A titanium hydride quantum dot functionalized chlorella biological composite material, a preparation method and application thereof

Abstract

The application discloses a kind of hydrogenated titanium quantum dots functionalized chlorella biological composite material and its preparation method and application, belong to the technical field of biomedical materials.The composite material is constructed by loading hydrogenated titanium quantum dots on the surface of chlorella modified by chitosan.The application utilizes the photosynthesis of chlorella to supply oxygen in situ, effectively relieves the hypoxic microenvironment of tumor, simultaneously utilizes the hydrogenated titanium quantum dots to generate reactive oxygen under the high-efficiency stimulation of ultrasound, realizes sonodynamic therapy.Both synergies, not only can directly kill tumor cells, but also can induce immunogenic cell death, activate the anti-tumor immune response of body, provide a kind of " hypoxia relief-high-efficiency killing-immune activation " multidimensional synergistic treatment new strategy for breast cancer and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a titanium hydride quantum dot-functionalized Chlorella biocomposite material, its preparation method and application. Background Technology

[0002] Ultrasound (US)-mediated sonodynamic therapy (SDT) is a rapidly growing treatment method derived from photodynamic therapy. It induces local tumor tissue damage by activating a sonosensitive agent to generate reactive oxygen species (ROS). SDT is highly controllable, non-invasive, and has deep tissue penetration, making it suitable for in situ tumor treatment. Furthermore, SDT can trigger immunogenic cell death (ICD), promoting the release of large amounts of immunogenic substances from tumor cells, allowing more T cells to infiltrate the tumor, and enhancing anti-tumor immunotherapy. Titanium hydride (TiH) 1.924 Compared to traditional organic photosensitizers, SDT is more easily activated by external stimuli (such as light, ultrasound, and microwaves) and can be used for photocatalysis and acoustic catalysis, exhibiting highly efficient ROS generation capabilities. Therefore, SDT combined with immunotherapy shows great promise for the treatment of breast cancer.

[0003] While SDT therapy can temporarily increase oxygen levels at the tumor site, it is far from sufficient for the long-term, severely hypoxic tumor microenvironment and cannot completely reverse the immunosuppressive microenvironment. Therefore, this invention introduces a microorganism—Chlorella (… Chlorella Chl (microalgae) exhibits good biocompatibility. Under specific light wavelengths, it produces oxygen through photosynthesis, alleviating local tumor hypoxia, reducing tumor immunosuppression, and enhancing the efficacy of dynamic therapy. Furthermore, the high content of bioactive substances (polysaccharides and polypeptides) in microalgae can enhance the phagocytic activity of macrophages, increase the cytotoxic effects of natural killer (NK) cells, and improve the penetration of immune cells, thus serving as an immune adjuvant to effectively enhance the immune response.

[0004] However, Chl photosynthetic oxygen production depends on specific wavelengths of light, and biological tissues exhibit significant light attenuation. Deep tumors, due to insufficient light exposure, suffer limited oxygen production and have a limited effect on improving hypoxia; TiH 1.924 The acoustic dynamic effect is influenced by the depth of ultrasound penetration and energy loss. In deep tumors, insufficient ultrasound energy reduces ROS generation efficiency, weakening the killing and immune activation effects. Therefore, single Chl or TiH... 1.924 Mediated sonodynamic-immunotherapy is difficult to achieve simultaneously deep tumor hypoxia relief, efficient killing and immune activation, and the overall anti-tumor efficacy has obvious shortcomings. There is an urgent need to build a composite delivery system that can integrate the advantages of both and overcome the limitations of single materials. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a titanium hydride quantum dot-functionalized Chlorella biocomposite material, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material includes the following steps: (1) Preparation of titanium hydride quantum dots: TiH2 powder was mixed with N-methylpyrrolidone, subjected to ultrasonic treatment, and the supernatant was collected by centrifugation. After washing, the precipitate was collected to obtain TiH2 quantum dots. 1.924 quantum dots; (2) Surface modification of Chlorella: The cultured Chlorella was stirred and reacted with chitosan solution at room temperature, washed and resuspended in PBS buffer to obtain surface-modified Chlorella. (3) Assembly of composite materials: The surface-modified Chlorella obtained in step (2) is combined with the TiH prepared in step (1). 1.924 Quantum dots were mixed, vortexed, and then centrifuged. The supernatant was discarded to obtain the titanium hydride quantum dot-functionalized Chlorella biocomposite material.

[0007] According to the method disclosed above, the present invention constructs a Chlorella / titanium hydride biocomposite material (Chl@TiH). 1.924 (abbreviated as CT) Figure 1 As shown), where TiH 1.924 Quantum dots attach to the Chl surface via electrostatic interactions. Under combined ultrasonic and laser stimulation, TiH... 1.924 It can efficiently trigger acoustic ROS bursts, directly killing tumors and inducing ICD; while Chl, through continuous photosynthesis and in-situ oxygen supply, fundamentally alleviates tumor hypoxia, relieves immunosuppression, and significantly improves ROS generation efficiency and immune cell infiltration levels. Simultaneously, Chl's innate immunomodulatory activity and TiH... 1.924 The mediated immunogenic death forms an immune cascade amplification, achieving a multi-dimensional synergistic anti-tumor effect of "hypoxia relief + efficient killing + immune activation", providing a highly promising new strategy for efficient and safe combination therapy of breast cancer.

[0008] Optionally, in step (1), the ratio of TiH2 powder to N-methylpyrrolidone is 100 mg : 20 mL.

[0009] Optionally, in step (1), the conditions for ultrasonic treatment are: ultrasonic power density 2-2.5 W / cm². 2 The ultrasonic frequency was 3 MHz, the pulse duty cycle was 20% (20% means 20% of the time is emitted and 80% of the time is paused), the ultrasonic processing time was 1 min, and the ultrasonic temperature was 15℃.

[0010] Optionally, in step (1), the washing is performed twice with anhydrous ethanol and centrifuged at 12,000 rpm for 10 min.

