Application of chemotherapeutic drug-microalgae embolism agent in preparation of drug for treating tumors by arterial embolism

Abstract

The invention discloses application of a chemotherapy drug-microalgae embolization agent in preparation of a drug for treating tumors through arterial embolization, belongs to the technical field of biological medicine, and particularly relates to the technical field of biological medicine manufacturing, and the chemotherapy drug-microalgae embolization agent is composed of a pharmacologically effective amount of chemotherapy drug and microalgae, the chemotherapeutic drug is electropositive in a physiological environment and is combined with the microalgae through electrostatic adsorption, and preferably, the mass ratio of the microalgae to the chemotherapeutic drug is 1: (0.1-10). The chemotherapy drug-microalgae embolization agent has pH responsiveness when releasing chemotherapy drugs, and can improve an acidic tumor microenvironment, so that the antitumor effect is synergistically enhanced; the chemotherapeutic drug, namely the microalgae embolization agent, also has good biocompatibility and biodegradability. The chemotherapeutic drug-loaded microalgae provided by the invention provides clinical transformation potential for malignant tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, especially the field of biopharmaceutical manufacturing technology, and specifically relates to the use of chemotherapy drugs—microalgae embolization agents—in the preparation of drugs for arterial embolization to treat tumors. Background Technology

[0002] Common treatments for liver cancer include local therapy, liver transplantation, surgical resection, and systemic therapy. Transarterial chemoembolization (TACE) is a recommended treatment for intermediate-to-advanced liver cancer. The unique vascular characteristics of liver cancer make TACE a safe and promising treatment option. Depending on the embolic agent, TACE is divided into conventional TACE and drug-eluting microsphere TACE. Conventional TACE primarily uses iodized oil-based chemotherapy drug emulsions, supplemented by particulate embolic agents. This emulsion is unstable, typically exhibiting poor physical stability and rapid phase separation, resulting in suboptimal embolization strength, rapid drug release in the body, increased systemic toxicity, and reduced treatment efficacy. Clinically used drug-eluting microspheres mainly include DC-Beads and HepaSpheres, which are typically loaded with anthracycline chemotherapy drugs and are non-degradable microspheres.

[0003] Chinese patent document CN 118987262A discloses a method for preparing drug-loaded embolized microalgae spheres for treating liver cancer. The method uses a drug-loaded microalgae cell solution containing polyvinyl alcohol (PVA) as the inner phase solution, a mixed solution containing polydimethylsiloxane (PDMS), dimethyl silicone oil, and a curing agent as the intermediate phase solution, and a PVA solution as the outer phase solution. Microfluidic technology is used to adjust the flow rates of the inner, intermediate, and outer phase solutions to achieve this. However, this drug-loaded embolized microalgae sphere requires a specific three-phase system and is essentially a PVA microsphere. Based on its composition, the microsphere is a composite artificial microsphere with non-degradable PDMS as the structural matrix, making it a typical non-degradable microsphere. Permanent embolization can lead to angiogenesis and acidification, potentially causing tumor invasion and metastasis, thus affecting treatment efficacy. Furthermore, the patent mentions that the drug-loaded embolized microalgae sphere acts as a drug carrier, but the microalgae only serve to transport the drug. Simply mixing the microalgae and drug components alone, relying solely on the microalgae cells, cannot produce drug-loaded microalgae spheres with embolizing effects. In Chinese patent CN118846195A, the drug-loaded microspheres in the dual-emulsion droplet method are also non-degradable microspheres, with a microalgal suspension containing an active drug within the drug-loaded microspheres. Furthermore, this method involves a complex preparation process, requiring not only precise microfluidic control but also the use of various chemical reagents. This increases production difficulty and process costs, and may lead to instability in mass production. Simultaneously, the chemical components used may potentially affect biocompatibility, increasing the risk of immune rejection or other adverse reactions, further limiting the clinical application prospects of this technology. Therefore, this technology still has significant room for improvement in terms of safety, preparation complexity, and cost control. Existing technology discloses a spirulina-based biodegradable carrier for the treatment of lung metastases from breast cancer via intravenous injection. While this approach utilizes the passive capture of microalgae by pulmonary capillaries to achieve targeting (dependent on the hydrodynamics of blood circulation), its technical essence is drug delivery rather than vascular embolization therapy. The paper clearly states that the microalgal carrier is ultimately biodegraded through renal clearance, exhibiting no significant toxicity and without any obvious inflammatory lesions or damage to the tissue. Therefore, this microalgae carrier is a mild carrier that needs to be rapidly metabolized in vivo (the article states that urine returned to pre-injection levels after 48 hours, indicating that the drug and carrier had been metabolized and cleared) to avoid causing additional damage. (Zhong D, Zhang D, Xie T, et al. Biodegradable Microalgae-Based Carriers for Targeted Delivery and Imaging-Guided Therapy toward Lung Metastasis of Breast Cancer[J].Small, 2020, 16(20):2000819).

