Preparation and application of MnO2 nano enzyme modified bionic nanoparticles loaded with Hb and Ce6

Abstract

The invention relates to the technical field of biomedical materials, in particular to a preparation method of MnO2 nano enzyme modified bionic nanoparticles loaded with Hb and Ce6, and the preparation method specifically comprises the following steps: S1, carrying out reduction treatment on bacterial outer membrane vesicles and hemoglobin to expose free sulfydryl; mixing the treated bacterial outer membrane vesicles with hemoglobin, and adding Ce6 for co-incubation, so as to prepare the bacterial outer membrane vesicles coated with an Hb-Ce6 complex; and S2, co-incubating the bacterial outer membrane vesicles coated with the Hb-Ce6 complex prepared in the step S1 and potassium permanganate, and inducing in-situ generation of manganese dioxide to obtain the bionic nanoparticles OMV-coated Hb-Ce6-Mn. The OMV-coated Hb-Ce6-Mn prepared by the invention can be used for killing tumors through combination of chemical power, photopower and immune activation, and has good dispersity and biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to the preparation and application of biomimetic nanoparticles modified with MnO2 nanozymes loaded with Hb and Ce6. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health. In recent years, tumor immunotherapy has shown great promise for clinical application due to its ability to activate the body's immune system and specifically eliminate tumor cells. However, the immunosuppressive properties of the tumor microenvironment often hinder the infiltration of immune cells and suppress the anti-tumor immune response, thus limiting its actual clinical efficacy.

[0003] Immunogenic cell death (ICD), a type of cell death that can induce a strong adaptive immune response, plays a crucial role in promoting antigen presentation, activating T cells, and enhancing tumor immune responses. Studies have shown that various tumor-killing methods, including chemotherapy, radiotherapy, photodynamic therapy (PDT), and chemodynamic therapy (CDT), can induce ICD. CDT relies on transition metal ions to catalyze the generation of highly reactive hydroxyl radicals from endogenous hydrogen peroxide (H2O2) in tumors, thereby killing tumor cells. PDT, on the other hand, uses photosensitizers to generate singlet oxygen under specific wavelengths of light, achieving tumor ablation. However, CDT is limited by insufficient H2O2 levels in tumor tissue, while PDT faces challenges such as hypoxia in solid tumors and limited light penetration depth. Furthermore, the high levels of antioxidant systems within tumor cells (such as glutathione and its peroxidase) can rapidly scavenge reactive oxygen species, weakening the induction efficiency of ICD.

[0004] Therefore, single-modality treatments often only induce limited ICD, failing to trigger a strong and sustained systemic immune response. Currently, there is an urgent need for multimodal combination strategies that synergistically enhance immune activation. The development of nanomedicine provides a significant opportunity to overcome these bottlenecks. Multifunctional nanocarriers can achieve drug delivery, microenvironment regulation, and immune activation on a single platform, thereby significantly improving therapeutic effects. Among these, biologically derived nanoparticles have attracted considerable attention due to their natural biocompatibility and inherent immunomodulatory capabilities.

[0005] Outer membrane vesicles (OMVs) are nanovesicles naturally secreted by Gram-negative bacteria, rich in pathogen-associated molecular patterns, which can efficiently activate immune cells such as dendritic cells and enhance anti-tumor immune responses. OMVs also possess excellent delivery capabilities, natural targeting, and immune adjuvant functions, making them an ideal platform for constructing multimodal ICD combination therapy systems. OMV-based systems hold the promise of integrating CDT, PDT, and immune activation functions within a single carrier. The various reactive oxygen species released by CDT and PDT can synergistically induce ICD in tumor cells; while OMVs further enhance adaptive immune responses by mobilizing the host immune system. The synergistic effect of these three elements achieves multidimensional regulation of the tumor microenvironment and enhances overall tumor-killing efficiency.