[0011] Optionally, in step (2), the volume ratio of the cultured Chlorella to the chitosan solution is 4:1; the concentration of the cultured Chlorella is 1×10⁻⁶. 7 The concentration of the chitosan solution is 1 mg / mL.

[0012] Optionally, in step (2), the stirring reaction conditions are: 200 rpm and 4 h.

[0013] Optionally, in step (3), the surface-modified Chlorella and TiH 1.924 The volume ratio of quantum dots is 2:1, in which TiH 1.924 The concentration of quantum dots was 5 mg / mL.

[0014] A titanium hydride quantum dot-functionalized Chlorella biocomposite material prepared by the above preparation method.

[0015] A pharmaceutical composition comprising the above-mentioned titanium hydride quantum dot-functionalized Chlorella biocomposite material and a pharmaceutically acceptable carrier.

[0016] The above-mentioned titanium hydride quantum dot-functionalized Chlorella biocomposite material is used in the preparation of drugs for tumor treatment.

[0017] Optionally, the tumor treatment is a combination of sonodynamic therapy and immunotherapy, specifically, under the combined stimulation of ultrasound and a 660 nm wavelength laser, the titanium hydride quantum dot-functionalized Chlorella biocomposite material generates reactive oxygen species and induces immunogenic cell death.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: This invention achieves a synergistic effect of sonodynamic therapy and immunotherapy by constructing a titanium hydride quantum dot-functionalized Chlorella biocomposite material. This composite material utilizes the in-situ oxygen production of Chlorella through photosynthesis, effectively alleviating the hypoxic microenvironment in the tumor site, thereby significantly improving the efficiency of reactive oxygen species production by titanium hydride quantum dots under ultrasound stimulation and enhancing the direct killing ability against tumor cells. Simultaneously, this system can induce strong immunogenic cell death, effectively activating dendritic cells and promoting the infiltration and activation of cytotoxic T lymphocytes and helper T cells, thereby initiating a powerful systemic anti-tumor immune response. Furthermore, this biocomposite material exhibits good biocompatibility and blood safety, with no significant toxicity to normal cells, demonstrating highly effective and safe anti-tumor therapeutic effects. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram illustrating the preparation of the CT scan and its anti-tumor mechanism in this invention; Figure 2 Chl, Chl-CS, TiH 1.924 And the Zeta potential of CT; Figure 3 (a) is TiH 1.924 (a) TEM image of Chl, scale bar: 25 nm; (b) TEM image of Chl, scale bar: 1 μm; (c) TEM image of CT, scale bar: 500 nm; (d) TEM image of CT, scale bar: 2 nm; (e) EDS image of C, P, Ti, O and N in CT, scale bar: 2.5 μm. Figure 4 X-ray diffraction for CT scans; Figure 5 Images of Chl and CT cells cultured on BG11 solid medium; Figure 6 O2 production in Chl cells and CT cells at 660 nm; Figure 7 The absorption spectra of DPBF after treatment with different components; Figure 8 (a) represents the survival rate of 4T1 cells after treatment with different components, (b) represents the survival rate of 4T1 cells after treatment with different concentrations of CT, and (c) represents the survival rate of L929 cells after treatment with different concentrations. Figure 9 (a) shows ROS fluorescence images after treatment with different components, scale bar: 100 μm; (b) shows representative ROS flow cytometry images after treatment with different components. Figure 10 (a) shows the fluorescence images of live / dead cells after different component treatments, scale bar: 100 μm; (b) shows the quantitative analysis of the number of live cells in 4T1 cells after different treatments, n = 3. Figure 11 The apoptosis status of cells after treatment with different components; Figure 12 Mitochondrial membrane potential fluorescence images after treatment with different components. Scale bar: 25 μm. Figure 13Hemolysis assays were performed after treatment with different concentrations of CT. PC: positive, NC: negative, n = 3. Figure 14 (a) is a schematic diagram of the in vivo experimental protocol, (b) is the body weight of mice in different groups, n = 5, (c) is the tumor volume of mice in different groups, n = 5, (d) is the tumor weight of mice in different groups, n = 5, and (e) is an image of the removed tumor, n = 5. Figure 15 H&E staining of tumors and major organs after different treatments, scale bar: 200 μm; Figure 16 TUNEL, HIF-1α, CRT, HMGB1 and CD4 / 8 staining for tumor sections from different groups; Figure 17 The blood routine parameters of mice in each group after different treatments, n = 3; Figure 18 (a) represents different processing methods for CD11c. + CD80 in tumor-draining lymph nodes + CD86 + The representative flow cytometry plot, (b) is CD11c + CD80 in tumor-draining lymph nodes + CD86 + The quantitative results, n = 5, (c) represent CD4 in the spleen. + (CD3 + CD4 + Flow cytometry analysis of T cells, (d) shows CD4+ in the spleen. + (CD3 + CD4 + Quantitative results of T cells, n = 5, (e) represents CD8+ in the spleen. + (CD3 + CD8 + Flow cytometry analysis of T cells, (f) shows CD8+ in the spleen. + (CD3 + CD8 + Quantitative results of T cells, n = 5, (g) represents CD4+ in tumor tissue. + (CD3 + CD4 + Flow cytometry analysis of T cells, (h) represents CD4+ in tumor tissue. + (CD3 + CD4 + Quantitative results of T cells, n = 5, (i) represents CD8+ in tumor tissue. + (CD3 + CD8+ Flow cytometry analysis of T cells, (j) represents CD8+ in tumor tissue. + (CD3 + CD8 + Quantitative results of T cells, n = 5; Figure 19 (a) represents CD4 in the spleen. + CD69 + Flow cytometry analysis of T cells, (b) showing CD4+ in the spleen. + CD69 + Quantitative results of T cells, n = 5, (c) represents CD8+ in the spleen. + CD69 + Flow cytometry analysis of T cells, (d) shows CD8+ in the spleen. + CD69 + Quantitative results of T cells, n = 5. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, "room temperature" in this invention refers to 20-30℃.

