Colloid self-assembly crystal monolayer film with photodynamic and immunoregulation functions, preparation method and application

Abstract

The invention discloses a colloid self-assembly crystal monolayer film with photodynamic and immunoregulation functions, a preparation method and application, the colloid self-assembly crystal monolayer film (BCCe6) is composed of SiO2-MnO2 particles and PSC-Ce6 particles, and a photosensitizer is chlorin e6. The diameter of the SiO2 particles is 4 to 6 m, and the diameter of the PSC particles is 300 to 500 nm; the density of the SiO2-MnO2 particles is 2.19 to 2.65 g / cm < 3 >, and the density of the carboxylated polystyrene particles modified by the photosensitizer is 0.96 to 1.05 g / cm < 3 >; the surface structure of the colloid self-assembly crystal monolayer film is a regular hexagonal close-packed structure, and the depth of a groove is 300-900 nm. The colloidal self-assembly crystal single-layer film can slowly release Mn < 2 + > ions and release ROS under the stimulation of near-infrared light, and efficient sterilization and macrophage phenotype rapid conversion are achieved through the cooperation of a surface physical structure and a chemical effect. The colloidal self-assembled crystal monolayer film is placed on a bacterial infected wound, and is removed after being irradiated by infrared light for 5-10 minutes, so that the healing of the wound can be accelerated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a colloidal self-assembled crystal monolayer with photodynamic and immunomodulatory functions, its preparation method, and its application. Background Technology

[0002] Bacterial infections have a significant impact on clinical treatment outcomes and pose considerable risks to patient health, often hindering recovery. Globally, approximately 700,000 people die from infections each year, with over 60% of these cases related to biofilms, which can lead to conditions such as chronic bacterial prostatitis, chronic otitis media, and osteomyelitis. Despite significant advances in infection control with antibiotics, antifungals, and antivirals, the overuse of broad-spectrum antibiotics has led to the emergence of multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Methicillin-resistant Staphylococcus aureus MRSA) and carbapenem-resistant Enterobacteriaceae ( Carbapenem-resistant Enterobacteriaceae Bacterial infections, such as bacterial vegetative-associated pneumonia (BVA) and cystic inflammatory response (CIRS), pose significant challenges to clinical diagnosis and treatment. Furthermore, the high tolerance of bacteria within biofilms to antibiotics and host defense mechanisms leads to chronic and persistent infections, exacerbating global health problems. To combat "superbugs," research focus is shifting towards alternative strategies such as phage therapy, antimicrobial peptides, photodynamic therapy (PDT), and nanostructured surfaces. In bacterial-infected wounds, a rapid immune response is crucial for wound healing and long-term tissue regeneration; otherwise, irreversible tissue damage may occur. In this proof-of-concept study, we designed colloidal self-assembled crystals (cSACs) based on manganese dioxide (MnO2)-modified silica and dihydroporphyrin E6 (chlorine 6, Ce6)-modified polystyrene particles, endowing them with bactericidal and immunomodulatory effects. Under near-infrared (NIR) irradiation, Ce6 undergoes a photodynamic reaction, generating reactive oxygen species (ROS), especially singlet oxygen, which can rapidly induce bacterial death. Mn is continuously released during the degradation of MnO2. 2+ It can act as an immune adjuvant, stimulating innate immune responses and preventing infection recurrence. This bifunctional binary colloidal crystal (BCC), named BCCe6, is designed to clear bacteria and control excessive inflammation in bacterial-infected wounds. Furthermore, the unique biophysical signals emitted by BCCe6 can trigger specific mechanotransduction in surrounding cells, including immune cells. This research inspires innovation in biomaterials and advances tissue engineering and biomedicine. Summary of the Invention

