Optoacoustic dual-response cn microalgae nanomaterial and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine nanomaterials, and specifically discloses a photoacoustic dual-response CN microalgae nanomaterial, a preparation method and application thereof, and the material comprises a photosynthetic microalgae carrier and CN nanoparticles loaded on the surface of the photosynthetic microalgae carrier; the CN nanoparticles are cobalt telluride nanoparticles modified by amino double-end polyethylene glycol, and the cobalt telluride nanoparticles have sound heat conversion performance and thermoelectric catalytic performance; and the photosynthetic microalgae carrier is a living microalgae with photosynthetic oxygen production function. The application adopts the photoacoustic dual-response CN microalgae nanomaterial, the preparation method and application thereof, the nanomaterial uses living photosynthetic microalgae as a biological carrier and an in-situ oxygen production unit, loads cobalt telluride nanoparticles modified by amino double-end polyethylene glycol, forms a photoacoustic dual-response composite treatment system, and can realize efficient treatment of metastatic melanoma through multi-mechanism synergy.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a photoacoustic dual-response CN microalgae nanomaterial, its preparation method and application. Background Technology

[0002] Metastatic melanoma is a clinically recognized highly aggressive and easily metastatic malignant skin tumor, and it is also one of the most lethal skin cancers. Currently, complete surgical resection remains the preferred core treatment for melanoma. However, most patients present with subcutaneous microinvasive lesions and occult micrometastases, making it difficult to completely remove all tumor tissue during surgery. Residual microtumor lesions can become the source of postoperative recurrence and distant metastasis, which is the core contributing factor to the high incidence of tumor-related deaths in patients with advanced melanoma.

[0003] While conventional clinical treatments often employ local surgical resection combined with systemic chemotherapy, targeted drugs, or various adjuvant therapies to reduce the probability of postoperative tumor recurrence and metastasis, existing comprehensive treatment systems still have significant shortcomings, making it difficult to effectively curb the proliferation of residual tumor cells and distant dissemination after surgery. Therefore, achieving efficient removal of melanoma lesions in situ, long-term control of postoperative recurrence and metastasis, and simultaneously enhancing tumor immunogenicity and systematically modifying the suppressive tumor microenvironment (TME) are key scientific challenges that urgently need to be addressed in the current clinical treatment of metastatic melanoma.

[0004] Reactive oxygen species (ROS), as core signaling molecules regulating cellular redox homeostasis, participate in a series of pathological processes, including tumorigenesis, proliferation, invasion, and metastasis. When intracellular ROS accumulates to a critical concentration, it directly attacks key biomolecules such as nucleic acids, structural proteins, and membrane lipids, completely disrupting cellular redox homeostasis and initiating oxidative stress-mediated tumor cell apoptosis or necrosis. Compared to normal tissue cells, tumor cells are chronically under high oxidative stress and are more sensitive to ROS overload damage. Based on this biological characteristic, targeted regulation of local ROS levels in tumors can precisely and selectively kill tumor tissue with lower toxicity to surrounding healthy tissues. In recent years, various nanodynamic therapy platforms built based on energy conversion mechanisms have been developed to directionally enhance the efficiency of ROS generation within lesions, significantly reducing systemic toxicity while strengthening anti-tumor efficacy, attracting widespread attention in the scientific research field.

[0005] Thermoelectric functional materials can sense local temperature fluctuations and efficiently convert thermal energy into electrical energy, thereby catalyzing the conversion of molecular oxygen into a large amount of ROS, providing a new approach for tumor treatment. However, singlet oxygen (… 1The efficient generation of ROS (reactive oxygen species) such as oxygen (O2) depends on a sufficient supply of oxygen (O2) to the tumor site. However, solid tumors generally have a hypoxic microenvironment, which can limit the therapeutic effect of oxygen-dependent ROS. To further amplify the efficiency of ROS generation and enhance the therapeutic effect, the photosynthetic microorganism Chlorella vulgaris (CV) has come into the researchers' view. Under continuous irradiation with a 660nm laser, Chlorella vulgaris can stably carry out photosynthetic reactions, continuously releasing oxygen in situ at the tumor lesion, effectively alleviating the hypoxic inhibitory microenvironment, and continuously replenishing O2 substrates, thus becoming an oxygen-dependent ROS generator. 1 The large-scale generation of O2 provides ample raw material support.

[0006] Besides ROS oxidative killing, the tumor microenvironment contains various stromal cells and immune cells, among which tumor-associated macrophages (TAMs) are the most prevalent and crucial immune cell subset, deeply involved in the entire process of tumor proliferation, invasion, metastasis, and the construction of the immunosuppressive microenvironment. Based on activation phenotype and functional differences, TAMs are mainly divided into M1-like macrophages and M2-like macrophages; M2-like TAMs are the dominant group. These macrophages can secrete large amounts of matrix proteases, immunosuppressive cytokines, and tumor growth factors, comprehensively helping tumor cells evade immune system surveillance and killing. They are a core barrier hindering the establishment of anti-tumor immune responses, thus becoming a highly promising target for immunotherapy. Previous studies have confirmed that compared to other antigen-presenting cells such as dendritic cells, M2-type tumor-associated macrophages have significantly higher cysteine ​​protease activity in their lysosomes. Effectively inhibiting this protease activity can significantly improve the efficiency of tumor antigen cross-presentation and fully activate cytotoxic CD8. + T cell-mediated specific anti-tumor immunity. Meanwhile, the tumor microenvironment is rich in L-cysteine ​​(L-Cys), a metabolite that not only meets the metabolic demands of rapid tumor cell proliferation but also continuously drives macrophage polarization towards the immunosuppressive M2 phenotype. Therefore, targeted intervention of the cysteine ​​metabolic pathway in the tumor microenvironment has become an important research direction for reshaping the tumor immune microenvironment.

