A d-pi-a sensitized i / ii type photodynamic carbon dot based on m1 type macrophage membrane and a preparation method and application thereof

Abstract

This invention relates to a type I / II photodynamic carbon dot sensitized by D-π-A and encapsulated in an M1 macrophage membrane, its preparation method, and its application. The type I / II photodynamic carbon dot sensitized by D-π-A and encapsulated in an M1 macrophage membrane consists of an M1 macrophage membrane and carbon dots with a D-π-A configuration. The M1 macrophage membrane further encapsulates carbon dots with a D-π-A configuration. Compared with existing technologies, this invention is the first to design near-infrared excited D-π-A sensitizing carbon dots, which can be used as type I / II photosensitizers for hypoxia-modulated photodynamic immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a type I / II photodynamic carbon dot sensitized by D-π-A and nearly based on M1 macrophage membrane, its preparation method and application. Background Technology

[0002] Photodynamic therapy (PDT) and photothermal therapy (PTT) utilize specific wavelengths of light to activate photosensitizers or photothermal agents at the tumor site, precisely killing tumor cells through the generation of cytotoxic reactive oxygen species (ROS) or local thermal effects. Both can trigger an immune response for photoimmunotherapy, possessing the potential to establish long-term immune memory. However, PTT requires a high temperature of 50-60°C to effectively induce immunogenic cell death (ICD), easily damaging surrounding normal tissues; while PDT, although triggering ICD at lower light levels, has limited efficacy due to its oxygen-dependent type II mechanism in the hypoxic tumor microenvironment. Currently, the PDT / PTT combination strategy, which combines PDT with milder PTT to synergistically improve tumor hypoxia and enhance efficacy, has attracted attention. However, existing combination systems still have key drawbacks: on the one hand, the optimal excitation wavelengths of PDT and PTT agents are often inconsistent, requiring the use of different lasers and increasing treatment complexity; on the other hand, even with single near-infrared laser excitation, high-power irradiation or highly efficient photothermal agents are often required, potentially causing vascular damage, disrupting tumor blood supply and oxygen sources, thus exacerbating hypoxia and limiting the effect of PDT.

[0003] To overcome the limitations of phototherapy time (PDT) caused by tumor microenvironment hypoxia, a novel type I / II dual-mode photosensitizer has been developed. It can switch between an oxygen-dependent type II mechanism and an oxygen-independent type I mechanism based on local oxygen concentration, making it suitable for treating both normoxic and hypoxic tumors. Currently, most such photosensitizers are based on inorganic semiconductor nanomaterials, which, while exhibiting good photostability, generally suffer from short absorption / excitation wavelengths, short excited-state lifetimes (nanosecond-level), and potential metal toxicity and poor biocompatibility. Therefore, developing a single-component type I / II photosensitizer that combines near-infrared response, tunable photothermal properties, high photostability, and biocompatibility, while being metal-free, to meet the demands of highly efficient photoimmunotherapy remains a significant challenge.

[0004] Carbon dots (CDs), as metal-free zero-dimensional semiconductors, possess electronic, optical, and catalytic properties that can be flexibly tunable through molecular engineering, allowing heteroatom-containing functional groups to be incorporated into their structure and surface. Due to their high photostability, excellent biocompatibility, good water solubility, and tunable luminescence properties, carbon dots have been widely used as stable, renally excretable therapeutic agents for bioimaging and image-guided sonodynamic, photothermal, and type I / II photodynamic therapy. However, all reported carbon dots as type I / II photosensitizers suffer from two major limitations: first, the excited-state lifetime is extremely short (approximately nanoseconds), resulting in low triplet yields and limiting photodynamic therapy efficacy; second, most carbon dots have absorption bands located in the ultraviolet-visible region, and although a few near-infrared response systems have been reported, the required light intensity (0.8-1.0 W / cm²) is limited. 2 This far exceeds the maximum permissible radiation dose to skin and tissue at a wavelength of 808 nm (0.33 W / cm²). 2 This restricts its safe application. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one of the following problems of existing nano-photosensitizers: low ROS generation efficiency due to the limitations of the tumor hypoxic microenvironment, low triplet state yield due to the extremely short excited state lifetime, and the required light intensity far exceeding the maximum allowable irradiation dose. Therefore, this invention provides a D-π-A sensitized type I / II photodynamic carbon dot based on M1 macrophage membrane encapsulation, its preparation method and application, aiming to enhance the photodynamic tumor treatment effect by mildly alleviating tumor hypoxia through photothermal stimulation.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a type I / II photodynamic carbon dot based on D-π-A sensitization wrapped in an M1 type macrophage membrane, which is composed of an M1 type macrophage membrane and carbon dots with a D-π-A configuration, wherein the M1 type macrophage membrane is further wrapped with carbon dots having a D-π-A configuration.

