ViFSP1 combined sorafenib ferroptosis enhanced liver cancer treatment system based on bionic nanoparticles as well as preparation method and application of viFSP1 combined sorafenib ferroptosis enhanced liver cancer treatment system

Abstract

The invention relates to a viFSP1 and sorafenib combined ferroptosis enhanced liver cancer treatment system based on bionic nanoparticles as well as preparation and application of the viFSP1 and sorafenib combined ferroptosis enhanced liver cancer treatment system, belongs to the field of electrochemical energy storage and new energy materials, and discloses a bionic nano delivery system for combined use of an FSP1 inhibitor viFSP1 and sorafenib. Ferroptosis is induced by targeting liver cancer cells and M2 type tumor-related macrophages so as to improve the treatment effect of sorafenib, and the immunosuppressive tumor microenvironment is effectively reversed, so that an efficient treatment method of liver cancer is realized.

Description

Technical Field

[0001] The present invention belongs to the field of electrochemical energy storage and new energy materials, and specifically relates to a viFSP1 combined with sorafenib ferroptosis-enhanced liver cancer treatment system based on bionic nanoparticles, and its preparation and application. Background Art

[0002] 1. Prior art related to the present invention

[0003] Hepatocellular Carcinoma (HCC) is a malignant tumor with high morbidity and mortality worldwide. Current treatments for HCC mainly include surgical resection, radiotherapy, chemotherapy, and targeted drug therapy. However, although these treatments have achieved certain results, they still face many challenges, especially the high recurrence, metastasis, and immune escape of HCC.

[0004] 1. Current status and problems of sorafenib in the treatment of liver cancer

[0005] Sorafenib is an oral multi-target tyrosine kinase inhibitor and has become one of the standard treatment options for advanced liver cancer. It can exert anti-tumor effects by inhibiting tumor angiogenesis and inhibiting the proliferation of tumor cells. However, in clinical applications, the therapeutic effect of sorafenib is limited, mainly due to the immunosuppressive effect of the tumor microenvironment and the drug resistance of tumor cells. Specifically, the tumor microenvironment (TME) of liver cancer includes tumor-associated macrophages (TAMs), tumor-infiltrating immune cells and other immunosuppressive factors, which promote tumor immune escape, resulting in poor treatment effects.

[0006] 2. The role of FSP1 in liver cancer immune escape

[0007] Studies have shown that ferroptosis inhibitory protein 1 (FSP1) is an important mechanism for tumor cells to cope with ferroptosis. High expression of FSP1 inhibits the occurrence of ferroptosis, thereby helping tumor cells resist cell death caused by therapeutic drugs. In liver cancer, overexpression of FSP1 has been shown to be closely related to tumor drug resistance. Especially during the treatment of sorafenib, FSP1 reduces the sensitivity of tumor cells to sorafenib by inhibiting ferroptosis. Therefore, FSP1 has become a key target in immunotherapy of liver cancer.

[0008] 3. Application research of FSP1 inhibitors

[0009] Although FSP1 is considered a promising therapeutic target, current research still has limitations. Some studies have attempted to target FSP1 through small molecule inhibitors, but the therapeutic effects of these inhibitors are still affected by the complexity of the tumor microenvironment and immune escape mechanisms. The use of a single FSP1 inhibitor has certain limitations in improving the sensitivity of tumor cells to chemotherapeutic drugs. In addition, the delivery system of FSP1 inhibitors still has deficiencies in terms of targeting, drug stability and therapeutic effect.

[0010] 4. Attempts and challenges of combined therapy

[0011] Based on the relationship between FSP1 and the immune microenvironment, this study attempts to use FSP1 inhibitors in combination with sorafenib in order to enhance the therapeutic effect of sorafenib on tumors. Currently, some studies have used nano-drug carrier systems to co-deliver FSP1 inhibitors and chemotherapy drugs. Such systems help improve the targeting and biocompatibility of drugs. However, existing combined treatment options generally have the following problems:

[0012] a) Drug release control problem: Most existing drug delivery systems rely on static release mechanisms, that is, the drug release rate is difficult to dynamically control, which leads to unstable drug concentration in the tumor microenvironment, affecting the continuity and effectiveness of treatment.

