A drug-loaded embolized microalgae ball for treating liver cancer, its preparation method and application

Abstract

The application discloses a drug-loaded embolization microalgae ball for resisting liver cancer and a preparation method and application thereof, and belongs to the technical field of biomedical materials, and the preparation method comprises the following steps: (1) mixing microalgae cells and drugs to prepare a drug-loaded microalgae cell solution; the microalgae cells are chlorella vulgaris cells or synechococcus cells; (2) using the drug-loaded microalgae cell solution containing polyvinyl alcohol as an inner phase solution, using a mixed solution containing polydimethylsiloxane, dimethyl silicone oil and a curing agent as an intermediate phase solution, using a polyvinyl alcohol solution as an outer phase solution, and using microfluidic technology to adjust the flow rates of the inner phase solution, the intermediate phase solution and the outer phase solution, so that the drug-loaded embolization microalgae ball for resisting liver cancer is prepared. The method is simple in process, the prepared drug-loaded embolization microalgae ball is uniform in particle size, good in sphericity, has excellent biocompatibility and biodegradability, and has a good treatment effect on liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a drug-loaded embolized microalgae ball for treating liver cancer, its preparation method, and its application. Background Technology

[0002] Hepatic artery chemoembolization (HACE) is a commonly used non-surgical interventional treatment for tumors. It typically involves injecting embolic materials into the blood supply arteries to the tumor tissue, blocking blood flow, inhibiting tumor growth, and releasing high concentrations of radiotherapy and chemotherapy drugs locally to kill tumor cells. This method has advantages such as being minimally invasive, safe, and targeted, with high treatment efficiency and low toxicity. However, existing embolic materials have certain limitations, such as drug-loaded embolic microspheres. Beads, due to their non-uniform particle size and tendency to break down within blood vessels, may lead to poor treatment outcomes or other complications. Therefore, it is necessary to optimize the shape of the embolic material and improve its particle size uniformity.

[0003] A variety of embolization materials are used in hepatic artery chemoembolization, including traditional iodized oil, gelatin, sodium alginate, and microspheres. Among them, microspheres are suitable for selective embolization of vessels with specific diameters and the full cross-section of easily occluded vessels, with more reliable embolization effects. Furthermore, microspheres can be used as a drug delivery system to achieve sustained release of drugs at the tumor site, resulting in better therapeutic effects.

[0004] Chinese patent document CN102585258A discloses a gelatin embolization microsphere, its preparation method, and its application. In this invention, a gelatin aqueous solution is first prepared, then added to an oil phase containing an emulsifier. After stirring, the solution is cooled, stirred again, and allowed to stand, allowing the formed microspheres to settle. The oil phase is then removed by pouring, and the surface of the microspheres is repeatedly washed with acetone to remove any remaining oil phase. The microspheres are then filtered out. Finally, the obtained microspheres are added to a crosslinking agent aqueous solution and stirred. After reaction and washing, gelatin embolization microspheres are obtained. These gelatin embolization microspheres are regular spherical in shape, and their degradation time and elasticity are more suitable for clinical applications. However, animal-derived gelatin may cause allergic reactions, and the multi-step preparation process increases costs, time, and the risk of errors.

[0005] Chinese patent document CN110354299A discloses a traditional Chinese medicine embolization microsphere for interventional treatment of mid-to-late stage liver cancer and its preparation method. The microsphere is composed of the following components by mass percentage: Bletilla striata polysaccharide 75.48%–81.99%, sodium cantharidate 0.5%–1.5%, lecithin 2.5%–3.0%, cholesterol 0.01%–0.02%, and 1,2-propanediol 15%–20%, with each component comprising 100% of the total mass. The microsphere is prepared by encapsulating sodium cantharidate nano-flexible liposomes with Bletilla striata polysaccharide as the backbone material, followed by emulsification, cross-linking, and sterilization. However, the extraction and standardization of traditional Chinese medicine components are challenging, potentially leading to batch-to-batch inconsistencies and affecting the stability of the active ingredients during storage and in vivo, which could impact drug activity. Summary of the Invention

[0006] This invention provides a method for preparing drug-loaded embolized microalgae balls for treating liver cancer. The drug-loaded embolized microalgae balls prepared by this method have uniform particle size distribution and good biocompatibility, which can solve the problems of uneven distribution and ectopic embolization of existing embolization materials in liver cancer treatment, and improve the treatment effect.

