Anti-tumor heterojunction functionalized chlorella as well as preparation method and application thereof

Abstract

The invention discloses an anti-tumor heterojunction functionalized chlorella as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The heterojunction functionalized chlorella is obtained by loading aminated black phosphorus nanosheets and antimonene quantum dots on chlorella cells. BP-NH2 and AM are obtained through a method of combining water bath ultrasound and cell disrupter ultrasound, AM and Chl are co-incubated to obtain AM (at) Chl, obvious color change is observed, and then AM (at) Chl is connected with BP-NH2 to obtain BP-NH2 (at) AM (at) Chl. Through verification, BP-NH2 (at) AM (at) Chl has good capability of generating hydroxyl free radicals, successful construction of the heterojunction avoids compounding of electron-hole pairs, prolongs the service life of electrons and holes, improves PDT capability, shows a good inhibition effect on tumor cells, and has good tumor treatment potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-tumor heterojunction functionalized Chlorella, a preparation method thereof, and an application thereof. Background Art

[0002] Hypoxia is a common characteristic of solid tumors. Studies have shown that the hypoxic environment in tumors promotes cell proliferation, forms heterogeneous vascular structures, and triggers lymphatic system dysfunction. This series of changes can lead to tumor metastasis, induce angiogenesis, and confer multidrug resistance in tumor cells. Furthermore, the hypoxic tumor microenvironment is a key factor in the development of resistance to various therapeutic approaches. To improve tumor hypoxia, various mitigation measures have been proposed, primarily categorized as physical oxygenation and chemical oxygenation. Physical oxygenation involves dissolving oxygen in a highly oxygen-soluble medium (such as perfluorocarbons), but this method suffers from low delivery efficiency and the risk of medium leakage. Chemical oxygenation utilizes endogenous cellular substances (H2O2) to transport catalase or catalase-like materials to the tumor site, where they decompose H2O2 to produce oxygen. However, catalase is easily inactivated in physiological environments, hindering long-term tumor suppression. Furthermore, the safety of the ions released by catalase-like materials requires further investigation. In summary, it is necessary to find a material that is physiologically stable, has strong oxygen production capacity, good biocompatibility, and can effectively treat tumors and improve the hypoxic environment.

[0003] Engineered microorganisms (including bacteria, viruses, microalgae, and fungi) combine microorganisms with functional materials through the comprehensive application of various technical means to enhance the natural anti-tumor activity of microorganisms and alleviate treatment limitations. The application of engineered microorganisms in tumor treatment has become a promising treatment method. Chlorella (Chl) is a photosynthetic microorganism with extremely high photosynthetic efficiency (approximately 8%). It can continuously produce O2 under light conditions and is considered an ideal "oxygen supply motor." In addition, the contents of Chlorella can release immunostimulatory substances to stimulate the body's immune response. Therefore, Chlorella can be used as an excellent material to improve the hypoxic environment and continuously regulate the hypoxic tumor microenvironment.

[0004] Photodynamic therapy (PDT) is a non-invasive treatment method in which photosensitizers react with O2 molecules under laser irradiation to produce highly cytotoxic reactive oxygen species (ROS) to kill tumor cells. PDT is a promising tumor treatment method due to its strong specificity, good controllability and minimal toxic side effects. However, the hypoxic tumor microenvironment, limited light penetration depth and tumor immunosuppression are three major obstacles to the application of PDT. Improving the hypoxic environment can not only enhance the effect of PDT in producing ROS, but also enhance the response of the immune system. Both photosynthesis and PDT treatment of Chlorella require light of a specific wavelength, but the penetration of light in tissues is limited. How to improve the oxygen production capacity of Chlorella under limited light conditions and the ability of PDT to produce reactive oxygen species has become a key issue in current research. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-tumor heterojunction functionalized Chlorella and its preparation method and application to solve the problems existing in the above-mentioned prior art. After modification with AM and BP-NH2, the oxygen production capacity of Chl is significantly enhanced, the hypoxic environment is improved, the anti-tumor efficacy is enhanced, and it has good tumor treatment potential.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an anti-tumor heterojunction functionalized Chlorella, which is obtained by loading amino-modified black phosphorus nanosheets and antimonene quantum dots onto Chlorella cells.

[0008] The present invention also provides a method for preparing the heterojunction functionalized Chlorella, comprising the following steps:

[0009] Step 1: Grind and disperse antimony powder in N-methylpyrrolidone, sonicate in a water bath and a cell disruptor, collect the supernatant by centrifugation, and centrifuge the supernatant again to collect the precipitate to obtain antimonene quantum dots;

[0010] Step 2: Grind and disperse black phosphorus in N-methylpyrrolidone, sonicate in a water bath and with a cell disruptor, and collect the supernatant by centrifugation. The supernatant is centrifuged again to collect the precipitate, which is black phosphorus nanosheets. The black phosphorus nanosheets are mixed with polyethyleneimine, stirred for 1 hour, and then arginine is added. After centrifugation, the amino-modified black phosphorus nanosheets are obtained.

[0011] Step 3, co-incubating the antimonene quantum dots and Chlorella and then centrifuging to obtain Chlorella that has engulfed the quantum dots;

[0012] Step 4: After vortexing the aminated black phosphorus nanosheets and tannic acid, the Chlorella vulgaris that has engulfed the quantum dots is added and vortexed again. Then, ferric chloride is added and vortexed again. The mixture is centrifuged to obtain the heterojunction functionalized Chlorella vulgaris.

[0013] Optionally, in step 1, the centrifugation condition is 3000 rpm for 20 min, and the re-centrifugation condition is 13000 rpm for 10 min.

[0014] Optionally, in step 2, the centrifugation condition is 7000 rpm for 10 min, and the centrifugation condition is 12000 rpm for 10 min.

[0015] Optionally, in step 2, the ratio of the black phosphorus nanosheets, polyethyleneimine and arginine is 1:10:3.

[0016] Optionally, in step 3, the concentration of the antimonene quantum dots is 50 μg / mL, and the concentration of the Chlorella vulgaris is 1×10 7 cell / mL.

[0017] Optionally, in step 4, the concentration of the amino-modified black phosphorus nanosheets is 0.2 mg / mL, the concentration of the tannic acid is 24 mM, and the concentration of the ferric chloride is 24 mM;

[0018] The time for vortexing, re-vortexing and continued vortexing was 30 seconds.