[0007] In the prior art, for example, the invention patent application with application number CN202510821593.6 discloses a nanoengineered microalgae for photoimmunotherapy of melanoma. The resulting functionalized microalgae drug delivery system, under 635nm red light irradiation, produces oxygen (O2) and H2 through photosynthesis and photocatalysis, respectively, to address the hypoxic tumor microenvironment and activate the body's immunity. However, this system relies on only a single photoresponse mode, has limited tissue penetration depth, and is difficult to effectively cover deep metastatic lesions; moreover, it lacks the ability to actively regulate the metabolic pathways of the tumor microenvironment, and its anti-tumor immune activation effect needs to be improved.

[0008] Therefore, it is of great significance to develop a multifunctional integrated treatment system that can simultaneously achieve efficient killing of deep tumors, continuous improvement of the tumor hypoxic microenvironment, significant improvement of ROS generation efficiency, and systemic activation of the anti-tumor immune response. Summary of the Invention

[0009] The purpose of this invention is to provide a photoacoustic dual-response CN microalgae nanomaterial, its preparation method and application. This nanomaterial uses living photosynthetic microalgae as a biological carrier and in-situ oxygen production unit, and loads cobalt telluride nanoparticles modified with amino-terminated polyethylene glycol to form a photoacoustic dual-response composite therapeutic system, which can achieve highly efficient treatment of metastatic melanoma through multi-mechanism synergy.

[0010] To achieve the above objectives, the present invention provides a photoacoustic dual-response CN microalgae nanomaterial, comprising a photosynthetic microalgae carrier and CN nanoparticles loaded on the surface of the photosynthetic microalgae carrier; the CN nanoparticles are obtained by modifying cobalt telluride nanoparticles with amino-terminated polyethylene glycol, and the cobalt telluride nanoparticles have acoustic-thermal conversion properties and thermoelectric catalytic properties; the photosynthetic microalgae carrier is a living microalgae with photosynthetic oxygen production function.

[0011] Preferably, the cobalt telluride nanoparticles have an average particle size of 80~100nm and a Zeta potential of +2mV to +7mV; the amino-terminated polyethylene glycol is NH2-PEG-NH2 with a molecular weight of 2000~5000Da.

[0012] Preferably, the hydrated particle size of the CN nanoparticles is 90~150nm, and the Zeta potential is +10mV to +30mV.

[0013] Preferably, the photosynthetic microalgae carrier is Chlorella vulgaris; the CN nanoparticles are loaded onto the surface of Chlorella vulgaris through electrostatic adsorption.

[0014] The CN nanoparticles also possess catalase-like and L-cysteine ​​oxidase-like activities. The photoacoustic dual-response CN microalgae nanomaterials can generate acoustic-thermal and thermo-electrocatalytic effects under ultrasonic stimulation, producing singlet oxygen. Under 660nm laser irradiation, the photosynthetic microalgae carrier can produce oxygen through photosynthesis.

[0015] This invention also provides a method for preparing photoacoustic dual-response CN microalgae nanomaterials, comprising the following steps: S1. Cobalt telluride nanoparticles were prepared by high-temperature pyrolysis. S2. The cobalt telluride nanoparticles obtained in S1 were surface-modified with amino-terminated polyethylene glycol to obtain CN nanoparticles. S3. The CN nanoparticles obtained in S2 are incubated and loaded with photosynthetic microalgae to obtain photoacoustic dual-response CN microalgae nanomaterials.

[0016] Preferably, S1 is as follows: Cobalt precursor and tellurium precursor were mixed in an oleylamine system and reacted at 280-320℃ for 50-70 min. After centrifugation, washing and vacuum drying, cobalt telluride nanoparticles were obtained. The cobalt precursor was prepared by reacting cobalt(II) acetylacetone in oleylamine at 160-200°C for 20-40 min. The tellurium precursor was prepared by dissolving tellurium particles in trioctylphosphine at 120-160°C.

[0017] Preferably, S2 is as follows: Cobalt telluride nanoparticles were dispersed in ultrapure water and sonicated for 20-40 min. NH2-PEG-NH2 was added in the same mass ratio as the cobalt telluride nanoparticles, and the mixture was stirred at room temperature in the dark for 10-14 h. CN nanoparticles were obtained by centrifugation, washing, and freeze-drying.

[0018] Preferably, S3 is as follows: Take photosynthetic microalgae in the logarithmic growth phase, add CN nanoparticles, make up to volume with sterile water, and incubate in a constant temperature shaker for 1-2 hours under light-protected conditions; centrifuge to remove supernatant, resuspend and wash with sterile water to obtain photoacoustic dual-response CN microalgae nanomaterials.

[0019] The present invention also provides the application of photoacoustic dual-response CN microalgae nanomaterials in the preparation of antitumor drugs, wherein the tumor is metastatic melanoma.

[0020] Preferably, the photoacoustic dual-response CN microalgae nanomaterials exert antitumor effects under the synergistic stimulation of ultrasound and laser; the ultrasound frequency is 1MHz and the power is 0.3~1.0W / cm². 2 The laser has a wavelength of 660nm and a power of 0.5~2.0W / cm². 2 .

[0021] Preferably, the antitumor effect includes at least one of the following mechanisms: Acoustic ablation directly kills tumor cells; Thermoelectrocatalysis generates reactive oxygen species that induce oxidative damage in tumor cells; Photosynthetic oxygen production and catalase-like activity synergistically alleviate the hypoxic microenvironment of tumors; L-cysteine ​​oxidase activity consumes L-cysteine ​​in the tumor microenvironment and inhibits glutathione synthesis. It promotes the polarization of tumor-associated macrophages from M2 to M1, thereby activating the anti-tumor immune response.

[0022] Therefore, the present invention employs the above-mentioned photoacoustic dual-response CN microalgae nanomaterial, its preparation method, and its application, with the following beneficial effects: (1) This invention constructs a photo-acoustic dual-response nanoengineered microalgae therapy system, which combines acoustic-thermal nanoparticles (CN) with photosynthetic live Chlorella vulgaris (CV) to achieve synergistic antitumor effects through multiple mechanisms. Under ultrasound stimulation, CN nanoparticles can generate heat locally in the tumor and produce periodic temperature fluctuations. On the one hand, this directly causes thermal damage to the tumor through acoustic-thermal therapy; on the other hand, it continuously and efficiently generates heat through thermoelectric catalytic reactions. 1 O2 induces oxidative damage that kills tumor cells.