[0003] The purpose of this invention is to provide a method for accelerating the healing of bacterial infection wounds through photodynamic immunomodulatory colloidal self-assembled crystals. By combining the dual functions of photodynamic sterilization and immunomodulatory timing of binary colloidal self-assembled crystals (BCCe6), this method addresses the problem of delayed healing in bacterial infection wounds caused by drug resistance, biofilm formation, and immune imbalance. While traditional photodynamic therapy has antibacterial effects, it lacks the ability to dynamically regulate macrophage polarization; and single immunomodulatory materials are insufficient for rapid pathogen clearance. BCCe6, through its unique ordered topological structure and controllable ion release, simultaneously achieves highly efficient sterilization and macrophage phenotypic conversion (M0 → M1 → M2) under near-infrared light (NIR) activation. Specifically, manganese dioxide (MnO2) modified silica particles (5μm) degrade and release Mn²⁺ in the weakly acidic infection microenvironment. + Activating the cGAS / STING pathway drives early M1 polarization to clear bacteria; while Ce6-modified polystyrene particles (400nm) generate reactive oxygen species (ROS) under NIR irradiation to disrupt biofilms, and their periodic groove topology promotes late-stage M2 macrophage recruitment through the integrin / PYK2 mechanotransmission pathway, accelerating tissue regeneration.

[0004] According to a first aspect of the present invention, a colloidal self-assembled crystal monolayer (BCCe6) with photodynamic and immunomodulatory functions is provided, wherein the colloidal self-assembled crystal monolayer is composed of SiO2-MnO2 particles and carboxylated polystyrene particles modified with a photosensitizer, wherein the photosensitizer is dihydroporphyrin e6. The photosensitizer-modified carboxylated polystyrene particles are PSC-Ce6 particles; The diameter of the SiO2-MnO2 particles is 4-6µm, and the diameter of the photosensitizer-modified carboxylated polystyrene particles is 300-500nm. The density of the SiO2-MnO2 particles is 2.19-2.65 g / cm³. 3 The density of the photosensitizer-modified carboxylated polystyrene particles is 0.96-1.05 g / cm³. 3 ; The silica particles in the colloidal self-assembled crystal monolayer film have a close-packed hexagonal structure with a particle center spacing of 5-7µm and a groove depth of 300-900nm.

[0005] Based on the above technical solution, the biocompatibility of the colloidal self-assembled crystal monolayer membrane with photodynamic and immunomodulatory functions meets the following requirements: NIH3T3 fibroblast survival rate >98% (detected by CCK-8 and cell live / dead staining assays); macrophage cytotoxicity thresholds: Ce6 ≤ 50 μg / mL, MnO2 ≤ 32 μM; in vivo degradation cycle is 7 days (MnO2 on day 7). 2+The residual amount is <10%, and the material maintains photodynamic stability during degradation (singlet oxygen yield decay <5%).

[0006] According to a second aspect of the present invention, a method for preparing a colloidal self-assembled crystal monolayer with photodynamic and immunomodulatory functions is provided, the method comprising the following steps: Step 1, Preparation of SiO2-MnO2 particles: SiO2 particles are mixed with (3-aminopropyl)triethoxysilane and amination is performed to obtain amination-treated SiO2 particles; BSA is mixed with EDC and NHS and reacted in reaction I to obtain BSA with activated carboxyl groups, and the amination-treated SiO2 particles are added and reacted in reaction II to obtain SiO2-BSA particles; KMnO4 is then added, and MnO2 nanoshells are deposited through in-situ biomineralization with BSA to obtain SiO2-MnO2 particles; Step 2, Preparation of PSC-Ce6 particles: PSC particles, EDC and NHS are mixed and reacted in reaction I to obtain activated PSC particles. Then, photosensitizer Ce6 is added and reacted in reaction II to obtain PSC-Ce6 particles. Step 3, Preparation of colloidal self-assembled crystal monolayer: The SiO2-MnO2 particles obtained in Step 1 and the PSC-Ce6 particles obtained in Step 2 are mixed and dispersed in water to form a mixed solution. After evaporation-induced self-assembly in a closed region, an ordered crystal structure is formed. Then, after heat treatment at 150~200℃ for 5-10 minutes or stabilization treatment with toluene solution, a colloidal self-assembled crystal monolayer, namely BCCe6, is obtained.