[0019] The present invention also provides the use of the heterojunction functionalized Chlorella in preparing anti-tumor drugs.

[0020] The present invention also provides an anti-tumor drug comprising the heterojunction functionalized Chlorella.

[0021] Optionally, it further comprises pharmaceutically acceptable excipients.

[0022] The present invention discloses the following technical effects:

[0023] The present invention utilizes a top-down preparation method, combining waterbath sonication and cell disruptor sonication to produce BP-NH2 and AM. AM and Chl are co-incubated to produce AM@Chl, which exhibits a distinct color change. AM@Chl is then linked to BP-NH2 to produce BP-NH2@AM@Chl. The size and zeta potential of BP-NH2@AM@Chl and its components were investigated. The drug-loading capacity of Chlorella vulgaris, the activity of various modified Chlorella species, the in vitro activity, cytotoxicity, and reactive oxygen species production of BP-NH2@AM@Chl, and the in vivo antitumor activity of BP-NH2@AM@Chl were also investigated, as detailed below.

[0024] (1) The characterization results of BP and AM showed that the potential of BP was -22.93±0.85mV, and the surface charge became 22.67±1.27mV after amination treatment. TEM showed that there was no obvious difference in the morphology of BP-NH2 structure, which was a lamellar structure with a particle size of about 150nm; the surface charge of AM was -20±0.72mV, and TEM showed a point-like distribution with a size of less than 10nm, which was consistent with the characteristics of quantum dots.

[0025] (2) Characterization of BP-NH2@AM@Chl: After modification with AM and BP-NH2, the charge changed from -29.43±0.57 mV to -21.13±0.67 mV. Both TEM and SEM could observe the loading of nanosheets on Chlorella, indicating the successful preparation of BP-NH2@AM@Chl.

[0026] (3) The colonies of Chl modified with AM and BP-NH2 showed no significant difference from those of pure Chl, indicating that the modified Chl still had good activity. Further testing of its in vitro activity showed that after modification with AM and BP-NH2, the oxygen production of BP-NH2@AM@Chl increased, confirming that the heterojunction constructed by the present invention can significantly enhance the oxygen production capacity of Chl. In addition, hydroxyl radical generation experiments showed that BP-NH2@AM@Chl has a good ability to generate hydroxyl radicals.

[0027] (4) BP-NH2@AM@Chl cell experiments showed that under 660nm near-infrared light irradiation, Chl cells generated O2 through photosynthesis. At the same time, the successful construction of the heterojunction avoided the recombination of electron-hole pairs, extended the lifespan of electrons and holes, improved the PDT ability, generated a large amount of ROS, and showed a good inhibitory effect on tumor cells.

[0028] (5) In vivo anti-tumor experiments with BP-NH2@AM@Chl showed that the tumor weight and volume of the BP-NH2@AM@Chl+660 group decreased significantly, indicating that it has a good tumor inhibitory effect. In addition, no significant damage was caused to the main organs, blood routine, body weight, and skin of the mice in each treatment group, indicating good biocompatibility. In addition, tumor tissue homogenates were collected at different treatment days, and the coating experiment showed that Chlorella has a good degradation effect inside the tumor, high biosafety, and good tumor treatment potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 Schematic diagram of the experimental process of Example 1;

[0031] Figure 2 Morphological characterization of BP, BP-NH2 and AM; a: Schematic diagram of the preparation of BP, BP-NH2 and AM; bc: TEM images of BP; de: TEM images of BP-NH2; fg: TEM images of AM;

[0032] Figure 3 Results of transmission electron microscopy and scanning electron microscopy; a: TEM image of Chl; bc: TEM image of BP-NH2@AM@Chl; d: SEM image of Chl; e: SEM image of BP-NH2@AM@Chl; f: actual image of co-incubation of AM and Chl; gh: SEM EDS images of Chl and BP-NH2@AM@Chl; i: TEM EDS image of BP-NH2@AM@Chl;

[0033] Figure 4 is the hydrated particle size distribution of BP, BP-NH2 and AM;

[0034] Figure 5 The potential of different components;

[0035] Figure 6 UV absorption diagram of different components;

[0036] Figure 7 Energy band analysis of BP-NH2, AM and Chl; ac: valence band diagram of BP-NH2, AM and Chl; df: band gap diagram of BP-NH2, AM and Chl calculated by Kubelka-Munk equation; g: principle diagram of photoexcited electron transfer in BP-NH2@AM@Chl;

[0037] Figure 8 is the X-ray diffraction pattern of different components;

[0038] Figure 9 The standard curves of UV absorption of Chl (a) and BP-NH2 (b);

[0039] Figure 10 Chl, AM@Chl, and BP-NH2@AM@Chl cultured on BG11 solid medium;

[0040] Figure 11 The O2 production of Chl, AM@Chl and BP-NH2@AM@Chl under light conditions;

[0041] Figure 12The degradation of MB after treatment with BP-NH2@AM@Chl at different times (a) and different concentrations (b);

[0042] Figure 13 Live-dead staining images of 4T1 cells treated with different methods;

[0043] Figure 14 Figure 2 shows the fluorescence images of reactive oxygen species in 4T1 cells treated with different methods (a) and the fluorescence quantitative analysis of reactive oxygen species in 4T1 cells treated with different methods (b).

[0044] Figure 15 Figure 1 shows the in vivo anti-tumor results of BP-NH2@AM@Chl; a: Treatment plan for tumor-bearing mice; b: In vitro images of tumors in tumor-bearing mice; c: Changes in tumor volume in tumor-bearing mice treated with different treatment groups over 14 days; d: In vitro weight of tumors in tumor-bearing mice;

[0045] Figure 16 Tumor tissues were stained with H&E and TUNEL;

[0046] Figure 17 Routine blood analysis of tumor-bearing mice after treatment for different treatment groups;

[0047] Figure 18 Pathological examination of major organs of tumor-bearing mice after treatment with different treatment groups (a) and body weight changes within 14 days (b);