[0007] The second technical solution of the present invention provides a method for preparing D-π-A sensitized type I / II photodynamic carbon dots based on M1 macrophage membranes as described in one of the above technical solutions, comprising the following steps: S1. Indocyanine green (IGC) and p-phenylenediamine are mixed and reacted under high temperature and high pressure. After filtration and dialysis, carbon dots P-CD with D-π-A configuration are obtained. S2. Macrophages and lipopolysaccharide (LPS) were co-cultured to obtain M1 macrophages. The M1 macrophages were then lysed to obtain the M1 macrophage membrane (MM). S3. Mix and extrude the P-CD obtained in step S1 and the MM obtained in step S2 to obtain type I / II photodynamic carbon dots sensitized by D-π-A based on M1 type macrophage membrane, which is P-CD@MM.

[0008] In some specific embodiments, in step S1, the mass ratio of indocyanine green to p-phenylenediamine is 1:2.

[0009] In some specific embodiments, in step S1, the temperature of the high-temperature and high-pressure reaction is (130~160)℃, the pressure is greater than or equal to 10 bar, and the time is (3~6) h.

[0010] The filtration process is as follows: the product obtained from the reaction under high temperature and high pressure is squeezed through a filter membrane of (0.1~10) μm using a syringe; The dialysis process is as follows: using a dialysis bag of (3000D~3500D), the filtered product is dialyzed for (30~40) hours.

[0011] As a more preferred embodiment, in step S1, the temperature of the high-temperature and high-pressure reaction is 150°C, the pressure is 10 bar, and the time is 4 hours. The filtration process is as follows: the product obtained from the reaction under high temperature and high pressure is squeezed through a 0.22 μm filter membrane using a syringe; The dialysis process is as follows: using a 3500D dialysis bag, the filtered product is dialyzed for 36 hours.

[0012] In some specific embodiments, in step S2, the ratio of macrophages to lipopolysaccharide is 2 × 10⁻⁶. 5 Cells: (0.1~0.3) μg.

[0013] As a more preferred embodiment, in step S2, the ratio of macrophages to lipopolysaccharide is 2 × 10⁻⁶. 5 0.1 μg per cell.

[0014] In some specific implementations, in step S2, the co-cultivation conditions are: 37°C, 5% CO2, humidity greater than 95%, and time is (20~26) h; The lysis process is as follows: M1 macrophages are suspended in lysis buffer, sonicated on ice at 80W for 15-25 min, centrifuged at 4°C for 25-35 min, and the supernatant is collected to obtain the M1 macrophage membrane MM.

[0015] As a more preferred method, in step S2, the co-cultivation conditions are: 37°C, 5% CO2, humidity greater than 95%, and time of 24 hours. The lysis process was as follows: M1 macrophages were suspended in lysis buffer, sonicated on ice at 80W for 20 min, centrifuged at 4℃ for 30 min, and the supernatant was collected to obtain the M1 macrophage membrane (MM).

[0016] In some specific implementations, in step S3, the mass ratio of P-CD to MM is 1:2.

[0017] In some specific embodiments, in step S3, 1 mL of a 0.5 mg / mL P-CD aqueous solution and 1 mL of a 1 mg / mL MM are mixed and extruded.