[0013] b) Immunosuppressive microenvironment: Although some studies have attempted to improve the efficacy of drugs through combined treatment strategies, most treatment strategies have failed to effectively reverse the tumor's immunosuppressive microenvironment. In particular, the presence of M2 tumor-associated macrophages (TAMs) makes it often impossible for single drug treatment to overcome the tumor's immune escape mechanism.

[0014] c) Targeting issues of combination drugs: Current combination therapies often fail to achieve complete targeting during drug delivery, resulting in increased distribution of drugs in non-target areas, causing side effects and reducing treatment efficiency.

[0015] 5. Biocompatibility and safety issues of existing technologies

[0016] Although nanotechnology is widely used in the delivery of tumor drugs, many materials used in existing nanocarrier systems (such as metal nanoparticles, polymers, etc.) may have cumulative toxicity problems in the body, and long-term use will affect the biocompatibility and safety of drugs. These problems have also become the bottleneck for the existing drug delivery system to be widely used in clinical practice.

[0017] 2. Disadvantages and Shortcomings of Existing Technologies

[0018] 1. Limited drug efficacy: Traditional treatments such as sorafenib have limited effects in the treatment of liver cancer, mainly due to the immunosuppression in the tumor microenvironment, high expression of FSP1 and drug resistance. Existing technologies have failed to effectively solve the dual problems of FSP1 inhibition and immune escape, resulting in unsatisfactory efficacy.

[0019] 2. The single application of FSP1 inhibitors is not effective enough: Although FSP1 inhibitors are considered to be a potential target for tumor treatment, the efficacy of single use of FSP1 inhibitors is still unstable, especially when used in combination with chemotherapy drugs such as sorafenib, its effect is restricted by the complexity of the tumor microenvironment.

[0020] 3. Combination therapy lacks synergistic mechanism: Although some studies have tried to treat by combining FSP1 inhibitors with other drugs, the existing combination therapy schemes have not fully exerted the synergistic effect. The existing drug delivery system lacks a dynamic and controllable drug release mechanism and fails to overcome the problem of tumor immunosuppressive microenvironment.

[0021] 4. Insufficiency of drug delivery system: Most existing drug delivery systems adopt passive delivery mode, which has poor drug targeting. Especially in liver cancer, the tumor microenvironment is complex, and drugs often cannot be accurately located in the tumor area, thus affecting the treatment effect. Some systems also have problems such as uneven drug release and too long or too short drug retention time.

[0022] 5. Biocompatibility and toxicity issues: Many existing nanocarrier materials have biotoxicity issues, especially quantum dots or metal nanoparticles containing heavy metal components. These issues limit the clinical application of existing technologies and require further optimization to improve biocompatibility and safety. Summary of the invention

[0023] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide a viFSP1 combined with sorafenib ferroptosis-enhanced liver cancer treatment system based on bionic nanoparticles and its preparation and application. The present invention provides a liver cancer treatment method based on the combined use of FSP1 inhibitor (viFSP1) and sorafenib, which enhances the therapeutic effect of sorafenib on liver cancer cells, overcomes the problems of tumor resistance and immunosuppressive microenvironment in the current treatment regimen, and ultimately improves the treatment effect of liver cancer and improves the patient's prognosis. Specifically, it includes:

[0024] 1. Enhance the therapeutic effect of sorafenib: By combining the use of FSP1 inhibitors (viFSP1), the sensitivity of sorafenib to liver cancer cells can be enhanced, the drug resistance of tumor cells can be overcome, and its anti-tumor efficacy can be improved.