[0048] Figure 19 Results of the allergic effect of BP-NH2@AM@Chl; a: Images of tumor-bearing mice treated with different treatment groups; b: In vivo survival of Chl in tumor homogenate on BG11 solid culture medium after treatment on the 1st and 14th days. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] The present invention introduces photosynthesis, the most efficient and intelligent self-oxygen supply system in nature, as a whole to overcome the hypoxic microenvironment of tumors. On this basis, through surface assembly and internal phagocytosis of photoactive nanoparticles BP-NH2 and AM, heterojunction structures based on the photosynthetic system of Chlorella cells are constructed on the surface and inside of Chlorella cells, respectively, to enhance Chlorella photosynthesis while providing a multi-mode synergistic anti-tumor effect. BP-NH2 is coupled with the original photosynthetic system in Chlorella cells to construct a heterojunction, which promotes the migration of excited electrons and holes of BP-NH2 and chlorophyll in Chlorella cells to the CB of BP-NH2 and the VB of Chlorella chlorophyll, respectively, and simultaneously and synergistically enhances the oxygen production capacity of Chlorella photosynthesis and the photodynamic production of BP-NH2. 1 O2 capacity; AM is coupled with the original photosynthetic system in the Chlorella cells to construct a heterojunction, which promotes the recombination of the excited electrons of chlorophyll in the Chlorella cells with the holes of AM, so that the excited electrons gather in the CB of AM, effectively improving the reduction capacity of the original excited electrons, thereby enhancing the ability of Chlorella to produce O2 through photosynthesis. See the following example (schematic diagram as shown in the figure) for details. Figure 1 shown).

[0055] Example 1

[0056] 1. Experimental reagents

[0057] The main experimental reagents used in the present invention are shown in Table 1.

[0058] Table 1 Experimental reagents and materials

[0059]

[0060]

[0061] 2. Preparation of BP@AM@Chl

[0062] 2.1 Cultivation of Chl

[0063] Place the newly arrived algae in a sterile conical flask and culture in an incubator under low light conditions (12h light / 12h dark, 25°C). For expansion, prepare sterile BG11 medium (components (g / L): sodium nitrate 1.5g, dipotassium hydrogen phosphate trihydrate 0.04g, magnesium sulfate heptahydrate 0.075g, calcium chloride dihydrate 0.036g, citric acid 0.006g, ammonium ferric citrate 0.006g, EDTA 0.001g, sodium carbonate 0.02g, boric acid 0.00286g, manganese chloride monohydrate 0.00181g, zinc sulfate heptahydrate 0.000222g, copper sulfate pentahydrate 0.000079g, sodium molybdate dihydrate 0.00039g, cobalt nitrate hexahydrate 0.000049g), inoculate the cultured Chlorella vulgaris at a ratio of algae liquid:medium of 1:5. Place the expanded Chlorella in an incubator for further culture.

[0064] Preparation of 2.2AM and BP-NH2

[0065] An appropriate amount of antimony powder was thoroughly ground and dispersed in NMP to form a 20 mg / mL mixed solution. The mixed solution was sonicated in a water bath for 6 h and then sonicated in a cell disruptor for 12 h (ultrasound power: 1100 W). The solution was centrifuged at 3000 rpm for 20 min to remove large nanosheets. The supernatant was collected and centrifuged again at high speed (13000 rpm for 10 min) to collect the precipitate to obtain AM (antimonene quantum dots).

[0066] The blocky black phosphorus was fully ground under a nitrogen atmosphere and dispersed in NMP to form a mixed solution of 2 mg / mL. The mixed solution was ultrasonicated in a water bath for 2 hours and then ultrasonicated for 6 hours using a cell disruptor (ultrasound power: 1100w). The large particles of BP were removed by centrifugation at 7000rpm for 10 minutes, and the supernatant was collected and centrifuged again at high speed (12000rpm for 10 minutes) to collect the precipitate, which was BP (black phosphorus nanosheets). An appropriate amount of BP and PEI were mixed in pure water, and L-arg (the ratio of BP, PEI and arginine: 1:10:3) was added after vigorous stirring for 1 hour. After reacting for 24 hours, the precipitate was collected by centrifugation (12000rpm for 10 minutes) to obtain amino-containing BP (BP-NH2).

[0067] Preparation of 2.3BP-NH2@AM@Chl

[0068] The chlorella that engulfed quantum dots was obtained by co-incubation. AM (50 μg / mL) and Chl (1×10 7The cells were incubated at 200 rpm for 24 hours and then centrifuged to obtain quantum dot-engorged Chlorella vulgaris (AM@Chl). BP-NH2 (0.2 mg / mL) and tannic acid (24 mM) were vortexed for 30 seconds, followed by the addition of AM@Chl and a further vortexing of 30 seconds. Then, ferric chloride (24 mM) was added and vortexed for 30 seconds. Finally, the solution was centrifuged at 5000 rpm for 10 minutes to collect the precipitate, yielding BP-NH2@AM@Chl.

[0069] Preparation of 2.4BP-NH2@Chl

[0070] BP-NH2 (0.2 mg / mL) and tannic acid (24 mM) were vortexed for 30 seconds and then added to Chlorella vulgaris (1×10 7 The mixture was vortexed for 30 seconds, and then ferric chloride (24 mM) was added and vortexed for 30 seconds. Finally, the mixture was centrifuged at 5000 rpm for 10 minutes to collect the precipitate to obtain BP-NH2@Chl.

[0071] 3. Characterization of BP-NH2@AM@Chl

[0072] 3.1 Morphological characterization

[0073] Freshly prepared AM, BP, BP-NH2, gradient-dehydrated Chl, and BP-NH2@AM@Chl were dispersed in anhydrous ethanol. An appropriate volume of the solution was slowly dripped onto a copper grid. After the ethanol evaporated, the morphology was observed using TEM. An appropriate amount of sample was dripped onto tin foil, and the surface morphology of the material was observed using SEM. EDS was used to identify and quantify the elemental composition and chemical state of the material.

[0074] 3.2 Hydration particle size and potential detection

[0075] Freshly prepared AM, BP, BP-NH2, Chl, AM@Chl and BP-NH2@AM@Chl were ultrasonically dispersed in pure water, and the hydrated particle size and potential were measured using a Malvern laser particle size analyzer. The same material was measured three times in parallel.

[0076] 3.3UV detection

[0077] Appropriate amounts of AM, BP-NH2, tannic acid, Chl, AM@Chl, and BP-NH2@AM@Chl were dispersed in water, and the UV absorption of each material at 200-800 nm was detected using a UV spectrophotometer.