[0006] Therefore, developing a multifunctional biomimetic nanosystem with OMV as its core platform, capable of achieving a triple synergistic effect of chemodynamic therapy, photodynamic therapy, and immune activation, will provide an important technical path for breaking through the tumor immunosuppression barrier and improving overall treatment efficacy. Summary of the Invention

[0007] To overcome the shortcomings of tumor microenvironment, such as immunosuppression, hypoxia, and low photosensitivity, this invention designs a method for preparing biomimetic nanoparticles with bacterial outer membrane vesicles as the core platform, loaded with Hb and Ce6 to form a core-shell structure, and simultaneously modified with manganese dioxide (MnO2) nanozymes. The specific preparation steps are as follows: First, a mixture of reduced bacterial outer membrane vesicles, Hb, and photosensitizer Ce6 is mixed and incubated to obtain bacterial outer membrane vesicles (OMV@Hb-Ce6) coated with Hb-Ce6 complex. Then, potassium permanganate is added to OMV@Hb-Ce6 for in-situ incubation to generate biomimetic nanoparticles (OMV@Hb-Ce6-Mn) through in-situ MnO2 generation. The OMV@Hb-Ce6-Mn prepared in this application can kill tumors through a combination of chemodynamics, photodynamics, and immune activation.

[0008] The first objective of this invention is to provide a method for preparing biomimetic nanoparticles modified with MnO2 nanozymes loaded with Hb and Ce6, the preparation method specifically including the following steps: S1. The bacterial outer membrane vesicles and hemoglobin were reduced to expose free sulfhydryl groups; then the treated bacterial outer membrane vesicles were mixed with hemoglobin and Ce6 was added for co-incubation to obtain bacterial outer membrane vesicles coated with Hb-Ce6 complex. S2. The bacterial outer membrane vesicles coated with the Hb-Ce6 complex obtained in step S1 are co-incubated with potassium permanganate to induce the in-situ generation of manganese dioxide, thereby obtaining biomimetic nanoparticles OMV@Hb-Ce6-Mn.

[0009] Furthermore, the specific steps of the reduction treatment are as follows: using a reducing agent to act on bacterial outer membrane vesicles and hemoglobin, wherein the reducing agent is tris(2-carboxyethyl)phosphonic acid hydrochloride and glutathione, respectively.

[0010] The above design disrupts the disulfide bonds between OMV and hemoglobin surface proteins, thereby fully exposing free sulfhydryl groups.

[0011] Preferably, the reduction treatment reaction time is 37 °C and the time is 30 min.

[0012] By limiting the reaction temperature and time of the reduction process, the reduction efficiency is ensured while avoiding irreversible damage to the protein structure of OMV and Hb.

[0013] Preferably, the mass ratio of bacterial outer membrane vesicles to hemoglobin is 1:2.

[0014] The Hb-Ce6 complex formed at this ratio can uniformly coat the surface of OMV, forming a stable core-shell structure.

[0015] Preferably, the incubation temperature in step S1 is 37 °C and the incubation time is 2 h.

[0016] Further, step S2 specifically involves adding a 100 mmol / L potassium permanganate solution to the bacterial outer membrane vesicle solution coated with the Hb-Ce6 complex, stirring for 2 min, then adding a 1 mol / L sodium hydroxide solution, and continuing stirring at room temperature for 30 min to obtain biomimetic nanoparticles OMV@Hb-Ce6-Mn. These steps promote the uniform deposition of MnO2 on the OMV surface.

[0017] Preferably, after obtaining the bacterial outer membrane vesicles coated with the Hb-Ce6 complex in step S1 and the biomimetic nanoparticles OMV@Hb-Ce6-Mn in step S2, they are purified by centrifugation using a 100 kDa MWCO ultrafiltration tube with a centrifugation force of 12,000 × g and a centrifugation time of 15 min.

[0018] Preferably, the photosensitizer Ce6 is introduced into a mixed solution of bacterial outer membrane vesicles and hemoglobin via a solution containing dimethyl sulfoxide (DMSO) and added at pH 7.4 to promote uniform loading of Ce6 in the OMV and Hb complex system and enhance its photodynamic activity.

[0019] The second objective of this invention is to provide the application of biomimetic nanoparticles OMV@Hb-Ce6-Mn in the preparation of antitumor immunotherapy drugs.