[0025] 2. Reversing the tumor immunosuppressive microenvironment: By targeting liver cancer cells and M2 tumor-associated macrophages (TAMs), using FSP1 inhibitor (viFSP1) and sorafenib to induce ferroptosis, reduce immunosuppressive factors, improve the immune microenvironment, and enhance the immune system's ability to clear tumors.

[0026] 3. Provide a new drug delivery platform: By designing a new biomimetic nanoparticle delivery system, the targeting and biocompatibility of drugs are improved, so that FSP1 inhibitors (viFSP1) and sorafenib can be accurately delivered to tumor cells and TAMs, maximizing their therapeutic effects.

[0027] 4. Improve the safety and controllability of treatment: By optimizing the drug delivery system, ensure the stability and continuity of drug release, reduce drug side effects, and improve the safety of treatment.

[0028] The specific scheme adopted by the present invention is:

[0029] In the first aspect, the present invention provides a viFSP1 combined with sorafenib ferroptosis-enhanced liver cancer treatment system based on bionic nanoparticles, wherein the liver cancer treatment system is targeted nanoparticles jointly loaded with viFSP1 and sorafenib, wherein the nanoparticles are jointly loaded with viFSP1 and sorafenib using responsive release factors, and are prepared by using delivery factors as the delivery system and modifying the surface of the nanoparticles with targeted polypeptides.

[0030] Furthermore, the responsive release factor is a GSH-responsive polymer DS-PLGA, the delivery factor is a biomimetic nanoparticle; and the targeting polypeptide is an M2 TAMs binding peptide.

[0031] In a second aspect, the present invention provides a method for preparing the liver cancer treatment system, comprising the following steps: Step 1, dissolution of DS-PLGA polymer and addition of drugs: using a mixture of ethyl acetate and dichloroethane to dissolve DS-PLGA to form a DS-PLGA solution; mixing a Sv mixture dissolved in dichloromethane with the DS-PLGA solution; the Sv refers to viFSP1 and sorafenib; Step 2, ultrasonic treatment and emulsification: placing the solution obtained in step 1 in an ice water bath for ultrasonic treatment to form a uniform emulsion; Step 3, mixing with PVA solution: slowly adding the emulsion obtained in step 2 to the 3% PVA solution, and continue to perform ultrasonic treatment to ensure that the emulsion and PVA solution are fully mixed; using a rotary evaporator to remove the organic solvent under low temperature conditions to form a nanoparticle suspension; Step 4, centrifugation and precipitate collection: collect the precipitate Sv@P by low-temperature centrifugation, wash the precipitate and resuspend it in PBS for later use; Step 5: Surface modification of nanoparticles: (1) Preparation and modification of HCC membranes: The nanoparticles were surface-modified with M2-type TAMs-binding peptides to obtain M2pep-modified HCC membranes, namely M-M2p. (2) Forming nanoparticles: mixing M-M2p with Sv@P; (3) The mixture was co-extruded to obtain M2pep-targeted Sv-loaded DS-PLGA nanoparticles, namely Sv@PM-M2p.

[0032] As a further optimization of the above preparation method, in step 1, the volume ratio of ethyl acetate and dichloromethane in the DS-PLGA solution is 3:7; the concentration of the DS-PLGA solution is 10 mg / mL; the mass ratio of sorafenib and viFSP1 is 1.4:1; the mass ratio of DS-PLGA to Sv is 1:1, 2.5:1, 5:1, 7.5:1 or 10:1.

[0033] As a further optimization of the above preparation method, in step 2, the operating conditions of the ultrasonic treatment are: 180 W ultrasonic power, 3s on / 1s off duty cycle, and lasting for 5 minutes.

[0034] As a further optimization of the above preparation method, in step 5, the specific method for preparing and modifying the HCC membrane is: taking 100 μg of DSPE-PEG-M2pep and mixing it with 1 mg of HCC membrane, incubating at 4°C for 30 minutes; and extruding the mixture through a 0.2 μm polycarbonate membrane.

[0035] As a further optimization of the above preparation method, in step five, the mixing mass ratio of the M-M2p and Sv@P is 1:1, 2.5:1, 5:1, 7.5:1, and 10:1.