[0078] 3.4 Energy band analysis

[0079] The band structures of BP-NH2, AM and Chl were detected by X-ray photoelectron spectroscopy (XPS) and ultraviolet diffuse reflectance.

[0080] 3.5 X-ray diffraction (XRD)

[0081] In order to evaluate the prepared crystals, X-ray diffraction was carried out at room temperature using a Rigaku Ultima VI X-ray diffraction instrument.

[0082] 4. Load rate measurement

[0083] Chl standard curve drawing: take an appropriate amount of Chl to obtain Chl solutions of different concentrations (0.025, 0.05, 0.1, 0.2, 0.3, 0.4 mg / mL), use UV-Vis to measure the absorbance of the samples to obtain the standard curve.

[0084] BP-NH2 standard curve drawing: Take an appropriate amount of BP-NH2 to obtain BP solutions of different concentrations (5, 10, 20, 40, 60, 80, 160 μg / mL), use UV-Vis to measure the absorbance of the samples, and obtain the standard curve.

[0085] As described in "2. Preparation of BP-NH2@AM@Chl," the stirred product was centrifuged and the supernatant collected. The BP-NH2 loading efficiency was calculated according to the following formula: M1 is the mass of the initial BP-NH2 added, M2 is the mass of the BP-NH2 in the supernatant after centrifugation, and M3 is the mass of the carrier Chl.

[0086]

[0087] 5. Determination of Chlorella Activity

[0088] In order to study the effects of different modifications on the activity of Chl, Chl, AM@Chl and BP-NH2@AM@Chl were coated on sterile BG11 solid plates and cultured in an artificial light incubator. The growth of Chl was recorded by photographing after two weeks.

[0089] 6. In vitro activity of BP-NH2@AM@Chl

[0090] 6.1 Generation of hydroxyl radicals

[0091] BP-NH2@AM@Chl([Chl]=0.25×10 7 The suspension of cells / mL was mixed with MB (1 mg / mL) and H2O2 and exposed to 660 nm laser irradiation (0.1 W / cm 2 ), and the absorbance of the sample was measured by UV-Vis within 30 min. The solutions with different Chl concentrations (0.25, 0.5, 1, 2×10 7cells / mL) were mixed with MB and H2O2 and exposed to 660 nm laser irradiation (0.1 W / cm 2 ) for 30 min, centrifuge and collect the supernatant, and use UV-Vis to measure the absorbance of the sample.

[0092] 6.2 In vitro O2 production

[0093] Preparation of Chl, AM@Chl and BP-NH2@AM@Chl ([Chl] = 2 × 10 7 Before detection, the mixed solution was placed in the dark for 1 hour to allow the solution to reach a stable hypoxic state. 2 ) irradiate the mixed solution and use a dissolved oxygen meter to record the dissolved oxygen content within 30 minutes.

[0094] 7. Cell experiments

[0095] 7.1 Cell culture

[0096] 4T1 (breast cancer cells) were used as a tumor cell model and cultured in RPMI 1640 medium (containing 1% (v / v) penicillin-streptomycin, 10% (v / v) fetal bovine serum) in a carbon dioxide cell culture incubator at 37°C and 5% humidity.

[0097] 7.2 Live-dead staining

[0098] After 4T1 cells were cultured to the logarithmic phase, the cell suspension (1 mL, about 2 × 10 5 Cells / well) were seeded into 12-well plates. After culturing for 24 h, the old culture medium was removed and serum-free culture medium containing different drugs was added. The experimental groups included (1) Control group; (2) Chl group; (3) Chl+660 group; (4) AM@Chl group; (5) AM@Chl+660 group; (6) BP-NH2@AM@Chl group; (7) BP-NH2@AM@Chl+660 group, ([Chl] = 1×10 7 For the 660 nm treatment group, after incubation with the drug for 12 h, a laser at 0.1 W / cm 2 Irradiate with a power of 100 nm for 20 minutes. Wash the cells three times with PBS. Then, add 12 μL of AO / EB working solution (AO:EB:PBS ratio of 1:1:8) to each well. Gently shake the plate to ensure even coverage of the cell bottom. After staining for 5 minutes, observe and image using a fluorescence microscope.

[0099] 7.3 Intracellular ROS

[0100] After 4T1 cells were cultured to the logarithmic phase, the cell suspension (1 mL, about 3 × 10 5 Cells / well) were seeded into 6-well plates. After culturing for 24 h, the old culture medium was removed and serum-free culture medium containing different drugs was added. The experimental groups included (1) Control group; (2) Chl group; (3) Chl+660 group; (4) AM@Chl group; (5) AM@Chl+660 group; (6) BP-NH2+660 group; (7) BP-NH2@Chl+660 group; (8) BP-NH2@AM@Chl group; (9) BP-NH2@AM@Chl+660 group, ([Chl] = 1×10 7 For the 660 nm treatment group, after incubation with the drug for 12 h, a laser at 0.1 W / cm 2 Irradiate with a power of 100 nm for 20 min. Remove the drug-containing medium and wash the cells with PBS. Add 2 mL of PBS solution containing DCFH-DA (10 μM) to each well. Incubate in the dark for 1 h before observation and imaging using a fluorescence microscope.

[0101] 8. Anti-tumor effect in vivo

[0102] 8.1 Establishment of tumor-bearing mouse model

[0103] Animal experiments were conducted in strict compliance with animal welfare and ethical standards. The acceptance agency is the Laboratory Animal Welfare and Ethics Committee of Zhejiang Ocean University, acceptance number 2024116. Five-week-old BALB / c female mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. All samples and instruments were sterilized by ultraviolet irradiation for 1 hour before the experiment. All mice were killed by cervical dislocation after the experiment. After 7 days of adaptive feeding, 100 μL of 4T1 cells (10 cells per mouse) were injected subcutaneously into the right dorsal region of the mice. 6 cells).

[0104] 8.2 In vivo anti-tumor effects

[0105] The tumor volume was approximately 80 mm 3 The tumor-bearing mice were randomly divided into 7 groups (4 mice in each group): (1) Control group; (2) Chl group; (3) Chl+660 group; (4) AM@Chl group; (5) AM@Chl+660 group; (6) BP-NH2@AM@Chl group; (7) BP-NH2@AM@Chl+660 group. Different materials were injected into the tumor of mice ([Chl] = 1×10 7 In groups (3), (5), and (7), 660 nm laser was used to irradiate the tumor site (0.1 W / cm 2, 20 min). The mouse body weight and tumor size were recorded every other day, and the tumor volume (V) was calculated using the following formula, where a = the widest part of the tumor and b = the narrowest part of the tumor.