[0018] In some specific embodiments, in step S3, the extrusion process is as follows: P-CD and MM are mixed and extruded using an extruder through an aqueous phase filter of (100~200) nm.

[0019] As a preferred embodiment, in step S3, the extrusion process is as follows: P-CD and MM are mixed and extruded using an extruder through a 100 nm aqueous phase filter.

[0020] The third technical solution of the present invention is to provide the application of D-π-A sensitized type I / II photodynamic carbon dots based on M1 macrophage membrane encapsulation in the preparation of antitumor photosensitizing agents, as described in one of the above technical solutions.

[0021] In some specific embodiments, the photosensitizing agent is also used in conjunction with near-infrared excitation.

[0022] In some specific embodiments, the near-infrared excitation parameters are: wavelength of 808 nm and power of (0.1~0.33) W / cm². 2 .

[0023] This invention is the first to design near-infrared excited D-π-A sensitizing carbon dots (CDs) as type I / II photosensitizers for hypoxia-modulated photodynamic immunotherapy. Through a molecular engineering combinatorial strategy, three types of carbon dots (O-CDs, M-CDs, and P-CDs) with different regional modifications were synthesized. Among them, P-CDs with the D-π-A configuration exhibited a significantly prolonged triplet lifetime (36.32 ms) and the highest type I / II reactive oxygen species (ROS). 1 O2 and O2• - The generation efficiency of the D-π-D type M-CDs is the highest (69.6%), but its photothermal conversion efficiency is the lowest (32.2%). In contrast, the D-π-D type M-CDs, which lack a clear acceptor, exhibit the highest photothermal conversion efficiency (69.6%) but weaker photodynamic performance, further revealing the mechanism by which the D-π-A structure enhances photodynamic performance by stabilizing ultra-long lifetime charge transfer states.

[0024] At the application level, the P-CD@MM particles prepared by this invention, formed by P-CDs encapsulated by M1-type macrophage membranes, can actively target tumors and prolong their in vivo retention, while simultaneously alleviating tumor hypoxia through a mild photothermal synergy. This system operates at low laser power density (0.30 W / cm²). 2 At 808 nm, it can effectively induce immunogenic cell death and reprogram tumor-associated macrophages into the M1 anti-tumor phenotype, thereby reversing the immunosuppressive microenvironment and achieving satisfactory therapeutic effects on primary and metastatic tumors. This invention highlights the key role of D-π-A sensitized carbon dots in enhancing near-infrared photoimmunotherapy through triplet stabilization, charge transfer regulation, and hypoxia relief.

[0025] Compared with the prior art, the present invention has the following advantages: The present invention relates to D-π-A sensitized type I / II photodynamic carbon dots encapsulated in M1 macrophage membranes, which possess both type I / II photodynamic properties and a mild near-infrared photothermal effect, thus evoking a strong immune response and showing great potential for application in the treatment of tumors. Attached Figure Description

[0026] Figure 1 a) is the simulated structure of P-CD; b) is the O 1s XPS fine spectrum of P-CD; c) is the N 1s XPS fine spectrum of P-CD; d) is the TEM image and particle size distribution of P-CD (inset); e) is the HR-TEM image of P-CD; f) is the TEM image of P-CD@MM.

[0027] Figure 2 a) shows the time-resolved PL spectrum of O-CD and the corresponding fitting curve; b) shows the time-resolved PL spectrum of M-CD and the corresponding fitting curve; c) shows the time-resolved PL spectrum of P-CD and the corresponding fitting curve; d) shows the time-resolved PL spectrum of P-CD under normal oxygen conditions. 1 O2 generation performance test; e) O2 generation of P-CD under normal oxygen conditions. •- Performance testing was conducted; f) P-CD was tested in an oxygen-deficient environment. 1 O2 generation performance test; g) O2 generation of P-CD under hypoxic conditions. •- Generate performance tests.

[0028] Figure 3 a) represents P-CD under different laser powers (808 nm, 0.1-0.4 W / cm). 2 a) Photothermal performance testing of P-CD at different concentrations (0.1 mg / mL-0.3 mg / mL); c) Calculation of photothermal conversion efficiency of P-CD.