[0023] (2) This invention utilizes the photosynthetic oxygen production function of Chlorella and the catalase-like activity of CN nanoparticles to form a dual oxygen supply system. Chlorella can continuously release oxygen through photosynthesis under 660nm laser irradiation, while CN nanoparticles can decompose hydrogen peroxide accumulated in the tumor microenvironment to produce oxygen in situ. The two work synergistically to effectively alleviate the hypoxic microenvironment of the tumor, making it an oxygen-dependent system. 1 The abundant generation of O2 provides sufficient substrate, significantly enhancing thermoelectric catalytic efficiency.

[0024] (3) The CN nanoparticles of the present invention have both L-cysteine ​​oxidase-like catalytic activity and can specifically consume free L-cysteine ​​in the tumor microenvironment, disrupt the redox metabolic balance between tumor cells and macrophages, and promote the polarization and enrichment of tumor-associated macrophages from immunosuppressive M2 type to anti-tumor M1 type; at the same time, by downregulating the activity of lysosomal cysteine ​​protease, they enhance the antigen cross-presentation function of M2-like macrophages and fully activate CD8 through multiple pathways. + T-cell-led systemic anti-tumor immune response.

[0025] (4) The preparation method of the present invention is simple and controllable. The resulting composite nanomaterial has good water dispersibility, colloidal stability and biocompatibility. It can simultaneously inhibit the growth of primary and metastatic melanoma lesions and induce long-term systemic anti-tumor immune response, providing a new treatment strategy for the comprehensive prevention and treatment of metastatic malignant melanoma.

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0027] Figure 1 This is a scanning electron microscope (SEM) image of the CoTe nanoparticles prepared in Example 1 of the present invention; Figure 2The X-ray powder diffraction (XRD) pattern of the CoTe nanoparticles prepared in Example 1 of this invention; Figure 3 This is a scanning electron microscope (SEM) image of CN nanoparticles prepared in Example 1 of the present invention; Figure 4 This is a comparison of the dynamic light scattering (DLS) particle sizes of CoTe nanoparticles and CN nanoparticles prepared in Example 1 of this invention. Figure 5 The Zeta potential comparison diagram of CoTe nanoparticles and CN nanoparticles prepared in Example 1 of the present invention (n=3). Figure 6 The images are scanning electron microscope (SEM) images of the CV Chlorella and CV@CN composite material prepared in Example 1 of the present invention, wherein (a) is CV Chlorella and (b) is the CV@CN composite material; Figure 7 The image shows the energy dispersive spectroscopy (EDS) elemental analysis of the CV@CN composite nanomaterial prepared in Example 1 of this invention, where (a) is a SEM image, (b) is C element, (c) is O element, (d) is Co element, and (e) is Te element. Figure 8 The Fourier transform infrared (FTIR) spectra of CV, CN, and CV@CN prepared in Embodiment 1 of the present invention are shown. Figure 9 A comparison diagram of the zeta potentials of CV, CN, and CV@CN prepared in Example 1 of the present invention (n=3); Figure 10 A comparison of the dynamic light scattering (DLS) particle sizes of CV, CN, and CV@CN prepared in Example 1 of this invention; Figure 11 Ultrasonic heat generation curves of CN nanoparticles at different concentrations prepared in Example 1 of the present invention (n=3). Figure 12 The image shows the acoustic and thermal cycling stability test results of CN nanoparticles prepared in Example 1 of this invention. Figure 13 Comparison of acoustic and thermal properties of CV, CN and CV@CN prepared in Example 1 of the present invention (n=3); Figure 14 Comparison of oxygen production capacity of Chlorella vulgaris prepared in Example 1 of the present invention under different wavelength light sources (n=3). Figure 15 The oxygen production capacity of CV@CN prepared in Example 1 of the present invention under different power 660nm laser irradiation (n=3) is shown. Figure 16A comparison of the oxygen production capacity of CV, CN and CV@CN prepared in Example 1 of the present invention under 660nm laser (1.0W) irradiation (n=3). Figure 17 The pyroelectric current detection diagrams of CN and CV@CN prepared in Embodiment 1 of the present invention are shown. Figure 18 The degradation diagrams (n=3) of DPBF by CV, CN and CV@CN prepared in Example 1 of the present invention under different stimulation conditions are shown, where (a) is CV, (b) is CN and (c) is CV@CN; Figure 19 The electron spin resonance (ESR) detection images of CV, CN, and CV@CN prepared in Embodiment 1 of the present invention are shown. Figure 20 The graphs (n=3) show the catalytic oxygen production capacity of CV@CN prepared in Example 1 of this invention under different pH conditions, where (a) is pH=5.5, (b) is pH=6.5, and (c) is pH=7.4. Figure 21 A summary chart of the catalytic oxygen production capacity of CV@CN prepared in Example 1 of the present invention under different pH conditions (n=3). Figure 22 The graph shows the degradation capacity of CV@CN prepared in Example 1 of this invention for L-cysteine ​​under different pH conditions, where (a) is pH=5.5, (b) is pH=6.5, and (c) is pH=7.4. Figure 23 A summary chart of the L-cysteine ​​degradation capacity of CV@CN prepared in Example 1 of the present invention under different pH conditions (n=3). Figure 24 This is a graph showing the enhancement of L-cysteine ​​degradation by CV@CN under different pH conditions under ultrasonic stimulation according to the present invention; Figure 25 The cytotoxicity test results of CN nanoparticles prepared in Example 1 of this invention are shown in the figure (n=3). Figure 26 The cytotoxicity test diagram of Chlorella vulgaris prepared in Example 1 of the present invention (n=3); Figure 27 The cytotoxicity test diagram of CV@CN prepared in Example 1 of the present invention (n=3); Figure 28 The killing effect of CV@CN on B16F10 tumor cells under different treatment conditions is shown in Example 1 of this invention (n=3, P<0.05, P<0.01, P<0.001). Figure 29The images show fluorescence images of B16F10 cells prepared by CV@CN under different treatment conditions in Example 1 of this invention, where (a) is the detection of ROS generation by DCFH-DA staining, and (b) is the detection of live / dead cells by Calcein-AM / PI staining. Figure 30 These are representative images of tumor resection in mice in different treatment groups using CV@CN prepared in Example 1 of the present invention. Figure 31 This is a bioluminescence imaging image of CV@CN prepared in Example 1 of the present invention in a melanoma brain metastasis model; Figure 32 This is a photograph of a solid tumor in a melanoma brain metastasis model prepared by CV@CN in Example 1 of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0030] Example 1 A photoacoustic dual-response CN microalgae nanomaterial is prepared by the following method: (1) Preparation of cobalt telluride (CoTe) nanoparticles: In this embodiment, a high-temperature pyrolysis method was used, and the specific steps are as follows: Take a clean three-necked flask, add 20 mL of oleylamine solution, then add 1 mmol of cobalt(II) acetylacetonate, and stir thoroughly to disperse it evenly; continuously introduce argon gas into the system as a protective gas to remove air and prevent oxidation of the raw materials and side reactions; raise the system temperature to 180 °C at a uniform heating rate, and react at a constant temperature for 30 min to obtain a uniform and stable Co precursor solution.