[0020] Furthermore, the biomimetic nanoparticles OMV@Hb-Ce6-Mn undergo oxygenation treatment before use, which involves introducing oxygen (O2) for 15 minutes.

[0021] The biomimetic nanoparticles obtained by this invention have good dispersibility and biocompatibility, and have the advantages of improving oxygen in the tumor microenvironment, efficiently generating reactive oxygen species, activating immunity, and having no side effects.

[0022] In summary, the beneficial effects of the present invention are as follows: This invention discloses a method for preparing biomimetic nanoparticles based on bacterial outer membrane vesicles combined with nanozymes and photodynamic therapy. The preparation method specifically includes: first, amplifying and culturing *E. coli*, and isolating bacterial outer membrane vesicles (OMVs) from the collected bacterial solution; then, mixing and incubating a reduced OMVs solution and a Hb solution with a photosensitizer Ce6 to obtain Hb-Ce6 complex-coated bacterial outer membrane vesicles (OMV@Hb-Ce6); finally, co-incubating OMV@Hb-Ce6 with potassium permanganate (KMnO4) and reducing it to obtain biomimetic nanoparticles (OMV@Hb-Ce6-Mn) with bacterial outer membrane vesicles as the core platform, loaded with Hb and Ce6 to form a core-shell structure, and simultaneously surface-modified with manganese dioxide (MnO2) nanozymes.

[0023] The biomimetic nanoparticles OMV@Hb-Ce6-Mn prepared by this invention have an EPR effect and can accumulate at the tumor site. At the same time, the Hb-Ce6 shell can effectively shield the antigen sites on the surface of OMVs, avoid antibody-mediated rapid clearance, prolong the in vivo circulation time and enhance the accumulation effect on tumor tissue.

[0024] The biomimetic nanoparticles OMV@Hb-Ce6-Mn of this application can continuously release Mn in the tumor microenvironment. 2+ And produce oxygen, Mn 2+ Further, it undergoes a Fenton-like reaction with H2O2 to generate hydroxyl radicals (•OH), while Ce6 catalyzes the production of singlet oxygen under 660nm laser irradiation. 1 O2) enables ROS to synergistically induce oxidative damage and death in tumor cells, thereby achieving the goal of killing tumors through a triple function of chemodynamics, photodynamics, and immune activation.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] Figure 1Transmission electron micrographs of OMVs (left), OMV@Hb-Ce6 (middle) and OMV@Hb-Ce6-Mn (right); Figure 2 Particle size (left) and potential (right) of OMVs, OMV@Hb-Ce6 and OMV@Hb-Ce6-Mn; Figure 3 The manganese ion release curve of OMV@Hb-Ce6-Mn in H2O2 environment; Figure 4 The concentration change curve of oxygen generated by OMV@Hb-Ce6-Mn in H2O2 environment; Figure 5 This demonstrates the effect of OMV@Hb-Ce6-Mn generating •OH in an H2O2 environment; Figure 6 Electron spin resonance (ESR) detection for singlet oxygen generated by OMV@Hb-Ce6-Mn; Figure 7 Confocal imaging of ROS generation in 4T1 cells after treatment with different drugs; Figure 8 Flow cytometry analysis of OMV@Hb-Ce6-Mn killing 4T1 tumor cells; Figure 9 The changes in tumor volume in mice of different groups after treatment with different drugs; Figure 10 The activation status of DCs in tumor tissues of mice in different groups after different drug treatments. Detailed Implementation

[0027] To facilitate a clearer understanding of the present invention, it will be further described below with reference to specific embodiments and accompanying drawings. Furthermore, unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0028] Experiment 1: Preparation of biomimetic nanoparticles OMV@Hb-Ce6-Mn

[0029] 1. Preparation of bacterial outer membrane vesicles coated with Hb-Ce6 complex (OMV@Hb-Ce6).

[0030] Prepare Escherichia coli bacterial suspension and hemoglobin. The Escherichia coli (Escherichiacoli BL21) bacterial suspension was ultracentrifuged to obtain bacterial outer membrane vesicles (OMVs). Subsequently, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and glutathione (GSH) were added to 1.5 mg of OMV and 3 mg of hemoglobin (Hb), respectively, and reacted at 37 °C for 30 min to expose the sulfhydryl groups of both.