[0029] Figure 4 a) Comparison of cell viability of 4T1 and NIH-3T3 cells treated with P-CD@MM; b) Comparison of cell viability of 4T1 cells treated with P-CD@MM under laser irradiation (808 nm, 0.3 W / cm2, 5 min) or without irradiation; c) Live and dead staining experiments of 4T1 cells after different treatments; d) Reactive oxygen species staining experiments of 4T1 cells after different treatments.

[0030] Figure 5 a) shows the change in primary tumor volume after intravenous injection of P-CD@MM; b) shows the change in distal tumor volume after intravenous injection of P-CD@MM; c) shows the change in mouse body weight during tumor treatment; d) shows the survival rate of mice after different treatments.

[0031] Figure 6 a) represents the changes in dendritic cell maturation in lymphoid tissue after different treatment methods; bc) represents the changes in T cell content in the primary tumor; de) represents the changes in macrophage content of different phenotypes in the primary tumor. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0034] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0035] Example 1: This embodiment provides a method for preparing M1 macrophage membrane-encapsulated carbon dots with a D-π-A configuration (P-CD@MM), which includes the following steps: (1) 5 mg of indocyanine green (IGC) and 10 mg of p-phenylenediamine were dissolved in 10 mL of ethanol. The solution was transferred to a 25 mL PTFE-lined autoclave and reacted at 150 °C and 10 bar for 4 h. The reactor was then cooled to room temperature. Large particles were removed through a 0.22 μm membrane and then dialyzed for 36 h using a dialysis bag (3500D) to obtain P-CD.

[0036] (2) RAW264.7 cells were seeded in 6-well plates at 2 × 10⁻⁶ wells. 5 Cells were cultured at 100 μg / mL per well for 24 h, followed by the addition of lipopolysaccharide (2 μL, 100 μg / mL) to each well. The cells were then co-cultured for another 24 h (37°C, 5% CO2, humidity >95%) to obtain M1 macrophages. Cells were suspended in lysis buffer (Beyotime, P0038) and sonicated on ice (80 W, 20 min). The solution was centrifuged at 4°C for 30 min, and the supernatant was collected to obtain the M1 macrophage membrane (MM).

[0037] (3) 1 mL of 0.5 mg / mL P-CD aqueous solution and 1 mL of 1 mg / mL M1 macrophage membrane were transferred into syringes, and then extruded sequentially through a 100 nm aqueous phase filter using an extruder to form P-CD@MM conjugates. Finally, the P-CD@MM solution was centrifuged to obtain purified P-CD@MM.

[0038] like Figure 1 As shown in Figure a, the simulated structure of P-CD is obtained, with the edges of the obtained P-CD modified by hydroxyl groups (D: donor) and pyridine nitrogen (A: acceptor). Figure b shows the fine 1s XPS spectrum of O in P-CD, revealing that O in P-CD mainly exists in the form of hydroxyl groups. Figure c shows the fine 1s XPS spectrum of N in P-CD, revealing that N in P-CD mainly exists in the form of pyridine nitrogen. Figure d shows the TEM image and particle size distribution of P-CD (inset), with a particle size distribution of 1.38 ± 0.40 nm.

[0039] The image shown is an HR-TEM image of the P-CD, revealing a lattice spacing of approximately 0.21 nm belonging to graphitic carbon.

[0040] Figure f shows a TEM image of P-CD@MM, with a particle size of approximately 150 nm.

[0041] Test Example 1: Evaluation of Type I / II Photodynamic Performance of P-CDs: (1) The fluorescence lifetimes of O-CD (carbon dots synthesized from o-phenylenediamine as a precursor), M-CD (carbon dots synthesized from m-phenylenediamine as a precursor) and P-CD (carbon dots synthesized from p-phenylenediamine as a precursor) were detected by time-resolved fluorescence spectroscopy. 3 mL of each of the three carbon dots aqueous solutions with a concentration of 100 μg / mL was placed in a cuvette, and their fluorescence decay lifetime at 808 nm was detected by time-resolved fluorescence spectroscopy.