[0007] Based on the above technical solution, the mass mixing ratio of SiO2 particles, (3-aminopropyl)triethoxysilane, BSA, and KMnO4 in step 1 is 5~15:10~100:5~15:0.32~1.58; The conditions for the amination reaction are as follows: The amination reaction is carried out at a temperature of 18~25℃; The amination reaction takes 8-24 hours. The conditions for the in-situ biomineralization method are as follows: The temperature for the in-situ biomineralization method is 18~25℃; The in-situ biomineralization process takes 20-30 minutes.

[0008] Based on the above technical solution, the percentage of Mn atoms in the SiO2-MnO2 particles obtained in step 1 is 1.5-2.0 at%, and the Zeta potential is |-14mV|~|-16mV|.

[0009] Based on the above technical solution, the mass mixing ratio of carboxylated polystyrene particles, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and photosensitizer Ce6 in step 2 is 10~20:10~20:20~30:1~2; The conditions for reaction I in step 2 are as follows: The temperature for reaction I is 18~25℃; The reaction time for reaction I is 0.5–4 hours; The conditions for reaction II in step 2 are as follows: The temperature for reaction II is 18~25℃; The reaction time for reaction II is 16-24 hours.

[0010] Based on the above technical solution, the loading of photosensitizer Ce6 in the PSC-Ce6 particles obtained in step 2 is 19~20μg / mL, and the Zeta potential is |-24mV|~|-26mV|.

[0011] Based on the above technical solution, the self-assembly conditions in step 3 are as follows: The concentration of both types of self-assembled particles is 1%, i.e., 10 mg / ml; The self-assembly temperature is 18~25℃; The self-assembly time is 24~48h.

[0012] According to a third aspect of the present invention, an application of a colloidal self-assembled crystal monolayer membrane in accelerating the healing of bacterial infection wounds is provided. The colloidal self-assembled crystal monolayer membrane is implanted into the bacterial infection wound site, and after near-infrared irradiation, the release of BCCe6 ions and topological signaling synergistically regulate the macrophage phenotype conversion from M1 to M2, thereby promoting wound healing.

[0013] Based on the above technical solution, the conditions for near-infrared irradiation are as follows: The wavelength of the near-infrared irradiation is 600~700nm, preferably 660nm; The power density of the near-infrared irradiation is 50~100mW / cm². 2 The preferred value is 100mW / cm 2 ; The near-infrared irradiation time is ≥10 minutes, preferably 15 minutes.

[0014] Based on the above technical solution, for skin defects infected with Staphylococcus aureus or drug-resistant Escherichia coli, a wound healing rate of >95% can be achieved within 9 days, and on day 7: M2 macrophages (CD206) +The proportion of collagen deposition increased significantly (>2 times higher than the control group); the amount of collagen deposition increased by more than 2 times (confirmed by Masson staining); the inflammatory factors IL-6 and IL-17 decreased to baseline levels, while the level of the anti-inflammatory factor IL-10 increased.

[0015] Beneficial effects (1) The technical solution disclosed in this invention can efficiently and synergistically promote antibacterial and immune regulation, under near-infrared light (NIR, preferably 660nm) irradiation (power density 100mW / cm²). 2 Within 15 minutes, BCCe6 generates reactive oxygen species (ROS) through the Ce6 photosensitizer, achieving a bactericidal rate of >92% against Staphylococcus aureus and Escherichia coli, realizing photodynamic sterilization and effectively disrupting biofilm structures. Immunological timing and regulation: In the early stages, a weakly acidic infection microenvironment (pH 5.0-6.5) and high levels of reduced glutathione (GSH ≥ 10 mM) trigger MnO2 degradation, releasing Mn... 2+ Activation of the cGAS / STING pathway upregulates TBK1 and IRF3 gene expression, driving macrophage M1 polarization (CD86). + Cell proportion increased by >2-fold), rapidly clearing pathogens; in the later stage: the surface periodic groove topology (groove depth 400nm) downregulates vinculin and lamin A / C through the integrin α5 / FAK / PYK2 pathway, promoting YAP nuclear translocation and synergistically inducing macrophages to first convert from M0 to M1 phenotype, and then to M2 phenotype (CD206). + The cell percentage increased significantly on day 7.

