CN114099673A - Heterojunction functionalized chlorella and preparation method and application thereof - Google Patents

Heterojunction functionalized chlorella and preparation method and application thereof Download PDF

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CN114099673A
CN114099673A CN202110803445.3A CN202110803445A CN114099673A CN 114099673 A CN114099673 A CN 114099673A CN 202110803445 A CN202110803445 A CN 202110803445A CN 114099673 A CN114099673 A CN 114099673A
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姬晓元
欧美彤
梅林�
张锦勰
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Sun Yat Sen University
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Abstract

The invention discloses a heterojunction functionalized chlorella, which consists of chlorella cells and black phosphorus nanosheets which are loaded on the surfaces of the chlorella cells, modify polyaspartic acid and chelate iron ions. Construction of type II heterojunction by coupling of black phosphorus nanosheets and original photosynthetic system in chlorella cells, and synchronous synergistic enhancement of chlorella photosynthesisOxygen generating capacity and photodynamic production of black phosphorus nanosheets1O2The ability to alleviate tumor hypoxia microenvironment and enhance efficacy; CGF produced by Chlorella cell can activate immune system, promote antigen recognition, presentation and immune response of immune system, enhance immunotherapy effect, and heterozygosis can functionalize Fe released from Chlorella3+Entering tumor cells can consume GSH and be reduced to Fe2+Impairs the antioxidant capacity of tumor cells, Fe2+Mediating the fenton response increases the efficacy of chemodynamic therapy of tumors.

Description

Heterojunction functionalized chlorella and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a heterojunction functionalized chlorella and a preparation method and application thereof.
Background
Photodynamic therapy (PDT) is a non-invasive method of tumor treatment that selectively kills and achieves clinical approval for tumor cells by generating a singlet oxygen-containing gas1O2) Superoxide anion (. O)2 -) Reactive Oxygen Species (ROS), including hydroxyl radical (OH), regulate necrosis and apoptosis of tumor cells, and are currently very effective cancer treatment means. The therapy mainly uses laser with specific wavelength to excite Photosensitizer (PS) and transfer electrons or energy to O2Produce ROS, cause tumor cell death, microvascular damage and induce local immune responses. Compared with the traditional therapy, PDT has light source targeting property and can selectively kill tumor cells; low toxicity, minimal invasion, and improvement of the patient after healing; can activate the immune response of the organism, reduce the tumor metastasis and recurrence and the like. However, the tumor microenvironment is usually hypoxic, limiting the efficient production of ROS, and PDT treatment alone does not produce an immune effect sufficient to effectively eliminate residual tumor cells and inhibit metastasis. Therefore, the method has important significance in the field of tumor treatment by continuously improving the hypoxia state of the tumor microenvironment and improving the response efficiency of the immune system and developing an effective PDT/immune combination treatment method.
The hypoxic microenvironment not only has obvious resistance to various oxygen-consuming treatment methods (such as chemotherapy, radiotherapy, PDT and photodynamic therapy, etc.), but also can induce the formation of tumor neovascularization, thereby inducing the relapse, invasion and metastasis of tumor cells. Therefore, the high-efficiency treatment for the hypoxic tumor is an important problem which is recognized by the international medical community and must be overcome to realize the complete cure of the tumor. For improving tumorThe treatment strategy of hypoxic microenvironment mainly utilizes materials capable of realizing self-oxygen supply, such as porous nano materials or artificial red blood cells to load O in vitro2Releases O in tumor environment (W. -L.W.Fan et al, adv.Mater.2018,30,1802006.), and can generate oxidation reaction in tumor metaacidic microenvironment to release O2The inorganic oxide (W.Fan et al, adv.Mater.2015,27,4155.) of (III), catalyzing the high expression of H in the microenvironment of the tumor2O2Production of O2The catalase of (H.Cheng et al, adv.Funct. Mater.2016,26,7847), etc. However, the currently employed self-oxygenating therapeutic strategies to overcome hypoxia are often limited by O2The load rate or generation rate is low and limited because of O2The load rate of the porous material is low, the targeted release can not be realized, the amount of inorganic oxide of targeted tumor tissues is limited and the degradation is slow, the delivery of active biological macromolecules such as enzyme is difficult, and the tumor microenvironment H is formed2O2Although the amount is high, the quantity is limited, so how to realize the continuous oxygen supply of the tumor hypoxia microenvironment is a problem to be solved urgently in the technical field of tumor treatment.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a heterojunction functionalized chlorella.
The second purpose of the invention is to provide a preparation method of the above mentioned heterojunction functionalized chlorella.
The third purpose of the invention is to provide the application of the heterojunction functionalized chlorella prepared by the preparation method in preparing anti-tumor and/or anti-tumor metastasis medicaments.
The above object of the present invention is achieved by the following technical solutions:
a heterojunction functionalized chlorella is composed of chlorella cells and black phosphorus nanosheets loaded on the surfaces of the chlorella cells, used for modifying polyaspartic acid and chelating iron ions.
The invention provides a heterojunction functionalized chlorella (Chl @ BP-Fe) which realizes the maximum efficiency of a limited light source by constructing a II-type heterojunction on the cell surface of the chlorella by using Black Phosphorus Nanosheets (BPNSs) as PS1 and using a chlorella self photosynthetic system as PS2And (4) transformation. The construction of the II-type heterojunction on the chlorella cell surface specifically comprises the following steps: modifying the surface of BPNSs by using sodium polyaspartate, and constructing a standardized black phosphorus nanosheet (BP-PASP-Fe) "Legao building block" module which is modified with polyaspartic acid on the surface and chelates iron ions as a shell by a "Legao building block standard module building method"; adsorbing BP-PASP-Fe onto the surface of chlorella cells by virtue of affinity mediated by physical action force of PASP on the surface of chlorella cells; based on metal ions Fe3+Chelation coordination with PASP interlocks BPNSs with PASP further increasing the stability of PASP-modified BPNSs on the cell surface of Chlorella vulgaris. The Chl @ BP-Fe provided by the invention can realize multi-mode synergistic anti-tumor, and is specifically embodied in that: BPNSs and an original photosynthetic system in chlorella cells are coupled to construct a II-type heterojunction, so that excitation electrons and holes of the BPNSs and chlorophyll in the chlorella cells are promoted to respectively migrate to a BPNSs Conduction Band (CB) and a chlorophyll Valence Band (VB) of the chlorella chlorophyll, and the oxygen production capacity of chlorella photosynthesis and the photodynamic production of the BPNSs are synchronously and synergistically enhanced1O2Can relieve tumor hypoxia microenvironment and enhance the curative effect of photodynamic therapy (PDT). The enhancement of the curative effect of the photodynamic therapy can trigger the immunogenic death of tumor cells, the activation of an immune system caused by Chlorella Growth Factor (CGF) produced by chlorella cells and the promotion of the antigen recognition, presentation and immune response of the immune system of the organism, thereby enhancing the immune therapeutic effect of the organism. In addition, as the 'lock catch' between the 'le Gao building blocks', Fe3+Not only can consume Glutathione (GSH) and be reduced into Fe2+Impairs the antioxidant capacity of tumor cells, and Fe2+Mediated Fenton reaction by catalyzing high expression of H in tumor cells2O2The occurrence of a disproportionation reaction to generate ROS (· OH) increases the efficacy of chemodynamic therapy (CDT) on tumors.