[0026] All raw materials used in this invention were purchased commercially. Chlorella (Chl) was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences; TiH2 (99%), N-methylpyrrolidone (NMP), chitosan (degree of deacetylation >95%), 1,3-diphenylisobenzofuran (DPBF), and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Shanghai Maclean Biotechnology Co., Ltd. The CCK-8 assay kit was purchased from Beyotime Biotechnology Co., Ltd., Shanghai, China; the AM / PI double staining kit and the Annexin V-FITC apoptosis assay kit were produced by Beijing Solarbio Science & Technology Co., Ltd.; and all cell surface marker immunoassay antibodies were purchased from BioLegend, San Diego, USA.

[0027] The technical solution of the present invention will be further illustrated by the following embodiments.

[0028] Example 1 A method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material includes the following steps: (1) Cultivation of Chlorella Chl algal strains were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. Under aseptic conditions, the algal strains were placed in sterile culture medium, sealed with breathable sealing film and kraft paper, and then placed in a light incubator for cultivation. The light cycle was set to alternate between 12 hours of light and 12 hours of darkness to simulate natural light duration, and the cultivation temperature was 25℃. When the cultured algal strains turned dark green, they were expanded into sterilized and cooled BG-11 culture medium at a ratio of 1:5 using Chl stock solution.

[0029] (2) TiH 1.924 Preparation TiH2 (100 mg) and NMP (20 mL) were sonicated (2.5 W / cm²). 2 Quantum dots were obtained by processing at 3 MHz, 20% duty cycle, 1 min processing time, and 15℃. Subsequently, TiH... 1.924 Centrifuge (3000 rpm, 5 min) to collect the supernatant, wash twice with anhydrous ethanol (12000 rpm, 10 min), collect the precipitate, freeze-dry for quantitative analysis and use.

[0030] (3) Preparation of CT Take 2 mL of Chl (1×10⁻⁶) cultured in step (1). 7 The mixture (1 mg / mL) of TiH2O was stirred at room temperature (200 rpm, 4 h) with chitosan (0.5 mL, 1 mg / mL), washed twice with PBS (3500 rpm, 5 min), and resuspended in 2 mL of PBS. Then, 1 mL (5 mg / mL) of TiH2O was added. 1.924 Quantum dots, vortex oscillations cause Chl cells and TiH 1.924 The quantum dots were thoroughly mixed. Finally, the suspension was centrifuged (3500 rpm, 5 min), and the supernatant was discarded to obtain CT, namely, titanium hydride quantum dot-functionalized Chlorella biocomposite material.

[0031] I. Material Characterization (1) TEM analysis The morphology of Chl (which, as mentioned here and in subsequent discussions, refers to the Chlorella cultured in step (1) of Example 1) and CT was observed using TEM. Chl and CT were dropped onto the surface of a copper mesh, dried, and then their morphology was observed using TEM at an appropriate magnification. Simultaneously, the elemental distribution of CT was analyzed using EDS.

[0032] (2) Particle size and zeta potential analysis Take a small amount of Chl and TiH 1.924 The samples were diluted with CT to an appropriate concentration, and the surface zeta potential and hydrated particle size were analyzed using a Malvern laser particle size analyzer. All samples were measured in triplicate.

[0033] (3) XRD analysis The freeze-dried CT powder was subjected to XRD analysis. The scanning angle range was the conventional 10-90 degrees, and the scanning speed was set to 2° / min.

[0034] Figure 2 Chl, Chl-CS, TiH 1.924 The Zeta potential of Chl and CT; as shown in the figure, after surface modification with chitosan (CS), the surface potential of Chl increased from -24.8 mV to 20.6 mV. The higher positive charge enhances its ability to target tumor cell mitochondria. TiH 1.924 The surface potential of CT is -16.4 mV, and it generates CT via electrostatic adsorption with chitosan-modified Chl. The surface potential of CT is -10.6 mV, and this potential change verifies the interaction between Chl and TiH. 1.924 The successful combination of these elements also endows it with the ability to circulate within the body for extended periods.

[0035] Figure 3 (a) is TiH 1.924(a) TEM image of Chl, scale bar: 25 nm; (b) TEM image of Chl, scale bar: 1 μm; (c) TEM image of CT, scale bar: 500 nm; (d) TEM image of CT, scale bar: 2 nm; (e) EDS image of C, P, Ti, O and N in CT, scale bar: 2.5 μm. Figure 3 As shown in Figure a, TiH prepared by liquid phase exfoliation method 1.924 Quantum dots exhibit uniform size and morphology, with an average particle size of approximately 5 nm. For example... Figure 3 As shown in Figure b, the Chl surface is composed of cell walls with smooth, wrinkled surfaces, and is loaded with TiH. 1.924 Later, there were obvious granules ( Figure 3 (c) and TEM showed obvious lattice striations on the Chl cell wall, such as ( Figure 3 The lattice spacing is 0.275 nm. Further EDS analysis of the CT surface elemental composition revealed that C, O, N, and P elements are uniformly distributed within Chl ( d). Figure 3 (e) These elements originate from Chl, while Ti elements are distributed around Chl, indicating that Chl is related to TiH. 1.924 The successful combination of TiH was demonstrated in the above summary, based on its morphology and chemical composition. 1.924 Successful load and successful CT construction.

[0036] Figure 4 X-ray diffraction of CT; XRD pattern of CT and TiH 1.924 (JCPDS No. 25-0983) is consistent, which also indicates that TiH 1.924 It was successfully loaded onto the surface of Chl cells.

[0037] II. CT in vitro bioactivity detection To investigate TiH 1.924 The effect of modification on Chl activity was investigated using a plate culture method for comparative growth experiments. First, sterile BG11 solid culture medium was prepared, sterilized, and poured onto solid plates for later use. Chl suspensions of consistent concentration were then mixed with TiH... 1.924 The modified Chl composite system (CT) was uniformly coated onto the aforementioned BG11 solid plates, which were then placed in an artificial light incubator and incubated under the following conditions: light intensity 3500 lx, light / dark cycle 12 h / 12 ​​h, and incubation temperature 25℃. Photos were taken of each plate on day 0 and day 10 of incubation to compare the growth of Chl and CT.