[0004] Currently, the clinical control rate of liver cancer is not satisfactory, and developing a safer and more effective embolization agent remains a challenge. An ideal embolization agent should possess characteristics such as controllable degradation, good biocompatibility, precise embolization capability, drug loading and release capacity, low biohazard and environmental friendliness, and ease of preparation and operation. Existing reports (Yun-Ping Chen et al., Recent Advances on Polymeric Beads or Hydrogels as Embolization Agents for Improved Transcatheter Arterial Chemoembolization (TACE). Frontiers in Chemistry, 7 (2019), p. 408) indicate that the success of TACE largely depends on the design of the embolization agent. An ideal embolization agent should simultaneously meet the following requirements: 1) rapid and effective intra-arterial injection to block blood supply; 2) release of embedded anticancer drugs for local chemotherapy; 3) post-treatment degradation to prevent thrombosis; and 4) the ability to impair tumor angiogenesis. However, current technology has not reported the use of chemotherapeutic drugs—microalgae embolization agents—for the delivery of chemotherapeutic drugs in arterial embolization. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing the use of a chemotherapy drug-microalgae embolizing agent in the preparation of drugs for arterial embolization therapy of malignant tumors. Unexpectedly, this invention has found that direct administration of the chemotherapy drug-microalgae as an embolizing agent can achieve arterial embolization. Furthermore, the release of the chemotherapy drug is better achieved in the tumor microenvironment at pH 6.5. The presence of microalgae in the chemotherapy drug-microalgae further improves the acidic tumor microenvironment, not only allowing the chemotherapy drug to exert its effects more effectively but also further inhibiting the proliferation and metastasis of tumor cells. In this invention, the microalgae are not merely a drug carrier but work synergistically with the chemotherapy drug to achieve physical embolization, block tumor blood supply, and targeted therapy.

[0006] Chemotherapy drugs-microalgae embolization agents create emboli within tumor angiogenesis, blocking blood supply to the tumor through vascular occlusion. The embolization agent directly enters the tumor's feeding arteries, physically blocking the vascular lumen, causing tumor necrosis due to lack of nutrients and oxygen. Simultaneously, microalgae possess pH-regulating capabilities, improving the acidic tumor microenvironment. Therefore, the chemotherapy drug-microalgae embolization agent exerts a synergistic anti-tumor effect while also contributing to the overall therapeutic effect. However, current research is limited to targeted delivery and has not truly achieved the holistic effect of chemotherapy drugs-microalgae embolization agents.

[0007] The specific technical solution adopted in this invention is as follows: The first objective of this invention is: A chemotherapy drug-microalgae embolizing agent is provided for use in the preparation of arterial embolization drugs for the treatment of tumors. The chemotherapy drug-microalgae embolizing agent is composed of a pharmacologically effective amount of chemotherapy drug and microalgae. The chemotherapy drug is positively charged under physiological conditions and binds to the microalgae through electrostatic adsorption.

[0008] Preferably, the chemotherapy drug-microalgae embolizing agent has the function of increasing the pH value of the tumor's acidic microenvironment.

[0009] In some embodiments of the present invention, the tumor is a malignant liver tumor, a malignant bile duct tumor, or a malignant lung tumor; preferably, the tumor is a malignant liver tumor or a malignant bile duct tumor.

[0010] In this invention, a chemotherapy drug-microalgae embolizing agent actively embolizes the arterial system. Through physical induction of local vascular embolism, it exhibits targeting and stability within the blood vessels. Specifically, the embolizing agent migrates through the blood via hemodynamics, moving in a hydrodynamic direction to distal arterial vessels, embolizing the tumor arterial lumen, causing local blood flow interruption, and subsequently inducing tumor ischemia. This prevents the tumor in the corresponding blood supply area from obtaining sufficient oxygen and nutrients, thus treating pathological conditions requiring blood flow restriction, ultimately leading to tumor damage or necrosis. Therefore, the chemotherapy drug-microalgae embolizing agent possesses precise predictability. Through arterial embolization, including but not limited to hepatic artery embolization and bronchial artery embolization, the chemotherapy drug-microalgae embolizing agent can treat malignant tumors such as liver cancer, bile duct cancer, and lung cancer.

[0011] The present invention is also effective in treating malignant tumors of the bile duct and lung, mainly due to the embolic agent's specific particle size (e.g., 100-500 micrometers) physically blocking the tumor's blood supply arteries and new blood vessels. An acidic tumor microenvironment (lactic acid buildup) is a common hallmark feature of most solid tumors (including lung cancer, bile duct cancer, etc.).

[0012] In this chemotherapy drug-microalgae embolization agent, the microalgae possess an inherent negative surface charge, enabling them to bind with positively charged chemotherapy drugs through non-covalent electrostatic interactions. This process is simple and demonstrates significant potential for drug delivery. Furthermore, unlike polyvinyl alcohol microspheres (CN118987262A), the microalgae in this invention contain abundant nutrients; for example, *Spirulina platensis* is rich in phycocyanin and unsaturated fatty acids, exhibiting anti-inflammatory and anti-tumor effects. The microalgae can precisely embolize the nutrient arteries of malignant tumors, controlling the slow release of chemotherapy drugs. By utilizing the inherent fluorescence properties of microalgae, in vivo distribution monitoring can be achieved. The chemotherapy drug-microalgae embolization agent exhibits pH sensitivity during chemotherapy drug release, and the microalgae simultaneously improve the acidic microenvironment of the tumor, thereby synergistically enhancing the anti-tumor effect. The microalgae possess excellent biocompatibility and biodegradability, thus providing potential for repeated treatment of malignant tumors.

[0013] In this patent, the embolic agent works by physically blocking the lumen of blood vessels, causing local blood flow interruption, which in turn induces tumor ischemia, ultimately leading to tumor damage or necrosis. This patent requires the microalgae to remain in the blood vessels for a sufficient period of time to block blood flow and cause tumor necrosis before complete degradation.

