber of a Transwell. After 24 hours, the DCs were collected and the expression of CD80, CD86, and MCH-II was detected by flow cytometry. Simultaneously, 100 μL of the DC culture supernatant was collected and IL-6 and TNF-α were detected by ELISA. Figure 16 and 17 As shown, after co-culturing tumor cells treated with Mn@mYhDh@CM light with DCs, 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 induced by photothermal reaction can effectively mature DC cells and activate the body's anti-tumor immune response.

[0081] Example 17

[0082] Mice with tumors were divided into four groups. The groups were injected via tail vein with PBS, manganese chloride solution, Mn@mYhDh@CM, and Mn@mYhDh@CM plus light treatment, respectively. All solutions were injected in 100 μL at 1 day, 4 days, and 8 days. The group receiving laser treatment was irradiated for 2 minutes at 1 day, 4 days, and 8 days. After 14 days, the mice were sacrificed, and their tumor tissue was aseptically removed, minced, ground, and filtered through a 400-mesh filter to obtain a cell suspension. Lymphocytes were then isolated using various animal tumor-infiltrating tissue lymphocyte isolation kits. Finally, T lymphocyte suspensions were obtained using nylon hair column chromatography. The obtained T cells were labeled with CD4 / CD8 antibodies, and CD4+ T cells and CD8+ T cells in the tumor tissue were quantitatively analyzed by flow cytometry. Results are as follows: Figure 18 , 19 As shown in Figure 20, the highest expression level of CD4 / CD8 in the Mn@mYhDh@CM plus light irradiation group is due to the combined stimulation of dendritic cell maturation by the immunogenic death of tumor cells induced by photothermal therapy and the activation of the CGAS-STING pathway, which enhances the ability of dendritic cells to present antigens, thereby enhancing the activation of T cells and promoting the typing of cytotoxic T cells.

[0083] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A chiral cyclic peptide-manganese ion coordination nanoassembly, characterized in that, The nanoassemblies are Mn@ L- Y D- h L- D D- h, via chiral cyclic peptide L- Y D- h L- D D- The chiral cyclic peptide is obtained by coordination of h with manganese ions; wherein, the chiral cyclic peptide is... L- Y D- h L- D D- h selects the optimal chiral peptides based on their characteristics, thereby regulating the chirality of cyclic peptides.

2. The chiral cyclic peptide-manganese ion coordination nanoassembly according to claim 1, characterized in that, The characteristics include internal circulation stability, cell entry efficiency, and immune activation ability.

3. A method for preparing a coordination nanoassembly of a chiral cyclic peptide and manganese ions, wherein the coordination nanoassembly as described in any one of claims 1-2 is prepared, characterized in that, Includes the following steps: S1: Different chiral cyclic peptides were dissolved in ultrapure water, and manganese chloride solution was added dropwise. The mixture was then stirred, centrifuged, washed, and dried to obtain the nano-assemblies 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 screen for chiral nanoassemblies with the best in vitro cell entry efficiency, strongest immune activation ability, and strongest in vivo circulation stability, Mn@ L- Y D- h L- D D- h-peptide was used as a subsequent material, in which the ratio of chiral cyclic peptide to manganese chloride solution was 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, the mixture was stirred, centrifuged, washed, and dried to obtain oxidized nanoassemblies Mn@m L- Y D- h L- D D- h, of which the amount of tyrosinase used is 0.1-0.15 mL; S3: Transfer Mn@m from 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. After centrifugation, washing, and drying, cell membrane-coated oxidized nanoassemblies Mn@m were obtained. L- Y D- h L- D D- h@CM, where the cell membrane and Mn@m L- Y D- h L- D D- The mass ratio of h is 1:2 to 1:1.

8.

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

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

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

7. The method for preparing the coordination nanoassemblies of chiral cyclic peptides and manganese ions 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 chiral cyclic peptide-manganese ion coordination nanoassembly, using the coordination nanoassembly as described in claim 1, characterized in that, Used to prepare drugs for treating melanoma.

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