[0038] Figure 5 Images of Chl and CT cells cultured on BG11 solid medium are shown. Further investigation is needed to determine the optimal loading of TiH on Chl cells.1.924 To determine whether algae maintained their biological activity after colony formation, the study evaluated cell viability using algal proliferation and colony formation as parameters. In the experiment, Chl and CT algae were spread on BG11 solid plates and cultured in an artificial light incubator, and the growth of algal colonies was observed and compared. Figure 5 As shown, the algal colonies on the solid agar plates treated with CT showed no significant difference from those in the Chl group, exhibiting good growth and stable proliferation capacity. This indicates that TiH... 1.924 The surface modification and loading process of quantum dots did not significantly inhibit the cell structure and physiological activity of Chl. After loading, Chl still maintained excellent biological activity and normal proliferation capacity, which provided an important guarantee for the subsequent system to exert photosynthetic oxygen production and immune regulation functions.

[0039] III. CT-based in vitro O2 production detection To assess the oxygen production capacity of Chl and CT, a dissolved oxygen meter was used to measure their oxygen production. 10 mL Chl and CT solutions were prepared separately (Chl = 1 × 10⁻⁶). 6 The solution was then purged with nitrogen gas, and dissolved oxygen levels (Chl and CT solutions at 660 nm (0.1 W / cm³) were recorded using a dissolved oxygen meter over 30 min with stirring in the dark. 2 O2 generated under laser irradiation was recorded every 30 seconds.

[0040] Figure 6 This study investigated the O2 production of Chl and CT cells at 660 nm. To assess the oxygen production capacity of CT cells, dissolved oxygen levels in Chl and CT cells were measured using a dissolved oxygen meter, thus exploring their photosynthetic oxygen production performance. Figure 6 The oxygen production curves show that Chl cells exposed to 660nm laser light can effectively perform photosynthesis and produce a large amount of O2. Furthermore, the oxygen production of the same number of Chl cells in the CT group is significantly higher than that in the pure Chl group; this improvement can be attributed to TiH. 1.924 Synergistic effect of quantum dots and Chl, TiH 1.924 Quantum dots can optimize light energy capture and transfer efficiency, reduce light energy loss, and provide microenvironment regulation for Chl photosynthesis, thereby significantly enhancing the photosynthetic oxygen production capacity of the CT system.

[0041] IV. In vitro ROS detection The ROS production capacity of different groups (PBS, Chl, CT, CT+US, CT+US+660) of biomaterials was detected using the ROS probe 1,3-diphenylisophenylfuran (DPBF). DPBF probe solution was added to each group's suspension (200 μg / mL) to a final concentration of 20 μg / mL, followed by measurement at a wavelength of 660 nm and a power of 0.1 W / cm².2 The mixed system was irradiated with a laser for 5 min. The degree of DPBF degradation was detected by UV-Vis spectrophotometer to characterize the ROS production level of each group.

[0042] Figure 7 The absorption spectra of DPBF after treatment with different components are shown. Based on the excellent ROS-producing characteristics of CT, the generation of ROS was detected using a DPBF fluorescent probe. Figure 7 As shown, more pronounced ROS generation was observed in the CT+US+660 nm laser group. This result is mainly attributed to Chl continuously providing sufficient O2 through photosynthesis, providing a key substrate for acoustodynamic ROS production. Meanwhile, TiH... 1.924 The functional coupling of quantum dots and Chl can optimize energy transfer efficiency, reduce energy loss, further enhance the ability of ultrasound-induced ROS generation, and achieve synergistic enhancement of oxygen production and acoustic dynamic effects.

[0043] V. Intracellular ROS Detection The experiment used DCFH-DA to detect intracellular ROS production. First, a 1 mg / mL DCFH-DA probe was prepared by dissolving DCFH-DA in DMSO, and then diluted to a 10 μM working solution before the experiment. 4T1 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured for 24 h. After cell attachment, 2 mL of PBS, Chl, CT, CT+US, and CT+US+660 culture medium were added, respectively. 12 h after drug administration, the culture medium was discarded, and the cells were washed twice with PBS. Finally, DCFH-DA (10 μM) was added and incubated for 30 min. After washing once with PBS, fluorescence images were observed using an inverted fluorescence microscope.

[0044] Figure 9 (a) shows ROS fluorescence images after treatment with different components, scale bar: 100 μm; (b) shows representative flow cytometry plots of ROS after treatment with different components. This invention uses the DCFH-DA reactive oxygen species fluorescent probe to detect ROS in 4T1 cells; stronger fluorescence indicates higher ROS concentration. Figure 9 As shown in Figure a, compared with CT, the fluorescence intensity of the CT+US group was slightly enhanced, which is because TiH 1.924 The ability of cells to produce ROS was enhanced under US stimulation. The CT+US+660 nm group showed the strongest fluorescence signal, which is due to TiH 1.924 When synergistically combined with Chl, Chl produces more O2 during photosynthesis and, in conjunction with SDT, promotes ROS generation, effectively alleviating the hypoxic microenvironment within tumors. Flow cytometry analysis of intracellular ROS yielded similar results (e.g., Figure 9As shown in b), the CT+US+660 nm group had a 44% concentration, which was twice that of the PBS group.

[0045] VI. Live and Dead Staining Experiment 4T1 cells were fed at a rate of 2 × 10 5 Seeds were planted at a density of cells / well in 12-well plates. After 24 h of culture, the plates were treated with 2 mL of PBS (100 μg / μL), US, Chl (100 μg / μL), CT (100 μg / μL), CT+US (100 μg / μL), and CT+US+660 (100 μg / μL) for 6 h, respectively. Finally, the plates were treated with SDT (2.5 W / cm²). 2 3 MHz, 20% duty cycle) for 1 min and laser treatment (0.1 W / cm²) 2 Stain with PBS (660 nm) for 5 min. Then wash twice with PBS, add 1 mL of AM and PI for 15 min, wash twice more with PBS, and finally observe live cells (green fluorescence) and necrotic cells (red fluorescence) under a fluorescence microscope.