[0014] Chemotherapy drugs that are positively charged under physiological conditions can be any chemical entity with antitumor activity, such as alkylating agents, antimetabolites, or topoisomerase inhibitors; they can also be drug molecules with different charge-modifying groups, such as alkylamine derivatives or quaternary ammonium compounds. The charge-modifying groups are preferably covalently bonded to specific sites on the drug's core (such as hydroxyl, carboxyl, or aromatic rings). The positively charged groups can include primary amines (-NH2), secondary amines (-NHR), tertiary amines (-NR2), or quaternary ammonium groups (-N...). + R3X - Positively charged chemotherapy drugs include doxorubicin, daunorubicin, doxorubicin, irinotecan, cisplatin, etc.

[0015] In some embodiments of the present invention, the microalgae are a type of tiny single-celled or multicellular photosynthetic microorganisms that typically grow in aquatic environments and can synthesize organic matter using light energy, carbon dioxide, and water through photosynthesis. The microalgae portion can be any microalgae capable of electrostatically adsorbing positively charged chemotherapeutic drugs to achieve vascular embolization. In some embodiments of the present invention, preferably, the mass ratio of the microalgae to the chemotherapeutic drug is 1:0.1-10 on a dry weight basis. When the mass ratio of microalgae to chemotherapeutic drug is less than 1:10, the chemotherapeutic drug far exceeds the loading limit of the microalgae, resulting in a low encapsulation rate and waste; when the mass ratio of microalgae to chemotherapeutic drug is greater than 1:0.1, the binding of the microalgae to the chemotherapeutic drug is unsaturated, resulting in a low drug loading rate and affecting the efficacy of the chemotherapeutic drug. Preferably, the mass ratio of the microalgae to the chemotherapeutic drug is 1:0.25-4; more preferably, the mass ratio of the microalgae to the chemotherapeutic drug is 1:0.5-2; most preferably, the mass ratio of the microalgae to the chemotherapeutic drug is 1:1.

[0016] In some embodiments of the present invention, the diameter of the chemotherapy drug-microalgae embolizing agent is 20-2000 micrometers. When the diameter of the drug-microalgae embolizing agent is less than 20 micrometers, the drug-microalgae embolizing agent can pass directly through blood vessels without causing embolism. When the diameter of the drug-microalgae embolizing agent is greater than 2000 micrometers, it cannot enter the tumor neovascularization through arterial branches to cause embolism. Preferably, the diameter of the chemotherapy drug-microalgae embolizing agent is 100-500 micrometers.

[0017] In some embodiments of the present invention, the microalgae are selected from any one or more of the following phyla: Chlorophyta, Euglenophyta, Charophyta, Chrysophyta, Xanthophyta, Diatomophyta, Dinophyta, Cyanophyta, Brownophyta, and Rhodophyta; preferably, the microalgae are selected from the following phyla: Cyanophyta, Dinophyta, and Chlorophyta; more preferably, the microalgae are selected from the following phyla: Cyanophyta.

[0018] In some embodiments of the present invention, the microalgae are selected from any one or more of Nostoc, Spirulina, Noctiluca scintillans, and Volvox.

[0019] In some embodiments of the present invention, the chemotherapeutic drug is selected from any one or more of platinum-based drugs, anthracyclines, fluorouracil drugs, and topoisomerase inhibitors; preferably, the chemotherapeutic drug is selected from any one or more of platinum-based drugs, anthracyclines, and topoisomerase inhibitors; more preferably, the chemotherapeutic drug is selected from anthracyclines.

[0020] In some embodiments of the present invention, the chemotherapeutic agent is selected from doxorubicin, daunorubicin, doxorubicin, irinotecan, cisplatin, or pharmaceutically acceptable salts thereof.

[0021] In some embodiments of the present invention, the chemotherapeutic drug is selected from chemical entities with antitumor activity or drug molecules with charge-modifying groups; preferably, the charge-modifying group is selected from positively charged groups; more preferably, the positively charged group is selected from any one or more of primary amine, secondary amine, tertiary amine or quaternary ammonium groups.

[0022] In some embodiments of the present invention, an embolic agent prepared from doxorubicin and Spirulina platensis, wherein the mass ratio of doxorubicin to Spirulina platensis is 1:1, is obtained as an doxorubicin-Spirulina platensis embolic agent with an encapsulation rate of 80%, and the tumor embolization therapeutic effect is successfully achieved after injection.

[0023] The second objective of this invention is to provide a one-step method for preparing a chemotherapy drug—a microalgae embolizing agent, the specific implementation steps of which are as follows: Chemotherapy drugs were mixed with microalgae to prepare a solution, which was then shaken overnight at room temperature in the dark. The mixture was centrifuged, the supernatant was removed, and the chemotherapy drug-microalgae embolizing agent was collected. In the preparation method of the chemotherapy drug-microalgae embolization agent, the microalgae and the chemotherapy drug, by weight, preferably have a mass ratio of microalgae to chemotherapy drug in the range of 1:0.1-10; more preferably, the mass ratio of microalgae to chemotherapy drug is 1:0.25-4; even more preferably, the mass ratio of microalgae to chemotherapy drug is 1:0.5-2; and most preferably, the mass ratio of microalgae to chemotherapy drug is 1:1.