[0036] In a third aspect, the present invention provides the use of the liver cancer treatment system or the liver cancer treatment system prepared by the preparation method in the preparation of liver cancer treatment drugs.

[0037] The invention solves several key problems in the treatment of liver cancer by combining FSP1 inhibitor (viFSP1) with sorafenib and combining with a bionic nanoparticle delivery system, and has the following significant advantages and beneficial effects:

[0038] 1. Enhance the therapeutic effect of sorafenib and overcome drug resistance

[0039] Sensitization effect: The present invention can effectively enhance the therapeutic effect of sorafenib on liver cancer by combining FSP1 inhibitor (viFSP1) with sorafenib. FSP1 inhibitor (viFSP1) restores the sensitivity of tumor cells to sorafenib-induced ferroptosis by inhibiting the function of FSP1, thereby overcoming the drug resistance of tumor cells. This synergistic effect significantly improves the effect of treatment and solves the problem of drug resistance and insufficient efficacy when sorafenib is used alone in the treatment of liver cancer.

[0040] 2. Reverse the tumor immune microenvironment and enhance the effect of immunotherapy

[0041] Reversal of immune microenvironment: This invention targets M2 tumor-associated macrophages (TAMs) and uses FSP1 inhibitor (viFSP1) and sorafenib to induce ferroptosis, which not only enhances the therapeutic effect of the drug on tumors, but also reverses the tumor immune escape phenomenon. M2 TAMs are immunosuppressive factors in the tumor microenvironment. Targeting these immune cells can improve the immune system's recognition and clearance of tumors, thereby significantly improving the treatment effect and improving the tumor's immunotherapy response.

[0042] 3. Accurately target liver cancer tissue and reduce side effects

[0043] Targeted delivery: Through the bionic nanoparticle delivery system, the present invention can accurately target liver cancer cells and M2 TAMs and deliver drugs to the tumor area. This targeting not only increases the concentration of drugs in tumor cells and increases the therapeutic effect, but also significantly reduces the accumulation of drugs in non-target areas, avoids the side effects existing in traditional drug delivery systems, reduces damage to normal tissues, and improves the safety of treatment.

[0044] 4. Dynamic drug release mechanism to improve treatment continuity

[0045] Intelligent release control: The present invention adopts a GSH-responsive drug release mechanism to dynamically control the release of drugs according to the concentration changes of GSH in the tumor microenvironment. This mechanism ensures that the drug is released only in the tumor area and is continuously released in the tumor area, maintaining the drug effect for a longer period of time. Compared with the traditional static release system, the dynamic drug release mechanism of the present invention can maximize the therapeutic effect of the drug and avoid uneven drug release.

[0046] 5. Improve biocompatibility and ensure the safety of long-term treatment

[0047] Biocompatibility and stability: The nanoparticles use low-toxic, degradable materials (such as DS-PLGA), and the biocompatibility of the drug delivery system is improved by PEG modification. This design reduces the clearance of the drug on the immune system, prolongs the drug's circulation time in the body, and ensures the stability of the drug, making it suitable for long-term treatment. In this way, the drug delivery system of the present invention has better safety and lower side effects, making it more suitable for clinical application.

[0048] 6. Provide new treatment options with broad clinical application prospects

[0049] Comprehensive therapeutic effect: The present invention combines FSP1 inhibitor (viFSP1) with sorafenib, combined with a precise drug delivery system, which not only improves the therapeutic effect of liver cancer, but can also be extended to the treatment of other types of tumors. Since the mechanism of action of FSP1 inhibitor (viFSP1) is to regulate the ferroptosis pathway, it can be used in combination with other chemotherapy drugs, immunotherapy drugs or targeted drugs, providing new ideas and solutions for the treatment of various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a transmission electron microscope photograph of the biomimetic nano-delivery system (called Sv@PM-M2p) in the implementation of the present invention.