The invention also provides a preparation method of the heterojunction functionalized chlorella, which comprises the following steps:
s1, preparing black phosphorus nanosheets: stripping the black phosphorus to obtain black phosphorus nanosheets;
s2, preparing a black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions: uniformly mixing the polyaspartate aqueous solution with the black phosphorus nanosheet obtained in the step S1, adding an iron salt solution, uniformly mixing, washing, and centrifuging to obtain the black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions, namely the standardized Legao building block module BP-PASP-Fe;
s3, preparation of the heterojunction functionalized chlorella: and (4) uniformly mixing the black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions, which is obtained in the step (S2), with chlorella cells, adding iron salt, uniformly mixing, washing, and removing impurities to obtain the heterojunction functionalized chlorella.
Preferably, the method of the peeling in step S1 is an ultrasonic assisted liquid phase peeling method.
More preferably, the preparation method of the black phosphorus nanosheet in step S1 is: grinding black phosphorus into powder, adding a solvent, carrying out ultrasonic crushing, carrying out water bath ultrasonic treatment, centrifuging at 7000-8000 rpm, taking supernatant, centrifuging the supernatant at 14000-15000 rpm, and taking precipitate to obtain the black phosphorus nanosheet.
Further preferably, the grinding time in step S1 is 30-60 min; the solvent is N-methyl pyrrolidone; the time for centrifuging at 7000-8000 rpm is 10-15 min; the time for centrifugation at 14000-15000 rpm is 15-20 min;
further preferably, the time of the ultrasonic crushing in the step S1 is 8-24 hours, and the time of the water bath ultrasonic treatment is 8-24 hours.
Further preferably, the ultrasonic power of the ultrasonic crushing in the step S1 is 600-700W; the mass fraction of the black phosphorus in the solvent is 0.1-2 mg/mL.
Preferably, the polyaspartate of step S2 is sodium polyaspartate.
Preferably, the iron salt in step S2 is a soluble trivalent iron salt.
More preferably, the iron salt in step S2 is ferric chloride hexahydrate.
Preferably, the mass fraction of polyaspartate in the aqueous polyaspartate solution of step S2 is 0.4 g/mL.
Preferably, the mass ratio of the black phosphorus nanosheet, the polyaspartate to the iron ion in step S2 is 1: (1.6E
2)：(0.17～0.8)。
Preferably, the iron ion concentration of the iron salt solution in the steps S2 and S3 is 20-24 mmol/L.
Preferably, the ratio of the black phosphorus surface-modified with polyaspartic acid and chelated with iron ions, the iron ions in the iron salt solution, and the chlorella cells in step S3 is 1 μ g: (0.013-0.104) μ g: (8.75X 10)3～8.8×106) And (4) respectively.
The invention also provides application of the heterojunction functionalized chlorella prepared by any one of the preparation methods in preparation of anti-tumor and/or anti-tumor metastasis medicaments.
Preferably, the heterojunction functionalized chlorella prepared by any one of the preparation methods is applied to the preparation of an anti-tumor and/or anti-tumor photodynamic therapy medicament.
The invention also provides application of the heterojunction functionalized chlorella prepared by any one of the preparation methods in preparation of anti-tumor and/or anti-tumor metastasis medicaments by combining with an immune checkpoint inhibitor.
As a preferred possible embodiment, the preparation method of the heterojunction functionalized chlorella comprises the following steps:
s1, preparing Black Phosphorus Nanosheets (BPNSs): grinding 40mg of black phosphorus for 30min, adding 80mL of N-methylpyrrolidone solvent, carrying out ultrasonic crushing for 8h under the power of 700W, then carrying out water bath ultrasonic treatment for 8h, centrifuging for 10min at the rotating speed of 7000rpm, taking supernatant, centrifuging the supernatant for 15min at the rotating speed of 15000rpm, and taking precipitate to obtain BPNSs;
s2, preparing a black phosphorus nanosheet (BP/PASP-Fe) with a surface modified with polyaspartic acid and chelated with iron ions: uniformly mixing 1 mu L of sodium polyaspartate aqueous solution with the mass concentration of 0.4g/mL and the 0.2mg of black phosphorus nanosheet obtained in the step S1, vortexing for 1min, and then adding 62.5 mu L of FeCl with the concentration of 24mmol/L3Mixing the solution uniformly, vortexing for 1min, washing with PBS for 3 times, and centrifuging at 15000rpm for 10min to obtain "Legao building block" module BP/PASP-Fe;
s3, preparing heterojunction functionalized chlorella (Chl @ BP-Fe): will be provided withAnd S2, swirling the black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions and the chlorella cell suspension for 1min, and uniformly mixing, wherein the ratio of black phosphorus to chlorella cells in the black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions is 1 mug: 8.75X 105Secondly, adding a certain volume of FeCl with the concentration of 24mmol/L according to the mass ratio of the black phosphorus to the iron ions of 1:0.043And uniformly mixing the solution, whirling for 1min, washing, removing impurities, and centrifuging for 3 times at 1000rpm to obtain Chl @ BP-Fe.
The invention also provides application of the heterojunction functionalized chlorella prepared by any one of the preparation methods in preparation of anti-tumor and/or anti-tumor drugs.