[0024] In the preparation method of the chemotherapy drug-microalgae embolizing agent, the diameter of the chemotherapy drug-microalgae embolizing agent is 20-2000 micrometers. When the diameter of the chemotherapy drug-microalgae embolizing agent is less than 20 micrometers, it can pass directly through blood vessels without causing embolism. When the diameter of the chemotherapy drug-microalgae embolizing agent is greater than 2000 micrometers, it cannot enter the tumor neovascularization through arterial branches to cause embolism.

[0025] In the preparation method of chemotherapy drug-microalgal embolization agent, the microalgae are selected from any one or more of the following phyla: Chlorophyta, Euglena, Charophyta, Chrysophyta, Xanthophyta, Diatoms, Dinophyta, Cyanobacteria, Brown Algae, and Rhodophyta; preferably, the microalgae are selected from any one or more of Nostoc, Spirulina, Noctiluca scintillans, Caesalpinia, and Volvox.

[0026] In some embodiments of the present invention, the microalgae in the chemotherapeutic drug-microalgae embolization agent accelerate the release of chemotherapeutic drugs in an in vitro simulated weakly acidic microenvironment; the release of chemotherapeutic drugs from this microalgae carrier is acid-responsive.

[0027] In some embodiments of the present invention, the microalgae in the chemotherapeutic drug-microalgae embolization agent have the effect of improving the pH of the tumor microenvironment. Tumor cells usually obtain energy through glycolysis, a process that produces a large amount of acidic metabolites such as lactic acid, resulting in a significantly lower extracellular pH of the tumor microenvironment compared to normal tissues. Unlike conventional TME carriers, SP has the function of raising the pH of the tumor microenvironment, disrupting the acidic metabolic advantage of tumor cells, and inhibiting their proliferation and invasion. The improvement of the tumor microenvironment (gradually returning to normal physiological pH, avoiding further acidification) can also moderately restore the function of immune cells, alleviate immunosuppression, and at the same time avoid the problem of other degradable embolization agents (such as PLGA) degrading and producing lactic acid, further aggravating the acidity of the TME, thus improving treatment safety.

[0028] In some embodiments of the present invention, the pH-regulating ability of the microalgae in the chemotherapy drug-microalgae embolizing agent originates from its inherent components and active substances released during degradation, with the core being the neutralization of H in the environment. + (Hydrogen ions) thereby increase the pH value of the acidic system and enhance the anti-tumor effect through synergistic effect.

[0029] In some embodiments of the present invention, the microalgae in the chemotherapy drug-microalgae embolizing agent contain chlorophyll and exhibit fluorescence in a fluorescence channel (excitation wavelength: 560 nm, emission wavelength: 600-680 nm), enabling in vivo tracking through fluorescence imaging.

[0030] In some embodiments of the present invention, the microalgae in the chemotherapeutic drug-microalgae embolization agent are biodegradable and biosafety.

[0031] In some embodiments of the present invention, the microalgae in the chemotherapeutic drug-microalgae embolization agent can be used to treat orthotopic liver cancer in rats.

[0032] This invention provides a chemotherapy drug-microalgae embolization agent, which is used to prepare a drug for arterial embolization to treat tumors. The drug is composed of a pharmacologically effective amount of chemotherapy drug and microalgae. The chemotherapy drug is positively charged under physiological conditions and binds to the microalgae through electrostatic adsorption. The mass ratio of the microalgae to the chemotherapy drug is 1:0.1-10.

[0033] In this invention, the term "SP" refers to Spirulina platensis.

[0034] In this invention, the term "DOX" refers to doxorubicin.

[0035] In this invention, the term "DOX-SP" refers to *Spirulina platensis* loaded with doxorubicin.

[0036] In this invention, the term "chemotherapeutic drug-microalgae embolization agent" refers to a chemotherapeutic drug embolization agent based on biodegradable microalgae.

[0037] The beneficial effects of this invention are: (1) The chemotherapy drug-microalgae embolizing agent provided by the present invention is prepared by a "one-step method" and achieves an encapsulation rate of 88.46%. The raw materials are simple and easy to obtain, the preparation method is simple, the encapsulation rate is high, and large-scale production can be realized.

[0038] (2) This invention utilizes a chemotherapy drug-microalgae embolization agent for tumor embolization therapy. Direct administration of the chemotherapy drug-microalgae as an embolization agent after injection achieves the same embolization effect, and also enables better release of the chemotherapy drug-microalgae embolization agent within the tumor blood vessels. It exhibits excellent anti-tumor effects in treating liver cancer via arterial embolization and has great potential for clinical translation. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the mechanism of the doxorubicin-loaded Spirulina platensis embolization agent (DOX-SP) used for arterial embolization therapy of malignant tumors as described in the examples; Figure 2 This is a schematic diagram of the preparation process of doxorubicin-loaded Spirulina platensis (DOX-SP) described in the examples; Figure 3 Images of Spirulina platensis (SP) and Spirulina platensis loaded with doxorubicin (DOX-SP) described in the examples include bright-field microscopy, fluorescence microscopy, and scanning electron microscopy images. Figure 4 Fluorescence spectra, ultraviolet spectra, and potential statistics of doxorubicin, Spirulina platensis, and Spirulina platensis loaded with doxorubicin; Figure 5 Statistical graphs showing the encapsulation efficiency and drug loading rate of different concentrations of doxorubicin on Spirulina platensis; Figure 6 The cumulative release curves of doxorubicin from Spirulina platensis loaded with doxorubicin at different time points in solutions with pH 6.5, 7, and 7.5; Figure 7 Statistical graph of cell viability after 24 hours of incubation with different concentrations of Spirulina platensis, doxorubicin, and Spirulina platensis preparation loaded with doxorubicin, N1S1 cells, and McA-RH7777 cells.