[0051] Figure 2 This is a diagram showing the results of a drug combination experiment on liver cancer cells and M2 macrophages in vitro; the figure shows the cell survival of sorafenib, viFSP1 alone and in combination.

[0052] Figure 3 The results of the in vivo Sv@PM-M2p treatment experiment are shown in Figure 1. The groups are: G1-PBS, G2-SF, G3-SF+vF, G4-Sv@PM, G5-Sv@PM-M2p; the sizes of liver cancer tumors in nude mice in different treatment groups are shown. DETAILED DESCRIPTION

[0053] The task of the present invention is to design and implement a combined treatment system, combining FSP1 inhibitor (viFSP1) and sorafenib, and to achieve the above purpose through the following innovative tasks:

[0054] 1. Develop an efficient combination therapy: Develop a new treatment method that combines the FSP1 inhibitor (viFSP1) with sorafenib, using the sensitizing effect of viFSP1 to overcome the resistance of tumor cells to sorafenib, thereby improving the effect of liver cancer treatment.

[0055] 2. Construct a precisely targeted drug delivery system: Design and synthesize a biomimetic nanoparticle delivery system to ensure that FSP1 inhibitor (viFSP1) and sorafenib can be efficiently delivered to liver cancer cells and M2 TAMs, and precisely released in the tumor microenvironment, thereby enhancing the drug's targeting and therapeutic effect.

[0056] 3. Reversing the tumor immunosuppressive environment: Using the combined effects of FSP1 inhibitor (viFSP1) and sorafenib, targeted therapy is performed on M2 TAMs in the tumor microenvironment to induce ferroptosis, thereby reversing tumor immune escape, improving the immunosuppressive environment, and enhancing the immune system's ability to eliminate tumors.

[0057] 4. Improve the biocompatibility and safety of drugs: In the design of drug delivery systems, non-toxic biomimetic nanoparticle carriers are used to ensure that the delivery system has high biocompatibility, reduce toxicity problems in traditional drug delivery systems, and provide safer treatment options.

[0058] 5. Optimize drug release control mechanism: Design a system that can dynamically regulate drug release to ensure continuous drug release in the tumor area to improve the therapeutic effect and avoid the problems of unstable release rate and fluctuating therapeutic effect in traditional drug delivery systems.

[0059] The present invention provides a more effective liver cancer treatment plan by combining FSP1 inhibitor (viFSP1) and sorafenib, overcoming various limitations of existing treatments; a new combined treatment platform has been developed, combining precise targeted delivery and immune microenvironment reversal strategies to optimize the therapeutic effect, release control and safety of the drug, ultimately improving the survival rate and quality of life of liver cancer patients.

[0060] The present invention provides a liver cancer treatment method based on the combined use of FSP1 inhibitor (viFSP1) and sorafenib and a drug delivery system thereof. The specific technical scheme is as follows:

[0061] 1. Combined therapy system:

[0062] The present invention proposes a treatment scheme of using a FSP1 inhibitor (viFSP1) in combination with sorafenib. The FSP1 inhibitor (viFSP1) can restore the sensitivity of tumor cells to ferroptosis induced by sorafenib by inhibiting FSP1, thereby overcoming the drug resistance of tumor cells to sorafenib and enhancing the anti-tumor effect of sorafenib. Specifically, the application of FSP1 inhibitor (viFSP1) can effectively reduce the inhibitory effect of FSP1 on ferroptosis of tumor cells, and synergistically enhance the therapeutic efficacy of sorafenib on liver cancer cells. At the same time, this combination therapy can also improve the patient's immune response and enhance the therapeutic effect by changing the tumor immune microenvironment and reversing the role of tumor-associated macrophages (TAMs) in immune escape.

[0063] 2. Drug delivery system:

[0064] To ensure the precise delivery of FSP1 inhibitor (viFSP1) and sorafenib, the present invention designs a biomimetic nanoparticle delivery system. The system co-loads FSP1 inhibitor (viFSP1) and sorafenib in a glutathione (GSH)-responsive polymer and is functionally modified by an M2 tumor-associated macrophage (TAMs) binding peptide (M2pep) to achieve precise targeting of liver cancer tissues and immune cells.