[0046] Figure 10 (a) shows fluorescence images of live / dead cells after treatment with different components, scale bar: 100 μm; (b) shows the quantitative analysis of the number of live cells in 4T1 cells after different treatments, n = 3. This invention evaluated the killing ability of CT on 4T1 cells using live / dead staining. Figure 10 As shown, the US and Chl groups alone showed no cytotoxicity to tumor cells, while the CT+US group exhibited significantly enhanced 4T1 cell killing ability (increased red fluorescence). This is mainly due to the TiH... 1.924 Conventional SDT mediated by CT promotes the generation of intracellular ROS, which then exert toxicity and kill cells. However, when subjected to dual stimulation with US and 660 nm, almost all tumor cells in the CT+US+660 nm group died, confirming its good anti-tumor killing ability.

[0047] VII. Cytotoxicity Experiments 4T1 and L929 cells were respectively fed with 5×10 3 Seeds were planted at a density of cells / well in 96-well plates. After 12 h of incubation, the plates were treated with 0.1 mL of PBS, US, Chl, CT, CT+US, and CT+US+660 for 12 h, respectively, at 2.5 W / cm². 2 SDT processing was performed for 1 min at 3 MHz and 20% duty cycle, and the concentration was 0.1 W / cm². 2After 660 nm laser treatment for 5 min, the culture medium was removed, and 0.1 mL of prepared CCK8 detection solution was added and incubated for 30 min. The absorbance at OD540 nm was then measured.

[0048] Figure 8 (a) shows the survival rate of 4T1 cells after treatment with different components; (b) shows the survival rate of 4T1 cells after treatment with different concentrations of CT; and (c) shows the survival rate of L929 cells after treatment with different concentrations. The in vitro cytotoxicity results of CT are as follows: Figure 8 As shown in Figures a and b, the tumor cell survival rate in the CT+US group was 34.4%, which can be attributed to the killing effect of SDT on tumor cells. The 4T1 cell survival rate in the CT+US+660 nm group decreased to 23.72%, significantly lower than the control group (PBS), indicating that CT, under the dual effects of US and 660 nm laser irradiation, enhanced the killing effect on 4T1 tumor cells. The cytotoxicity results of CT on L929 normal cells are shown below. Figure 8 As shown in Figure c, CT exhibits low levels of cytotoxicity. Even after co-incubating L929 cells with a high concentration of CT (300 μg / mL) for 24 h, the cell survival rate still exceeded 90%, indicating that CT has no significant toxicity to normal cells and only has the ability to kill 4T1 tumor cells.

[0049] VIII. Apoptosis Experiment 4T1 cells were loaded at 5 × 10 5 Seeds were planted at a density of cells / well in 6-well plates. After 24 h of culture, the plates were treated with 2 mL of PBS, US, Chl, CT, CT+US, and CT+US+660 for 10 h, respectively. The culture was then maintained at 2.5 W / cm². 2 SDT processing was performed for 1 min at 3 MHz and 20% duty cycle, and the concentration was 0.1 W / cm². 2 After treatment with a 660 nm laser for 5 min, cells were digested with trypsin, washed with PBS, resuspended in 100 μL Annexin V Binding Buffer, and stained with 5 μL Annexin V-FITC and 5 μL PI in the dark for 30 min. Finally, fluorescence signals were detected by flow cytometry.

[0050] Figure 11 Apoptosis was observed after treatment with different components. The toxicity of CT to 4T1 cells was assessed using the Annexin V-FITC / PI apoptosis detection kit. Figure 11As shown, CT primarily induced a late-stage apoptosis in cells. The late-stage apoptosis rate in the CT+US+660nm group was 28.4%, which was 1.88 times that of the CT group. These results indicate that the CT composite system, under the synergistic stimulation of ultrasound and light, can effectively trigger the apoptosis program in 4T1 cells, and the significant increase in the late-stage apoptosis rate fully demonstrates its highly efficient tumor cell killing activity.

[0051] IX. Mitochondrial membrane potential experiment 4T1 cells were fed at a rate of 2 × 10 5 Seeds were planted at a density of cells / well in 12-well plates. After 12 h of incubation, the plates were treated with 2 mL of PBS, US, Chl, CT, CT+US, and CT+US+660 for 6 h, respectively. The plates were then incubated at 2.5 W / cm². 2 SDT processing was performed for 1 min at 3 MHz and 20% duty cycle, and the concentration was 0.1 W / cm². 2 The sample was treated with a 660 nm laser for 5 min. After 6 h of treatment, it was washed twice with PBS, stained with 0.5 mL of prepared JC-1 staining working solution in the dark for 30 min, and then washed. Finally, fluorescence images were captured using an inverted fluorescence microscope.

[0052] Figure 12 Images of mitochondrial membrane potential fluorescence after treatment with different components. Scale bar: 25 μm.

[0053] A decrease in mitochondrial membrane potential is a typical characteristic of early apoptosis. To verify the effect of CT on changes in mitochondrial membrane potential, this invention used the JC-1 fluorescent probe to detect mitochondrial membrane potential. The results are as follows: Figure 12 The results showed that the mitochondria in the normal PBS control group had a high negative charge, and JC-1 entered the mitochondria as a polymer, exhibiting strong red fluorescence. However, in the CT+US+660 nm group, when cells underwent extensive apoptosis, mitochondrial depolarization occurred, resulting in a decrease in negative charge, and JC-1 existed as a monomer in the cytoplasm, exhibiting enhanced green fluorescence. This indicates that CT can effectively identify and target tumor cell mitochondria, reducing the mitochondrial membrane potential of tumor cells.

[0054] 10. Hemolysis test Mouse blood was collected, and red blood cells were collected by centrifugation (1000 rpm, 5 min). The cells were washed three times with PBS and diluted to prepare a 2% red blood cell suspension. The 2% red blood cell suspension was then incubated with PBS (negative control), 1% Triton X-100 solution (positive control), and CT (25, 50, 100, 150, 200 μg / mL) for 1 h. After centrifugation, the absorbance of the supernatant was measured at 540 nm, and the hemolysis rate (the relative ratio of absorbance between the sample group and the control group) was calculated using standard methods.