[0040] Figure 8 Cell flow cytometry and apoptosis rate statistics of control group, different concentrations of Spirulina platensis, doxorubicin, Spirulina platensis preparation loaded with doxorubicin, and N1S1 cells after incubation for 24 hours.

[0041] Figure 9 Cell viability and mortality fluorescence images of the control group, Spirulina platensis, doxorubicin, Spirulina platensis preparation loaded with doxorubicin, and McA-RH7777 cells after incubation for 24 hours.

[0042] Figure 10 Microscopic and scanning electron microscope images of Spirulina platensis at different time points after in vitro degradation.

[0043] Figure 11 Images of the liver at different time points following hepatic artery embolization with Spirulina obtusifolia. Image A: Fluorescence image and hematoxylin-eosin staining image; Image B: Transmission electron microscopy image. Scale bar = 5 µm.

[0044] Figure 12 Digital subtraction angiography images before and after administration of DOX-SP embolizing agent via the hepatic artery; Figure 13 The images show the fluorescence signals of chemotherapy drugs in major organs at different time points after administration of doxorubicin emulsion (DOX-iodized oil) and DOX-SP embolization agent via the hepatic artery, as well as the statistical graph of DOX fluorescence intensity. Figure 14 The image shows the DOX fluorescence signal and fluorescence intensity statistics of liver tumors after administration of iodized oil doxorubicin emulsion (DOX-iodized oil) and DOX-SP embolization agent via the hepatic artery. Figure 15 A statistical graph showing plasma DOX concentrations at different time points after administration of iodized oil doxorubicin emulsion (DOX-iodized oil) and DOX-SP embolization agent via the hepatic artery; Figure 16 The pH changes at different time points after adding Spirulina platensis to solutions with initial pH of 5, 6, and 7.

[0045] Figure 17 pH fluorescence probe detection and fluorescence intensity statistics of liver tumors before surgery and after DOX-SP embolization via the hepatic artery; Figure 18 This is a schematic diagram of an animal model experiment for liver cancer. Figure 19 The following is a statistical chart showing the weight changes during the treatment period for the control group, the hepatic artery iodized oil embolization group, the hepatic artery doxorubicin emulsion (DOX-iodized oil) embolization group, the hepatic artery spirulina embolization group, and the hepatic artery DOX-SP embolization group. Figure 20 Liver MRI images on days 7 and 14 post-treatment, including the control group, the hepatic artery iodized oil embolization group, the hepatic artery iodized oil doxorubicin emulsion (DOX-iodized oil) embolization group, the hepatic artery spirulina embolization group, and the hepatic artery DOX-SP embolization group. Figure 21The images and tumor size statistics of liver tumors on the 14th day after treatment are shown in the control group, the hepatic artery iodized oil embolization group, the hepatic artery doxorubicin emulsion (DOX-iodized oil) embolization group, the hepatic artery spirulina embolization group, and the hepatic artery DOX-SP embolization group. Figure 22 Immunohistochemical images (Ki-67, TUNEL, CD31) of tumors in the control group, the hepatic artery iodized oil embolization group, the hepatic artery doxorubicin emulsion (DOX-iodized oil) embolization group, the hepatic artery spirulina embolization group, and the hepatic artery DOX-SP embolization group. Figure 23 Immunohistochemical graphs of tumors in the control group, the hepatic artery iodized oil embolization group, the hepatic artery iodized oil doxorubicin emulsion (DOX-iodized oil) embolization group, the hepatic artery spirulina embolization group, and the hepatic artery DOX-SP embolization group are statistically analyzed. Figure 24 Hematoxylin and eosin staining of major organs in rats after different time periods following Spirulina platensis embolization; Figure 25 The following figures show the results of routine blood tests and blood biochemical indicators of rats 14 days after treatment: control group, hepatic artery iodized oil embolization group, hepatic artery doxorubicin emulsion (DOX-iodized oil) embolization group, hepatic artery spirulina embolization group, and hepatic artery DOX-SP embolization group.

[0046] Figure 26 Statistical chart showing the encapsulation efficiency and drug loading rate of different concentrations of daunorubicin on Volvox. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0048] The following is a detailed description of the implementation method using doxorubicin (DOX)-Spirulina platensis (SP) embolization agent as an example: This invention utilizes Spirulina platensis (SP) as a drug carrier to effectively load doxorubicin (DOX), hereinafter referred to as DOX-SP embolization agent. The SP prepared by this invention has the ability to embolize the hepatic artery, can control the slow release of DOX, and exhibits pH responsiveness during DOX release, thus improving the acidic tumor microenvironment and synergistically enhancing the antitumor effect. Furthermore, after hepatic artery embolization, SP can be safely degraded in vivo without significant adverse reactions to blood and major organs, demonstrating good biocompatibility. Figure 1 and Figure 2 The optimal ratio of SP to DOX in the DOX-SP embolizing agent, by mass, is 1:0.125-4; the particle size range is 100-500 micrometers. This invention provides a safe, precise, and effective treatment method for liver cancer.

[0049] Example 1. Preparation of DOX-SP embolic agent 100 μg (dry weight) of SP was mixed with 1 mL of DOX solution (100 μg / mL), and the mixture was shaken overnight at room temperature in the dark. The mixture was then centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded to obtain the DOX-SP embolic agent. Bright-field and fluorescence field images were taken using an optical microscope, and the morphology was observed using a scanning electron microscope. The results showed that the DOX-SP embolic agent (…) was obtained. Figure 3 ).