[0065] Specifically, GSH-responsive polymers can control drug release according to changes in GSH concentration in the tumor microenvironment. Higher GSH levels on tumor cells and M2 TAMs will trigger the fragmentation of the polymer, thereby releasing FSP1 inhibitor (viFSP1) and sorafenib.

[0066] In addition, the surface of the nanoparticles is modified with targeting molecules (such as M2pep) that specifically target liver cancer cells and M2 TAMs, which enables the nanoparticles to efficiently recognize and bind to liver cancer cells and their immune cells, thereby improving the targeting and delivery efficiency of the drug.

[0067] 3. Dynamic drug release mechanism:

[0068] The drug delivery system of the present invention adopts a dynamic drug release mechanism, that is, the drug is released in the tumor microenvironment according to the change of GSH concentration. This mechanism ensures that the drug can be continuously and effectively released in the tumor area, and reduces the problems of uneven drug release and too long or too short drug retention time in the prior art. Through intelligent release control, the delivery system of the present invention can achieve sustained release of drugs in tumor cells, thereby improving the durability of treatment and enhancing the therapeutic effect.

[0069] 4. Biocompatibility and safety: The biomimetic nanoparticles used in the present invention are made of low-toxic materials (such as DS-PLGA) and modified with PEG to enhance biocompatibility, reduce the clearance of the immune system, and prolong the circulation time of the drug in the body. In this way, the drug can be safely and efficiently delivered to the tumor area and reduce side effects on normal tissues.

[0070] Compared with the prior art, the present invention has the following significant advantages:

[0071] 1. Sensitization therapeutic effect: The combined use of FSP1 inhibitor (viFSP1) and sorafenib can effectively overcome the resistance of tumor cells to sorafenib, significantly enhance the therapeutic effect of sorafenib, and solve the problem of drug resistance in the treatment of liver cancer.

[0072] 2. Reversal of immune microenvironment: The present invention targets M2 TAMs and tumor cells, induces ferroptosis and improves the tumor immune microenvironment, thereby reversing tumor immune escape, enhancing the immune system's tumor clearance effect, and improving the treatment effect.

[0073] 3. Precision targeted drug delivery: Through nanoparticles modified with M2-type TAMs-binding peptide (M2pep), drugs can be precisely delivered to liver cancer cells and tumor immune cells, thereby increasing the local concentration of drugs, reducing drug accumulation in non-target areas, and reducing side effects.

[0074] 4. Dynamic drug release and lasting therapeutic effect: The GSH-responsive release mechanism ensures the intelligent release of drugs in the tumor microenvironment, thereby improving the sustainability and stability of the therapeutic effect and avoiding therapeutic fluctuations caused by uneven drug release in existing technologies.

[0075] 5. Improve biocompatibility and safety: Nanoparticles are made of low-toxic materials and modified with PEG, which makes the drug delivery system have better biocompatibility, reduces the toxicity problems in traditional drug delivery systems, and ensures the safety of treatment.

[0076] Technical points that need to be protected:

[0077] 1. Combined treatment scheme: The core technical point of the present invention is to use FSP1 inhibitor (viFSP1) and sorafenib in combination to enhance the therapeutic effect of sorafenib on liver cancer. In particular, FSP1 inhibitor (viFSP1) can effectively overcome the drug resistance of tumor cells to sorafenib, thereby enhancing the sensitization treatment effect. Therefore, the synergistic effect of the combined use of FSP1 inhibitor (viFSP1) and sorafenib is an important technical point of the present invention.

[0078] 2. Drug delivery system: The present invention provides a biomimetic nanoparticle delivery system that co-loads FSP1 inhibitor (viFSP1) and sorafenib, and enables precise release of the drugs in the tumor microenvironment through a GSH-responsive polymer. The design of the drug delivery system ensures that the drugs can accurately target liver cancer cells and M2 TAMs, thereby improving the targeting of the drugs, reducing side effects, and improving the efficacy.