[0031] Weigh 1 mmol of Te particles and add them to 3 mL of trioctylphosphine (TOP) solution. After mixing thoroughly, transfer the mixture to an oil bath at 140 °C and stir magnetically for 30 min until the Te particles are completely dissolved, forming a transparent and homogeneous Te precursor solution.

[0032] After the Co precursor solution is prepared, the prepared Te precursor solution is slowly added to the three-necked flask, and the dropping rate is controlled to prevent the local concentration from being too high. After the dropping is completed, the system temperature is raised to 300℃ at a uniform heating rate and the reaction is kept at a constant temperature for 60 min to ensure that the crystal grows fully and the structure is perfect.

[0033] After the reaction was completed, heating was stopped and the system was allowed to cool naturally to room temperature. The reaction product was transferred to a centrifuge tube and washed by alternating centrifugation with cyclohexane and anhydrous ethanol at 11,000 rpm for 10 min. The washing was repeated 3 to 5 times to thoroughly remove unreacted raw materials, residual oleylamine, and surfactants. The washed product was placed in a vacuum drying oven and vacuum dried for 24 h to finally obtain dry and pure CoTe nanoparticles.

[0034] (2) Preparation of CN nanoparticles: In view of the defects of pure CoTe nanoparticles in aqueous environment, such as easy aggregation and sedimentation, poor dispersion stability and insufficient biocompatibility, this embodiment uses NH2-PEG-NH2 as a surface modification agent to coat and modify the surface of CoTe nanoparticles, thereby improving the water dispersion performance and biocompatibility of nanomaterials. The specific steps are as follows: The dried CoTe nanoparticles were accurately weighed and uniformly dispersed in ultrapure water to prepare a CoTe nanoparticle aqueous dispersion with a concentration of 1 mg / mL. The prepared dispersion was then subjected to ultrasonic treatment in an ultrasonic homogenizer for 30 minutes. The mechanical vibration of ultrasound effectively broke down the agglomeration forces between CoTe nanoparticles, dismantling their aggregated structure and allowing the originally easily agglomerated CoTe nanoparticles to be fully dispersed in the aqueous solution. This resulted in a uniform, stable, and non-agglomerated CoTe aqueous dispersion, providing a good reaction basis for subsequent surface modification reactions.

[0035] The CoTe aqueous dispersion was transferred to a clean beaker and continuously stirred at a constant speed on a magnetic stirrer to ensure uniform concentration and stability of the system. Then, NH2-PEG-NH2 was slowly added to the dispersion in a ratio equal to the mass of the CoTe nanoparticles. Continuous stirring promoted the full diffusion of the surfactant molecules, ensuring they fully contacted and bound to the active sites on the CoTe nanoparticle surface, and facilitating interfacial bonding reactions through coordination interactions. The entire reaction system was placed in a dark environment at room temperature and stirred continuously for 12 hours to ensure the surfactant molecules were fully modified and firmly bound to the CoTe nanoparticle surface, ultimately forming a uniform and stable modified layer.

[0036] After the reaction was completed at room temperature in the dark with stirring, the resulting mixture was centrifuged at 7000 rpm for 10 minutes, and the lower solid precipitate was collected. The precipitate was then washed and centrifuged repeatedly with ultrapure water, three times in total, to thoroughly remove unbound, free, residual amino-modified PEG molecules and other impurities, preventing interference from free reagents on the properties of the modified material. The final purified solid product was freeze-dried to completely remove moisture, yielding the amino-modified PEG-coated CoTe nanomaterial, labeled CN.

[0037] (3) Preparation of CV@CN composite nanomaterials: In this embodiment, natural microalgae Chlorella (CV) is used as a biological carrier. Relying on the unique surface physicochemical properties of Chlorella, combined with the physical adsorption effect and electrostatic interaction between nanomaterials and microalgae cells, CN nanomaterials are efficiently fixed and loaded on the surface of Chlorella. The preparation process is mainly divided into two core stages: microalgae domestication culture and gradient loading incubation. The specific steps are as follows: First, purified Chlorella vulgaris (species number FACHB-2338) was inoculated into a specialized liquid culture medium. Key parameters of the culture environment, such as light intensity, temperature, and aeration rate, were strictly controlled to provide suitable and stable culture conditions for the proliferation and growth of Chlorella vulgaris. Throughout the culture period, the optical density (OD value) of the algal solution was sampled and measured periodically to dynamically monitor the growth and proliferation status of Chlorella vulgaris. When the OD value of the algal solution stabilized at 1.0, it indicated that Chlorella vulgaris had entered the vigorous growth phase with optimal activity. At this point, a microalgal stock solution with excellent growth and high activity was selected as the substrate material for subsequent nanomaterial loading experiments.