[0050] Example 2. Validation of drug loading performance UV-Vis absorption spectroscopy and fluorescence spectroscopy were used to detect SP, DOX, and DOX-SP embolic agents, showing that the fluorescence characteristics of DOX-SP embolic agents were consistent with those of SP. The UV spectrum of DOX showed a characteristic peak at 480 nm, while the UV spectrum of SP showed characteristic peaks at 425 nm, 620 nm, and 670 nm. The UV spectrum of DOX-SP embolic agents also showed characteristic peaks at 480 nm, 620 nm, and 670 nm. The zeta potential of SP changed from -27.07 mV to -9.11 mV after DOX loading, indicating that the negatively charged SP interacted electrostatically with the positively charged DOX. Figure 4To investigate the drug loading capacity of SP for DOX, 1 mL of different concentrations of DOX (125, 250, 500, 1000, 2000, and 4000 μg / mL) were added to 9 mL of SP solution containing 1000 μg. The mixture was stirred at 200 rpm for 12 hours in the dark at room temperature. Finally, the mixture was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in ultrapure water to obtain the DOX-SP embolic agent. The drug loading efficiency and encapsulation efficiency at different DOX / SP material ratios were calculated using the DOX standard curve obtained from UV-Vis absorption spectroscopy and the absorbance of different supernatants. The electron microscopy results of the DOX-SP embolic agent are shown below. Figure 3 As shown in the figure. When the initial dose of DOX was 100 μg / mL, the encapsulation efficiency was as high as 88.46%. The results of encapsulation efficiency and drug loading are as follows. Figure 5 As shown in the figure. This experiment investigated the drug loading performance of a mixture system of DOX and SP at different concentrations. The results showed that within the range of SP:DOX mass ratio of 1:0.5 to 1:2, higher drug loading and encapsulation efficiency could be obtained simultaneously.

[0051] Example 3. In vitro drug release performance testing DOX-SP embolic agent was suspended in 10 mL of PBS at pH 6.5, 7, and 7.5. At each given time point, 1 mL of supernatant was collected and replenished with an equal volume of fresh PBS. The absorbance of the supernatant at 480 nm was measured using UV-Vis spectrophotometry combined with a DOX standard curve to calculate the cumulative DOX release at different time points. The in vitro release results of DOX-SP embolic agent are as follows: Figure 6 As shown, the DOX-SP embolic agent exhibits better DOX release efficiency in a weakly acidic environment with a pH of 6.5.

[0052] Example 4. Detection of the effect of DOX-SP embolization agent on liver cancer cells. To evaluate the in vitro cytotoxicity of DOX-SP embolization agents, different concentrations of SP, DOX, and DOX-SP embolization agents were incubated with McA-RH7777 and N1S1 cells for 24 hours, and cell viability was assessed using the CCK8 assay. Figure 7Cells co-cultured with SP showed higher survival rates, while those co-cultured with DOX and DOX-SP embolization agents showed lower survival rates, with survival gradually decreasing with increasing DOX concentration. At a SP concentration of 100 μg / mL, the survival rate of McA-RH7777 cells was 64.12±10.03%, and that of N1S1 cells was 72.24±5.06%, indicating good biocompatibility of SP. At a DOX concentration of 80 μg / mL, the survival rate of DOX-treated McA-RH7777 cells was only 3.15±0.74%, and that of DOX-SP embolization agents was only 16.03±1.43%. Similarly, the survival rates of N1S1 cells treated with DOX and DOX-SP embolization agents were 6.95±0.31% and 7.97±0.50%, respectively, with no statistically significant difference between the two groups. These results demonstrate that DOX-SP embolization agents have significant antitumor effects. To further confirm the antitumor effect of DOX-SP embolization, flow cytometry analysis showed that SP at a concentration of 100 μg / mL induced only 34.01 ± 0.63% apoptosis in N1S1 cells. Conversely, at a DOX concentration of 80 μg / mL, DOX-SP embolization induced an apoptosis rate of 96.69 ± 0.79% in N1S1 cells, comparable to that of DOX, indicating that DOX-SP embolization has a similar antitumor effect to DOX. Figure 8 The cytotoxicity of different drugs on McA-RH7777 cells was detected using the Calcein-AM / PI double staining method. Figure 9 Both the DOX group and the DOX-SP embolization group induced a large number of dead cells (red fluorescence), and no obvious live cells were observed (green fluorescence). Flow cytometry and live / dead cell staining results were consistent with the CCK8 results, confirming the in vitro antitumor activity of DOX-SP embolization against hepatocellular carcinoma.