[0016] (2) The technical solution disclosed in this invention can accelerate wound healing and tissue regeneration: i. Improved healing speed: In a mouse model of drug-resistant bacterial infection, the wound healing rate of the BCCe6+NIR treatment group was >95% within 9 days, which was significantly better than that of the control group; ii. Enhanced collagen deposition: Masson staining showed that the amount of collagen deposition increased by >2 times; iii. Shortened inflammatory cycle: Serum inflammatory factors (IL-6, IL-17) dropped to baseline levels on day 7, while the repair-promoting factor IL-10 increased, avoiding tissue damage caused by chronic inflammation.

[0017] (3) The self-assembled crystal material obtained by the technical solution disclosed in this invention has good safety and stability, i. excellent biocompatibility: NIH3T3 fibroblast survival rate >98% (CCK-8 assay), and macrophage toxicity threshold is much higher than the actual release concentration (Ce6≤50μg / mL, MnO2≤32μM); ii. controllable degradation: the in vivo degradation cycle is 7 days (MnO2 on day 7 is 7 days). 2+The residual amount is <10%, and the singlet oxygen production rate decreases by <5% during the degradation process, ensuring continuous efficacy; iii. Systemic safety: No pathological damage was found in the H&E staining of the major organs (heart, liver, spleen, lung, kidney).