[0106]

[0107] The mice were sacrificed on day 14, and the tumor tissues were collected, weighed, and photographed.

[0108] 8.3 Pathological Observation

[0109] Tumor tissue was collected and preserved in 4% paraformaldehyde tissue fixative. The tissue was dehydrated, embedded, and then frozen for sectioning. The sections were dewaxed and stained with hematoxylin-eosin (H&E) and TUNEL to assess tumor cell morphology and apoptosis.

[0110] 8.4 In vivo safety studies

[0111] After treatment, the main organs (heart, liver, spleen, lungs, and kidneys) of the mice were collected and preserved in 4% paraformaldehyde tissue fixative. The tissues were embedded, dewaxed, and stained with H&E to evaluate the effects of each treatment group on the tumor-bearing mouse tissues. To further investigate the in vivo safety of each treatment group, orbital blood was collected from each group of mice on day 14 of treatment for routine blood analysis.

[0112] The long-term activity of Chl in mouse tumors was analyzed. Tumor tissues were collected on the 1st and 14th day after intratumoral injection, homogenized and spread on BG11 solid plates, and cultured in an artificial light incubator. Photos were taken and recorded after one week.

[0113] 9. Results and Analysis

[0114] Characterization of 9.1BP-NH2@AM@Chl

[0115] 9.1.1 Morphological Characterization

[0116] The method for preparing BP-NH2 and AM is as follows Figure 2 As shown in a, BP and AM were obtained by mixed ultrasound using a water bath and a cell disruptor, and finally by differential centrifugation. BP is easily oxidized in the air, while amino-treated BP has higher stability and biosafety.

[0117] The transmission electron microscopy image of BP prepared by liquid phase exfoliation method using NMP as solvent is shown in the figure below. Figure 2 As shown in Figures bc, BP is in an irregular flake shape with uniform thickness. Some particles have a layered structure with a visible surface. The nanosheet size is about 140nm and the lattice spacing is 0.35nm. Figure 2As shown in Figure 5, the morphology of the nanosheets has not changed significantly, the particle size is about 150nm, and the lattice spacing is 0.34nm, and there is no significant change in the two. Figure 2 As shown in Figures f-g, AM is arranged in a dot-like pattern with a particle size of approximately 5 nm. Upon magnification, distinct lattice fringes are observed, with a lattice spacing of 0.3 nm, consistent with the lattice interval of quantum dots. In summary, TEM analysis demonstrates the successful synthesis of nanosheets and quantum dots.

[0118] Transmission electron microscopy and scanning electron microscopy were used to determine whether the two-dimensional material was successfully modified inside and outside Chl. Figure 3 As shown. The surface of pure Chl is a cell wall with smooth wrinkles. The surface of the modified BP-NH2@AM@Chl exhibits a distinct granular texture. Transmission electron microscopy reveals distinct lattice fringes on the Chl cell wall with a lattice spacing of 0.53 nm. This is likely due to the incorporation of AM@Chl, which alters the lattice spacing of BP-NH2. Further surface elemental analysis of BP-NH2@AM@Chl using EDS revealed that the phosphorus content on the surface of pure Chlorella was 2.5%. This increased to 4.5% after modification with BP-NH2, indicating successful loading of BP-NH2. Because AM was engulfed by Chlorella, surface elemental scanning of BP-NH2@AM@Chl revealed no Sb. Therefore, transmission EDS scanning of BP-NH2@AM@Chl revealed the presence of C, N, O, P, Fe, and Sb within BP-NH2@AM@Chl, indicating successful engulfment of AM by Chl. The actual picture of the co-incubation of AM and Chl shows that the color of AM changes from gray to dark green when co-incubated with Chl. After 24 h of co-incubation, the color returns to the emerald green of Chlorella, indicating that the phagocytosis is complete.

[0119] 9.1.2 Particle size distribution and potential analysis

[0120] The particle size distribution of BP, BP-NH2 and AM measured by dynamic light scattering (DLS) is shown in Figure 2. Figure 4 As shown in Figure 2, the hydrated particle size of BP is approximately 127 nm, slightly increasing after amino modification. The hydrated particle size of AM is approximately 110 nm. The discrepancy between the experimental results and the electron microscopy results may be due to the fact that DLS measures the particle size in water, detecting the aggregate diameter. Particles form a hydration layer in water, and some particles are polydispersed in water, resulting in a larger detected particle size than the actual size. AM has poor solubility in water, so the detected value may be an aggregate of multiple particles, which may be larger than the actual value.

[0121] Zeta potential results are as follows Figure 5As shown, the potential of BP was -22.93±0.85mV, which is within the normal potential range of BP (BP has phosphate groups on its surface, so it is negatively charged in water). To enhance the biocompatibility and stability of BP, BP was amino-treated, and the surface charge of BP-NH2 changed from negative to 22.67±1.27mV. This change in potential indicates the successful amino modification. The surface charges of AM and Chl were -20±0.72mV and -29.43±0.57mV, respectively. In addition, the surface charge of Chl after engulfing AM did not change significantly, while the surface charge of BP-NH2 was reduced to -21.13±0.67mV after the introduction of BP-NH2. This charge change once again confirmed that BP-NH2 was successfully loaded onto Chlorella.

[0122] 9.1.3 UV Analysis

[0123] The particles prepared in the experiment were tested using a UV-visible spectrophotometer. Figure 6 As shown, AM exhibits a distinct absorption peak at approximately 200 nm. Chl cells contain chloroplasts, and the pigment molecules within these chloroplasts absorb light in the UV-visible band. The UV spectrum reveals a characteristic peak for Chl at approximately 692 nm, which is observed in both AM@Chl and BP-NH2@AM@Chl, indicating the presence of Chl. BP-NH2 exhibits UV absorption near 300-600 nm. The absorption at 300 nm observed in BP-NH2@AM@Chl after the introduction of BP-NH2 is attributed to the tannic acid introduced when BP-NH2 was loaded onto AM@Chl.