[0055] Figure 13 Hemolysis assays were performed after treatment with different concentrations of CT. PC: positive, NC: negative, n = 3. Hemolysis assays are used to assess the blood compatibility of biomaterials. Figure 13 As shown, the hemolysis rate of CT at different concentrations was below the 5% clinical safety threshold stipulated by international standards. This indicates that the biomaterial meets the blood safety requirements for drug delivery systems.

[0056] XI. In vivo anti-tumor effects (1) Establishment of tumor-bearing mouse model Female BALB / c mice (5-6 weeks old) were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). Animal ethics approval was obtained from the Animal Ethics Committee of Zhejiang Ocean University (Approval Nos.: 2025089, 2025095). 4T1 cell suspension (1×10⁻⁶) was used. 6 A 4T1 tumor-bearing mouse model was established by injecting 100 μL PBS into the right leg root of mice.

[0057] (2) In vivo anti-tumor efficacy When the tumor volume reaches approximately 100 mm 3 Mice were randomly divided into 5 groups: PBS (control group), Chl group, CT group, CT+US group, and CT+US+660 group (Chl=1×10⁻⁶). 7 mice, TiH 1.924 =4 mg / kg). Intratumoral injection of the corresponding Chl and CT was administered. Six hours after injection, the light therapy group and ultrasound group received a 660 nm laser (0.1 W / cm²). 2 Irradiate the tumor site for 10 minutes at 2.5 W / cm². 2 SDT was performed at 3 MHz and 20% duty cycle. All treatments were administered on days 0, 2, 4, 6, 8, 10, 12, and 14, with tumor volume and body weight monitored every other day until day 15. Tumors were collected after euthanasia for tumor weight, histopathological examination, immunohistochemical evaluation of TUNEL, HIF-1α, HMGB1, and CRT markers, toxicity assessment of major organs by H&E staining, and serum biochemical analysis of orbital blood samples.

[0058] (3) Biosafety Fourteen days after treatment, blood samples were collected from each group of mice under section “2.15.2” and routine blood tests were performed using a blood analyzer.

[0059] 12. In vivo anti-tumor immunity To evaluate the immunotherapy mechanism of CT, tumor-bearing mice were treated with the same grouping and dosing regimens as in the anti-tumor efficacy study. When the 4T1 tumor reached approximately 100 mm...3 Intratumoral administration was initiated at this time. Seven days after treatment, the mice were euthanized, and the tumors, tumor-draining lymph nodes, and spleen were collected for comprehensive immunological analysis.

[0060] (1) Dendritic cell maturation Lymph nodes were placed on a 70 μm cell sieve and homogenized using a 5 mL syringe plunger to obtain a single-cell suspension. The suspension was washed with PBS, centrifuged (2000 rpm, 5 min), and resuspended in 100 μL PBS. Subsequently, the cells were stained at 4°C with CD86-PE (0.5 μL), CD80-PercpCy5.5 (0.5 μL), and CD11c-FITC (0.5 μL) in the dark for 30 min. The cells were washed twice with PBS, fixed and resuspended in 500 μL paraformaldehyde, and analyzed using flow cytometry.

[0061] (2) T cell activation Spleen tissue: The spleen was minced, ground, and sieved through a 70 μm sieve to obtain a cell suspension. 3 mL of erythrocyte lysis buffer was added for 3 min to lyse the cells and remove the erythrocyte lysate. 5 mL of PBS was added, and the cells were collected by centrifugation (2000 rpm, 5 min). The cells were washed twice, resuspended in 100 μL of PBS, and stained at 4°C with CD8a-PE (0.5 μL), CD4-PercpCy5.5 (0.5 μL), and CD3-FITC (0.5 μL) in the dark for 30 min. The cells were washed twice with PBS and fixed and resuspended in 500 μL of paraformaldehyde. The stained mixed cell suspension was immediately analyzed by flow cytometry.

[0062] Tumor tissue: 100 mg of non-marginal, non-necrotic tumor tissue was cut into small pieces and added to 3 mL of digestion solution (1 mL RPMI 1640 with 0.3 mg collagenase IV and 0.12 mg DNase I added). The mixture was incubated at 37°C with shaking for 30 min. The tumor cells were then ground, sieved through a 70 μm sieve, and centrifuged (2000 rpm, 5 min). The tumor supernatant was collected for subsequent experiments. The tumor cells were resuspended in 100 μL PBS, and then stained at 4°C with CD8a-PE (0.5 μL), CD4-PercpCy5.5 (0.5 μL), and CD3-FITC (0.5 μL) in the dark for 30 min. The cells were washed twice with PBS, fixed and resuspended in 500 μL paraformaldehyde, and analyzed by flow cytometry.

[0063] (3) Antigen-specific activation Spleen tissue was ground and passed through a 70 μm cell sieve, centrifuged (450 g, 5 min), and erythrocytes were lysed with 3 mL of erythrocyte lysis buffer for 2 min. After lysing, 5 mL of PBS was added, followed by centrifugation (450 g, 5 min). The precipitate was washed twice with PBS, resuspended in 100 μL of PBS, and stained with CD69-AF700 (1 μL), CD4-FITC (0.5 μL), and CD8-PE (0.5 μL) for 30 min (4℃). After washing twice with PBS, the precipitate was fixed with 500 μL of paraformaldehyde and analyzed by flow cytometry.