[0018] (4) The technical solution disclosed in this invention can solve key clinical problems, i. Overcoming antibiotic resistance: through physical topology inhibition of bacterial adhesion and photodynamic ROS sterilization, effectively clearing the biofilm of drug-resistant bacteria; ii. Immunological microenvironment remodeling: spatiotemporal coordination of macrophage polarization (M1 dominates the clearance of bacteria on day 3 → M2 dominates the promotion of repair on day 7), avoiding delayed healing caused by immune imbalance. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the entire process of preparing BCCe6 material in Example 1 of the present invention. Figure 2 The scanning electron microscope morphology and EDS analysis results of the BCCe6 material in Example 1 of this invention are shown. Figure 3 The zeta potential of the particles used in BCCe6 in Example 1 of this invention: PS, PSC, PSC-Ce6, SiO2 and SiO2-MnO2; Figure 4 A schematic diagram showing the initial bacterial adhesion and antibacterial adhesion ability of the BCCe6 material prepared in Example 1 of the present invention, and a schematic diagram showing the photo-response-regulated bactericidal activity after the formation of bacterial biofilm. Figure 5 SEM images of the BCCe6 material prepared in Example 1 of this invention after 2 hours of bacterial culture on the surface of *S. aurenus*; SEM images of the surface of *E. coli* after 2 hours of bacterial culture; quantitative analysis results of bacteria in three specific regions: on top of silicon particles (red), most bacteria are located on silicon particles, some are attached to Si and P regions (Si / P, green); bacteria are attached to both Si and P regions and only to P regions (blue); conventional SEM images of the morphology of *S. aurenus* and *Escherichia coli* after culture and near-infrared spectroscopy on the surface of *S. aurenus* (sputtered gold); conventional SEM images of the morphology of *S. aurenus* and *Escherichia coli* after culture and near-infrared spectroscopy on the surface of *E. coli* (sputtered gold); magnified SEM images show the microstructure and morphological damage of *S. aurenus* (yellow) and *E. coli* (green); Figure 6The activation results of bone marrow dendritic cells (BMDCs) on BCCe6 material in Test Example 2 of this invention include: a polarization diagram; expression of co-stimulatory molecules in BMDCs treated with BCCe6 material; quantitative analysis of BMDC maturation (CD80+ and CD86+); quantitative analysis of TNF-α in the culture supernatant of BMDCs cultured on BCCe6 material; quantitative analysis of IL-1β in the culture supernatant of BMDCs cultured on BCCe6 material; and quantitative analysis of IL-6 in the culture supernatant of BMDCs cultured on BCCe6 material. Figure 7 The results show the polarization of RAW264.7 macrophages in Test Example 2 of this invention, the immunofluorescence staining results of M1-like cells with different surfaces (F4 / 80+ / CD86+), and the results of quantitative analysis of macrophages using multicolor flow cytometry. Figure 8 For the analysis of the polarization performance of THP-1 macrophages on BCCe6 in Test Example 2 of the present invention (wherein, A: flow cytometry analysis of THP-1 polarization; B: representative colocalization confocal images of cell nucleus (blue), actin cytoskeleton (green) and 0.5 μm particles (red); Figure 9 This is a flowchart of the experimental design for establishing and treating a bacterial infection wound model using BCCe6 material prepared in Example 1 of the present invention and BCCe6+NIR. Figure 10 The results of dynamic wound healing in a mouse model of the BCCe6 material prepared in Example 1 of this invention and the effect of BCCe6+NIR on a bacterial infection wound model, and the wound bed closure marks within 9 days of each treatment. Figure 11 The dynamic results of BCCe6 material promoting wound healing in a mouse model of bacterial infection wounds prepared in Example 1 of this invention and BCCe6+NIR are shown. Figure 12 The in vivo degradation degree of BCCe6 material was evaluated in the BCCe6 material prepared in Example 1 of this invention and in the BCCe6+NIR model of bacterial infection wound. Figure 13 The BCCe6 material prepared in Example 1 of this invention and the H&E staining of the central lesion in a bacterial infection wound model using BCCe6+NIR were used. Figure 14 Masson staining image of the central lesion in a bacterial infection wound model using BCCe6 material prepared in Example 1 of this invention and BCCe6+NIR. Figure 15The BCCe6 material prepared in Example 1 of this invention and the BCCe6+NIR immunofluorescence staining of CD86 and CD206 in a bacterial infection wound model; Figure 16 The semi-quantitative analysis results of M1 fluorescence intensity at the lesion site in a bacterial infection wound model using BCCe6 material prepared in Example 1 of this invention and BCCe6+NIR. Figure 17 The semi-quantitative analysis results of fluorescence intensity of M1 and M2 at the lesion site in a bacterial infection wound model using BCCe6 material prepared in Example 1 of the present invention and BCCe6+NIR. Figure 18 In the BCCe6 material prepared for Example 1 of the present invention and the BCCe6+NIR model of bacterial infection wound, the serum IL-6 concentration of mice was measured by ELISA on day 3 and day 7. Figure 19 The concentration of serum IL-17 in mice on day 7 in the BCCe6 material prepared in Example 1 of this invention and the BCCe6+NIR model of bacterial infection wounds; Figure 20 The concentration of serum IL-10 in mice on day 7 in the BCCe6 material prepared in Example 1 of this invention and the BCCe6+NIR model of bacterial infection wounds; Figure 21 The results of blood biochemical analysis after 7 days in a bacterial infection wound model using BCCe6 material prepared in Example 1 of this invention and BCCe6+NIR. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0021] Unless otherwise specified, all raw materials and reagents used in the following examples were obtained through purchase.

