Synthesis method of algae integrated oxygen supply platform based on OLED photoelectric shielding material modification

Abstract

The invention relates to the technical field of photodynamic therapy, in particular to a synthesis method of an algae integrated oxygen supply platform based on OLED photoelectric shielding material modification, and the method comprises the following steps: step 1, selecting a photoelectric material based on pentaperylene diimide (PPD), including PPD derivatives modified by PPD-Se, PPD-Te and PPD-Br and containing heavy atoms as a core photosensitizer; step 2, mixing a photoelectric material based on PPD, a PEG derivative containing PPD-Se and a responsive chemical bond, and DSPE-PEG-S-S-NHH through a coprecipitation method, and stirring for 1-6 hours at the temperature of 30-70 DEG C to form PPD type photoelectric material nano-particles with the particle size within the range of 80-120 nm, namely PPD-based NPs; step three, pretreatment and compounding of green algae: chlorella, scenedesmus and chlamydomonas are used as oxygen supply carriers, the surface of the green algae is modified through carboxymethyl chitosan CMCS, and carboxyl groups are introduced; and step 4, combining the modified PPD type photoelectric material nano particles with the microalgae through amido bonds, so as to form the microalgae-photoelectric material composite system Alga (at) PPD-based.

Description

Technical Field

[0001] This invention relates to the field of photodynamic therapy technology, specifically a method for synthesizing an algae-integrated oxygen supply platform modified with OLED opto-shielding materials. Background Technology

[0002] Photodynamic therapy (PDT), a non-invasive tumor treatment, works by activating a photosensitizer through specific wavelengths of light. This activation causes the photosensitizer to transition from its ground state to an excited state. The excited-state photosensitizer then reacts with oxygen in the tumor microenvironment via energy or electron transfer pathways, generating highly cytotoxic reactive oxygen species (ROS), including singlet oxygen. 1 O2), superoxide anion radicals (O 2- •), hydroxyl radicals (·OH), etc., can oxidize and damage the DNA, proteins, lipids, and other biomolecules of tumor cells, thereby inducing apoptosis or necrosis of tumor cells. References: Hu T, Wang Z, Shen W, et al. Recent advances in innovative strategies for enhanced cancer photodynamic therapy[J]. Theranostics, 2021, 11(7): 3278. doi: 10.7150 / thno.54227 and Algorri JF, Ochoa M, Roldán-Varona P, et al. Light technology for efficient and effective photodynamic therapy: a critical review[J]. Cancers, 2021, 13(14): 3484. doi: 10.3390 / cancers13143484.

[0003] From the perspective of photosensitizer action mechanism, PDT can be divided into type I and type II mechanisms: In type I mechanism, the excited-state photosensitizer undergoes electron transfer with the substrate (such as proteins and nucleic acids in tumor cells), generating free radicals or free radical ions. These reactive species then react with oxygen to generate O2. 2- The type II mechanism involves excited-state photosensitizers directly transferring energy to ground-state oxygen (·, ·OH, etc.). 3 O2) transforms it into the main cytotoxic substances. 1O2, Ref: Zhao X, Liu J, Fan J, et al. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application[J]. Chemical Society Reviews, 2021, 50(6): 4185-4219. doi: 10.1039 / d0cs00173b. The commonly used photosensitizers in clinic and research can be divided into porphyrin (such as hematoporphyrin derivative Photofrin), phthalocyanine (such as zinc phthalocyanine ZnPc), rhodamine (such as Rose Bengal) and indocyanine green (ICG) according to the chemical structure, Ref: Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review[J]. Cancer research, 1989, 49(23): 6449-6465 and Lee C, Lim K, Kim S S, et al. Chlorella-gold nanorods hydrogels generating photosynthesis-derived oxygen and mild heat for the treatment of hypoxic breast cancer[J]. Journal of Controlled Release, 2019, 294: 77-90. doi: 10.1016 / j.jconrel.2018.12.011, but these traditional photosensitizers have significant defects: first, poor light stability, such as ICG is prone to photobleaching under light, leading to rapid decline in ROS generation efficiency with prolonged light exposure; second, the absorption spectrum is limited, most traditional photosensitizers have absorption peaks concentrated in the short-wavelength region of 400-600 nm (such as Soret band of porphyrin about 410 nm, Q band about 500-650 nm), and the penetration depth of short-wavelength light in biological tissue is limited (usually <2 mm), making it difficult to effectively activate photosensitizers in deep solid tumors, Ref: Hong L, Li J, Luo Y, et al.Recent advances in strategies for addressing hypoxia in tumor photodynamic therapy[J]. Biomolecules, 2022, 12(1): 81. doi: 10.3390 / biom12010081; Thirdly, tumor targeting is insufficient. Unmodified photosensitizers are easily enriched in normal tissues (such as skin and liver). After irradiation, they may cause phototoxic damage to normal tissues, such as skin redness, pigmentation, or even mucosal inflammation. Reference: Zhao X, Liu J, Fan J, et al. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application[J]. Chemical Society Reviews, 2021, 50(6): 4185-4219. doi:10.1039 / d0cs00173b.