[0007] The specific technical solution adopted is as follows:

[0008] A method for preparing drug-loaded embolized microalgae balls for treating liver cancer includes the following steps:

[0009] (1) A drug-loaded microalgae cell solution is prepared by mixing microalgae cells and drugs; wherein the microalgae cells are Chlorella proteoglycans cells or Synechocystis cells; wherein the microalgae cells can be ordinary microalgae cells or freeze-dried microalgae cells;

[0010] (2) Using a drug-loaded microalgae cell solution containing polyvinyl alcohol as the inner phase solution, a mixed solution containing polydimethylsiloxane, dimethyl silicone oil and curing agent as the intermediate phase solution, and a polyvinyl alcohol solution as the outer phase solution, the flow rates of the inner phase solution, intermediate phase solution and outer phase solution are adjusted by microfluidic technology to prepare the drug-loaded embolized microalgae ball for anti-liver cancer.

[0011] When microalgal cells are mixed with drugs, the microalgal cells adsorb the drugs through surface electrostatic interactions, achieving a loading effect. Microalgal cells provide a biocompatible and biodegradable platform for drug loading and sustained release. Furthermore, the high surface area to volume ratio of microalgal cells helps improve drug loading capacity and controlled release capabilities, prolonging the duration of drug action. Microalgal cells, such as *Chlorella protozoa* or *Synthia spp.*, grow rapidly and are easy to cultivate on a large scale. Moreover, microalgal cells can be designed with specific morphologies and sizes to meet different drug delivery needs. Additionally, their thinner cell walls facilitate drug loading and release.

[0012] The drugs mentioned include camrelizumab SHR-1210 or the peptide antagonist AUNP-12.

[0013] Preferably, when the microalgae cells are freeze-dried microalgae cells, the mass ratio of microalgae cells to drugs is 0.1-2:1.

[0014] Optionally, in step (2), polyvinyl alcohol is added to the drug-loaded microalgae cell solution from step (1) to obtain an inner phase solution, and the concentration of polyvinyl alcohol in the inner phase solution is controlled to be 0.2-10 wt%.

[0015] In the intermediate phase solution, the mass ratio of polydimethylsiloxane, dimethyl silicone oil and curing agent is 1:0.5-5:0.05-5; the mass concentration of polydimethylsiloxane is 3-30 wt%; the curing agent can be a silicone elastomer curing agent or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, etc.

[0016] The concentration of polyvinyl alcohol in the external phase solution is 0.2-10 wt%.

[0017] Specifically, in the microfluidic process, the inner phase solution and the intermediate phase solution are premixed using a T-channel, and then the premixed solution is mixed with the outer phase solution using the same T-channel. The flow rate of the inner phase solution is 1-100 μL / h, the flow rate of the intermediate phase solution is 2-200 μL / h, and the flow rate of the outer phase solution is 300-2000 μL / h. The resulting dual-emulsion droplets are collected in a 0.2-10 wt% polyvinyl alcohol aqueous solution to obtain the drug-loaded embolized microalgae spheres for anti-hepatocellular carcinoma. Using a polyvinyl alcohol aqueous solution to collect the dual-emulsion droplets ensures that unnecessary fusion between the collected droplets does not occur. Furthermore, under the specific process and parameters of this invention, the resulting microspheres exhibit better stability, are less prone to breakage in blood vessels, and can form microspheres of more uniform size.

[0018] The present invention also provides a method for preparing drug-loaded embolized microalgae spheres for anti-hepatocellular carcinoma, wherein the drug-loaded embolized microalgae spheres for anti-hepatocellular carcinoma are prepared. The particle size of these drug-loaded embolized microalgae spheres for anti-hepatocellular carcinoma is 50-200 μm, preferably 80-120 μm.

[0019] The present invention also provides the application of the drug-loaded embolized microalgae balls for anti-liver cancer in the preparation of liver cancer treatment products.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The method of the present invention uses microfluidic technology and a specific three-phase system to precisely control the particle size of drug-loaded embolized microalgae balls. The resulting drug-loaded embolized microalgae balls have uniform particle size and good sphericity, avoiding the problem of vascular blockage caused by uneven particle size. Furthermore, the drug-loaded embolized microalgae balls serve as drug carriers, enabling controlled release of drugs, increasing local drug concentration, and enhancing the therapeutic effect.