[0031] Thiol-exposed OMV and Hb were mixed together at a mass ratio of 1:2, and a dimethyl sulfoxide (DMSO) solution (pH 7.4) containing the photosensitizer Ce6 was added to obtain the final mixture. After thorough stirring, the mixture was incubated at 37 °C for 2 h. The mass ratio of Ce6 to OMV and Hb in the final mixture was 6:1:2. After incubation, the mixture was subjected to ultrafiltration and centrifugation (100 kDa MWCO, 12,000 × g, 15 min) to obtain purified Hb-Ce6 complex-coated core-shell bacterial outer membrane vesicles (OMV@Hb-Ce6).

[0032] 2. Preparation of biomimetic nanoparticles OMV@Hb-Ce6-Mn with "chemical dynamics-photodynamics-immune activation" functions.

[0033] The purified OMV@Hb-Ce6 solution was mixed with 200 μL of 100 mmol / L potassium permanganate (KMnO4) and stirred for 2 min. Then, 120 μL of 1 mol / L sodium hydroxide (NaOH) was added, and the mixture was stirred for another 30 min at room temperature to obtain the reaction solution.

[0034] The reaction solution was subjected to ultrafiltration and centrifugation (100 kDa MWCO, 12,000 × g, 15 min) to obtain biomimetic nanoparticles (OMV@Hb-Ce6-Mn) with bacterial outer membrane vesicles as the core platform, loaded with Hb and Ce6 to form a core-shell structure, and surface modified with manganese dioxide (MnO2) nanozymes.

[0035] The morphology of OMVs, OMV@Hb-Ce6, and OMV@Hb-Ce6-Mn was observed by transmission electron microscopy, and the results are as follows: Figure 1 As shown, all three types of nanoparticles exhibit typical spherical vesicle structures with regular morphology and clear background.

[0036] Dynamic light scattering (DLS) was used to determine the particle size and zeta potential of three types of nanoparticles. The results are as follows: Figure 2As shown, the particle size distribution of the three nanoparticles is mainly between 20-200 nm, with an average particle size of approximately 90 nm. Among them, the OMVs have a particle size of approximately 50 nm and a Zeta potential of approximately -20 mV. The average particle size of OMV@Hb-Ce6 and OMV@Hb-Ce6-Mn is approximately 80 nm, and their Zeta potentials are approximately -24 mV. The above results demonstrate the successful preparation of biomimetic nanoparticles (OMV@Hb-Ce6-Mn) with bacterial outer membrane vesicles as the core platform, loaded with Hb and Ce6 to form a core-shell structure, and surface-modified with manganese dioxide (MnO2) nanozymes.

[0037] Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to determine the amount of Mn released from biomimetic nanoparticles in an acidic buffer solution (pH 6.5). 2+ Content, the results are as follows Figure 3 As shown, OMV@Hb-Ce6-Mn can continuously release Mn under acidic conditions. 2+ Furthermore, the release of Mn increases significantly in the presence of H2O2. Dissolved oxygen analyzers were used to detect Mn. 2+ The release of oxygen during the generation process, and the results are as follows: Figure 4 As shown, OMV@Hb-Ce6-Mn can continuously generate oxygen in an acidic buffer solution containing H2O2. The ability of the biomimetic nanoparticles to catalyze the generation of hydroxyl radicals (•OH) from H2O2 was detected using a methylene blue (MB) probe, and the results are as follows. Figure 5 As shown, the absorbance of MB in the OMV@Hb-Ce6-Mn treated group gradually decreased in a solution containing H2O2, indicating that MB was degraded by •OH. Electron spin resonance (ESR) spectroscopy was used to detect the degradation. 1 The generation of O2 results in the following: Figure 6 As shown, OMV@Hb-Ce6-Mn solution was observed to contain [unclear - likely a typo, should be "660 nm laser irradiation"]. 1 O2 signal. The above results indicate that OMV@Hb-Ce6-Mn can continuously release Mn by reacting with H2O2 in the slightly acidic tumor microenvironment. 2+ And produce oxygen, Mn 2+ It can further undergo a Fenton-like reaction with H2O2 to generate hydroxyl radicals (•OH), while Ce6 catalyzes the production of singlet oxygen under 660 nm laser irradiation. 1 O2).