[0004] Besides the inherent defects of photosensitizers, the hypoxic microenvironment of tumors is the core bottleneck restricting the efficacy of photodynamic therapy (PDT). Due to disordered angiogenesis (such as vascular tortuosity and high permeability), insufficient blood perfusion, and vigorous metabolism of tumor cells (oxygen consumption rate is much higher than that of normal tissues), the partial pressure of oxygen in tumor tissues is usually lower than 5 mmHg (the partial pressure of oxygen in normal tissues is about 20-60 mmHg). Reference: Wan Y, Fu LH, Li C, et al. Conquering the hypoxia limitation for photodynamic therapy[J]. Advanced Materials, 2021, 33(39): 2103978. doi:10.1002 / adma.202103978. The generation of ROS in PDT is highly dependent on oxygen: for PDT with type II mechanism as the main component (such as PDT mediated by porphyrin and phthalocyanine photosensitizers), 1 O2 production directly depends on oxygen concentration; oxygen deficiency leads to... 1O2 production is sharply reduced; even for type I mechanism PDT, the subsequent reaction of free radicals with oxygen also consumes oxygen, which is also limited by hypoxia. Reference: Ming L, Cheng K, Chen Y, et al. Enhancement of tumor lethality of ROS in photodynamic therapy [J]. Cancer Medicine, 2021, 10(1): 257-268. doi:10.1002 / cam4.3496 and Maharjan PS, Bhattarai HK. Singlet oxygen, photodynamic therapy, and mechanisms of cancer cell death [J]. Journal of Oncology, 2022, 2022:7211485. doi:10.1155 / 2022 / 7211485. In addition, hypoxia induces tumor cells to highly express hypoxia-inducible factor-1a (HIF-1a), which not only promotes tumor angiogenesis to adapt to the hypoxic environment, but also up-regulates the expression of antioxidant substances such as glutathione (GSH) and superoxide dismutase (SOD), enhancing the tumor cell's ability to clear ROS and further reducing the efficacy of PDT.

[0005] Therefore, to solve the problem of hypoxia in PDT, researchers have proposed the use of biomaterial-mediated "endogenous oxygen supply" strategy. Green algae (such as Chlorella vulgaris) have become a very potential oxygen supply carrier due to their high photosynthetic capacity (can convert CO2 into O2 using light energy), excellent biocompatibility (no toxicity to major organs through in vivo experiments) and low immunogenicity. In recent years, some studies have combined green algae with photosensitizers to construct a "green algae + PDT" system. For example, Lee et al. combined Chlorella vulgaris with gold nanorods to enhance the efficacy of PDT for hypoxic breast cancer by generating O2 through green algae photosynthesis; Hong et al. tried to physically adsorb photosensitizers on the surface of green algae, hoping to alleviate tumor hypoxia by oxygen supply.

[0006] However, the existing "green algae + PDT" system still has defects that cannot be ignored: first, the release of oxygen is uncontrollable. After the green algae enter the body, whether they reach the tumor site or not, they will continue to perform photosynthesis to release O2. Excessive O2 in normal tissues may interfere with the local physiological environment, and the oxygen concentration in the tumor area cannot be targeted to be increased. Second, there is a lack of tumor-responsive release capability. Photosensitizers are connected to green algae through physical adsorption or non-specific binding, and cannot be specifically released in the tumor microenvironment (such as high GSH, weak acidic pH, and high ROS). This leads to insufficient enrichment of photosensitizers in tumors, and the residual photosensitizers in normal tissues are prone to phototoxicity. Third, the metabolic activity of green algae is easily disturbed. Non-specific modification of nanomaterials or photosensitizers in some studies can destroy the cell membrane structure of green algae and inhibit the activity of photosynthesis-related enzymes (such as Rubisco), resulting in a decrease in oxygen supply capacity. SUMMARY

[0007] The present application aims to combine the tumor microenvironment response characteristics, the high photosensitivity of PPD-based photoelectric materials, and the biological oxygen supply capacity of green algae. A tumor microenvironment-responsive green algae-photovoltaic shielding material modified photosensitizer composite system (Algae@PPD-Se-F) is designed. Through a three-level regulation mechanism of "nanometer shielding - microenvironment triggering - precise drug release and oxygen supply", the tumor-specific activation of photosensitizers and the targeted oxygen supply of green algae are realized. At the same time, the performance defects of traditional photosensitizers and the controllability problem of the existing "green algae + PDT" system are solved. Finally, the PDT efficacy is improved and the toxicity to normal tissues is reduced.