[0022] (2) The drug-loaded embolized microalgae balls provided by the present invention have good biocompatibility, reduce damage to normal tissues, and can be biodegraded after drug release, reducing the risk of long-term presence in the body and having a good therapeutic effect on liver cancer. Attached Figure Description

[0023] Figure 1 This is a morphological image of the drug-loaded embolized microalgae balls prepared in Example 1 for treating liver cancer.

[0024] Figure 2 The drug release kinetic curve is shown for the drug-loaded embolized microalgae balls prepared in Example 1 for treating liver cancer.

[0025] Figure 3 The image shows the hemolysis results of the drug-loaded embolized microalgae balls prepared in Example 1 for treating liver cancer.

[0026] Figure 4 The graph shows the blood biochemical analysis results of different animal model groups (Model group, lodine oil group, IRE group, VA group; IRE+VA group). BUN represents blood urea nitrogen; CRE represents creatinine; ALT represents alanine aminotransferase; AST represents aspartate aminotransferase; and D-Bil represents direct bilirubin.

[0027] Figure 5 HE staining results of histopathology for different animal model groups (Model group, lodine oil group, IRE group, VA group; IRE+VA group).

[0028] Figure 6 Statistical graphs of tumor size in different animal model groups (Model group, IRE group, VA group; IRE+VA group).

[0029] Figure 7 HE staining results and Ki67 immunohistochemistry results for different animal model groups (Model group, IRE group, VA group; IRE+VA group).

[0030] Figure 8 In vivo images of different animal model groups (Model group, IRE group, VA group; IRE+VA group).

[0031] Figure 9Imaging images showing the embolization effect of drug-loaded microalgae balls at different concentrations.

[0032] In the attached figure, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] The rat liver cancer (N1S1) cells used in the examples were purchased from ATCC China Cell Bank - ATCC Cell Resource Center, the peptide antagonist AUNP-12 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and camrelizumab SHR-1210 was provided by Jiangsu Hengrui Medicine Co., Ltd.

[0035] Example 1

[0036] (1) Chlorella proteoglycans cells were cultured in BG11 medium (BG11 medium was purchased from Shanghai Guangyu Biotechnology Co., Ltd.). After a certain period of culture at 25℃ under light (the medium was changed every 15 days, replacing 1 / 3 of the total volume of the medium), the Chlorella proteoglycans cell solution was centrifuged at 3000g for 10min, and the Chlorella proteoglycans cells were collected. Then, the cells were washed three times with deionized water and freeze-dried to obtain freeze-dried Chlorella proteoglycans cells. 0.5mg of freeze-dried Chlorella proteoglycans cells and 0.5mg of polypeptide antagonist AUNP-12 were mixed in water to load the drug and prepare drug-loaded microalgae cell solution.

[0037] (2) Preparation of the required fluid solutions for each phase: Polyvinyl alcohol was added to the drug-loaded microalgae cell solution in step (1) to obtain the inner phase solution, and the concentration of polyvinyl alcohol in the inner phase solution was controlled to be 5 wt%; polydimethylsiloxane, dimethyl silicone oil, and SYLGARD were added in a mass ratio of 1:2.33:0.33. TM 184 silicone elastomer curing agent (item number 1024001) was dissolved in distilled water to prepare an intermediate phase solution, in which the concentration of polydimethylsiloxane was 27.2 wt%; a 5 wt% polyvinyl alcohol solution was then used as the external phase solution; the aqueous phase solution was filtered with a 0.45 μm filter before use to prevent clogging of the microchannels.

[0038] (3) Pure water is first introduced into each channel of the microfluidic system. After the pure water fills the chip, the required solution is introduced. The external phase fluid, intermediate phase fluid and internal phase fluid prepared in the above steps are injected into the glass capillary microfluidic chip through a micro-injection pump. Specifically, in the microfluidic process, the internal phase solution and intermediate phase solution are pre-mixed using a T-channel, and then the pre-mixed solution is mixed with the external phase solution using a T-channel. The flow rate of the internal phase solution is 25 μL / h, the flow rate of the intermediate phase solution is 50 μL / h, and the flow rate of the external phase solution is 1000 μL / h. The double emulsion droplets obtained by the microfluidic process are collected in a 1 wt% polyvinyl alcohol aqueous solution to obtain the drug-loaded embolized microalgae balls for anti-liver cancer.