[0022] Example 1: Preparation of BCCe6 material Step 1, Preparation of SiO2-MnO2 particles: 10 mg of silica (SiO2) particles with a diameter of 5 μm were resuspended in 1 ml of anhydrous ethanol and mixed with 10% (3-aminopropyl)triethoxysilane (APTES) and amination treatment was carried out at 25 °C for 24 h. After the reaction was completed, the particles were washed three times with toluene and anhydrous ethanol and dried and fixed at 97 °C for 6-18 h for later use. 10 mg of bovine serum albumin (BSA) was mixed with 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysuccinimide (NHS) in 1 ml of morpholine ethanesulfonic acid (MES, pH 6.0) reaction buffer and reacted at 18-15 °C for 2 h to obtain BSA with activated carboxyl groups. The pH of the reaction system was then adjusted to 7.0, and 10 mg of amination- and heat-fixed SiO2 particles were added. The reaction was carried out for 18-24 h to obtain SiO2-BSA particles. Then, 20-100 μL of 100 mM KMnO4 solution was added, and MnO2 nanoshells were deposited by in-situ biomineralization with SiO2-BSA to obtain SiO2-MnO2 particles.

[0023] Step 2, Preparation of PSC-Ce6 particles: 10 mg of carboxylated polystyrene particles (400 nm in diameter, PS), 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 20 mg of N-hydroxysuccinimide (NHS) were mixed in 1 ml of morpholine ethanesulfonic acid (MES, pH 6.0) reaction buffer and reacted at 25°C for 2 h to obtain polystyrene particles with activated carboxyl groups (PSC). The pH of the reaction system was then adjusted to 7.0, and 1 mg of dihydroporphyrin e6 (photosensitizer Ce6) was added. The reaction was continued at 25°C for 18 h to obtain PSC-Ce6 particles. The PSC-Ce6 particles were prepared by... Figure 3 It can be seen that the Zeta potential of the PSC-Ce6 particles is |-25mV|.

[0024] Step 3, Preparation of self-assembled BCCe6 crystals: 95.08 μg of SiO2-MnO2 particles obtained in Step 1 and 32.89 μg of PSC-Ce6 particles obtained in Step 2 were dispersed in 150 μl of water (the distribution of the self-assembled particles is shown in Table 1; the density of the SiO2-MnO2 particles is 2.19-2.65 g / cm³). 3 The density of PSC-Ce6 particles is 0.96-1.05 g / cm³. 3 Evaporation-induced self-assembly of a sealed region at 25°C for 24 hours forms an ordered crystal structure. This is followed by heat treatment at 150°C for 5 minutes or stabilization with 1 ml of toluene solution to obtain self-assembled crystal BCCe6 (hexagonal close-packed crystal, i.e., BCCe6 material). A schematic diagram of the BCCe6 material preparation process is shown below. Figure 1 As shown.

[0025] Table 1 shows the physicochemical properties of self-assembled particles.

[0026] Test Example 1: Material Characterization A schematic diagram of the self-assembly process of the self-assembled crystal BCCe6 in Example 1 is shown below. Figure 1 As shown; the scanning electron microscope morphology and EDS analysis results of the BCCe6 material in Example 1 of this invention are as follows. Figure 2 As shown, by Figure 2 Scanning electron microscopy (SEM) images show that the inter-particle spacing of the BCCe6 material is 5-6 μm, and the groove depth is 300-900 nm. The zeta potentials of the particles—PS, PSC, PSC-Ce6, SiO2, and SiO2-MnO2—are as follows: Figure 3 As shown.

[0027] Test Example 2: In vitro antibacterial test The initial bacterial adhesion and bactericidal effect of the self-assembled crystal BCCe6 prepared in Example 1 are as follows: Figure 4-5 As shown, in the antibacterial performance test, Staphylococcus aureus (ATCC29213) and Escherichia coli (1×10⁻⁶) were tested. 8 CFU / mL was inoculated onto the surface of BCCe6 and incubated at 37°C for 2 hours. SEM observation showed that BCCe6 significantly reduced the adhesion density of the two bacteria, with Staphylococcus aureus and Escherichia coli decreasing by 29.9% and 23.7%, respectively. Furthermore, over 90% of the bacteria were localized within the grooves of the Ce6-modified PSC region. For the established bacterial biofilm, NIR irradiation (preferred wavelength 660 nm, 100 mW / cm²) was applied. 2 After treatment (15 min), SYTO9 / PI staining showed that the bacterial membrane structure disintegrated, and colony counting further confirmed that the sterilization rate exceeded 92%, with the survival rates of Staphylococcus aureus and Escherichia coli decreasing to 17.3% and 7.6%, respectively.