[0038] Secondly, after completing the microalgae culture pretreatment, a gradient loading incubation experiment of CN nanomaterials on the surface of Chlorella vulgaris was conducted. 1 mL of Chlorella vulgaris stock solution in excellent growth condition was precisely measured, and a fixed amount of CN nanomaterials was added. Then, sterile pure water was slowly added to adjust the volume of the reaction system to 10 mL, ensuring uniform concentration and a pure environment. The prepared algae-nanomaterial mixture was placed in a constant-temperature shaker and incubated in a dark environment for 1.5 hours. During the dark incubation, the loading reaction was completed using electrostatic adsorption within the system: Chlorella vulgaris exhibits a negative charge in aqueous solution, while the CN nanoparticles carry a positive charge. The two can rapidly combine through strong electrostatic interaction, allowing the free CN nanoparticles to be fully adsorbed and fixed on the surface of Chlorella vulgaris cells, achieving efficient loading of nanomaterials.

[0039] Next, after the incubation and loading reaction was complete, the mixed system was centrifuged to remove the clear supernatant, thus completely removing any unreacted and dispersed CN nanomaterials. The lower solid precipitate was collected and resuspended in sterile pure water. Finally, the purified composite precipitate was resuspended in 1 mL of sterile water and stored in a sealed container at 4°C for later use. Through the complete process of cultivation, loading, purification, and storage described above, the CV-loaded CN nanoparticle composite nanomaterial from Chlorella vulgaris was successfully prepared and labeled as CV@CN.

[0040] Test 1. Structural characterization of the CoTe nanoparticles obtained in Example 1: like Figure 1 As shown, the prepared CoTe nanoparticles exhibit a regular spherical structure; the particle size distribution is concentrated, with an average particle size of approximately 90 nm, and the size uniformity is good, with no large-area aggregation, which meets the ideal size range for nanomaterials to be used for in vivo delivery, tumor penetration, and cellular uptake.

[0041] like Figure 2 As shown, the characteristic diffraction peak positions and intensities of the CoTe nanoparticles perfectly match those of the standard card, with no impurities or shifts, indicating that the product has high purity, excellent crystallinity, and a single phase.

[0042] 2. Structural characterization of the CN nanoparticles obtained in Example 1: like Figure 3 As shown, unmodified CoTe nanoparticles exhibit some particle aggregation and poor overall dispersion; while CN nanoparticles modified with amino-modified PEG have a more uniform particle distribution, significantly improved interparticle aggregation, and exhibit superior dispersion characteristics.

[0043] like Figure 4 As shown, the hydrated particle size of CN nanoparticles increased slightly compared to the original CoTe nanoparticles. This was mainly due to the successful modification and coating of the CoTe nanoparticles with amino-modified PEG molecules, which formed a stable polymer coating layer on the outer layer of the particles and increased the hydrodynamic radius of the nanoparticles.

[0044] like Figure 5 As shown, pure CoTe nanoparticles exhibit only a weak positive charge on their surface, with a low potential value. In contrast, the positive charge intensity of CN nanoparticles after surfactant coating modification is significantly improved. This is because the surfactant molecules carry a large number of positively charged amino active groups, which can effectively improve the surface charge state of the particles after successful coating on the material surface.

[0045] 3. Structural characterization of the CV@CN composite nanomaterials obtained in Example 1: like Figure 6 As shown, pure CV Chlorella cells have a smooth surface, intact structure, and regular overall morphology without damage, wrinkles, or defects. The composite modified CV@CN Chlorella cells still maintain an intact morphology and clear, regular cell outline, without structural damage such as cell breakage, lysis, or collapse. At the same time, CN nanoparticles can be uniformly and densely attached to the surface of CV Chlorella cells, with a uniform distribution, and there are no problems such as large-area aggregation, local exposure of cell surface, or nanoparticle detachment.

[0046] like Figure 7 As shown, multiple characteristic elements such as Co, Te, C, and O can be effectively detected in the CV@CN composite nanomaterial. The types of each element are completely matched with the raw material element composition of Chlorella vulgaris and CN nanoparticles, proving that CN nanoparticles have been successfully loaded onto the surface of Chlorella vulgaris.

[0047] like Figure 8 As shown, in the infrared curve of the CV@CN composite nanomaterial, the characteristic functional group absorption peaks of CV Chlorella and CN nanomaterials can be clearly observed simultaneously. The characteristic absorption peaks did not shift, weaken or disappear significantly, and no new characteristic functional group peaks were generated. This indicates that CN nanoparticles and CV Chlorella are composited through physical interaction without chemical reaction or structural modification, and the chemical functional group structure of the raw materials is completely preserved.

[0048] like Figure 9 As shown, the CV@CN composite nanomaterial has a moderate hydrated particle size, with a uniform overall particle size distribution and concentrated dispersion range, indicating that the composite modified CV@CN has excellent dispersion effect in aqueous solution.

[0049] like Figure 10 As shown, the surface potential of CV@CN composite nanomaterials is stable and the charge characteristics are uniform, which enables the composite particles to generate good electrostatic repulsion in aqueous solutions and physiological environments, effectively inhibiting particle aggregation and sedimentation behavior, and ensuring long-term uniform dispersion of the system.