[0039] Example 2

[0040] The difference between the preparation method of the drug-loaded embolized microalgae balls for anti-liver cancer in this embodiment and that in Example 1 is that 1 mg of freeze-dried Synechocystis cells and 0.5 mg of camrelizumab SHR-1210 are mixed in water to load the drug and prepare the drug-loaded microalgae cell solution.

[0041] Sample Analysis

[0042] The drug-loaded embolized microalgae balls obtained in Example 1 for treating liver cancer were characterized and their performance analyzed, as follows:

[0043] (1) Morphological characteristics

[0044] Optical images of the drug-loaded embolized microalgae spheres were obtained through optical microscopy, as shown below. Figure 1 As shown, the particle size is uniformly distributed at around 100 μm, with good sphericity and uniform size. This indicates that the method of the present invention can produce microalgae spheres with consistent physical properties, which is beneficial for drug delivery and embolization effects.

[0045] (2) Drug release kinetics

[0046] Drug release kinetics experiments were conducted using a dialysis bag method at 37°C in phosphate buffer, simulating the in vivo release environment. Results are as follows: Figure 2 As shown, the drug release kinetic curves indicate that the drug-loaded embolized microalgae balls can control the drug release rate in a simulated in vivo environment, exhibiting good sustained-release characteristics. This helps maintain an effective drug concentration at the tumor site and prolong the duration of drug action. (3) Biocompatibility and safety assessment

[0047] The effects of drug-loaded embolized microalgae on blood cells were evaluated using a hemolysis assay. The results are as follows: Figure 3 As shown, the hemolysis test results indicate that the drug-loaded embolized microalgae balls have a safe effect on blood cells and do not cause significant hemolysis, indicating that they have good biocompatibility.

[0048] By injecting drug-loaded embolized microalgae balls into animal models, their distribution, metabolism, and potential toxicity in vivo were observed. Blood biochemical analysis and histopathological examination were used to evaluate the effects of the microalgae balls on major organs (such as heart, liver, spleen, lung, and kidney). The animal models were divided into five groups: Model: blank control group; iodine oil: iodized oil group, using conventional iodized oil for embolization; IRE: irreversible electroporation treatment group; VA: treatment group using drug-loaded embolized microalgae balls prepared in Example 1 for anti-hepatocellular carcinoma; IRE+VA: combined treatment group using irreversible electroporation and drug-loaded embolized microalgae balls prepared in Example 1 for anti-hepatocellular carcinoma.

[0049] Figure 4 In this context, BUN represents blood urea nitrogen; CRE represents creatinine; ALT represents alanine aminotransferase; AST represents aspartate aminotransferase; and D-Bil represents direct bilirubin. Figure 4 and Figure 5 Blood biochemical analysis and histopathological examination results showed that the drug-loaded embolized microalgae balls had little effect on major organs such as the heart, liver, spleen, lungs, and kidneys after injection, and did not cause significant pathological changes, further confirming its biosafety.

[0050] The results of tissue section staining showed that no obvious pathological damage or inflammatory reaction was observed in any of the observed organs, indicating that the microalgae pellets have good biocompatibility. Furthermore, no abnormal cell proliferation or tissue structural changes were observed, further confirming that the microalgae pellets do not cause significant tissue reactions or damage in vivo.

[0051] (4) Evaluation of the efficacy of liver cancer treatment

[0052] A liver cancer animal model was established, divided into four groups: Model (blank control group); IRE (irreversible electroporation) treatment group; VA (treatment group using drug-loaded embolized microalgae balls prepared in Example 1 for liver cancer treatment); and IRE+VA (combined treatment group using irreversible electroporation and drug-loaded embolized microalgae balls prepared in Example 1 for liver cancer treatment). The efficacy of drug-loaded embolized microalgae balls in liver cancer treatment was evaluated. The drug-loaded embolized microalgae balls were injected into the tumor site via the hepatic artery, and tumor growth was monitored regularly using imaging techniques (such as CT, MRI, or ultrasound). Simultaneously, pathological changes in tumor tissue and the distribution of drug-loaded embolized microalgae balls were assessed using HE staining and immunohistochemical analysis. Furthermore, the survival of the treated animals was observed to evaluate the treatment effect.