[0008] To achieve the above purpose, the present application provides the following technical scheme: a synthesis method of an algae integrated oxygen supply platform based on OLED photoelectric shielding material modification, which comprises the following steps:

[0009] Step one, select PPD-based photoelectric materials including PPD-Se, PPD-Te, and PPD-Br heavy atom modified PPD derivatives as core photosensitizers;

[0010] Step two, mix PPD-based photoelectric materials including PPD-Se with PEG derivatives containing responsive chemical bonds including DSPE-PEG-S-S-NH2 by co-precipitation, stir at 30-70°C for 1-6h, and form PPD-based photoelectric material nanoparticles (PPD-based NPs) with a particle size in the range of 80-120 nm;

[0011] Step three, pretreatment and compounding of green algae: use Chlorella vulgaris, Scenedesmus and Chlamydomonas reinhardtii as oxygen supply carriers, modify the surface of green algae with carboxymethyl chitosan (CMCS), and introduce carboxyl groups;

[0012] Step four, the modified PPD-based photoelectric material nanoparticles are combined with microalgae through amide bond to form a microalgae-photoelectric material composite system Algae@PPD-based.

[0013] Preferably, in step one, the photoelectric material has a wide spectrum of 280-700 nm absorption, which is suitable for the light requirements of different depth tissues, including shallow and deep tissues, excellent light stability, low light bleaching rate after long time light, which can avoid the rapid decay of photosensitive activity and efficient singlet oxygen 1 O2 generation capacity, quantum yield is usually between 0.008-0.02, and the heavy atoms introduced in its molecular structure include Se, Te, Br.

[0014] Preferably, in step two, the temperature range and stirring time ensure that the materials are fully mixed and reacted to form nanoparticles with good dispersibility and uniform particle size, meeting the subsequent requirements for compounding with microalgae; PPD-Se and DSPE-PEG-S-S-NH2 are co-precipitated at 40-60°C for 2-4 h to form PPD-Se nanoparticles with a particle size of about 98 nm, i.e. PPD-Se NPs.

[0015] Preferably, the photoelectric shielding material is modified: PPD-Se NPs are further coupled with PEG segment modified PPD-Se on the surface of PPD-Se, i.e. PPD-Se-PEG.

[0016] Preferably, the PEG segment has multiple key roles: first, it forms a "barrier" around PPD-Se through its good hydrophilicity and steric hindrance effect, which physically blocks the full contact of external light with PPD-Se, thereby shielding the photosensitive activity of PPD-Se; second, it enhances the hydrophilicity of PPD-Se NPs, making the nanoparticle surface more easily combined with the amino groups introduced on the surface of green algae modified by carboxymethyl chitosan through amide bond; third, the "stealth" property of PEG segment.

[0017] Preferably, in step three, Chlorella vulgaris, i.e. Chlorella vulgaris, has a diameter of usually 1-8 μm, Scenedesmus obliquus, i.e. Scenedesmus obliquus, has a diameter of generally 3-10 μm, and Chlamydomonas reinhardtii, i.e. Chlamydomonas reinhardtii, has a diameter of about 2-10 μm.

[0018] Preferably, in step four, the modified PPD-based photoelectric material nanoparticles, including PPD-Se-F NPs, are mixed with the selected microalgae in a buffer solution with a pH of 7.0-7.8, including phosphate buffer PBS and Tris-HCl buffer, and incubated at 30-60°C for 1-5 h, so that the PPD-based photoelectric material nanoparticles are anchored on the surface of the microalgae through a chemical bond, i.e., an amide bond, to form a microalgae-photoelectric material composite system Algae@PPD-based.

[0019] Preferably, the composite system has a particle size of generally 1.8-2.5 μm as characterized by TEM and a zeta potential of -20 to -30 eV; for example, the modified PPD-Se-F NPs are mixed with Chlorella in a phosphate buffer PBS with a pH of 7.4, and incubated at 30-60°C for 1-5 h, so that the PPD-Se-F NPs are anchored on the surface of the Chlorella through an amide bond to form an Algae@PPD-Se-F composite system, which has a particle size of about 2.1 μm and a zeta potential of -25.2 eV, and has good colloidal stability.

[0020] Based on the application of the algae integrated oxygen supply platform modified by the OLED photoelectric shielding material, the OLED photoelectric shielding material is given three functions of "photosensitive activity shielding", "green algae metabolism regulation" and "blood stability improvement", which breaks through the traditional application of the OLED photoelectric shielding material as a "carrier skeleton".

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] 1. Full tumor microenvironment response type "switch" design: for the first time, the photosensitizer (PPD-Se NPs) modified by the photoelectric shielding material and the disulfide bond is anchored on the surface of the green algae, and the photosensitizer activity and the photosynthesis of the green algae are specifically turned on through the "physical shielding" of the photoelectric shielding material and the "disulfide bond breaking" in response to GSH, which solves the problem of normal tissue damage caused by the "full activation" of the existing system.

[0023] 2. Multifunctional integrated application of photoelectric shielding material: the OLED photoelectric shielding material is given three functions of "photosensitive activity shielding", "green algae metabolism regulation" and "blood stability improvement", which breaks through the traditional application of the OLED photoelectric shielding material as a "carrier skeleton".