[0124] 9.1.4 Energy Band Analysis

[0125] The results are as follows Figure 7As shown, the valence band (VB) values of BP-NH2, AM, and Chl were 0.83 eV, 0.63 eV, and 0.31 eV, respectively. The band gaps (Eg) of BP-NH2, AM, and Chl were calculated using the Kubelka-Munk equation to be 1.45 eV, 1.97 eV, and 1.74 eV, respectively. The difference between Eg and VB represents the conduction band (CB) value. Further calculations revealed CB values of -0.62 eV, -1.34 eV, and -1.43 eV for BP-NH2, AM, and Chl, respectively. When the CB and VB of photosensitizers 1 and 2 form an interdigitated structure, a type II heterojunction is formed. Examination of the CB and VB of BP-NH2, AM, and Chl revealed that the structures between BP-NH2 and Chl, and between AM and Chl, conform to type II heterojunction structures. Under 660nm laser irradiation, BP-NH2, Chl and AM in Chl are simultaneously excited. In the heterojunction structure formed by the three, the electrons on the CB of Chlorella chlorophyll tend to be transferred to the CB of BP-NH2 and AM. At the same time, the holes on the VB of BP-NH2 and AM are transferred to the VB of Chl, fundamentally avoiding the recombination of electron-hole pairs. The photogenerated electron-hole pairs with extended lifetime can enhance the ability of BP-NH2@AM@Chl to produce oxygen and reactive oxygen species through light.

[0126] 9.1.5 XRD analysis

[0127] The XRD results of the prepared AM, BP-NH2, Chl and BP-NH2@AM@Chl are shown in Figure 8 As shown, the XRD pattern of AM is consistent with the hexagonal phase of antimony (JCPDS 35-0732), and that of BP-NH2 is consistent with BP-NH2 (JCPDS No. 73-1358). Chl has no obvious diffraction peaks. The BP-NH2@AM@Chl modified with AM and BP-NH2 exhibits characteristic patterns of both AM and BP-NH2, indicating successful encapsulation of AM and successful loading of BP-NH2. New diffraction peaks were observed at approximately 31° and 56°, likely due to the formation of a new crystal structure after loading BP-NH2 onto AM@Chl.

[0128] 9.2 Load rate detection

[0129] The UV standard curve of BP-NH2 is as follows Figure 9 As shown in a, the regression equation is y = 0.0085x + 0.0169, R 2 =0.9997, the linear relationship is good. The relationship between the amount of Chl and UV absorption is as follows Figure 9 As shown in b, the regression equation is y = 4.3731x + 0.0394, R 2=0.995, showing a good linear relationship. By detecting the remaining BP-NH2 content in the supernatant, the loading rate of BP-NH2 was found to be 56.67%.

[0130] 9.3 Chlorella activity analysis

[0131] The photo results are as follows Figure 10 As shown, the colonies of Chl on the solid plate treated with AM and BP-NH2 were similar to those of pure Chl, indicating that the Chl after the above modifications still had good activity and could grow normally.

[0132] In vitro activity analysis of 9.4BP-NH2@AM@Chl

[0133] 9.4.1 In vitro O2 production

[0134] Under 660nm near-infrared light irradiation, Chl can produce O2. The dissolved oxygen content change was detected by a dissolved oxygen meter to study the oxygen production capacity of AM@Chl and BP-NH2@AM@Chl. Figure 11 As shown, under 660nm excitation light, Chl, AM@Chl, and BP-NH2@AM@Chl can all photosynthesize and produce O2. AM@Chl and BP-NH2@AM@Chl with the same number of Chl cells produce more O2 than Chl alone. AM@Chl's superior O2 production is likely due to the formation of a heterojunction between Chl and AM after engulfing quantum dots, thereby enhancing O2 production. BP-NH2 can consume O2 under 660nm laser light by mediating PDT, but its O2 production is also higher than that of the Chl group with the same number of Chl cells. This indicates that BP-NH2@AM@Chl photosynthetically produces more O2 than it consumes. This is because AM and BP-NH2 can form heterojunctions within and on the surface of Chlorella, promoting the rapid separation of photogenerated charge carriers and enhancing the oxygen production capacity of Chlorella.

[0135] 9.4.2 Generation of Hydroxyl Radicals

[0136] The excellent oxygen production capacity of BP-NH2@AM@Chl lays the foundation for its PDT effect. MB is a commonly used ·OH detection agent. The MB characteristic absorption peak near 664nm can be used to detect the ·OH situation based on the changes in the characteristic peak. Figure 12 As shown in Figure a, as the irradiation time increases, the absorbance of the characteristic absorption peak of MB in the mixed solution continues to decrease, indicating that the Chl cells in BP-NH2@AM@Chl produce O2 through photosynthesis, which promotes BP-NH2 to exert PDT effect and generate ROS. Figure 12As shown in Figure b, with the increase of the number of Chl, the absorption peak of MB gradually decreases and the color of MB is almost transparent. This is because with the increase of the number of Chl, the amount of O2 produced by Chl photosynthesis and the amount of BP-NH2 inducing PDT increase, thus showing excellent ·OH generation ability.

[0137] 9.5BP-NH2@AM@Chl cell experimental analysis

[0138] 9.5.1 Live-dead staining results

[0139] Fluorescence results such as Figure 13 As shown, the Chl and AM@Chl groups alone were non-cytotoxic to 4T1 cells. However, after 660nm laser irradiation, their killing ability against 4T1 cells was enhanced. This is attributed to the 660nm laser promoting photosynthesis of Chl to produce O2, which in turn generates toxic radicals within the cells, thereby killing tumor cells. Cell death occurred in the BP-NH2@AM@Chl group. This is attributed to the fact that even in the absence of light, the charge on the BP-NH2 surface also transfers to produce ·OH, thereby killing tumor cells. When irradiated with 660nm laser, almost all cells in the BP-NH2@AM@Chl group died, confirming the excellent tumor cell killing ability of BP-NH2@AM@Chl+660.