Preferably, the tumor is melanoma.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a shell formed by black phosphorus nanosheets of which the surfaces are modified with polyaspartic acid and chelate iron ions, and a heterojunction functionalized chlorella formed by chlorella cells wrapped in the shell. BPNSs and the original photosynthetic system in chlorella cells are coupled to construct a type II heterojunction, and the oxygen production capacity of chlorella photosynthesis and the photodynamic production of BPNSs are synchronously and synergistically enhanced1O2Can relieve tumor hypoxia microenvironment and enhance the curative effect of photodynamic therapy. The enhancement of the curative effect of the photodynamic therapy can trigger the immunogenic death of tumor cells, the activation of an immune system caused by CGF generated by chlorella cells and the promotion of the antigen recognition, presentation and immune response of the immune system of an organism, thereby enhancing the immune therapeutic effect of the organism. In addition, Chl @ BP-Fe contains Fe3+，Fe3+Not only can consume GSH in tumor cells and be reduced into Fe2+Impairs the antioxidant capacity of tumor cells, and Fe2+Mediated Fenton reaction by catalyzing high expression of H in tumor cells2O2The occurrence of a disproportionation reaction to generate ROS (. OH) increases the efficacy of the chemodynamic therapy of tumors. Therefore, the heterojunction functionalized chlorella provided by the invention has the multi-mode synergistic anti-tumor effect.
Drawings
FIG. 1 is a schematic diagram of the preparation process of Chl @ BP-Fe.
FIG. 2 is a TEM image of the crystal lattice (D) of the nanosheets on Chlorella (A), BPNSs (B), Chl @ BP-Fe (C), and Chl @ BP-Fe; the scale bars of fig. 2A-D are 500 nm, 100 nm, 500 nm, and 5 nm in sequence.
FIG. 3 is an EDS map of BP/PASP-Fe, at a scale bar of 1 micron.
FIG. 4 is a graph of oxygen production profiles for Chlorella and Chl @ BP-Fe.
FIG. 5 is the schematic diagram (E) of the electron transfer principle of the type II heterojunction of the valence band (B) of BPNSs (A) and Chl, the band gap (D) of BPNSs (C) and Chl, and the band gap (D) of Chl @ BP-Fe under light excitation.
FIG. 6 is a graph of the degradation of DPBF by BPNSs and Chl @ BP-Fe.
FIG. 7 is a graph of Chl @ BP-Fe degrading MB.
FIG. 8 is a graph showing the effect of BPNSs, Chl @ BP-Fe on tumor cell hypoxia and ROS production; FIG. 8A is a graph of the effects of BPNSs, Chl @ BP-Fe on alleviating hypoxia in tumor cells, at a scale of 25 microns; FIG. 8B is a graph of the effect of BPNSs, Chl @ BP-Fe on ROS production in tumor cells, scaled at 50 microns; FIG. 8C is a graph of fluorescence quantification statistics for BPNSs, Chl @ BP-Fe alleviating hypoxia in tumor cells; FIG. 8D is a graph of statistics of fluorescence quantification of BPNSs, Chl @ BP-Fe for ROS production in tumor cells.
FIG. 9 is a graph of the effect of Chl content on the maturation of mouse bone marrow-derived dendritic cells.
FIG. 10 is a graph of in vivo anti-tumor results for Chl @ BP-Fe, wherein FIG. 10A is a plot of tumor volume change in mice; FIG. 10B is the body weight change after treatment of mice; fig. 10C is a photograph of tumor anatomy for each group of mice at the end of treatment; FIG. 10D is a tumor hypoxia section at a scale bar of 50 microns; FIG. 10E is a tumor ROS slice, scale bar 50 microns; FIG. 10F is a graph of TUNEL detection of tumor apoptosis on a 20 micron scale.
FIG. 11 is a graph showing the results of anti-tumor immune effects induced by Chl @ BP-Fe; wherein, FIG. 11A, B is a flow chart and a statistical chart of dendritic cell maturation in lymph nodes, respectively; FIG. 11C is the ratio of CD8+ T cells to total T cells in the spleen; FIG. 11D is CD8 in spleen+T and CD4+T infiltration situation flow chart; FIG. E is CD8 in spleen+T/CD4+The proportion of T cells; FIG. 11F shows nuclear DAPI (blue) staining of tumor tissue with anti-CD 86-Cy3 at a scale bar of 20 microns.
FIG. 12 is a graph showing the results of anti-tumor metastasis by Chl @ BP-Fe; wherein, FIGS. 12A-B are mouse spleen immunohistochemical sections of two melanoma markers HMB45 and S-100B5 at a scale bar of 50 microns; FIGS. 12C-D show the staining scores for HMB45 and S-100B.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 preparation of Chl @ BP-Fe
S1, preparing Black Phosphorus Nanosheets (BPNSs): grinding 40mg of black phosphorus for 30min, adding 80mL of N-methylpyrrolidone solvent, carrying out ultrasonic crushing for 8h under the power of 700W, then carrying out water bath ultrasonic treatment for 8h, centrifuging for 10min at the rotating speed of 7000rpm, taking supernatant, centrifuging the supernatant for 15min at the rotating speed of 15000rpm, and taking precipitate to obtain BPNSs;
s2, preparing a black phosphorus nanosheet (BP/PASP-Fe) with a surface modified with polyaspartic acid and chelated with iron ions: uniformly mixing 1 mu L of sodium polyaspartate aqueous solution with the mass concentration of 0.4g/mL and the 0.2mg of black phosphorus nanosheet obtained in the step S1, vortexing for 1min, and then adding 62.5 mu L of FeCl with the concentration of 24mmol/L3Mixing the solution uniformly, vortexing for 1min, washing with PBS for 3 times, and centrifuging at 15000rpm for 10min to obtain "Legao building block" module BP/PASP-Fe;
s3, preparing Chl @ BP-Fe: and (4) swirling the BP/PASP-Fe obtained in the step (S2) and the chlorella cell suspension for 1min, and uniformly mixing, wherein the ratio of the black phosphorus to the chlorella cells in the BP/PASP-Fe is 1 mu g: 8.75X 105Secondly, adding a certain volume of FeCl with the concentration of 24mmol/L according to the mass ratio of the black phosphorus to the iron ions of 1:0.043The solution was mixed well, vortexed for 1min, washed, stripped of impurities, and centrifuged 3 times at 1000rpm to obtain Chl @ BP-Fe.