[0053] Example 5. In vivo distribution and degradation detection of DOX-SP embolic agent To assess the degradability of DOX-SP embolization agent, the DOX-SP embolization agent was suspended in PBS containing 10% fetal bovine serum and incubated for 7, 14, and 28 days. Figure 10 Microscopic and scanning electron microscopic morphological observations showed that the DOX-SP embolic agent initially exhibited a complete helical shape. By day 7, the helical structure of the DOX-SP embolic agent began to break down, shortening in length to approximately 50 μm. By day 28, the helical structure further fragmented, decreasing in size to between 1 and 10 μm in length. These observations demonstrate that the DOX-SP embolic agent gradually breaks down and shrinks over time, proving its biodegradability. Fluorescence images following injection of the DOX-SP embolic agent into the hepatic artery showed its distribution within the liver. Figure 11No fluorescent signal of DOX-SP embolization agent was observed in extrahepatic organs, indicating no ectopic embolization. Over time, the DOX-SP embolization agent in the liver degraded, leading to a decrease in fluorescence signal. Liver pathological sections one hour post-procedure showed complete SP filling of the arteries. Yellow degradation products of DOX-SP embolization agent were detected in the liver on days 7 and 14, with no significant residue on day 28. Transmission imaging on day 14 showed that the liver had phagocytosed and cleared DOX-SP embolization agent fragments. No significant DOX-SP embolization agent residue was observed in hepatocytes on day 60, but significant thrombus formation was observed in the hepatic sinusoids, suggesting that DOX-SP embolization agent can promote thrombus formation, thereby preventing vascular recanalization. These results indicate that the embolization agent of this invention has excellent biodegradability. The embolization agent gradually disintegrates in vivo over time, breaking down from an intact helical structure (>50 μm) and completely degrading and disappearing within 28 days. Figure 10 , Figure 11 This avoids the risk of foreign body reaction caused by permanent residues of traditional embolizing agents, and no ectopic embolization was observed, confirming its biocompatibility.

[0054] Example 6. Embolizing ability of DOX-SP embolic agent on arteries The DOX-SP embolic agent used in this study had a diameter of 100–500 μm. To further verify the embolic effect of the DOX-SP embolic agent, digital subtraction angiography was performed on rats before surgery and after transhepatic artery embolization with Spirulina platensis. Figure 12 As shown, after injection of DOX-SP embolic agent into the hepatic artery, the contrast agent signal in the hepatic artery branches was significantly lower than that before embolization. This indicates that the contrast agent could not enter the hepatic artery branches after injection of DOX-SP embolic agent, and that DOX-SP embolic agent can achieve embolization of the hepatic artery. This demonstrates the great potential of DOX-SP embolic agent as a vascular embolic substance.

[0055] Example 7. Detection of the effect of DOX-SP embolic agent on DOX release The distribution of DOX in the rat liver and major organs was assessed using ex vivo fluorescence imaging. Fluorescence imaging of the rat liver and major organs was performed in the DOX channels (excitation wavelength: 480 nm, emission wavelength: 500-580 nm). Results are referenced... Figure 13In the DOX-SP embolization group, DOX signals were still visible near the tumor at 24 and 48 hours post-procedure, while the signal in the DOX-iodized oil group was very weak and almost undetectable during these time intervals. Compared to the DOX-iodized oil group, the DOX-SP embolization group showed significantly more DOX fluorescence at 24 hours post-embolization. This indicates that the DOX-SP embolization agent achieves a slower DOX release at the liver tumor site compared to iodized oil DOX emulsion (DOX-iodized oil). Tumor sections showed the presence of DOX signals (red) within tumor cells, and the fluorescence intensity in the DOX-SP embolization group was higher than that in the DOX-iodized oil group at 24 and 48 hours post-procedure. Figure 14 The difference in quantitative fluorescence between the two groups was statistically significant at 24 hours post-procedure. Plasma DOX concentration was assessed at different time intervals post-procedure. One hour after embolization, the DOX concentration in the DOX-iodized oil group was 247.35 ± 123.69 ng / mL, while that in the DOX-SP embolization group was 75.78 ± 26.51 ng / mL. Figure 15 Plasma DOX concentrations in both groups decreased over time, and were lower than those in the DOX-iodized oil group at all time points. Therefore, DOX-SP embolization can achieve sustained release of DOX from liver tumors and enhance the anti-tumor effect. These results indicate that, compared with traditional iodized oil emulsions, DOX-SP embolization achieves local sustained drug release and low systemic toxicity. At 24-48 hours post-embolization, the drug fluorescence intensity at the tumor site was significantly higher (…). Figure 13 , Figure 14 ), while the drug concentration in the plasma remained at a low level ( Figure 15 This effectively increases the local therapeutic concentration and reduces systemic side effects.

[0056] Example 8. Detection of the effect of SP on pH in vitro and in vivo SP samples were suspended in 5 mL of PBS at pH 5, 6, and 7 to simulate the tumor microenvironment, and the pH of the solution was measured at given time points. Results are referenced... Figure 16 SP can improve the acidic environment, and the pH of the solution tends to be neutral. To investigate the effect of SP embolization on tumors, we used a pH fluorescent probe to compare the pH value within the tumor. Figure 16 , Figure 17 Compared to preoperative levels, an increase in tumor pH was observed 24 hours after SP injection. This improved acidic environment not only facilitates the utilization of chemotherapy drugs and maintains their stability, but also further inhibits tumor cell proliferation and metastasis.