[0042] like Figure 2 As shown in the ac, P-CD exhibits a significantly extended triplet lifetime (36.32 ms), while O-CD and M-CD both have nanosecond lifetimes of 2.02 ns and 8.88 ns, respectively.

[0043] (2) By using 1,3-diphenylisobenzofuran (DPBF) and dihydrorhodamine-123 (DHR-123) as... 1 O2 and O2 •- The probe was used to detect the P-CD prepared in Example 1 under low-power near-infrared laser irradiation (808 nm, 0.3 W / cm²). 2 Normal and hypoxic environments 1 O2 and O2 •- Generation efficiency was assessed to evaluate its photodynamic performance.

[0044] like Figure 2 As shown in the figure, P-CD at a final concentration of 0.3 mg / mL under low-power near-infrared laser irradiation (808 nm, 0.3 W / cm²) 2 In an aerobic environment, a large amount of singlet oxygen can be generated. 1 O2) and superoxide anion (O2) •- The values ​​() correspond to type II and type I photodynamic properties, respectively, indicating that P-CDs possess excellent I / II photodynamic properties under normal oxygen conditions.

[0045] like Figure 2 As shown in the fd, P-CD at a final concentration of 0.3 mg / mL exhibited almost no inhibition of its type I photodynamic properties under hypoxic conditions, while its type II photodynamic properties were inhibited. Further verification of its type I photodynamic characteristics under hypoxic conditions was conducted to demonstrate its ability to effectively overcome the hypoxic tumor microenvironment.

[0046] (3) Further testing was conducted on P-CD at a final concentration of 0.3 mg / mL under near-infrared laser (808 nm) at different powers (0.1-0.4 W / cm²). 2 Photothermal properties under (e.g.) Figure 3 As shown in a.

[0047] (4) Further testing of different P-CD concentrations under low-power near-infrared laser irradiation (808 nm, 0.3 W / cm²) 2 Photothermal properties, such as Figure 3 As shown in b.

[0048] This test case comprehensively investigated the photothermal behavior of P-CD by varying the laser power and P-CD concentration, and further calculated its photothermal conversion efficiency. The P-CD (0.3 mg / mL) prepared in Example 1 was subjected to near-infrared laser irradiation (808 nm, 0.3 W / cm²). 2 It exhibits mild near-infrared photothermal properties, with a photothermal conversion efficiency η of 32.2%. Figure 3 As shown in c.

[0049] Test Case 2: Extracorporeal photodynamic therapy of P-CD@MM: (1) In this test case, the cell viability of cells treated with P-CD@MM and 808 nm laser was detected by the MTT method.

[0050] (1-1) Mouse breast cancer cells (4T1) and mouse embryonic fibroblast cell line (NIH-3T3 cells) were seeded into 96-well plates at a density of 5000 cells per well and cultured for 24 h. Then, P-CD@MM was added to achieve different final concentrations (0, 12.5, 25, 50, 75, 100 μg / mL) in the co-culture system and co-cultured for 4 h. The efficacy of P-CD@MM in in vitro sonodynamic therapy was then detected using MTT assay.

[0051] like Figure 4 As shown in Figure a, both 4T1 cells and NIH-3T3 cells remained viable after treatment with different concentrations of P-CD@MM, indicating that P-CD@MM alone is non-cytotoxic.

[0052] (1-2) Mouse breast cancer cells (4T1) were seeded into 96-well plates at a density of 5000 cells per well and cultured for 24 h. Then, P-CD@MM was added to achieve different final concentrations (0, 12.5, 25, 50, 75, 100 μg / mL) in the co-culture system, and co-cultured for 4 h. The cells were then treated with a laser (808 nm, 0.3 W / cm²). 2 Irradiation was performed for 5 minutes or without laser irradiation, and then the efficacy of P-CD@MM in extracorporeal sonodynamic therapy was detected using MTT reagent.