[0028] Test Example 3: Immune Cell Activation Experiment Activation of bone marrow dendritic cells (BMDCs) on BCCe6 material prepared in Example 1 Figure 6 ) and the polarization regulation effect of RAW264.7 macrophages ( Figure 7 ) and the polarization of THP-1 macrophages on BCCe6 ( Figure 8 ),like Figure 6-8 As shown, BCCe6 can efficiently activate the polarization and maturation of different types of immune cells and express corresponding markers.

[0029] Application Example 1: Effect on wound healing in cases of bacterial infection in vivo The effects of BCCe6 and BCCe6+NIR on bacterial infection wound models are as follows: Figure 9-21 As shown, during the animal model establishment phase, C57 / 6 male mice (10 weeks old) were used to construct a full-thickness skin defect on their backs, and inoculated with Staphylococcus aureus (1×10⁻⁶). 6 CFU / mL). The experimental groups were as follows: the control group (Ctrl) received a sterile PDMS substrate; the BCCe6 group received BCCe6 material; and the BCCe6+NIR group received NIR irradiation (preferred wavelength 660nm, 100mW / cm²) 24 hours after BCCe6 implantation. 2 (15 min). Healing effect assessment showed that on day 9, the wound closure rate of the BCCe6+NIR group exceeded 95%, significantly higher than the control group. Histological analysis showed that Masson staining confirmed a more than two-fold increase in collagen deposition in the BCCe6+NIR group; H&E staining showed the formation of new hair follicles and reduced inflammatory cell infiltration. Immunochromatographic control results showed that M1 macrophages (CD86) appeared in all groups on day 3. + ) recruitment, while the BCCe6+NIR group simultaneously showed M2 macrophages (CD206) recruitment. + By day 7, the proportion of M2 in the BCCe6+NIR group had significantly increased, serum pro-inflammatory factors IL-6 and IL-17 had decreased to baseline levels, while anti-inflammatory factor IL-10 had increased. Systemic safety assessment confirmed that no pathological damage was observed in the H&E staining of major organs (heart, liver, spleen, lung, and kidney).

[0030] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A colloidal self-assembled crystal monolayer membrane with photodynamic and immunomodulatory functions, characterized in that, The colloidal self-assembled crystal monolayer film is composed of SiO2-MnO2 particles and carboxylated polystyrene particles modified with a photosensitizer, wherein the photosensitizer is dihydroporphyrin E6. The photosensitizer-modified carboxylated polystyrene particles are PSC-Ce6 particles; The diameter of the SiO2-MnO2 particles is 4-6µm, and the diameter of the photosensitizer-modified carboxylated polystyrene particles is 300-500nm. The density of the SiO2-MnO2 particles is 2.19-2.65 g / cm³. 3 The density of the photosensitizer-modified carboxylated polystyrene particles is 0.96-1.05 g / cm³. 3 ; The silica particles in the colloidal self-assembled crystal monolayer film have a close-packed hexagonal structure with a particle center spacing of 5-7µm and a groove depth of 300-900nm.