[0050] 4. The acoustic and thermal properties of the CV@CN composite nanomaterials obtained in Example 1 were characterized: Under fixed ultrasound parameters (frequency 1MHz, power 0.5W / cm²), 2 (50% duty cycle) to test the acoustic-thermal heating effect of CN nanoparticles: Concentration dependence: such as Figure 11 As shown, when the concentration of CN aqueous solution is 5 mg / mL, it can rapidly heat up to 57°C by 31°C under ultrasonic stimulation, reaching the effective thermal ablation temperature; as the concentration decreases, the heating rate gradually decreases, and when the concentration is 0.1 mg / mL, it can still heat up to 40°C by 13°C, reaching the effective thermotherapy temperature threshold for tumors.

[0051] Cyclic stability: such as Figure 12 As shown, after five consecutive cycles of ultrasound stimulation with 5 mg / mL CN, the temperature rise can be stably increased to about 55°C without significant attenuation, indicating that the CoTe crystal structure is stable and the acoustic and thermal properties can be repeatedly triggered, making it suitable for multiple treatments.

[0052] Acoustic and thermal properties of composite nanomaterials: such as Figure 13 As shown, a pure CN solution of 0.8 mg / mL can rapidly heat up from 18°C ​​to 45°C. The acoustic and thermal heating range of the same concentration of CV@CN aqueous solution is slightly reduced due to the coating of Chlorella carrier, but it can still rapidly heat up from 16°C to 43°C, which meets the needs of tumor hyperthermia.

[0053] 5. The photosynthetic oxygen production performance of the CV@CN composite nanomaterials obtained in Example 1 was characterized: like Figure 14 As shown, comparing the oxygen production effects of Chlorella under different wavelengths of light, the results indicate that Chlorella vulgaris exhibits the highest oxygen production efficiency under 660nm laser irradiation. 10 minutes of laser stimulation can increase the oxygen concentration by 0.8 mg / mL, demonstrating rapid and efficient oxygen production, which can quickly reverse tumor hypoxia. Under continuous 660nm laser irradiation, the oxygen production response of Chlorella vulgaris is stable, continuous, and without attenuation, maintaining a high-oxygen state in the tumor area for an extended period.

[0054] like Figure 15 As shown in the figure, the oxygen production capacity test of CV@CN under laser irradiation with different powers shows that the oxygen production efficiency increases with increasing laser power. Figure 16 As shown, a comparison of the oxygen production capabilities of CV, CN, and CV@CN under 660nm laser irradiation with 1.0W power reveals that the CV@CN composite nanomaterial combines the photosynthetic oxygen production function of Chlorella with the catalase-like oxygen production function of CN, exhibiting the best oxygen production effect.

[0055] 6. The reactive oxygen species (ROS) generation performance of the CV@CN composite nanomaterials obtained in Example 1 was characterized: The verification was performed using 1,3-diphenylisobenzofuran (DPBF) degradation, electron paramagnetic resonance (ESR), and pyroelectric current testing. Pyroelectric current detection: such as Figure 17 As shown, under the external excitation condition of ultrasonic stimulation, the CV@CN composite nanomaterial can respond to the ultrasonic signal and generate a significant pyroelectric current, proving that the composite material has excellent pyroelectric catalytic performance and can efficiently realize the multi-energy conversion between acoustic energy, thermal energy and electrical energy.

[0056] DPBF degradation experiment: such as Figure 18As shown, under dark conditions, the DPBF degradation value of *Chlorella vulgaris* (CV) is approximately 0.1; after irradiation with a 660 nm laser, the DPBF degradation value is approximately 0.2. The DPBF degradation value of pure CN nanoparticles is approximately 0.5, but remains unchanged after irradiation with a 660 nm laser (CN has no photosynthetic oxygen production capacity). The DPBF degradation value of CV@CN is approximately 0.7, which increases to approximately 1.2 under 660 nm laser irradiation. 1 The significant increase in O2 generation indicates that photocatalysis can greatly enhance the efficiency of acoustic, thermo, and electrocatalytic oxygen production.

[0057] ESR detection: such as Figure 19 As shown, under the synergistic stimulation of US and Laser, the pure CV Chlorella and pure CN nanoparticle system only produced a weak [response]. 1 The O2 characteristic signal is significantly enhanced by CV@CN composite nanomaterials. 1 The O2 response signal indicates that the excellent photo-oxygenation characteristics of Chlorella vulgaris can have a highly efficient synergistic effect with the acoustic-thermal-electrocatalytic effect of CN, effectively amplifying the ROS generation level during the catalytic reaction process.

[0058] 7. The CAT enzyme activity of the CV@CN composite nanomaterials obtained in Example 1 was investigated: The catalase-like catalytic activity of the CV@CN composite system was tested to evaluate its ability to decompose H2O2 and produce oxygen. Figure 20 As shown, CN can efficiently catalyze the decomposition of H2O2 under ultrasonic stimulation, exhibiting a concentration-dependent effect. In environments with pH values ​​of 5.5, 6.5, and 7.4, the CAT-Like activity gradually decreases with decreasing CN nanoparticle concentration. Figure 21 As shown, the CAT-Like activity of the nanomaterial is optimal at pH 6.5, which can continuously generate oxygen and significantly increase the oxygen concentration of the system. In the weakly acidic environment of the tumor, the CAT-Like activity remains stable and can work synergistically with photosynthetic oxygen production to maximize the reversal of tumor hypoxia.

[0059] 8. The LCO enzyme activity of the CV@CN composite nanomaterials obtained in Example 1 was tested: The consumption of cysteine ​​was quantitatively detected using ultraviolet absorption spectroscopy. like Figure 22 As shown, in the reaction system where L-Cys and CV@CN coexist, a characteristic absorption peak of cysteine ​​appears at a wavelength of 425 nm; and with the extension of reaction time, the UV absorbance at 425 nm shows a significant decreasing trend, confirming that CV@CN can efficiently consume cysteine ​​in the system and has excellent LCO enzyme catalytic activity.

[0060] like Figure 23 As shown in the results of enzyme activity gradient experiments under different conditions, the LCO catalytic performance of CV@CN exhibits significant concentration dependence and pH response. The decrease in UV absorbance of the reaction system was most significant at an ambient pH of 7.4, indicating that CV@CN exhibits optimal LCO catalytic activity under this neutral environment.