[0053] like Figure 4 As shown in b, the survival rate of 4T1 cells after treatment with P-CD@MM and 808 nm laser irradiation is inversely proportional to the concentration of P-CD@MM.

[0054] (2) The cytotoxicity of the cells treated with P-CD@MM and 808 nm laser was further verified by live-dead staining experiments.

[0055] 4T1 cells will be stored at 2 × 10⁴ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates. These cells were treated with Control (100 μL PBS) and Laser (808 nm, 0.3 W / cm²) as well. 2 Cells were treated with P-CD@MM (100 μL of 1 mg / mL P-CD@MM) and P-CD@MM + Laser for 5 min. After 24 hours of culture, cells were stained with Calcein-AM (488 nm) and PI (561 nm), and fluorescence images of live and dead cells were captured by fluorescence microscopy. Live cells showed green fluorescence, and dead cells showed red fluorescence.

[0056] like Figure 4 As shown in Figure c, 4T1 cells exhibited red fluorescence after treatment with P-CD@MM and 808 nm laser, indicating that P-CD@MM and 808 nm laser treatment could lead to 4T1 cell death.

[0057] (3) The production of intracellular reactive oxygen species after treatment with P-CD@MM and 808 nm laser was verified by reactive oxygen species staining experiment.

[0058] 4T1 cells will be stored at 2 × 10⁴ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. These cells were treated with Control (100 μL PBS) and Laser (808 nm, 0.3 W / cm²) as well. 2 Cells were treated with P-CD@MM (100 μL of 1 mg / mL P-CD@MM) and P-CD@MM + Laser for 5 min. After 24 hours of culture, cells were stained with DCFH-DA and DAPI. Confocal microscopy was then used to capture confocal images to assess intracellular ROS levels.

[0059] DAPI is used for localization, and staining the cell nucleus results in a blue color. DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) itself is non-fluorescent. After entering the cell, it is hydrolyzed by intracellular esterases to generate DCFH (dichlorodihydrofluorescein). When ROS is present in the cell, DCFH is oxidized to DCF (2,7-dichlorofluorescein), which has strong green fluorescence. Its fluorescence intensity is proportional to the intracellular ROS level.

[0060] like Figure 4 As shown in d, 4T1 cells showed green fluorescence after being treated with P-CD@MM and 808 nm laser irradiation, indicating that P-CD@MM can generate a large amount of reactive oxygen species under 808 nm laser irradiation.

[0061] In summary, P-CD@MM alone has no cytotoxicity to NIH-3T3 and 4T1 cells, but under 808 nm laser irradiation, it can generate a large amount of reactive oxygen species and almost completely kill tumor cells.

[0062] Test Case 3: In vivo sonodynamic therapy of P-CD@MM: 100 μL (2 × 10⁻⁶) was subcutaneously implanted into the left and right axillae of 5-week-old mice. 6 Mouse breast cancer cells (4T1) were used until the tumor volume reached approximately 100 mm. 3 The mice were divided into 6 groups (n=5 per group): Control (with 100 μL PBS), Laser (808 nm, 0.3 W / cm²), and Laser (with PBS). 2 The following treatments were performed: P-CD (100 μL of 1 mg / mL P-CD), P-CD@MM (100 μL of 1 mg / mL P-CD@MM), P-CD+Laser, and P-CD@MM+Laser. Tumor volume was measured every other day, and mouse weight was recorded daily to evaluate the in vivo photodynamic therapy efficiency of P-CD@MM.

[0063] like Figure 5 As shown in the ad, after treatment with P-CD@MM+Laser, the volume of both primary and distant tumors in mice was significantly reduced, the body weight of mice remained essentially unchanged, and the survival time of mice was significantly prolonged. At the same time, P-CD@MM did not show obvious long-term toxicity in vivo.