[0064] Figure 14 (a) is a schematic diagram of the in vivo experimental protocol; (b) shows the body weight of mice in different groups, n = 5; (c) shows the tumor volume of mice in different groups, n = 5; (d) shows the tumor weight of mice in different groups, n = 5; and (e) shows an image of the removed tumor, n = 5. Figure 14 As shown in Figure a, a 4T1 tumor-bearing mouse model was established and treated with the drug. During the 14-day treatment period, the body weight of the mice in each group remained stable. Figure 14 In group b), there was no significant difference in body weight between the mouse groups. Tumor volume was effectively controlled during treatment. Figure 14 In the study of mouse tumor weight, the CT+US+660 nm group showed a significant tumor-suppressive effect. Figure 14 (d) Further analysis of tumor volume changes in each group of mice confirmed that the CT+US+660 nm group had the most significant anti-tumor effect. Analysis of tumor weight measurements and digital images of resected tumors in each group showed that the CT+US+660 nm group exhibited the most significant anti-tumor effect. Figure 14 (e).

[0065] Figure 15 H&E staining of tumors and major organs after different treatments, scale bar: 200 μm. H&E staining results of major organs 14 days after treatment showed no significant pathological changes in the major organs of mice in each treatment group, further demonstrating the good biocompatibility of CT+US+660 nm.

[0066] Figure 16 TUNEL, HIF-1α, CRT, HMGB1, and CD4 / 8 staining were performed on tumor sections from different groups. TUNEL staining results showed that the CT+US+660 nm group exhibited the strongest apoptotic effect. Figure 16 (a) This demonstrates that CT can effectively promote cell apoptosis, which is consistent with the results of flow cytometry. From the HIF-1α staining results ( Figure 16(b) It can be seen that the fluorescence expression of HIF-1α in the CT+US+660 nm group was significantly decreased, indicating that CT significantly alleviated the hypoxic microenvironment of the tumor under the stimulation of laser and SDT. CRT fluorescence staining results showed ( Figure 16 (c) Compared with the PBS group, the CT+US+660 nm group strongly stimulated tumor cells to undergo ICD, and CRT translocated to the cell surface, with a significant increase in fluorescence signal. HMGB1 fluorescence staining showed that HMGB1 was released from the cell nucleus into the extracellular microenvironment, with a significant increase in fluorescence signal, indicating a substantial increase in extracellular HMGB1 concentration. The fluorescence of the CT+US+660 nm group was significantly higher than that of the PBS group. Figure 16 (d). Simultaneously, immunofluorescence measurements indicated that, under combined SDT and immunotherapy, the CD4 / CD8 ratio in distal tumors... + The number of T cells has increased. Figure 16 (e) Promotes cellular autoimmunity. This indicates that CT can initiate a specific anti-tumor adaptive immune response, thereby inducing the proliferation and differentiation of cytotoxic T lymphocytes and precisely killing untreated live tumor cells.

[0067] Figure 17 The figures show the blood routine parameters of mice in each group after different treatments, n = 3. As can be seen from the figure, no significant changes were observed in the blood routine parameters of mice in each group after 14 days of treatment, further confirming that CT has good biocompatibility within the effective dose range.

[0068] Figure 18 (a) represents different processing methods for CD11c. + CD80 in tumor-draining lymph nodes + CD86 + The representative flow cytometry plot, (b) is CD11c + CD80 in tumor-draining lymph nodes + CD86 + The quantitative results, n = 5, (c) represent CD4 in the spleen. + (CD3 + CD4 + Flow cytometry analysis of T cells, (d) shows CD4+ in the spleen. + (CD3 + CD4 + Quantitative results of T cells, n = 5, (e) represents CD8+ in the spleen. + (CD3 + CD8 + Flow cytometry analysis of T cells, (f) shows CD8+ in the spleen. + (CD3 + CD8 +Quantitative results of T cells, n = 5, (g) represents CD4+ in tumor tissue. + (CD3 + CD4 + Flow cytometry analysis of T cells, (h) represents CD4+ in tumor tissue. + (CD3 + CD4 + Quantitative results of T cells, n = 5, (i) represents CD8+ in tumor tissue. + (CD3 + CD8 + Flow cytometry analysis of T cells, (j) represents CD8+ in tumor tissue. + (CD3 + CD8 + Quantitative results of T cells, n = 5.

[0069] To systematically evaluate the immunomodulatory efficacy of CT, a 4T1 tumor-bearing mouse model was used, and tumors were measured when the volume reached 100 mm. 3 At that time, anti-tumor immunotherapy was administered.

[0070] To investigate the synergistic antitumor and immune activation mechanism of the biocomposite material CT / US / ultrasound, this invention quantitatively detected the distribution and infiltration level of mature dendritic cells (DCs) in tumor-draining lymph nodes. CT, after being excited by a 660 nm laser, can efficiently generate reactive oxygen species (ROS) and induce immunogenic cell death in tumor cells, releasing a large amount of tumor-associated antigens. Simultaneously, it improves the local immunosuppressive microenvironment of the tumor, effectively promoting the proliferation and maturation of dendritic cells. As key professional antigen-presenting cells, mature DCs can efficiently present tumor-specific antigens and activate T lymphocytes, thereby initiating a systemic antitumor adaptive immune response. Quantitative flow cytometry analysis showed that the proportion of mature DCs in the tumor-draining lymph nodes of the CT+US+660 group reached 29.4%, which was 5.66 times higher than that of the PBS group (5.19%). Figure 18 (a) The CT+US group (23%) and the CT group (16.9%) also showed different degrees of increasing trend in the number of DCs.

[0071] Among T lymphocyte subsets, helper T cells (Th, CD3) + CD4 + CD8 - ) participate in immune regulation by secreting cytokines such as IL-2 and IFN-γ, while cytotoxic T lymphocytes (CTL / Tc, CD3) participate in immune regulation. + CD4 - CD8 +This invention directly mediates target cell killing by releasing granzyme and perforin. Flow cytometry was used to detect CD3 in spleen and tumor tissues from different treatment groups. + CD4 + (Th) and CD3 + CD8 + Dynamic changes in the (CTL) subpopulation. Flow cytometry results show ( Figure 18 In the CT+US+660 group (C and E), the proportion of Th cells in the spleen was 23.8%, and the proportion of CTLs was 30.6%, significantly higher than that in the PBS (Th: 4.03%, CTL: 6.13%), Chl (Th: 8.22%, CTL: 14.5%), CT (Th: 11.8%, CTL: 19.1%), and CT+US (Th: 18%, CTL: 24.9%) groups, demonstrating the strongest immune activation effect. In tumor tissue analysis ( Figure 18 In the CT+US group, the proportions of Th (22.9%) and CTL (20.5%) were significantly higher than those in the CT group (Th: 18.7%, CTL: 15.7%), which is attributed to the effect of US inducing TiH. 1.924 The release of ROS triggers related immunogenic cell death. The CT+US+660 group showed the highest proportions of Th (30.6%) and CTL (25.9%). This immune activation effect may stem from the cascade reaction of oxygen production to alleviate hypoxia, ROS killing tumor cells, and immunogenic cell death under the synergistic stimulation of ultrasound and light, which jointly drive the enhanced anti-tumor immune response.