[0061] like Figure 24 As shown, ultrasonic stimulation can further enhance the LCO enzyme catalytic performance of CV@CN. After applying ultrasonic external field stimulation, the characteristic UV absorbance of L-Cys at 425 nm of the system further decreased, proving that ultrasonic excitation can significantly improve the LCO enzyme activity of CV@CN and accelerate the consumption of cysteine.

[0062] 9. The biosafety of the CN nanoparticles, CV Chlorella, and CV@CN composite nanomaterials obtained in Example 1 was evaluated: like Figure 25 As shown, when the concentration of CN nanoparticles is in the range of 0~200 μg / mL, the cell survival rate is consistently above 80%, and CN nanoparticles within this concentration range have no significant toxic side effects on cells. Figure 26 As shown, when the concentration of Chlorella vulgaris (CV) is controlled between 0 and 2.96 × 10⁻⁶, 7 Within the range of / mL, the cell viability of the co-culture system was greater than 80%, indicating that *Chlorella vulgaris* exhibited no significant cytotoxicity or immunogenicity within this concentration range. For example... Figure 27 As shown, when the concentration of Chlorella vulgaris (CV) is 0~1.49×10⁻⁶, 7 At a concentration of 1 / mL, CV@CN showed a cell viability of over 80%.

[0063] Based on the above biosafety assessment results, 1.49 × 10⁻⁶ was selected. 7 / mL is the optimal safe working concentration for Chlorella in CV@CN composite nanomaterials.

[0064] 10. The antitumor effect of the CV@CN composite nanomaterials obtained in Example 1 was evaluated: (1) Cellular level anti-tumor detection: Different groups were set up to evaluate the killing effect on tumor cells, and were divided into 6 groups according to different treatment conditions: Ⅰ: PBS; II: Laser + US; III: CV@CN; IV: CV@CN+Laser; V: CV@CN+US; VI: CV@CN+Laser+US.

[0065] The US parameters are: frequency 1MHz, power 0.5W / cm². 2 50% duty cycle, 10 minutes; Laser parameters: wavelength 660nm, power 1W / cm² 2 Stimulation time: 10 minutes.

[0066] Test results: like Figure 28 As shown, compared with the CV@CN group, the cell survival rate of the CV@CN+Laser group was significantly reduced. This is mainly because the large amount of oxygen produced by Chlorella under light disrupts the survival environment of tumor cells. In the US and CV@CN combined treatment group, the survival rate of tumor cells was further reduced. Under the synergistic stimulation of laser and ultrasound, CV@CN showed a significant killing effect on cells, with a killing rate of up to 82%, confirming the excellent anti-tumor effect of photoacoustic therapy.

[0067] like Figure 29 As shown, ROS production in tumor cells was detected using the DCFH-DA fluorescent probe. Compared with the control group, the CV@CN stimulation group showed stronger green fluorescence, and the red hypoxic fluorescence signal of CV@CN was significantly reduced when laser stimulation was applied, indicating that CV@CN produced ROS under both US and laser stimulation. 1 O2 performance was enhanced, oxygen production was improved under laser stimulation, and ROS generation was also enhanced. Fluorescent staining of both live and dead cells also showed similar results to those observed in cell killing.

[0068] (2) Animal-level antitumor detection - in situ tumor therapy detection: A metastatic melanoma model was established using C57BL / 6J mice to evaluate the therapeutic efficacy of this regimen for both orthotopic and metastatic melanoma. An orthotopic melanoma model was established in mice, and tumors were allowed to reach 100 mm in size. 3 Treatment began at that time, and the mice were randomly divided into 6 groups: G1: PBS control group; G2: Laser + US simple stimulation group; G3: CV@CN drug-only group; G4: CV@CN+Laser drug-induced laser stimulation group; G5: CV@CN+US group receiving ultrasound stimulation with added medication; G6: CV@CN+Laser+US drug-treated group receiving laser and ultrasound stimulation.

[0069] The tumor-targeting composite nanozyme material prepared above was diluted to a certain concentration and injected into the tumor of mice in situ. The size of the tumor in the tumor-bearing mice was then recorded.

[0070] like Figure 30 As shown, compared with the control group, the tumors of experimental mice in the CV@CN, CV@CN+Laser, CV@CN+US, and CV@CN+Laser+US groups all showed a certain degree of inhibition. Compared with the non-stimulated CV@CN group, the tumor inhibition of experimental mice in the CV@CN+Laser, CV@CN+US, and CV@CN+Laser+US groups was stronger, and the tumors of mice in the CV@CN+Laser+US group showed a significant shrinkage trend.

[0071] (3) Animal-level anti-tumor detection - brain metastasis tumor treatment detection: A mouse model of melanoma brain metastasis was established by orally injecting B16F10-LUC cells into the brain and simultaneously constructing melanomas up to 100 mm in situ. 3 Mice were randomly divided into 6 groups, with the same grouping method as described above. The prepared tumor-targeting composite nanozyme material was diluted to a certain concentration and injected into the mouse tumor in situ to begin treatment. After treatment, the tumor in situ was removed, and the size of the brain tumor in the tumor-bearing mice was recorded.

[0072] like Figure 31 As shown, compared with the control group G1 and the stimulation-only group G2, the brain tumor metastasis of mice in groups G3, G4, G5, and G6 was inhibited to some extent; compared with the drug-treated but non-stimulated group G3, the tumor metastasis inhibition of mice in groups G4, G5, and G6 was stronger, and the brain tumors in group G6 mice showed a significant shrinkage trend. Figure 32 As shown, after dissecting the brain tumors in mice, the CV@CN+Laser+US group showed the best inhibitory effect on the brain tumors.

[0073] In summary, the CV@CN composite nanomaterials of this invention achieve anti-tumor therapy through the following multi-mechanism synergistic effect: (1) Acoustic-thermal ablation: Under ultrasonic stimulation, CN nanoparticles rapidly heat up the tumor local area through acoustic-thermal conversion effect (heating to 43~45℃ at a concentration of 0.8mg / mL), directly causing thermal damage to tumor cells.