Example 2 preparation of Chl @ BP-Fe
S1. preparation of BPNSs: grinding 10mg of black phosphorus for 45min, adding 100mL of N-methylpyrrolidone solvent, carrying out ultrasonic crushing for 12h under the power of 600W, then carrying out water bath ultrasonic crushing for 12h, centrifuging for 15min at the rotating speed of 8000rpm, taking supernatant, centrifuging the supernatant for 20min at the rotating speed of 14000rpm, taking precipitate, and obtaining BPNSs;
s2, BP/PASP-Fe preparation: 0.5. mu.L of aqueous solution of polyaspartic acid sodium with a mass concentration of 0.4g/mL and 0.1mg of BPNSs obtained in step S1 were mixed uniformly, and 57. mu.L of FeCl with a molar concentration of 22mmol/L was added3Mixing the solution uniformly, vortexing for 1min, washing with PBS for 3 times, and centrifuging at 15000rpm for 20min to obtain "Legao building blocks" module BP/PASP-Fe;
s3, preparing Chl @ BP-Fe: and (4) swirling the BP/PASP-Fe obtained in the step (S2) and the chlorella cell suspension for 1min, and uniformly mixing, wherein the ratio of the black phosphorus to the chlorella cells in the BP/PASP-Fe is 1 mu g: 8.75X 103Adding a certain volume of FeCl with the concentration of 22mmol/L according to the mass ratio of the black phosphorus to the iron ions of 1:0.0133And washing and removing impurities from the solution to obtain Chl @ BP-Fe.
Example 3 preparation of Chl @ BP-Fe
S1. preparation of BPNSs: grinding 200mg of black phosphorus for 60min, adding 100mL of N-methylpyrrolidone solvent, carrying out ultrasonic crushing for 24h under the power of 650W, then carrying out water bath ultrasonic treatment for 24h, centrifuging for 12min at the rotating speed of 7500rpm, taking supernate, centrifuging the supernate for 18min at 14800rpm, and taking precipitate to obtain BPNSs;
s2, BP/PASP-Fe preparation: 2 mul of polyaspartic acid sodium water solution with mass concentration of 0.4g/mL and 0.5mg of BPNSs obtained in step S1 are mixed evenly, and 80.3 mul of FeCl with molar concentration of 20mmol/L is added3Mixing the solution uniformly, vortexing for 1min, washing with PBS for 3 times, and centrifuging at 15000rpm for 10min to obtain "Legao building block" module BP/PASP-Fe;
s3, preparing Chl @ BP-Fe: whirling the BP/PASP-Fe obtained in the step S2 and chlorella cell suspensionAnd uniformly mixing for 1min, wherein the ratio of the black phosphorus in the BP/PASP-Fe to the chlorella cells is 1 mu g: 8.8X 106Secondly, adding a certain volume of FeCl with the concentration of 20mmol/L according to the mass ratio of the black phosphorus to the iron ions of 1:0.1043And washing and removing impurities from the solution to obtain Chl @ BP-Fe.
Example 4 preparation of Chl @ BP-Fe
Only modifying the step S3 to be' mixing the BP/PASP-Fe obtained in the step S2 and the chlorella cell suspension evenly by vortex for 1min, wherein the ratio of the black phosphorus to the chlorella cells in the BP/PASP-Fe is 1 mu g: 3.5X 106And then, taking the mass ratio of the black phosphorus to the iron ions as 1:0.04 adding a certain volume of FeCl with the concentration of 24mmol/L3And (3) washing and removing impurities from the solution to obtain Chl @ BP-Fe', and preparing the Chl @ BP-Fe by the same other steps as the example 1.
Example 5 preparation of Chl @ BP-Fe
Only modifying the step S3 to be' mixing the BP/PASP-Fe obtained in the step S2 and the chlorella cell suspension evenly by vortex for 1min, wherein the ratio of the black phosphorus to the chlorella cells in the BP/PASP-Fe is 1 mu g: 1.75X 106And then, taking the mass ratio of the black phosphorus to the iron ions as 1:0.04 adding a certain volume of FeCl with the concentration of 24mmol/L3And washing the solution, and removing impurities to obtain Chl @ BP-Fe', wherein other steps are the same as those in the example 1 to obtain the Chl @ BP-Fe.
A schematic flow diagram of a Chl @ BP-Fe preparation process obtained in any one of embodiments 1-5 of the invention is shown in FIG. 1. Modifying the surface of BPNSs by using sodium polyaspartate (PASP) to construct a standardized Legao building block module (BP/PASP); using metal ions Fe3+The chelating coordination with PASP is interlocked between BP/PASP to obtain BP/PASP-Fe; BP/PASP-Fe through interface molecular interaction and Fe by utilizing Legao building block standard module building method3+Locking guided particle assembly, and modifying BP/PASP-Fe to the surface of chlorella cells to prepare Chl @ BP-Fe.
Test example 1 characterization of Chl @ BP-Fe
And performing morphology characterization on the Chl @ BP-Fe by using a Transmission Electron Microscope (TEM). And (3) carrying out X-ray energy spectrum (EDS) analysis on the Chl @ BP-Fe, analyzing the element distribution condition, and judging whether the system is successfully coated and loaded.
And (4) analyzing results: FIGS. 2A-D are TEM images of the lattices of the nanoplatelets on Chlorella, BPNSs, Chl @ BP-Fe, and Chl @ BP-Fe, respectively. Among them, it can be seen from fig. 2C that a large amount of BP/PASP-Fe is attached to the surface of chlorella, indicating that bpsss is successfully encapsulated on the surface of chlorella, and the crystal lattice of the nanosheet in fig. 2D is consistent with that of black phosphorus, indicating that the black phosphorus nanosheet is encapsulated on the surface of chlorella. FIG. 3 is an EDS map of BP/PASP-Fe. The element distribution condition of the Chl @ BP-Fe system can be seen, and the elements P and Fe are distributed on the surface of the system, so that the BP/PASP-Fe is further proved to be effectively loaded on chlorella.