[0057] Example 9. Antitumor activity of DOX-SP embolization agent Rat hepatocellular carcinoma cells were inoculated under the liver capsule of SD rats to construct an orthotopic hepatocellular carcinoma animal model. Figure 18One week after modeling, the rats were divided into 5 groups: a control group, a group treated with hepatic artery iodized oil embolization, a group treated with hepatic artery iodized oil DOX emulsion embolization, a group treated with hepatic artery SP embolization, and a group treated with hepatic artery DOX-SP embolization. There were no significant differences in body weight among the groups during the treatment period. Figure 19 Tumor growth in each group was monitored by MRI at 1 and 2 weeks post-embolization. Figure 20 Fourteen days after treatment, blood was drawn from the tail vein of the rats for blood tests. After euthanasia, the liver and other major organs were removed for observation and analysis, and macroscopic photographs of the rat liver were recorded. Figure 21 Tumors were removed and measured. The differences in liver tumor volume growth rate among the groups were statistically significant (P<0.05). DOX-SP embolization effectively inhibited the progression of liver cancer. The effects of TUNEL, Ki-67, and CD-31 immunohistochemical staining on tissue apoptosis, anti-tumor proliferation, and angiogenesis were observed. Figure 22 and Figure 23 The control group had the highest cell proliferation index (87.33±2.05%), while the DOX-SP embolization group had the lowest (8±5.10%). Conversely, TUNEL expression was significantly higher in the DOX-SP embolization group than in other groups (P<0.05). Furthermore, compared to other treatment groups, CD-31 expression was significantly reduced in tumors treated with DOX-SP embolization. DOX-SP embolization exhibits anti-tumor effects, including promoting tumor necrosis, inhibiting angiogenesis, and suppressing cell proliferation.

[0058] Example 10. Biodegradability and biosafety of DOX-SP embolic agent DOX-SP embolization agent was injected into the liver of SD rats via the hepatic artery. Liver sections were stained with hematoxylin and eosin on days 7, 14, and 28 post-surgery. Results were as follows: Figure 24 No Spirulina platensis residue was found in the liver on day 28, indicating its biodegradability. Blood samples were collected from rats for complete blood count and blood biochemistry tests. Results are as follows. Figure 24 , 25 After administration, the main blood routine and blood biochemical indicators of rats were all within the normal range, indicating that DOX-SP embolization agent has good biosafety in rats.

[0059] Implementation Case 11 To investigate the drug loading capacity of *Volvoxella* for daunorubicin, different concentrations of daunorubicin were added to solutions containing 1000 μg of *Volvoxella*, and the mixture was stirred at 200 rpm in the dark for 12 hours at room temperature. Finally, the mixture was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in ultrapure water to obtain *Volvoxella* loaded with daunorubicin. The drug loading efficiency and encapsulation efficiency were calculated using a daunorubicin standard curve obtained from UV-Vis absorption spectroscopy and the absorbance of different supernatants. Results were referenced... Figure 26 When the initial dose of daunorubicin was 100 μg / mL, the encapsulation efficiency was as high as 87.23%.

[0060] Similar to Cases 1-10, daunorubicin-Volvoxel embolization achieved the same tumor embolization therapeutic effect as doxorubicin-Spirulina platensis embolization. The performance of daunorubicin-Volvoxel embolization in tumor embolization therapy was comparable to that of doxorubicin-Spirulina platensis embolization, demonstrating good therapeutic efficacy. This further validates the potential of chemotherapy drug-microalgae embolization agents in tumor treatment, laying the foundation for subsequent research and clinical application.

[0061] Those skilled in the art will understand that the steps, measures, and schemes in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted; furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted; furthermore, the steps, measures, and schemes in the prior art that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above-described embodiments are merely illustrative of several implementation methods of this disclosure, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent for the embodiments of this disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this disclosure, and these all fall within the protection scope of the embodiments of this disclosure. Therefore, the protection scope of the embodiments of this disclosure should be determined by the appended claims. As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and detail can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

[0062] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. Use of a chemotherapeutic drug-microalgae embolizing agent in the preparation of a medicament for treating tumors by arterial embolization, characterized in that, The chemotherapy drug-microalgae embolus is composed of a pharmacologically effective amount of chemotherapy drug and microalgae, the chemotherapy drug is positively charged in a physiological environment, and is combined with the microalgae through electrostatic adsorption.

2. Use according to claim 1, characterized in that, The tumor is a liver malignant tumor, a bile duct malignant tumor or a lung malignant tumor.

3. Use according to claim 1, characterized in that, The mass ratio of the microalgae to the chemotherapy drug is 1:0.1-10 by dry weight.

4. Use according to claim 1, characterized in that, The diameter of the microalgae is 20-2000 microns.

5. Use according to claim 1, characterized in that, The microalgae are selected from any one or more of Chlorophyta, Euglenophyta, Charophyta, Chrysophyta, Xanthophyta, Bacillariophyta, Dinophyta, Cyanophyta, Phaeophyta and Rhodophyta.

6. Use according to claim 1, characterized in that, The chemotherapy drug is selected from any one or more of platinum drugs, anthracycline drugs, fluorouracil drugs and topoisomerase inhibitor drugs.

7. Use according to claim 1, characterized in that, The chemotherapy drug is selected from adriamycin, daunorubicin, doxorubicin, irinotecan and cisplatin, or a pharmaceutically acceptable salt thereof.

8. Use according to claim 1, characterized in that, The chemotherapy drug is selected from a chemical entity with anti-tumor activity or a drug molecule with a charge-modifying group.

9. Use according to claim 8, characterized in that, The charge-modifying group is selected from a positively charged group.

10. A chemotherapeutic drug-microalgal embolization agent, characterized in that, The chemotherapy drug-microalgae embolus is used for preparing an arterial embolization treatment drug for tumors, the chemotherapy drug-microalgae embolus is composed of a pharmacologically effective amount of chemotherapy drug and microalgae, the chemotherapy drug is positively charged in a physiological environment, and is combined with the microalgae through electrostatic adsorption, and the mass ratio of the microalgae to the chemotherapy drug is 1:0.1-10.

Images