[0024] 3. Synergistic optimization of photoelectric material and biological oxygen supply: PPD-Se with wide spectrum absorption is selected as the photosensitizer, which is suitable for the light condition (545 nm) of the photosynthesis of the green algae, so as to realize the synergistic regulation of "light - oxygen supply - PDT", and the ROS generation efficiency is significantly enhanced through the heavy atom effect of Se atom and the oxygen supply of the green algae, which significantly enhances the PDT effect on the hypoxic tumor. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For the present application, 1,3-diphenyl isobenzofuran (DPBF) is used as a probe to measure the reactive oxygen species (ROS) quantum yield map of TPH, TPH-Se, PPD and PPD-Se under light-emitting diode (LED) irradiation at the same concentration, the light irradiation interval is 15 seconds, and the absorption wavelength is 410 nanometers;

[0026] Figure 2 For the present application, 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used as a free radical probe to evaluate the type I photodynamic activity map of TPH, TPH-Se, PPD and PPD-Se under light-emitting diode (LED) irradiation at the same concentration, the light irradiation interval is 15 seconds, and the absorption wavelength is 405 nanometers;

[0027] Figure 3 For the present application, the hydrodynamic map of TPH, TPH-Se, PPD and PPD-Se is determined by dynamic light scattering (DLS), and the diameters are about 190 nanometers, 142 nanometers, 190 nanometers and 164 nanometers, respectively;

[0028] Figure 4 For the present application, the zeta potential map before and after mPEG-2000 coated TPH, TPH-Se, PPD and PPD-Se;

[0029] Figure 5 For the present application, the CCK-8 cell survival rate map of 4T1 cells co-incubated with green algae (Algae), PPD-Se and Algae@PPD-Se for 48 hours (with or without LED light irradiation treatment);

[0030] Figure 6 For the present application, the flow cytometry JC-1 analysis map of 4T1 cells treated with green algae (Algae), PPD-Se and Algae@PPD-Se (with or without LED light irradiation);

[0031] Figure 7 For the present application, the hemolysis experiment map of Algae@PPD-Se at different concentrations. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0033] Example 1:

[0034] Core material selection and preparation

[0035] Photoelectric materials based on pentaperylene diimide (PPD), including PPD-Se, PPD-Te, PPD-Br, and other PPD derivatives modified with heavy atoms, are selected as core photosensitizers. These materials generally possess a broad spectral absorption of 280-700 nm (suitable for the illumination requirements of tissues at different depths, including shallow and deep tissues), excellent photostability (low photobleaching rate after long-term illumination, avoiding rapid decay of photosensitivity during illumination), and highly efficient singlet oxygen (…). 1 The O2 generation capacity (quantum yield is typically between 0.008 and 0.02) and the introduction of heavy atoms (including Se, Te, Br, etc.) into its molecular structure can enhance intersystem crossing (ISC) efficiency through the heavy atom effect, thereby further increasing the amount of reactive oxygen species (ROS) generated. Taking PPD-Se as an example, it has a broad spectral absorption range of 300-650 nm, excellent photostability, and an O2 generation quantum yield of 0.01263. The introduction of Se atoms significantly enhances ISC efficiency and effectively increases ROS generation, meeting the performance requirements of photosensitizers in photodynamic therapy. To impart tumor microenvironment responsiveness, PPD-Se was surface-modified with DSPE-PEG-SS-NH2 (distearate-phosphatidylethanolamine-polyethylene glycol-disulfide-amino). PPD-based optoelectronic materials (including PPD-Se) were mixed with PEG derivatives containing responsive chemical bonds (including DSPE-PEG-SS-NH2) via co-precipitation, and stirred at 30-70℃ for 1-6 h to form PPD-based optoelectronic nanoparticles (NPs) with a particle size in the range of 80-120 nm. This temperature range and stirring time ensured thorough mixing and reaction of the materials, resulting in well-dispersed, uniformly sized nanoparticles, meeting the requirements for subsequent composite with microalgae. Taking the coprecipitation reaction of PPD-Se with DSPE-PEG-SS-NH2 as an example, stirring at 40-60℃ for 2-4 h can form PPD-Se nanoparticles (PPD-Se NPs) with a particle size of about 98 nm. These nanoparticles have good dispersibility and can exist stably in the reaction system. The disulfide bonds (-SS-) on their surface can be specifically cleaved by the high concentration of GSH in tumors.

[0036] Modification of photoelectric shielding material: Taking PPD-Se NPs as an example, PPD-Se NPs are further modified on the surface by coupling PEG segments to form PPD-Se-PEG. PEG segments have multiple key roles: first, by its good hydrophilicity and steric hindrance effect, a layer of "barrier" is formed around PPD-Se, which physically blocks the full contact of external light with PPD-Se, thereby shielding the photosensitive activity of PPD-Se and preventing the incorrect activation of photosensitizer at non-tumor sites due to light; second, the hydrophilicity of PPD-Se NPs is enhanced, making the nanoparticle surface more easily bind to the amino groups introduced on the surface of green algae modified by carboxymethyl chitosan through amide bond, thereby improving the stability and efficiency of the combination of the two; third, the "stealth" property of PEG segments can reduce the immunogenicity and non-specific interaction of the composite system in the blood, improve the blood stability of the composite system, reduce the recognition and removal of the composite system by the reticuloendothelial system, and prolong its circulation time in the body.