[0038] Experiment 2: Study on the biological functions of biomimetic nanoparticles OMV@Hb-Ce6-Mn

[0039] 1. To investigate the ability of biomimetic nanoparticles to inhibit tumor cell growth in vitro.

[0040] Following the experimental method in Experiment 1, four types of nanoparticles, namely OMV@Hb, OMV@Hb-Ce6, OMV@Hb-Mn and OMV@Hb-Ce6-Mn, were prepared respectively. Oxygen was introduced into the nanoparticles for 10-20 min before use.

[0041] Tumor cells were prepared and randomly divided into five groups, which were treated with the following drugs: PBS (control group), OMV@Hb, OMV@Hb-Ce6, OMV@Hb-Mn, and OMV@Hb-Ce6-Mn. All groups were incubated with nanoparticles at 200 μg / mL (based on protein concentration) for 12 h (37℃), while the control group was incubated with an equal volume of PBS for 12 h (37℃). Subsequently, H2O2 was added to all groups at a final concentration of 100 μmol / L, and the cells were immediately irradiated with a 660 nm laser (power density 100 mW / cm²) for 1 min. After irradiation, the level of reactive oxygen species (ROS) generated in the tumor cells of each group was detected using the ROS fluorescent probe DCFH-DA.

[0042] The fluorescent probe DCFH-DA can penetrate the cell membrane and enter the cell. It is then converted into non-fluorescent DCFH by intracellular esterases and subsequently oxidized by ROS to become highly fluorescent DCF, which emits green fluorescence under a fluorescence microscope.

[0043] Experimental results are as follows Figure 7 As shown, the OMV@Hb-Ce6-Mn group exhibited the highest ROS fluorescence intensity, significantly higher than both the PDT (OMV@Hb-Ce6) and CDT (OMV@Hb-Mn) single groups, indicating that these biomimetic nanoparticles can be transmitted through Mn. 2+ It participates in a Fenton-like reaction (CDT) to produce •OH, while Ce6 catalyzes the production of… under laser irradiation. 1 O2 (PDT) and two mechanisms work together to induce a large accumulation of ROS.

[0044] 2. Study on the effect of biomimetic nanoparticles on killing 4T1 tumor cells in vitro.

[0045] Five treatment groups were set up: PBS (control group), OMV@Hb, OMV@Hb-Ce6, OMV@Hb-Mn, and OMV@Hb-Ce6-Mn. Each group of proteins (concentration of 200 μg / mL) was co-incubated with 4T1 tumor cells at 37℃ for 12 h. The control group was incubated with an equal volume of PBS (at 37℃) for 12 h. Then, H2O2 was added to each treatment group to a final concentration of 100 μmol / L, followed by irradiation with a 660 nm laser for 1 min. After irradiation, cells in each group were stained using an Annexin V-FITC / PI double staining kit, and the apoptosis rate was detected by flow cytometry.

[0046] The result is as follows Figure 8 As shown, all treatment groups exhibited certain cytotoxicity. Among them, the OMV@Hb-Ce6-Mn treatment group had the highest apoptosis rate (84.1%), which was significantly better than the PDT single group (OMV@Hb-Ce6) and the CDT single group (OMV@Hb-Mn). This indicates that the biomimetic nanoparticles OMV@Hb-Ce6-Mn can significantly induce tumor cell apoptosis through a synergistic ROS-mediated mechanism, demonstrating an enhanced tumor cell killing effect.

[0047] Experiment 3: Study on the in vivo antitumor effect of biomimetic nanoparticles OMV@Hb-Ce6-Mn

[0048] With the approval of the Laboratory Animal Management Ethics Committee of Longgang Central Hospital (Approval No.: Longer Medical Animal Ethics (2022) No. 0334), laboratory animals were used in this experiment.