[0072] Synchronous expansion of CTL and Th subsets was detected in both the spleen and peripheral tumor tissues. This result indicates that after effective uptake and cross-presentation of tumor-associated antigens by dendritic cells, the body can initiate an antigen-specific T-cell immune response. During this process, activated antigen-specific T lymphocytes not only proliferate and expand in peripheral immune organs (such as the spleen) but also undergo effective targeted migration and infiltration into the tumor parenchyma, thereby exerting immune surveillance and killing functions locally within the tumor. This result further confirms that the immune response induced by this treatment strategy has good systemic and tumor-targeting properties.

[0073] Figure 19 (a) represents CD4 in the spleen. + CD69 + Flow cytometry analysis of T cells, (b) showing CD4+ in the spleen. + CD69 + Quantitative results of T cells, n = 5, (c) represents CD8+ in the spleen. + CD69 +Flow cytometry analysis of T cells, (d) shows CD8+ in the spleen. + CD69 + Quantitative results of T cells, n = 5. This invention assesses the activation level of antigen-specific T cells by quantitatively analyzing the expression level of CD69. Flow cytometry analysis results ( Figure 19 Figures a and b) show that CD69 in the PBS group + The expression level was 4.55%, while the Chl group (12.4%) and CT group (18.1%) increased to 2.7 times and 3.9 times that of the PBS group, respectively. Among them, the CD69 expression level in the CT+US group was... + CD4 + The proportion of T cells was significantly increased to 25.1%, and after further treatment with 660 nm light, the proportion in the CT+US+660 group rose to 33.9%, a 7.5-fold increase compared to the PBS group. (In CD8...) + T cell subsets ( Figure 19 In the PBS group (c and d), the expression rate was 1.43%. After treatment with Chl (11.2%), CT (18.8%), and CT+US (26.7%), the expression was significantly increased, with the CT+US+660 group showing the highest CD69 expression. + CD8 + T cell expression was highest (37.1%). These results indicate that CT significantly accelerates the activation of antigen-specific T cells through a synergistic regulatory mechanism, especially after treatment with US and 660 nm, which enables rapid proliferation and functional enhancement of CTL subsets, providing a cellular basis for subsequent anti-tumor immune responses.

[0074] XIII. Statistical Methods Data were statistically analyzed and plotted using GraphPad Prism 9.5 software, flow cytometry data were analyzed using FlowJo 10.8.1 software, and images were analyzed using ImageJ software. All data are expressed as mean ± standard deviation and were analyzed using ANOVA and Tukey. Statistical significance was defined as follows:* P < 0.05, ** P < 0.01, *** P <0.001, **** P < 0.0001, ns indicates no significance.

[0075] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material, characterized in that, Includes the following steps: (1) TiH2 powder was mixed with N-methylpyrrolidone, subjected to ultrasonic treatment, and the supernatant was collected by centrifugation. After washing, the precipitate was collected to obtain TiH2 powder. 1.924 quantum dots; (2) The cultured Chlorella was stirred and reacted with chitosan solution at room temperature, washed and resuspended in PBS buffer to obtain surface-modified Chlorella. (3) The surface-modified Chlorella obtained in step (2) is combined with the TiH prepared in step (1). 1.924 Quantum dots were mixed, vortexed, and then centrifuged. The supernatant was discarded to obtain the titanium hydride quantum dot-functionalized Chlorella biocomposite material.

2. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (1), the ratio of TiH2 powder to N-methylpyrrolidone is 100 mg : 20 mL.

3. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (1), the conditions for ultrasonic treatment are: ultrasonic power density 2-2.5 W / cm². 2 The ultrasonic frequency was 3MHz, the pulse duty cycle was 20%, the ultrasonic treatment time was 1 min, and the ultrasonic temperature was 15℃.

4. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (1), the washing is performed twice with anhydrous ethanol and centrifuged at 12,000 rpm for 10 min.

5. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (2), the volume ratio of the cultured Chlorella to the chitosan solution is 4:1; the concentration of the cultured Chlorella is 1×10⁻⁶. 7 The concentration of the chitosan solution is 1 mg / mL.

6. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (2), the stirring reaction conditions are: 200 rpm and 4 h.

7. The method for preparing a titanium hydride quantum dot-functionalized Chlorella biocomposite material according to claim 1, characterized in that, In step (3), the surface-modified Chlorella and TiH 1.924 The volume ratio of quantum dots is 2:1, in which TiH 1.924 The concentration of quantum dots was 5 mg / mL.

8. A titanium hydride quantum dot-functionalized Chlorella biocomposite material prepared by the preparation method according to any one of claims 1-7.

9. A pharmaceutical composition, characterized in that, It includes the titanium hydride quantum dot-functionalized Chlorella biocomposite material of claim 8 and a pharmaceutically acceptable carrier.

10. The application of the titanium hydride quantum dot-functionalized Chlorella biocomposite material as described in claim 8 in the preparation of a drug for tumor treatment, characterized in that, The conditions for tumor treatment are as follows: under the combined stimulation of ultrasound and a laser with a wavelength of 660 nm, reactive oxygen species are generated and immunogenic cell death is induced by the titanium hydride quantum dot-functionalized Chlorella biocomposite material.

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