[0064] Test Example 4: In vivo immune activation of P-CD@MM: 100 μL (2 × 10⁻⁶) was subcutaneously implanted into the left and right axillae of 5-week-old female mice. 6 Mouse breast cancer cells (4T1) were used until the tumor volume reached approximately 100 mm. 3 The mice were divided into 6 groups (n=5 per group): Control (with 100 μL PBS), Laser (808 nm, 0.3 W / cm²), and Laser (with 808 nm PBS, 0.3 W / cm²). 2 The following treatments were performed: P-CD (100 μL of 1 mg / mL P-CD), P-CD@MM (100 μL of 1 mg / mL P-CD@MM), P-CD+Laser, and P-CD@MM+Laser. After 7 days of treatment, mice were euthanized, and their lymphoid tissue, spleen, primary tumor, and distant tumors were extracted for immunoassay.

[0065] like Figure 6As shown in the ae, after treatment with P-CD@MM+Laser, the dendritic cells (DCs) of mice matured significantly, activating a large number of CD4+ cells. + T cells and CD8 + T cells were activated, and macrophages were polarized towards the M1 phenotype, while M2 phenotype macrophages were reduced. This indicates that P-CD@MM+Laser has significant immune activation and immunomodulatory functions, can systematically enhance the cellular immune response in mice, and reshape the macrophage polarization phenotype, thus possessing potential anti-tumor application value.

[0066] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A type I / II photodynamic carbon dot based on D-π-A sensitized by M1 macrophage membrane encapsulation, characterized in that, It consists of an M1 type macrophage membrane and carbon dots with a D-π-A configuration, wherein the M1 type macrophage membrane is also wrapped with carbon dots with a D-π-A configuration.

2. A method for preparing D-π-A sensitized type I / II photodynamic carbon dots encapsulated in M1 macrophage membranes as described in claim 1, characterized in that, Includes the following steps: S1. Indocyanine green and p-phenylenediamine are mixed and reacted under high temperature and high pressure. The mixture is then filtered and dialyzed to obtain carbon dots P-CD with a D-π-A configuration. S2. Macrophages and lipopolysaccharide are co-cultured to obtain M1 macrophages. M1 macrophages are then lysed to obtain the M1 macrophage membrane MM. S3. Mix and extrude the P-CD obtained in step S1 and the MM obtained in step S2 to obtain type I / II photodynamic carbon dots sensitized by D-π-A based on M1 type macrophage membrane, which is P-CD@MM.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of indocyanine green to p-phenylenediamine is 1:

2.

4. The preparation method according to claim 2, characterized in that, In step S1, the temperature of the high-temperature and high-pressure reaction is (130~160)℃, the pressure is greater than or equal to 10 bar, and the time is (3~6) h; The filtration process is as follows: the product obtained from the reaction under high temperature and high pressure is squeezed through a filter membrane of (0.1~10) μm using a syringe; The dialysis process is as follows: using a dialysis bag of (3000D~3500D), the filtered product is dialyzed for (30~40) h.

5. The preparation method according to claim 2, characterized in that, In step S2, the ratio of macrophages to lipopolysaccharide is 2 × 10⁻⁶. 5 Cells: (0.1~0.3) μg.

6. The preparation method according to claim 2, characterized in that, In step S2, the co-cultivation conditions are: 37℃, 5% CO2, humidity greater than 95%, and time is (20~26) h; The lysis process is as follows: M1 macrophages are suspended in lysis buffer, sonicated on ice at 80W for 15-25 min, centrifuged at 4°C for 25-35 min, and the supernatant is collected to obtain the M1 macrophage membrane MM.

7. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of P-CD to MM is 1:

2.

8. The preparation method according to claim 2, characterized in that, In step S3, 1 mL of P-CD aqueous solution with a concentration of 0.5 mg / mL and 1 mL of MM with a concentration of 1 mg / mL are mixed and extruded.

9. The preparation method according to claim 2, characterized in that, In step S3, the extrusion process is as follows: P-CD and MM are mixed and extruded using an extruder through a (100~200) nm aqueous phase filter.

10. The application of type I / II photodynamic carbon dots sensitized by D-π-A and encapsulated in M1 macrophage membranes as described in claim 1 in the preparation of photosensitizing agents for antitumor purposes.

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