[0049] A breast cancer tumor-bearing model was established in Balb / c mice. The tumor-bearing mice were randomly divided into 5 groups. Each group received a drug via tail vein injection: PBS (control group), OMV@Hb, OMV@Hb-Ce6, OMV@Hb-Mn, and OMV@Hb-Ce6-Mn. The injection volume was 100 μL, and the drug dose was 100 μg per mouse. The control group was incubated with an equal volume of PBS (at 37℃) for 12 h. Six hours after each injection, the tumor site was irradiated with a 660 nm laser for 5 min at a power density of 100 mW / cm². Treatment was administered every 3 days for a total of 5 times. Tumor growth was observed and recorded in each group.

[0050] Experimental results are as follows Figure 9 As shown, the tumor volume in the PBS group mice continued to grow, reaching more than 500 mm³ on day 20; in contrast, the tumor growth in the OMV@Hb-Ce6-Mn group was significantly inhibited, with better results than the other treatment groups, indicating that it has good tumor-suppressing activity in vivo.

[0051] Three days after the last administration, the mice were euthanized, and tumor tissue was harvested to prepare a single-cell suspension for flow cytometry analysis. Results are as follows: Figure 10 As shown, the activation rate of DCs in the PBS group was less than 5%, while the activation rate of DCs in the OMV@Hb-Ce6-Mn treatment group was significantly increased, reaching approximately 15%. These results indicate that the biomimetic nanoparticles OMV@Hb-Ce6-Mn exhibit excellent immune activation capabilities and synergistic therapeutic effects in vivo.

[0052] The embodiments described above are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing biomimetic nanoparticles modified with MnO2 nanozymes loaded with Hb and Ce6, characterized in that, The preparation method specifically includes the following steps: S1. The bacterial outer membrane vesicles and hemoglobin were reduced to expose free sulfhydryl groups; then the treated bacterial outer membrane vesicles were mixed with hemoglobin and Ce6 was added for co-incubation to obtain bacterial outer membrane vesicles coated with Hb-Ce6 complex. S2. The bacterial outer membrane vesicles coated with the Hb-Ce6 complex obtained in step S1 are co-incubated with potassium permanganate to induce the in-situ generation of manganese dioxide, thereby obtaining biomimetic nanoparticles OMV@Hb-Ce6-Mn.

2. The preparation method according to claim 1, characterized in that, The specific steps of the reduction treatment are as follows: using a reducing agent to act on bacterial outer membrane vesicles and hemoglobin, wherein the reducing agent is tris(2-carboxyethyl)phosphonic acid hydrochloride and glutathione.

3. The preparation method according to claim 2, characterized in that, The reduction treatment reaction time is 37 °C for 30 min.

4. The preparation method according to claim 1, characterized in that, The mass ratio of bacterial outer membrane vesicles to hemoglobin is 1:

2.

5. The preparation method according to claim 4, characterized in that, The incubation temperature in step S1 is 37 ℃ and the incubation time is 2 h.

6. The preparation method according to claim 1, characterized in that, The S2 step specifically involves adding a 100 mmol / L potassium permanganate solution to the bacterial outer membrane vesicle solution coated with the Hb-Ce6 complex, stirring for 2 min, then adding a 1 mol / L sodium hydroxide solution, and continuing to stir at room temperature for 30 min to obtain biomimetic nanoparticles OMV@Hb-Ce6-Mn.

7. The preparation method according to any one of claims 1-6, characterized in that, After obtaining the bacterial outer membrane vesicles coated with the Hb-Ce6 complex in step S1, and after obtaining the biomimetic nanoparticles OMV@Hb-Ce6-Mn in step S2, both were purified by centrifugation using a 100 kDa MWCO ultrafiltration tube at a centrifugation force of 12,000 × g for 15 min.

8. The use of the biomimetic nanoparticles OMV@Hb-Ce6-Mn prepared by any one of claims 1-7 in the preparation of antitumor immunotherapy drugs.

9. The application according to claim 8, characterized in that, The biomimetic nanoparticles OMV@Hb-Ce6-Mn are oxygenated to load or carry oxygen.

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