[0140] 9.5.2 Intracellular ROS

[0141] DCFH-DA is a commonly used reactive oxygen species fluorescent probe that produces green fluorescence when combined with ROS. The stronger the fluorescence, the higher the ROS content. In order to further study the production of ROS, the BP-NH2+660 group and the BP-NH2@Chl+660 group were added to the group. The results are shown in Figure 2. Figure 14As shown, the Control, Chl, and AM@Chl groups exhibited almost no fluorescence, while the Chl+660 and AM@Chl+600 groups exhibited slightly enhanced fluorescence. This is likely due to 660nm laser irradiation, which caused photosynthesis of O₂ by Chl cells in the Chl group, generating trace amounts of superoxide anions and ·OH in the water. The stronger fluorescence observed in the AM@Chl+660 group can be attributed to the heterojunction formed between AM and Chl, which promotes Chl photosynthesis and produces more O₂, thereby generating more ·OH. The enhanced green fluorescence in the BP-NH₂+660 and BP-NH₂@Chl+660 groups under illumination is attributed to the generation of ROS by photodynamic action of black phosphorus under illumination. In contrast, the BP-NH₂@AM@Chl group also exhibited strong fluorescence in the absence of illumination. This is likely due to the fact that the Chlorella vulgaris, after ingesting the antimonene quantum dots, promoted charge transfer on the black phosphorus surface, transferring oxygen molecules through water molecules, and generating superoxide anions, which then converted into ·OH through the interconversion of free radicals. Compared with other groups, the BP-NH2@AM@Chl+660 group showed the strongest fluorescence intensity. This is because under the irradiation of 660nm laser, AM coupled with the original photosynthetic system in the Chlorella cells to construct a heterojunction, and the excited electrons and holes in BP-NH2 and Chlorella cells migrated to the CB of BP-NH2 and the VB of Chlorella chlorophyll respectively, reducing the hole-electron recombination and synergistically enhancing the oxygen production capacity of Chlorella photosynthesis and the photodynamic production of BP-NH2. 1 In summary, the BP-NH2@AM@Chl prepared by the present invention exhibits excellent ROS generation ability and can enhance the photodynamic reactive oxygen species generation ability of BP-NH2.

[0142] In vivo antitumor analysis of 9.6BP-NH2@AM@Chl

[0143] 9.6.1 In vivo anti-tumor analysis

[0144] Dosage regimen such as Figure 15 As shown in middle a, when the tumor volume of mice reached about 80 mm 3 The mice were randomly divided into 7 groups (n=4): (1) Control group; (2) Chl group; (3) Chl+660 group; (4) AM@Chl group; (5) AM@Chl+660 group; (6) BP-NH2@AM@Chl group; (7) BP-NH2@AM@Chl+660 group. Different preparations were injected into the tumor on day 0. The light group was irradiated with 660nm laser for 20min 4h after injection. The tumor volume and body weight of the mice were recorded every other day. The mice were killed on the 14th day after treatment. The experimental results are shown in Figure 2. Figure 15As shown in Figures b,d, and e, Chl effectively controlled tumor volume in the BP-NH2@AM@Chl+660 group compared to the control group. Tumor weight and images showed a significant decrease in tumor mass in the treated group, demonstrating a strong tumor-suppressing effect. While Chl and AM@Chl alone also exhibited some anti-tumor effects, attributed to Chl's ability to stimulate an immune response in mice, thereby suppressing tumor growth, the effect was limited. Notably, when given the same drugs, light-treated mice performed better than those not treated. This is attributed to light promoting O2 production by Chl implanted within the tumor, thereby improving the hypoxic environment within the tumor and inhibiting tumor growth. The BP-NH2@AM@Chl group exhibited superior tumor-suppressing ability compared to the Chl alone and AM@Chl groups. This is because the addition of AM and BP-NH2 enhances Chl's immune response to the tumor, thereby enhancing its tumor-suppressing effect. The BP-NH2@AM@Chl+660 group showed the best therapeutic ability among many groups, which was attributed to the fact that the addition of BP-NH2 and AM enhanced the ability of Chl to produce O2. At the same time, the improved hypoxic environment provided effective support for BP-NH2 to exert its PDT effect, thereby exerting good anti-tumor ability.

[0145] 9.6.2 Analysis of pathological results

[0146] The results of H&E and TUNEL staining were as follows Figure 16 As shown, the tumor cells in the Control group were tightly arranged, with intact cell morphology and nuclei. Although the nuclear structure between the tumor cells in the Chl group was still obvious, vacuoles appeared between the tumor cells and a small amount of cell apoptosis was observed by TUNEL staining, indicating that Chl alone can have a certain anti-tumor effect on tumor cells, but the effect is limited. The H&E staining results of the BP-NH2@AM@Chl+660 group clearly showed that the tumor cell tissue structure was ablated, the cell morphology was lost, and a large number of blanks appeared. TUNEL staining also showed that a large number of apoptotic cells appeared in this group, further demonstrating the excellent anti-tumor effect of BP-NH2@AM@Chl. Under the same dosing conditions, the light-treated groups of mice were better than those without light treatment, and the staining results were consistent with the tumor images, indicating that the addition of light can promote Chl to play an anti-tumor role.

[0147] 9.6.3 In vivo safety analysis

[0148] Blood test results such as Figure 17As shown, there were no significant differences between most groups. Compared to the control group, the RBC count in the BP-NH2@AM@Chl+660 group was slightly different but remained within normal levels. WBC counts, which correlate with inflammation levels, were significantly lower in the BP-NH2@AM@Chl+660 group compared to the control group, indicating that BP-NH2@AM@Chl+660 treatment alleviated inflammation in mice. The experimental results showed that none of the formulations had any significant effect on the mice.

[0149] After 14 days of treatment, the main organs of the mice (heart, liver, spleen, lung, and kidney) were collected and the effects of each preparation on the main organs were evaluated by H&E staining. Figure 18 As shown in Figure a, the BP-NH2@AM@Chl+660 group showed no obvious histological abnormalities compared to the Control group and did not cause serious adverse reactions in vivo. The safety of each group was evaluated by measuring and recording the weight changes of tumor-bearing mice every two days. Figure 18 As shown in middle b, the body weights of mice in each group fluctuated within a relatively stable range, indicating that each preparation had no obvious toxic side effects on the mice and could ensure their good quality of life.