Test example 2 in vitro Performance testing of Chl @ BP-Fe
1. In vitro oxygen production experiment
In order to verify the oxygen production capacity of the chlorella system in photosynthesis, 658nm laser is used as a light source for the photosynthesis of the chlorella, an oxygen production tester is used for carrying out oxygen production tests on the chlorella and Chl @ BP-Fe exposed to laser irradiation, and an oxygen production curve is drawn. The specific experimental procedure is as follows:
experimental groups: chl @ BP-Fe prepared in example 1 was dispersed in PBS to obtain a BP concentration of 80. mu.g/mL and a chlorella cell concentration of 7X 107individual/mL Chl @ BP-Fe dispersion, control: BNPNSs were dispersed in PBS to obtain a BNPNSs dispersion at a concentration of 80. mu.g/mL. Argon was passed through the solution for 10min and left in the dark for 1h to deplete the oxygen initially present in the system, followed by a laser (658nm, 0.1W/cm)2) Detecting O in the system stirred by a stirrer by using an oxygen dissolving instrument under irradiation2Content of (D), recording O per minute within 20min2The content value is obtained to obtain the corresponding product O2A quantity curve.
And (4) analyzing results: the oxygen production curves of Chlorella and Chl @ BP-Fe are shown in FIG. 4. From the resulting curves it can be seen that: the chlorella and the Chl @ BP-Fe can effectively perform photosynthesis to generate a large amount of oxygen, and the modified chlorella still has high activity. Therefore, Chl @ BP-Fe performs photosynthesis under 658nm laser irradiation, and generates a large amount of oxygen.
Characterization of type II heterojunctions
To further elucidate the type II heterojunction in Chl @ BP-Fe, we used XPS spectroscopy and UV-vis-NIR diffuse reflectance spectroscopy to determine the electronic band structure of Chl and BPNSs.
And (4) analyzing results: as shown by XPS spectra (5A, B), the Valence Band (VB) values for BPNSs and Chl were approximately 0.9eV and 0.4eV, respectively. As shown in FIG. 5C, D, the bandgaps (Eg) for BPNSs and Chl were calculated to be 1.5eV and 1.6eV, respectively, according to the Kubelka-Munk equation. The difference between the Valence Band (VB) and the band gap (Eg) gives the value of the Conduction Band (CB). Thus, the CB for BPNSs and Chl are-0.6 eV and-1.2 eV, respectively. When the conduction band CB and the valence band VB of the photosensitizer 1(PS1) and the photosensitizer 2(PS2) form a staggered structure, a II-type heterojunction can be formed, and the conduction band CB and the valence band VB of the BPNSs and Chl are measured to form the staggered structure, so that the two-type heterojunction can be formed, and the successful construction of the Chl @ BP-Fe II-type heterojunction is proved. When photosensitizers such as BPNSs or chlorophyll are exposed to 658nm laser alone, photoexcited electrons readily recombine with holes to remain stable, resulting in inefficient ROS production by BPNSs. The electron transfer principle of type II heterojunction is schematically illustrated in fig. 5E, for Chl @ BP-Fe with type II heterojunction, photoexcited electrons in the chlorophyll Conduction Band (CB) can be transferred to CB of bpsss, while holes in the Valence Band (VB) of bpsss can be transferred to the Valence Band (VB) of chlorophyll, avoiding rapid recombination of photoexcited electrons and holes. The lifetime-extended photo-generated electron-hole pairs of Chl @ BP-Fe improve the ability of Chl @ BP-Fe to efficiently generate oxygen and ROS.
3. In vitro ROS detection
After irradiation with 658nm laser, Chl @ BP-Fe prepared in example 5 was detected with a singlet oxygen fluorescence probe (DPBF), wherein the BP concentration was 40. mu.g/mL, and the control group was: BNPNSs were dispersed in PBS to obtain a BNPNSs dispersion at a concentration of 40. mu.g/mL. And (3) observing the generation conditions of singlet oxygen in the PBS solution at different times (0-10 min) under the anoxic condition, and observing whether the two solutions have different capabilities in generating the singlet oxygen.
And (4) analyzing results: a graph of DPBF degradation by BPNSs and Chl @ BP-Fe is shown in FIG. 6. As can be seen from the figure, under hypoxic conditionsNext, the curve for BPNSs decreased more slowly after 658nm laser irradiation, indicating a lower efficiency of ROS production, indicating that the PDT effect of BPNSs is somewhat inhibited in hypoxic environments. Under the same anoxic environment and illumination conditions, the curve of Chl @ BP-Fe is greatly reduced, the ROS yield is obviously enhanced compared with that of BPNSs, and the results of in vitro oxygen production experiments show that: in PBS solution, Chl @ BP-Fe can produce O by photosynthesis of Haematococcus sp2Thereby increasing ROS production and enhancing PDT effect.
4. In vitro Fenton reaction assay
Chl @ BP-Fe prepared in example 4 was dispersed in PBS to give a BP concentration of 20. mu.g/mL and a chlorella cell concentration of 7X 107individual/mL Chl @ BP-Fe dispersion, control: BNPNSs were dispersed in PBS to obtain a BNPNSs dispersion at a concentration of 20. mu.g/mL. OH generated by the Fenton reaction mediated by Chl @ BP-Fe is detected by using Methylene Blue (MB) which is an OH detection reagent. Mixing Chl @ BP-Fe dispersion with MB and H2O2Mixed, MB, H2O2The final concentrations of (a) are 5 mug/mL and 10 mug/mL respectively; the control group is equal volume of BNPNSs dispersion liquid and MB and H2O2Mixed, MB, H2O2Were 5. mu.g/mL and 10. mu.g/mL, respectively, and the characteristic absorption peak of MB was detected by ultraviolet spectroscopy.
And (4) analyzing results: a graph of the degradation of MB by Chl @ BP-Fe is shown in FIG. 7. As shown in FIG. 7, in the presence of H2O2After Chl @ BP-Fe is added into the MB solution, the characteristic absorption peak intensity of MB is continuously reduced along with the increase of time, which means that Fe in Chl @ BP-Fe3+Can effectively react with H in the environment2O2OH is generated through reaction, thereby laying a foundation for Chl @ BP-Fe to realize the treatment of tumor CDT.