2. The method for preparing a colloidal self-assembled crystalline monolayer film according to claim 1, characterized in that, The preparation method includes the following steps: Step 1, Preparation of SiO2-MnO2 particles: SiO2 particles are mixed with APTES and amination is performed to obtain amination-treated SiO2 particles; BSA is mixed with EDC and NHS and reacted in reaction I to obtain BSA with activated carboxyl groups, and the amination-treated SiO2 particles are added and reacted in reaction II to obtain SiO2-BSA particles; then KMnO4 is added and MnO2 nanoshells are deposited through in-situ biomineralization with BSA to obtain SiO2-MnO2 particles; Step 2, Preparation of PSC-Ce6 particles: PSC particles, EDC and NHS are mixed and reacted in reaction I to obtain activated PSC particles. Then, photosensitizer Ce6 is added and reacted in reaction II to obtain PSC-Ce6 particles. Step 3, Preparation of colloidal self-assembled crystal monolayer: The SiO2-MnO2 particles obtained in Step 1 and the PSC-Ce6 particles obtained in Step 2 are mixed and dispersed in water to form a mixed solution. After evaporation-induced self-assembly in a closed region, an ordered crystal structure is formed. Then, after heat treatment at 150~200℃ for 5-10 minutes or stabilization treatment with toluene solution, a colloidal self-assembled crystal monolayer, namely BCCe6, is obtained.

3. The preparation method according to claim 2, characterized in that, In step 1, the mass mixing ratio of SiO2 particles, APTES, BSA, and KMnO4 is 5~15:10~100:5~15:0.32~1.

58. The conditions for the amination reaction are as follows: The amination reaction is carried out at a temperature of 18~25℃; The amination reaction takes 8-24 hours; The conditions for the in-situ biomineralization method are as follows: The temperature for the in-situ biomineralization method is 18~25℃; The in-situ biomineralization process takes 20-30 minutes.

4. The preparation method according to claim 2, characterized in that, The percentage of Mn atoms in the SiO2-MnO2 particles obtained in step 1 is 1.5-2.0%, and the Zeta potential is |-12mV|~|-18mV|.

5. The preparation method according to claim 2, characterized in that, The mass mixing ratio of PSC particles, EDC, NHS, and photosensitizer Ce6 in step 2 is 10~20:10~20:20~30:1~2; The conditions for reaction I in step 2 are as follows: The temperature for reaction I is 18~25℃; The reaction time for reaction I is 0.5–4 hours; The conditions for reaction II in step 2 are as follows: The temperature for reaction II is 18~25℃; The reaction time for reaction II is 16-24 hours.

6. The preparation method according to claim 2, characterized in that, The photosensitizer Ce6 loading in the PSC-Ce6 particles obtained in step 2 is 19~20μg / mL, and the Zeta potential is |-22mV|~|-28mV|.

7. The preparation method according to claim 2, characterized in that, The self-assembly conditions in step 3 are as follows: The weight percentage concentration of both types of self-assembled particles is 1%, i.e., 10 mg / ml; The self-assembly temperature is 18~25℃, preferably 23℃; The self-assembly time is 12~48h, preferably 24h.

8. The application of the colloidal self-assembled crystalline monolayer membrane prepared by any one of claims 2 to 7 in the healing of bacterial infection wounds, characterized in that, Colloidal self-assembled crystal monolayer membranes were applied to bacterial wound sites in rats, and then removed after near-infrared irradiation for 5-10 minutes. The topological structure and Mn content of the colloidal self-assembled crystal monolayer membrane surface were analyzed. 2+ Ion release and ROS release can rapidly activate M0 macrophages to polarize into the M1 pro-inflammatory phenotype, eliminate bacterial infection as early as possible, and promote the conversion of newly recruited M0 macrophages into the M2 repair phenotype, thus promoting wound healing.

9. The application according to claim 8, characterized in that, The conditions for near-infrared irradiation are as follows: The wavelength of the near-infrared irradiation is 600~800nm, preferably 660nm; The power density of the near-infrared irradiation is 50~200mW / cm². 2 The preferred value is 100mW / cm 2 ; The near-infrared irradiation time is 5-20 minutes, preferably 15 minutes.

10. The application according to claim 8, characterized in that, Bacterial infection wounds are skin defects caused by Staphylococcus aureus (drug-resistant bacteria) or Escherichia coli (normal bacteria).

Images