[0037] Pre-treatment and complexation of green algae: Microalgae with photosynthetic ability are selected as oxygen carriers, including Chlorella (including Chlorella vulgaris, usually 1-8 μm in diameter), Scenedesmus (including Scenedesmus obliquus, usually 3-10 μm in diameter), Chlamydomonas (including Chlamydomonas reinhardtii, about 2-10 μm in diameter), etc. These microalgae have high photosynthetic ability (convert CO2 to O2 using light energy), good biocompatibility and low immunogenicity, and can provide endogenous oxygen for photodynamic therapy and relieve the hypoxic microenvironment of tumors. Taking Chlorella vulgaris as an example, its diameter is about 2-5 μm, and it has high photosynthetic efficiency. In vivo experiments have verified that it has no toxicity to major organs, and as an oxygen carrier, it can effectively supplement oxygen to the tumor area. The surface of the green algae is first modified by carboxymethyl chitosan (CMCS) to introduce amino groups; then the modified PPD-based photoelectric material nanoparticles (including PPD-Se-F NPs) are mixed with the selected microalgae in a buffer solution (including phosphate buffer PBS, Tris-HCl buffer, etc.) at pH 7.0-7.8, and incubated at 30-60°C for 1-5 h. PPD-based photoelectric material nanoparticles are anchored on the surface of microalgae through chemical bonds (including amide bonds) to form a microalgae- photoelectric material composite system (Algae@PPD-based). The particle size of the composite system is usually in the range of 1.8-2.5 μm (TEM characterization), and the zeta potential is between -20 and -30 eV (to ensure that the composite system has good colloidal stability and avoids aggregation). For example, PPD-Se-F NPs modified with PPD-Se-F NPs and Chlorella are mixed in a phosphate buffer (PBS) at pH 7.4, incubated at 30-60°C for 1-5 h, and PPD-Se-F NPs are anchored on the surface of Chlorella through amide bonds to form an Algae@PPD-Se-F composite system. The particle size is about 2.1 μm (TEM characterization), and the zeta potential is -25.2 eV, with good colloidal stability.

[0038] Example 2:

[0039] System working mechanism (tumor microenvironment response process)

[0040] In vivo delivery stage: After the Algae@PPD-base composite system (including Algae@PPD-Se-F, Algae@PPD-Te-F, etc.) is injected into the body through intravenous, peritumoral or intraperitoneal injection, etc., the photoelectric shielding material on the surface of PPD-based NPs (including PPD-Se-F NPs, PPD-Te-F NPs) and the responsive PEG derivative (including DSPE-PEG-S-S-NH2, mPEG-SS-COOH, etc.) together form a dense "physical barrier": on the one hand, it shields the photosensitive activity of PPD-based photoelectric materials, and even if it is exposed to external light, it cannot effectively generate ROS before reaching the tumor site; on the other hand, it inhibits the photosynthesis of microalgae (avoids excessive oxygen supply at non-tumor sites to interfere with the physiological environment of normal tissues), while increasing the blood half-life of the system (extended to 5-9h, higher than 1-4h of unmodified microalgae), increasing the circulation time of the system in the body to improve the enrichment rate at the tumor site.

[0041] Tumor site enrichment and triggering: When the system reaches the tumor site, specific stimulating factors in the tumor microenvironment (including high concentration of GSH, about 8-25mM, normal tissue about 0.1-1.2mM; or weakly acidic pH, about 6.0-6.8, normal tissue about 7.2-7.4; or high ROS concentration, about 100-500nM, normal tissue about 10-50nM) can specifically cleave the responsive chemical bonds (including disulfide bonds (-S-S-), borate ester bonds (-O-B-O-), or acetal bonds (-O-C-O-), etc.) on the surface of PPD-based NPs, resulting in the shedding of PPD-based NPs from the surface of microalgae: ① The shed PPD-based NPs lose the shielding of photoelectric materials and are activated under specific wavelength light (520-570nm LED or laser) to generate O2 efficiently; ② The "physical barrier" on the surface of microalgae disappears, photosynthesis is restored, and O2 is generated by CO2 in the tumor tissue and light to supplement oxygen for PDT and relieve tumor hypoxia (dissolved oxygen concentration is increased to 7-11mg / L, higher than 1-4mg / L of untreated tumors). 1 O2; ② The "physical barrier" on the surface of microalgae disappears, photosynthesis is restored, and O2 is generated by CO2 in the tumor tissue and light to supplement oxygen for PDT and relieve tumor hypoxia (dissolved oxygen concentration is increased to 7-11mg / L, higher than 1-4mg / L of untreated tumors).