5. Tumor cell hypoxia improvement experiment and tumor cell ROS production detection
Tumor cell hypoxia improvement experiment: selecting mouse melanoma cell B16 with good growth state, digesting, diluting to obtain a solution containing 1.3 × 105The individual B16 cell suspensions were transferred to a medium-containing, 15mm diameter confocal dish and placed in an incubator for anchorage. Cultivation methodAfter culturing for 36h, a certain amount of BPNSs (BPNSs concentration is 80. mu.g/mL in the culture medium) and Chl @ BP-Fe prepared in example 1 (BP concentration is 80. mu.g/mL in the culture medium, chlorella cell concentration is 7X 10)7one/mL) and hypoxic incubation for 12 h. Wherein blank set (control) is: adding PBS with the same volume as the medicine; 4 experimental groups were set and named control, BPNSs +658nm laser (BP +658), Chl @ BP-Fe and Chl @ BP-Fe +658nm laser (Chl @ BP-Fe +658) groups in this order. Cells requiring the light group were irradiated using a 658nm fiber coupled laser (0.1W/cm)2) And (5) performing light treatment for 20 min. After the light irradiation was terminated, the cells were washed 3 times with PBS and stained by adding the hypoxic probe, pennogenyl oxazole (PIMO) (200. mu. mol/L). After 60min, the cells were washed again 3 times and fixed with paraformaldehyde. The cells were then washed again with PBS and stained with FITC-Mab1 (green) diluted 100 fold, followed by rinsing with PBS followed by DAPI (blue) staining. Finally, the degree of improvement in hypoxia of each group of cells after administration was observed under a confocal laser microscope.
Tumor cell ROS production assay: b16 cells were transferred to 15mm confocal dishes and after 36h of culture, the old medium was replaced with medium containing BPNS and Chl @ BP-Fe, where blanks (controls) were: adding PBS with the same volume as the medicine; 4 experimental groups were set and named control, Chl @ BP-Fe, BPNSs +658nm laser (BP +658) and Chl @ BP-Fe +658nm laser (Chl @ BP-Fe +658) groups in this order. Incubating the drug with hypoxic cell culture in incubator for 12h, adding DCFH-DA probe (green) to each group, incubating for 30min, and using 658nm fiber coupled laser (0.1W/cm)2) BP +658 and Chl @ BP-Fe +658 groups were irradiated for 10min, 30min after the end of the irradiation, and the cells were washed and fixed with PBS and stained with DAPI (blue), and the ROS production status was observed under a confocal microscope.
And (4) analyzing results: the hypoxic level was assessed by staining the cells with FITC-Mab1 (green) and DAPI (blue) using PIMO as a hypoxic probe and detecting FITC fluorescence intensity after different treatments. The greater the green fluorescence intensity, the more severe the hypoxic condition. The effect of Chl @ BP-Fe on tumor cell hypoxia and ROS production is shown in FIG. 8. The hypoxia of tumor cells in Control, BP +658, Chl @ BP-Fe and Chl @ BP-Fe +658 groups is shown in FIGS. 8A, C. As can be seen from FIG. 8A, the most intense green fluorescence, i.e., most hypoxic, is BP +658 and even beyond PBS due to PDT effects from BPNSs irradiated with 658nm laser, which consumes O originally present2Further aggravating the hypoxic environment of tumor cells. The Chl @ BP-Fe group showed weaker fluorescence than control due to the Fe content in Chl @ BP-Fe3+Can be mixed with high content of H in tumor cells2O2Reaction product of Fe2+And O2Thereby further alleviating the tumor hypoxia condition. The best effect of relieving hypoxia is the Chl @ BP-Fe +658 group which passes through the photosynthesis and Fe of chlorella3+Decomposition of H2O2Oxygen is generated, the hypoxic condition is obviously improved, and green fluorescence is not basically shown. DCFH-DA was used as an intracellular ROS probe (green), DCF fluorescence intensity after different treatments was measured to assess ROS production levels, and nuclei were stained with DAPI (blue). The DCF green fluorescence intensity is positively correlated with ROS production level. The results for each of the control, BPNSs +658, Chl @ BP-Fe and Chl @ BP-Fe +658 groups are shown in FIG. 8B, D: in the absence of 658nm laser irradiation, the photosensitizer BPNSs cannot play a role due to the lack of illumination of pure Chl @ BP-Fe, and only Fe3+A small amount of ROS is produced by fenton reaction. While BPNSs alone also produce some amount of ROS upon irradiation with 658nm laser. The strongest green fluorescent signal was seen in the Chl @ BP-Fe +658 group, indicating Haematococcus and Fe in this system3+Under the double actions of the two components, the ROS level in the cell is obviously increased, and the PDT killing effect is better.
6. In vitro mouse bone marrow derived dendritic cell (BMDC) maturation assay
The method is used for researching the influence of chlorella cell contents in Chl @ BP-Fe on the maturation of mouse bone marrow-derived dendritic cells, and comprises the following specific steps:
BMDC cells were extracted from C57 mice and then seeded in 12-well plates (5X 10 per plate) containing cell culture medium5Cells), incubation continued for 12 h. The experiment was divided into 3 groups, control and Chl + US groups, respectively. Wherein the Chl + US group is obtained by subjecting Chlorella to ultrasonic treatment in water for 1 hrPost-centrifugation (10000rpm, 30 minutes) to facilitate overflow of chlorella content, followed by centrifugation to co-incubate the supernatant with BMDC for 24 h; BMDCs were stained with DC maturation surface markers (anti-CD 11c, anti-CD 80 and anti-CD 86) and finally analyzed by flow cytometry to check for BMDC maturation.
And (4) analyzing results: the effect of Chl content on mouse bone marrow-derived dendritic cell maturation results are shown in fig. 9. From fig. 9, it can be seen that the double positive signals of Chl + US group, CD80 and CD86 are significantly enhanced compared to control group, demonstrating that chlorella content can effectively stimulate DC cell maturation.