[0053] Imaging studies monitoring tumor growth showed that both IRE therapy alone and drug-loaded microalgae embolization significantly inhibited tumor growth compared to the model group. Figure 6 and Figure 7The combined treatment group, which used IRE combined with drug-loaded embolized microalgae balls for embolization, showed a more significant tumor-suppressive effect, effectively inhibiting tumor growth. This demonstrates the superiority of the combined treatment. Imaging results after interventional injection of drug-loaded embolized microalgae balls showed that the microalgae balls effectively achieved vascular embolization, blocking the blood supply to the tumor and thus inhibiting tumor growth.

[0054] (5) Evaluation of in vivo retention and embolization effect

[0055] The persistence and dynamic behavior of drug-loaded embolic microalgae in vivo were evaluated by observing different time points. Fluorescence imaging was used to monitor the persistence time of the drug-loaded embolic microalgae in vivo. Figure 8 As shown, microalgae aggregation in liver tumors was still observed 14 days after administration; and the fluorescence intensity did not change significantly, indicating good embolization effect. Different concentrations of drug-loaded embolized microalgae were injected into animals using interventional techniques at an injection rate of 0.1 mL / min and an injection volume of 0.2 mL. Imaging was performed, and the results are as follows. Figure 9 As shown, a significant vascular embolization effect was observed. Angiography results after injecting different concentrations of drug-loaded embolic microalgae balls via interventional methods showed that the embolization effect increased with increasing concentration of the drug-loaded embolic microalgae balls. At concentrations of 10 mg / ml and higher, the microalgae balls effectively achieved vascular embolization, blocking the blood supply to the tumor and thus inhibiting tumor growth.

[0056] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing drug-loaded embolic microalgal spheres for use against liver cancer, characterized in that, The preparation method comprises the following steps: (1) mixing microalgae cells and a drug to obtain a drug-loaded microalgae cell solution; the microalgae cells are Chlorella pyrenoidosa cells or Synechococcus sp. cells; the drug is carimab SHR-1210 or polypeptide antagonist AUNP-12; (2) using a drug-loaded microalgae cell solution containing polyvinyl alcohol as an inner phase solution, a mixed solution containing polydimethylsiloxane, dimethyl silicone oil and a curing agent as an intermediate phase solution, and a polyvinyl alcohol solution as an outer phase solution, adjusting the flow rates of the inner phase solution, the intermediate phase solution and the outer phase solution by using microfluidic technology to obtain the drug-loaded embolic microalgae balls for treating liver cancer; In step (2), polyvinyl alcohol is added to the drug-loaded microalgae cell solution of step (1) to obtain the inner phase solution, and the concentration of polyvinyl alcohol in the inner phase solution is controlled to be 0.2-10 wt%; In the intermediate phase solution, the mass ratio of polydimethylsiloxane, dimethyl silicone oil and the curing agent is 1:0.5-5:0.05-5, and the mass concentration of polydimethylsiloxane is 3-30 wt%; In the outer phase solution, the concentration of polyvinyl alcohol is 0.2-10 wt%.

2. The process for the preparation of drug loaded embolizing microalgal spheres for use against liver cancer as claimed in claim 1, wherein, When the microalgae cells are freeze-dried microalgae cells, the mass ratio of the microalgae cells to the drug is 0.1-2:

1.

3. The process for the preparation of drug loaded embolizing microalgal spheres for use against liver cancer as claimed in claim 1 wherein, In the microfluidic process, the inner phase solution and the intermediate phase solution are pre-mixed by using a T-shaped channel, and then the pre-mixed solution is mixed with the outer phase solution by using a T-shaped channel; the flow rate of the inner phase solution is 1-100 μL / h, the flow rate of the intermediate phase solution is 2-200 μL / h, and the flow rate of the outer phase solution is 300-2000 μL / h; the obtained double emulsion droplets are collected in a 0.2-10 wt% polyvinyl alcohol aqueous solution to obtain the drug-loaded embolic microalgae balls for treating liver cancer.

4. The drug-loaded embolic microalgae balls for treating liver cancer prepared by the preparation method of any one of claims 1-3.

5. The drug-loaded embolic microalgae balls for treating liver cancer of claim 4 in the preparation of liver cancer treatment products.

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