[0042] Enhanced PDT Therapy and Microenvironment Regulation: In the Algae@PPD-based composite system of this method, O2 generated by photosynthetic microalgae (including Chlorella and Scenedesmus from the Chlorophyta phylum, and Synechococcus from the Cyanophyta phylum) enhances the PDT efficacy of PPD-based photoelectric materials (including PPD-Se, PPD-Te, PPD-Br, etc.) through multi-dimensional effects. First, as a core auxiliary substrate of the photodynamic mechanism, O2 can simultaneously improve the ROS generation efficiency of both type I and type II photodynamic mechanisms: For PPD-based photoelectric materials dominated by the type II mechanism, O2 is singlet oxygen (… 1 O2) is a direct substrate generated; sufficient O2 can enable... 1O2 generation efficiency is significantly improved, which in turn increases the total ROS production by 1.5-3.5 times compared to the condition without additional oxygen supply; for PPD derivatives with a higher proportion of type I mechanism (including PPD and porphyrin complex derivatives), O2 can also participate in the oxidation reaction of free radicals (including semiquinone free radicals) to generate active species such as superoxide anion radicals (O2⁻・), further supplementing the ROS production, and adapting to the mechanism requirements of different PPD-based photosensitizers. Second, the continuous oxygen supply of microalgae can improve the hypoxic microenvironment of tumors, thereby down-regulating the expression level of the hypoxia-inducible factor family (including HIF-1α, HIF-2α, etc.). Western blot or immunohistochemical detection shows that the relative expression of HIF-1α protein can be reduced by 50%-80%, and the relative expression of HIF-2α protein can be reduced by 45%-75%; the down-regulation of HIF family proteins can reduce the synthesis and activity of tumor cell antioxidant defense system-related substances (including but not limited to GSH, SOD, catalase (CAT), etc.), weaken the tumor cell's ability to clear and tolerate ROS, and reduce its resistance to PDT from a mechanism perspective, while also inhibiting HIF-mediated abnormal tumor angiogenesis and reducing the risk of tumor metastasis. Finally, the high-concentration ROS generated through the above mechanisms can induce tumor cell death through multiple synergistic pathways to enhance the anti-tumor effect and cover the differences in the death sensitivity of different types of tumor cells: on the one hand, ROS can attack the mitochondrial membrane of tumor cells, damage the mitochondrial membrane integrity, cause mitochondrial membrane depolarization (reduction of red-green fluorescence ratio or fluorescence intensity by 35%-65% through JC-1, TMRM, etc. Fluorescent probe staining detection), promote the release of cytochrome C, Smac / DIABLO, etc. Promoting factors, and activate the caspase family of proteases (including caspase-3, caspase-6, caspase-9, etc.) cascade reaction, ultimately inducing tumor cell apoptosis; on the other hand, part of ROS (including・OH) directly damages the lipid bilayer of the tumor cell membrane, leading to increased cell membrane permeability, content leakage, and triggering cell necrosis, while also attacking tumor cell DNA, causing DNA chain breaks, base modifications, and other damage, which, if beyond the capacity of the DNA repair system, will activate the DNA damage response pathway p53, further promoting cell apoptosis or senescence; through flow cytometry, TUNEL staining, etc. Method detection, the total death rate (apoptosis rate + necrosis rate) of different tumor cells (including breast cancer cells, melanoma cells, colorectal cancer cells, etc.) is increased to 70%-90%, which is significantly higher than the PDT treatment effect without oxygen supply.

[0043] Example 3:

[0044] Key experimental verification method

[0045] Photosensitizer activity detection: Single-state oxygen detection probes (including DPBF (1,3-diphenylisobenzofuran), SOSG (single-state oxygen sensor green), ABDA (9,10-anthracenediyl-bis(methylene)-diethylmalonate), etc.) are used as probes, and the oxygen generation amount of PPD-based photoelectric materials (including PPD-Se) under different conditions (with or without tumor microenvironment response substances, with or without light) is detected by ultraviolet spectrophotometer (the detection wavelength is adjusted according to the characteristics of the probe in the range of 400-430 nm) and fluorescence spectrophotometer (the detection wavelength is adjusted according to the characteristics of the probe in the range of 510-540 nm) respectively, so as to evaluate the photosensitive performance of the photoelectric materials. Taking PPD-Se detection as an example, DPBF and SOSG are used as probes, and the oxygen generation amount of PPD-Se under the conditions of with or without GSH, with or without light is detected by ultraviolet spectrophotometer (410 nm) and fluorescence spectrophotometer (525 nm) respectively. 1 O2 generation amount, and the results show that the oxygen generation amount of PPD-Se under the conditions of with GSH and with light is significantly improved. 1 O2 generation amount, and the results show that the oxygen generation amount of PPD-Se under the conditions of with GSH and with light is significantly improved. 1 O2 generation amount, and the results show that the oxygen generation amount of PPD-Se under the conditions of with GSH and with light is significantly improved.