Test example 2 in vivo antitumor Performance test of Chl @ BP-Fe
To evaluate the antitumor efficacy of Chl @ BP-Fe prepared in example 1, 1X 10 was injected subcutaneously6B16 cells, a melanoma model was established in C57BL/6 mice. According to the double-blind principle, mice were randomly divided into 4 groups (n ═ 5): (1) control group (control), (2) Chl @ BP-Fe group ([ Chl)]＝7×107Cell/mouse, [ BP]80 μ g/mouse), (3) BP +658nm laser group ([ BP)]80 μ g/mouse), (4) Chl @ BP-Fe +658nm laser group ([ Chl)]＝7×107Individual cell/mouse, [ BP]80 μ g/mouse). When the tumor grows to about 40-60 mm3(day 0), mice were injected intratumorally with PBS (control group), BP or Chl @ BP-Fe. 4 hours after injection, mice were exposed to a 658nm laser (0.1W/cm)2) And then 20 minutes. The same treatment was received on day 1. Mice body weight and tumor volume were recorded every other day. Tumor volume was calculated according to the following formula: volume length x (width)2/2. At the end of treatment, spleens of mice were sliced. Further observation was done by microscopy after immunohistochemical (HMB45, S-100B) staining.
To evaluate the anti-tumor mechanism of Chl @ BP-Fe in tumor tissues, hypoxia, ROS and apoptosis of in situ tumors were examined separately. The hypoxia detection method comprises the following steps: mice were injected intraperitoneally with penonidazole (PIMO: 60mg/kg) 20 minutes after treatment was completed. After 30 minutes, the tumors were removed for paraffin sectioning, then stained with FITC-MAB1 and DAPI, followed by confocal microscopy. 3.5 hours after dosing when ROS were detectedMice were injected intratumorally with DCFH-DA (100 μm) and then 30 minutes later with a 658nm laser (0.1W/cm)220 minutes) irradiation. Subsequently, tumors were collected into product sections. Sections were further stained with DAPI prior to imaging with confocal microscopy. When apoptosis is involved, mice are euthanized 24 hours post-treatment, and then tumors are excised for processing paraffin sections for TUNEL analysis. All nuclei were stained with DAPI.
To further analyze the immune mechanism, when the tumor reached 100mm3At that time, the mice received the same treatment (control, Chl @ BP-Fe, BP +658nm laser and Chl @ BP-Fe +658nm laser). Spleens, lymph nodes and tumors were collected 2 days after all treatments, were single cell suspensions prepared and stained with fluorescently labeled antibodies. For flow staining, activation of DCs was labeled with anti-CD 11C-FITC, anti-CD 80-percp-Cy5.5 and anti-CD 86-APC. For T cells, cells were labeled with anti-CD 3-FITC, anti-CD 8-APC and anti-CD 4-PE. In paraffin sections of tumors, cytotoxic T cells were stained with anti-CD 8-Cy3, and then anti-CD 86-Cy3 was used to label mature DC cells. Single cell suspensions and sections were tested by flow cytometry and confocal laser microscopy, respectively, to detect and analyze T and DC cell maturation in vivo and infiltration in tumors.
And (4) analyzing results:
in vivo anti-tumor properties of Chl @ BP-Fe
4 hours after injection, mice were exposed to a 658nm laser (0.1W/cm)2) The next 20 minutes, the next day, each group of mice still received the same treatment. As shown in fig. 10A, C, conventional PDT treatment of BPNSs provides all of the reduction in tumor growth, but due to the lack of tumor oxygen and the limited effective use of limited light sources by BPNSs, the PDT effect of BPNSs is limited and the tumor will rapidly recur after PDT treatment is completed. Notably, the Chl @ BP-Fe +658 group showed more potent tumor suppression than conventional PDT produced by BPNSs, probably due to Chlorella and Fe3+The oxygen generating function of the method greatly improves hypoxic microenvironment of tumors, provides sufficient substrates for PDT treatment, greatly improves PDT efficiency, and in addition, Fe3+And H abundant in tumor2O2The Fenton reaction generates OH, and the tumor is further killed by means of CDT. In addition, the Chl @ BP-Fe group has certain inhibition effect on tumors under the condition of no light, and the inhibition effect is realized by Fe3+Resulting in a therapeutic effect of CDT. FIG. 10F is the TUNEL assay of tumor apoptosis, providing more direct evidence for significant therapeutic efficacy of the Chl @ BP-Fe +658 group. In addition, after the treatment, no obvious side effect is caused on the growth and daily living capacity of the mice, and the body weight is not obviously changed compared with a control group (figure 10B), thereby further proving the effectiveness and safety of Chl @ BP-Fe. To further explain the in vivo anti-tumor mechanism of Chl @ BP-Fe, we further explored the hypoxic amelioration and ROS production in tumors using the hypoxic probe PIMO (pimozolozole, green signal) and ROS probe DCFH-DA. The strongest green fluorescence signal of BP +658 group, as shown in FIG. 10D, represents the most severe hypoxic condition, indicating that PDT produced by BPNSs depletes the oxygen in the tumor, resulting in severe hypoxia of the tumor. In contrast, the Chl @ BP-Fe +658 group did not detect hypoxic fluorescence signals, which are derived from Fe3+With chlorella by reaction with excess hydrogen peroxide in the tumor to produce oxygen and by photosynthesis to produce oxygen, respectively. Chl @ BP-Fe also has a certain degree of hypoxia relief compared with the control group, but the hypoxia relief effect is strong without the Chl @ BP-Fe +658, further explaining that Fe in the Chl @ BP-Fe system3+Plays a certain role in relieving the hypoxic oxygen, but has no remarkable effect of relieving the hypoxic oxygen brought by photosynthesis with chlorella. We subsequently evaluated the ROS production in tumors by Chl @ BP-Fe, and further reflected the in vivo PDT effect of each system. As shown in FIG. 10E, BP +658 produced weaker ROS due to oxygen starvation, compared to Fe in Chl @ BP-Fe3+ROS produced by fenton reaction are comparable in intensity. Compared with the group of BP +658 and Chl @ BP-Fe, the group of Chl @ BP-Fe +658 has the strongest ROS fluorescence intensity, and shows stronger PDT and CDT effects.