[0150] Photosensitizers can cause skin allergic reactions when exposed to high-intensity light. Ideally, photosensitizers should be completely metabolized in the body after a few days of treatment. The safety of BP-NH2@AM@Chl photosensitizer in mice was further studied. After treatment, the mice were exposed to sunlight for 10 minutes and then photographed to record their skin conditions. Figure 19 As shown in Figure a, there was no obvious allergic reaction in the mouse skin compared with the control group. In order to test the activity of Chl cells in vivo, tumors on day 1 and day 14 were collected to make tumor homogenates and then smeared on sterile BG11 solid plates. The experimental results are shown in Figure 4. Figure 19 As shown in middle b, algae colonies appeared in all three groups on day 1, indicating that the Chl cells injected into the tumor were still active. No algae colonies appeared in the tumor homogenates collected on day 14, indicating that the Chl cells had been eliminated in the body. The above results indicate that BP-NH2@AM@Chl is an ideal photosensitizer with good biocompatibility and safety.

[0151] Based on the above experimental results, the present invention utilizes a top-down preparation method, combining waterbath sonication and cell disruptor sonication to produce BP-NH2 and AM. AM and Chl were co-incubated to produce AM@Chl, where a distinct color change was observed. The AM@Chl block was then connected to BP-NH2 to produce BP-NH2@AM@Chl. The size and zeta potential of BP-NH2@AM@Chl and its components were investigated. The drug-loading capacity of Chlorella vulgaris, the activity of Chlorella after different modifications, the in vitro activity, cytotoxicity, and reactive oxygen species production of BP-NH2@AM@Chl, and the in vivo anti-tumor ability of BP-NH2@AM@Chl were also investigated. The details are as follows.

[0152] (1) The characterization results of BP and AM showed that the potential of BP was -22.93±0.85mV, and the surface charge became 22.67±1.27mV after amination treatment. TEM showed that there was no obvious difference in the morphology of BP-NH2 structure, which was a lamellar structure with a particle size of about 150nm; the surface charge of AM was -20±0.72mV, and TEM showed a point-like distribution with a size of less than 10nm, which was consistent with the characteristics of quantum dots.

[0153] (2) Characterization of BP-NH2@AM@Chl: After modification with AM and BP-NH2, the charge changed from -29.43±0.57 mV to -21.13±0.67 mV. Both TEM and SEM could observe the loading of nanosheets on Chlorella, indicating the successful preparation of BP-NH2@AM@Chl.

[0154] (3) The colonies of Chl modified with AM and BP-NH2 showed no significant difference from those of pure Chl, indicating that the modified Chl still had good activity. Further testing of its in vitro activity showed that after modification with AM and BP-NH2, the oxygen production of BP-NH2@AM@Chl increased, confirming that the heterojunction constructed by the present invention can significantly enhance the oxygen production capacity of Chl. In addition, hydroxyl radical generation experiments showed that BP-NH2@AM@Chl has a good ability to generate hydroxyl radicals.

[0155] (4) BP-NH2@AM@Chl cell experiments showed that under 660nm near-infrared light irradiation, Chl cells generated O2 through photosynthesis. At the same time, the successful construction of the heterojunction avoided the recombination of electron-hole pairs, extended the lifespan of electrons and holes, improved the PDT ability, generated a large amount of ROS, and showed a good inhibitory effect on tumor cells.

[0156] (5) In vivo anti-tumor experiments with BP-NH2@AM@Chl showed that the tumor weight and volume of the BP-NH2@AM@Chl+660 group decreased significantly, indicating that it has a good tumor inhibitory effect. In addition, no significant damage was caused to the main organs, blood routine, body weight, and skin of the mice in each treatment group, indicating good biocompatibility. In addition, tumor tissue homogenates were collected at different treatment days, and the coating experiment showed that Chlorella has a good degradation effect inside the tumor, high biosafety, and good tumor treatment potential.

[0157] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An anti-tumor heterojunction functionalized Chlorella, characterized in that: It is obtained by loading amino-modified black phosphorus nanosheets and antimonene quantum dots onto Chlorella cells.

2. The method for preparing heterojunction functionalized Chlorella according to claim 1, wherein: The following steps are involved: Step 1: Grind and disperse antimony powder in N-methylpyrrolidone, sonicate in a water bath and a cell disruptor, collect the supernatant by centrifugation, and centrifuge the supernatant again to collect the precipitate to obtain antimonene quantum dots; Step 2: Grind and disperse black phosphorus in N-methylpyrrolidone, sonicate in a water bath and with a cell disruptor, and collect the supernatant by centrifugation. The supernatant is centrifuged again to collect the precipitate, which is black phosphorus nanosheets. The black phosphorus nanosheets are mixed with polyethyleneimine, stirred for 1 hour, and then arginine is added. After centrifugation, the amino-modified black phosphorus nanosheets are obtained. Step 3, co-incubating the antimonene quantum dots and Chlorella and then centrifuging to obtain Chlorella that has engulfed the quantum dots; Step 4: After vortexing the aminated black phosphorus nanosheets and tannic acid, the Chlorella vulgaris that has engulfed the quantum dots is added and vortexed again. Then, ferric chloride is added and vortexed again. The mixture is centrifuged to obtain the heterojunction functionalized Chlorella vulgaris.

3. The preparation method according to claim 2, wherein In step 1, the centrifugation condition is 3000 rpm for 20 min, and the centrifugation condition is 13000 rpm for 10 min.

4. The preparation method according to claim 2, wherein In step 2, the centrifugation condition is 7000 rpm for 10 min, and the re-centrifugation condition is 12000 rpm for 10 min.

5. The preparation method according to claim 2, wherein In step 2, the ratio of the black phosphorus nanosheets, polyethyleneimine and arginine is 1:10:

3.

6. The preparation method according to claim 2, wherein In step 3, the concentration of antimonene quantum dots is 50 μg / mL, and the concentration of Chlorella vulgaris is 1×10 7 cell / mL.

7. The preparation method according to claim 2, wherein In step 4, the concentration of the amino-modified black phosphorus nanosheets is 0.2 mg / mL, the concentration of the tannic acid is 24 mM, and the concentration of the ferric chloride is 24 mM; The time for vortexing, re-vortexing and continued vortexing was 30 seconds.

8. Use of the heterojunction functionalized Chlorella according to claim 1 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The invention comprises the heterojunction functionalized Chlorella according to claim 1.

10. The drug according to claim 9, characterized in that Also contains pharmaceutically acceptable excipients.

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