[0046] Green algae photosynthesis detection: The oxygen release rate of microalgae-photoelectric material composite system (including Algae@PPD-Se-F) under the treatment of tumor microenvironment response substances (including GSH, acidic pH, high ROS, etc.) is monitored by dissolved oxygen meter, the light condition is selected as light source (including LED lamp, laser, etc.) with wavelength in the range of 520-570 nm, and the power density is adjusted in the range of 80-120 mW / cm 2 2, and the dissolved oxygen concentration is recorded every 3-8 min, so as to evaluate the oxygen supply capacity of the composite system under different conditions. Taking Algae@PPD-Se-F composite system as an example, the oxygen release rate thereof under the treatment of with or without GSH is monitored by dissolved oxygen meter, the light condition is 545 nm LED (power density 100 mW / cm 2 2), and the dissolved oxygen concentration is recorded every 5 min, and the results show that the oxygen release rate of the composite system under the treatment of with GSH is significantly improved.

[0047] Cell level verification: 4T1 breast cancer cells are used as a model, the uptake of PPD-Se by cells is observed by confocal laser scanning microscope (CLSM) (545 nm excitation, 610 nm emission); Calcein-AM / PI staining and flow cytometry are used to detect the apoptosis rate of cells; and the expression levels of HIF-1α and caspase-3 are detected by Western blot.

[0048] Animal level verification: BALB / c mice 4T1 subcutaneous tumor model is constructed, and the microalgae-photoelectric material composite system (including Algae@PPD-Se-F) is given by peritumoral injection or intravenous injection, the concentration of the composite system is in the range of 40-80 μg / mL, the injection volume is 150-250 μL; the tumor site is irradiated by a light source (including LED lamp, laser, etc.) with a wavelength in the range of 520-570 nm every 1-3 days, the irradiation time is 5-15 min, and the tumor volume change is continuously monitored for 10-20 days to evaluate the inhibitory effect of the composite system on the tumor. Taking the peritumoral injection of the Algae@PPD-Se-F composite system as an example, the injection concentration is 60 μg / mL, the injection volume is 200 μL, the 545 nm LED is irradiated for 10 min every 2 days, and the tumor volume change is monitored for 14 consecutive days. The results show that the composite system can effectively inhibit tumor growth; the tumor necrosis and hypoxia improvement are evaluated by H&E staining and immunofluorescence staining (HIF-1α antibody).

[0049] In summary, the Algae@PPD-Se-F composite system of the method relies on the "tumor microenvironment response - photosensitizer activation - biological oxygen supply" synergistic mechanism to solve the core pain points of existing PDT and "green algae + PDT" systems from multiple dimensions. In terms of solving the PDT hypoxia bottleneck, animal experiment data shows that the system can increase the dissolved oxygen concentration in tumor tissue from 2.3 mg / L to 9.8 mg / L, reduce the HIF-1α protein expression level by 68%, and the ROS generation amount (SOSG fluorescence intensity) is 2.7 times that of the traditional PDT group and 1.8 times that of the unmodified "green algae + PDT" group; immunofluorescence staining results further confirm that it mediates 1 O2in the tumor tissue has a wider distribution range and can cover the deep layer area, and the tumor necrosis area accounts for 75%, which is significantly higher than the 30% of the traditional PDT group and the 45% of the unmodified "green algae + PDT" group. In terms of solving the problem of non-targeted and normal tissue toxicity of the system, in vitro experiments show that when there is no GSH (not reaching the tumor site), the ROS generation amount of the system is only 5% of the activated state; in animal experiments, the skin phototoxicity score of the mice in its group is only 0.5 (0-4 scale), which is much lower than the 2.5 of the traditional PDT group and the 1.8 of the unmodified "green algae + PDT" group, and H&E staining shows that there is no obvious inflammatory infiltration or tissue necrosis in the heart, liver and other major organs, and the blood biochemical indicators (ALT, AST, BUN, Cr) are all within the normal range, and the biological safety is excellent.

[0050] Meanwhile, the Algae@PPD-Se-F complex system greatly improves the in vivo stability and the persistence of therapeutic effect. Quantitative data show that the blood half-life of the system is 7.2 h, which is 3.1 times that of the unmodified green algae, and the drug concentration in the tumor reaches a peak at 6 h after injection and is maintained at a high level for 24 h, which is significantly longer than the 12 h of the unmodified system; the results of the 14-day animal experiment show that the tumor volume inhibition rate of the system is 82% and there is no obvious recurrence, while the tumor volume inhibition rates of the PBS, simple green algae and simple PPD-Se control groups are only 5%, 20% and 35% respectively, and all have different degrees of recurrence, fully proving the significant advantages of the system in the persistence of therapeutic effect and the ability to resist recurrence.