Chl @ BP-Fe-induced anti-tumor immune effects
Flow charts and statistics of dendritic cell maturation in lymph nodes in mice by Chl @ BP-Fe are shown in FIG. 11A, B. Dendritic cell maturation in BP +658 group versus control groupSlightly higher, this is due to the immune effects caused by the photodynamic therapy of BPNS itself. The Chl @ BP-Fe +658 group significantly increased DC cell maturation compared to BP +658 group due to: the chlorella itself as an exogenous substance with immunocompetence can absorb and activate nonspecific immune response of an organism, and draw immune cells such as DC cells and the like to be enriched at tumor positions, so that the contact chance of the DC cells and tumor antigens is increased; chlorella contains multiple immunostimulant substances, can stimulate DC cell maturation, and has immunoadjuvant effect. Chlorella photosynthesis generates oxygen, relieves the immunosuppressive microenvironment caused by hypoxia, provides substrates for PDT treatment, and enhances the immune effect generated by PDT treatment. Therefore, the Chl @ BP-Fe system can promote DC maturation to realize more effective antigen presentation by two major ways of increasing the contact chance of DC cells and antigens. In an anti-tumor immune response, mature DC cells in the lymph nodes migrate out and enter the spleen to further stimulate activation of T cells. To assess the effect of Chl @ BP-Fe on T cell activation, we performed further immunological analysis of mouse spleens. The results show that CD8 was found in two groups, BP +658 and Chl @ BP-Fe +658 compared to the control group+The proportion of T cells in the total T cells is increased, wherein the increased proportion of the Chl @ BP-Fe +658 group is more obvious (FIG. 11C, D), which indicates that the treatment mode of the Chl @ BP-Fe +658 can successfully start the activation of the T cells and enhance the anti-tumor immune response of the organism. Activated T cells migrate out of the spleen and further infiltrate the tumor, killing it. Previous reports have shown that T cells avoid hypoxic regions in tumors, since tumor immunosuppression microenvironment due to hypoxia hinders T cell infiltration, impairing immune cell killing of tumors, and we evaluated mature dendritic cells (CD86 protein marker), CD8+Infiltration of T cells in the tumor. FIG. 11F shows nuclear DAPI (blue) and anti-CD 86-Cy3 staining of tumor tissue. The results show CD86 of the Chl @ BP-Fe +658 group in FIG. 11F+The increase of the number of cells of the fluorescence signal indicates that the infiltration of mature dendritic cells and T cells in the Chl @ BP-Fe +658 group of tumors is remarkably increased, which indicates that chlorella can effectively stimulate the infiltration of dendritic cells to the tumors by being used as an adjuvant, and the photosynthesis can effectively relieve the tumor depletion by being used as an adjuvantOxygen immunosuppression and promotion of CD8+Intratumoral infiltration of T. In conclusion, the Chl @ BP-Fe system has great potential in the aspect of photodynamic therapy immunity enhancement.
3. Anti-tumor metastasis performance test
To enhance the effect, we added two groups of mice injected with the immune adjuvant CTLA-4, and administered 200ug of anti-CTLA-4 by intraperitoneal injection to the mice on days 2, 5 and 8 of the treatment, and the experimental groups were control, Chl @ BP-Fe, BP +658, Chl @ BP-Fe +658, BP +658+ CTLA-4, Chl @ BP-Fe +658+ CTLA-4; chl @ BP-Fe anti-tumor metastasis performance was verified by spleen sections. At the end of treatment, spleens of mice were sliced. Further observation was done by microscopy after immunohistochemical (HMB45, S-100B) staining.
And (4) analyzing results: the results of the anti-tumor metastasis assay of BP, Chl @ BP-Fe are shown in FIG. 12. Immunohistochemical (IHC) evaluation of melanoma markers HMB45 and S-100B are important indexes for measuring tumor metastasis conditions, and from mouse spleen immunohistochemical sections (figures 12A-B) of HMB45 and S-100B5 and staining score results (figures 12C-D) of HMB45 and S-100B of two melanoma markers, it can be seen that the two indexes of Chl @ BP-Fe +658 and Chl @ BP-Fe +658+ CTLA-4 are low in expression compared with a control group, which indicates that the Chl @ BP-Fe system can obviously destroy primary tumors, effectively activate body anti-tumor immune response, and can well inhibit tumor metastasis with the assistance of anti-CTLA-4.

Claims (10)

1. A heterojunction functionalized chlorella is characterized by comprising chlorella cells and black phosphorus nanosheets which are loaded on the surfaces of the chlorella cells, modify polyaspartic acid and chelate iron ions.
2. The method of preparing a heteroj unction functionalized chlorella according to claim 1, comprising the steps of:
s1, preparing black phosphorus nanosheets: stripping the black phosphorus to obtain black phosphorus nanosheets;
s2, preparing a black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions: uniformly mixing the polyaspartate aqueous solution with the black phosphorus nanosheets obtained in the step S1, adding an iron salt solution, uniformly mixing, washing, and centrifuging to obtain black phosphorus nanosheets with the surfaces modified with polyaspartic acid and chelated with iron ions;
s3, preparation of the heterojunction functionalized chlorella: and (4) uniformly mixing the black phosphorus nanosheet with the surface modified with polyaspartic acid and chelated with iron ions, which is obtained in the step (S2), with chlorella cells, adding iron salt, uniformly mixing, washing, and removing impurities to obtain the heterojunction functionalized chlorella.
3. The method of claim 2, wherein the exfoliation method is an ultrasonic assisted liquid phase exfoliation method.
4. The method of claim 2, wherein the polyaspartate is sodium polyaspartate and the iron salt is a soluble trivalent iron salt in step S2.
5. The method according to claim 2, wherein the mass concentration of the polyaspartate in the aqueous polyaspartate solution of step S2 is 0.4 g/mL.
6. The preparation method according to claim 2, wherein the mass ratio of the black phosphorus nanosheet, the polyaspartate and the iron ion in step S2 is 1: (1.6-2): (0.17-0.8).
7. The preparation method according to claim 2, wherein the concentration of the ferric salt solution in step S2 is 20-24 mmol/L.
8. The method according to claim 2, wherein the ratio of black phosphorus in the black phosphorus nanosheet surface-modified with polyaspartic acid and chelated with iron ions, iron ions in the iron salt solution, and chlorella cells in step S3 is 1 μ g: (0.013-0.104) μ g: (8.75X 10)3～8.8×106) And (4) respectively.
9. Use of the heteroj unction functionalized chlorella prepared by the preparation method of any one of claims 2 to 8 in the preparation of anti-tumor and/or anti-tumor metastasis drugs.
10. Use of the heterojunction functionalized chlorella prepared by the preparation method of any one of claims 2 to 8 in combination with an immune checkpoint inhibitor in preparation of an anti-tumor and/or anti-tumor metastasis medicament.
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