[0074] (2) Thermoelectric catalysis of ROS production: Ultrasonic-induced periodic temperature fluctuations drive CN nanoparticles to generate a pyroelectric effect, promoting electron-hole pair separation and catalyzing the conversion of molecular oxygen into ROS. 1 O2 and other ROS induce oxidative damage in tumor cells. Photosynthetic oxygen production and CAT-like catalytic oxygen production provide sufficient O2 substrates for thermoelectrocatalysis, enabling the cascade amplification of ROS.

[0075] (3) Dual oxygen supply to reverse hypoxia: Chlorella continuously produces oxygen through photosynthesis under 660nm laser irradiation (increases by 0.8mg / mL in 10min), while CN’s CAT-Like activity catalyzes the decomposition of H2O2 in the tumor microenvironment to produce oxygen. The dual oxygen supply synergistically reverses the hypoxic microenvironment of the tumor.

[0076] (4) LCO-Like metabolic regulation: CN's LCO-Like activity specifically consumes L-cysteine ​​enriched in the tumor microenvironment, inhibits glutathione synthesis, breaks down the tumor cell's antioxidant defense system, and amplifies the ROS killing effect.

[0077] (5) Antitumor immune activation: L-cysteine ​​consumption drives TAMs to polarize from the immunosuppressive M2 type to the antitumor M1 type. At the same time, it enhances the antigen cross-presentation function of M2-like TAMs by downregulating the activity of lysosomal cysteine ​​proteases and activates CD8. + T-cell-led systemic anti-tumor immune response.

[0078] Therefore, the present invention adopts the above-mentioned photoacoustic dual-response CN microalgae nanomaterial, its preparation method and application. The nanomaterial uses living photosynthetic microalgae as a biological carrier and in-situ oxygen production unit, and loads cobalt telluride nanoparticles modified with amino-terminated polyethylene glycol to form a photoacoustic dual-response composite therapeutic system, which can achieve efficient treatment of metastatic melanoma through multi-mechanism synergy.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A photoacoustic dual-response CN microalgae nanomaterial, characterized in that: The invention includes a photosynthetic microalgae carrier and CN nanoparticles loaded on the surface of the photosynthetic microalgae carrier; the CN nanoparticles are cobalt telluride nanoparticles modified with amino-terminated polyethylene glycol, and the cobalt telluride nanoparticles have acoustic-thermal conversion properties and thermoelectric catalytic properties; the photosynthetic microalgae carrier is a living microalgae with photosynthetic oxygen production function.

2. The photoacoustic dual-response CN microalgae nanomaterial according to claim 1, characterized in that: The cobalt telluride nanoparticles have an average particle size of 80~100nm and a zeta potential of +2mV to +7mV; the amino-terminated polyethylene glycol is NH2-PEG-NH2 with a molecular weight of 2000~5000Da.

3. The photoacoustic dual-response CN microalgae nanomaterial according to claim 1, characterized in that: The CN nanoparticles have a hydrated particle size of 90~150nm and a zeta potential of +10mV to +30mV.

4. The photoacoustic dual-response CN microalgae nanomaterial according to claim 1, characterized in that: The photosynthetic microalgae carrier is Chlorella vulgaris; the CN nanoparticles are loaded onto the surface of Chlorella vulgaris through electrostatic adsorption.

5. A method for preparing photoacoustic dual-response CN microalgae nanomaterials as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Cobalt telluride nanoparticles were prepared by high-temperature pyrolysis. S2. The cobalt telluride nanoparticles obtained in S1 were surface-modified with amino-terminated polyethylene glycol to obtain CN nanoparticles. S3. The CN nanoparticles obtained in S2 are incubated and loaded with photosynthetic microalgae to obtain photoacoustic dual-response CN microalgae nanomaterials.

6. The method for preparing a photoacoustic dual-response CN microalgae nanomaterial according to claim 5, characterized in that, S1 specifically refers to: Cobalt precursor and tellurium precursor were mixed in an oleylamine system and reacted at 280-320℃ for 50-70 min. After centrifugation, washing and vacuum drying, cobalt telluride nanoparticles were obtained. The cobalt precursor was prepared by reacting cobalt acetylacetone in oleylamine at 160-200°C for 20-40 min. The tellurium precursor was prepared by dissolving tellurium particles in trioctylphosphine at 120-160°C.

7. The method for preparing a photoacoustic dual-response CN microalgae nanomaterial according to claim 5, characterized in that, S2 specifically refers to: Cobalt telluride nanoparticles were dispersed in ultrapure water and sonicated for 20-40 min. NH2-PEG-NH2 was added in the same mass ratio as the cobalt telluride nanoparticles, and the mixture was stirred at room temperature in the dark for 10-14 h. CN nanoparticles were obtained by centrifugation, washing, and freeze-drying.

8. The method for preparing a photoacoustic dual-response CN microalgae nanomaterial according to claim 5, characterized in that, S3 specifically refers to: Take photosynthetic microalgae in the logarithmic growth phase, add CN nanoparticles, make up to volume with sterile water, and incubate in a constant temperature shaker for 1-2 hours under light-protected conditions; centrifuge to remove supernatant, resuspend and wash with sterile water to obtain photoacoustic dual-response CN microalgae nanomaterials.

9. The application of the photoacoustic dual-response CN microalgae nanomaterial as described in any one of claims 1 to 4 in the preparation of antitumor drugs, characterized in that: The tumor is a metastatic melanoma.

10. The application according to claim 9, characterized in that: The photoacoustic dual-response CN microalgae nanomaterials exert anti-tumor effects under the synergistic stimulation of ultrasound and laser; the ultrasound frequency is 1MHz and the power is 0.3~1.0W / cm. 2 The laser has a wavelength of 660nm and a power of 0.5~2.0W / cm². 2 .

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