[0051] It should be noted that, in this document, the terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed or inherent to such a process, method, article or device.

[0052] Although embodiments of the present application have been shown and described, it will be understood by those having ordinary skill in the art that various changes, modifications, substitutions and alterations can be made hereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing an algae-integrated oxygen supply platform modified with OLED optoelectronic shielding materials, characterized in that, The method includes the following steps: Step 1: Select optoelectronic materials based on pentaperyl diimide (PPD), including PPD-Se, PPD-Te, and PPD-Br derivatives modified with heavy atoms, as core photosensitizers; Step 2: PPD-based optoelectronic materials, including PPD-Se, are mixed with PEG derivatives containing responsive chemical bonds, including DSPE-PEG-SS-NH2, by co-precipitation and stirred at 30-70℃ for 1-6 h to form PPD-based optoelectronic material nanoparticles with a particle size in the range of 80-120 nm, i.e. PPD-based NPs. Step 3, Pretreatment and compounding of green algae: Chlorella, Scenedesmus and Chlamydomonas are used as oxygen carriers, and the surface of green algae is modified with carboxymethyl chitosan (CMCS) to introduce carboxyl groups. Step four involves combining the modified PPD-based optoelectronic material nanoparticles with microalgae via amide bonds to form the microalgae-optoelectronic material composite system Algae@PPD-based.

2. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 1, characterized in that: In step one, the optoelectronic material possesses a broad spectral absorption range of 280-700 nm, adapting to the lighting requirements of tissues at different depths, including both shallow and deep tissues. It also exhibits excellent photostability, low photobleaching rate after prolonged illumination, and can prevent rapid decay of photosensitive activity during illumination, while providing highly efficient singlet oxygen. 1 O2 generation capacity, with a quantum yield typically between 0.008 and 0.02, and the heavy atoms introduced into its molecular structure include Se, Te, and Br.

3. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 1, characterized in that: In step two, the temperature range and stirring time ensure that the materials are fully mixed and reacted to form nanoparticles with good dispersibility and uniform particle size, which meets the requirements for subsequent composite with microalgae. The co-precipitation reaction of PPD-Se and DSPE-PEG-SS-NH2 is stirred at 40-60℃ for 2-4 h to form PPD-Se nanoparticles with a particle size of about 98 nm, namely PPD-Se NPs.

4. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 3, characterized in that: Modification of optoelectronic shielding materials: PPD-Se modified by further coupling PEG segments to PPD-Se on its surface, i.e. PPD-Se-PEG.

5. The method for synthesizing an algae-integrated oxygen supply platform based on OLED photoelectric shielding material modification according to claim 4, characterized in that: PEG segments have multiple key functions: First, through their good hydrophilicity and steric hindrance effect, they form a "barrier" around PPD-Se, physically blocking external light from fully contacting PPD-Se, thereby shielding the photosensitivity of PPD-Se. Secondly, it enhances the hydrophilicity of PPD-Se NPs, making it easier for the surface of nanoparticles to bind with the amino groups introduced on the surface of green algae after modification with carboxymethyl chitosan through amide bonds; thirdly, it utilizes the "stealth" properties of PEG segments.

6. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 1, characterized in that: In step three, Chlorella vulgaris typically has a diameter of 1-8 μm, Scenedesmus obliquus typically has a diameter of 3-10 μm, and Chlamydomonas reinhardtii typically has a diameter of 2-10 μm.

7. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 1, characterized in that: In step four, the modified PPD-based optoelectronic nanoparticles, including PPD-Se-F NPs, are mixed with selected microalgae in a buffer solution at pH 7.0-7.8, including phosphate-buffered saline (PBS) and Tris-HCl buffer. The mixture is then incubated at 30-60°C for 1-5 h. The PPD-based optoelectronic nanoparticles are anchored to the surface of the microalgae through chemical bonds, specifically amide bonds, forming the microalgae-optoelectronic composite system Algae@PPD-based.

8. The method for synthesizing an algae-integrated oxygen supply platform based on OLED opto-shielding material modification according to claim 7, characterized in that: The particle size of this composite system is typically in the range of 1.8-2.5 μm, as characterized by TEM, and the zeta potential is between -20 and -30 eV. Taking the modified PPD-Se-F NPs and Chlorella composite as an example, they are mixed in phosphate-buffered saline (PBS) at pH 7.4 and incubated at 30-60°C for 1-5 h. The PPD-Se-F NPs are anchored to the surface of Chlorella via amide bonds to form an Algae@PPD-Se-F composite system with a particle size of about 2.1 μm and a zeta potential of -25.2 eV, exhibiting good colloidal stability.

9. The application of an integrated algae oxygen supply platform modified with OLED opto-shielding materials, based on the synthesis method described in any one of claims 1 to 8, characterized in that, Breaking away from the traditional application of optoelectronic shielding materials as merely "carrier skeletons," this invention endows them with three functions: "photosensitive active shielding," "green algae metabolic regulation," and "enhanced blood stability."

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