[0079] 3. Dynamic drug release mechanism: The present invention adopts a GSH-responsive drug release mechanism to control the release of drugs according to the concentration changes of GSH in the tumor microenvironment. This mechanism can ensure the continuous release of drugs in the tumor area and reduce the problem of uneven drug release rate in traditional drug delivery systems, thereby improving the therapeutic effect.

[0080] 4. Nanoparticle surface modification technology: The bionic nanoparticles of the present invention are modified with M2 TAMs binding peptide (M2pep) to ensure that the drug can accurately target liver cancer cells and immune cells, especially M2 TAMs, thereby enhancing the accumulation of the drug in the tumor microenvironment and improving the therapeutic effect.

[0081] 5. Biocompatibility and safety: Nanoparticles are made of low-toxic materials and modified with PEG to improve biocompatibility and stability, reduce the clearance of the immune system, and thus ensure the stability and long-term biocompatibility of the drug system in the body.

[0082] Alternative solutions:

[0083] Although the technical solution of the present invention effectively solves some technical problems in existing treatment methods, some alternative solutions can also be considered, as follows:

[0084] 1. Use of FSP1 inhibitor (viFSP1) alone: ​​If sorafenib is not considered, the combined use of FSP1 inhibitor (viFSP1) and other chemotherapy drugs or immunotherapy drugs can be considered. This scheme may alleviate the immunosuppressive microenvironment and improve the therapeutic effect of other drugs, but its effect may not be as good as the combined use of viFSP1 and sorafenib in the present invention, because sorafenib itself has angiogenesis inhibition effect, which can fundamentally inhibit tumor growth.

[0085] 2. Alternative delivery systems: Although the present invention uses a biomimetic nanoparticle delivery system, other types of drug delivery systems may also be used, such as:

[0086] Liposomes: Liposomes are widely used as drug carriers in targeted drug delivery systems. They have good biocompatibility and can circulate in the body for a long time.

[0087] Dendrimers: Dendrimers can also be used as drug carriers due to their high branching and controllable structure, with high drug loading capacity and targeting.

[0088] Although these alternatives can provide similar therapeutic effects to the present invention in some aspects, they may not be as good as the GSH-responsive polymer nanoparticle system in terms of the accuracy, stability and sustained release of drugs.

[0089] 3. Alternative drug release control mechanisms: The present invention uses a GSH-responsive drug release mechanism, which can regulate the release rate of the drug according to the change in GSH concentration in the tumor microenvironment. However, the following alternative drug release control mechanisms can also be used:

[0090] pH-responsive release system: Many tumor microenvironments are acidic. By utilizing this property, pH-responsive polymers can be designed to release drugs in an acidic environment.

[0091] Thermal responsive release system: Due to poor blood flow in the tumor area, the temperature is often higher than that of normal tissue. Thermal responsive polymers can also release drugs when the local temperature changes.

[0092] 4. Alternatives to targeting peptides: In the present invention, M2-type TAMs binding peptide (M2pep) is used to target tumor-associated macrophages (TAMs). However, other targeting molecules can also be considered:

[0093] Anti-tumor antibodies: For example, using monoclonal antibodies that target surface markers on liver cancer cells, drugs can be directed to tumor cells.

[0094] Small molecule targeting ligands: Small molecule ligands that target specific receptors of cancer cells (such as EP-CAM) can also be used as alternative targeting molecules to enhance the targeting of drugs.

[0095] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the examples. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. If the specific technology or conditions are not specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0096] Reagents:

[0097] 1. FSP1 inhibitor (viFSP1): synthesize or purchase commercial FSP1 inhibitors (such as viFSP1).

[0098] 2. Sorafenib (SF): Purchase the commercialized drug Sorafenib.

[0099] 3. GSH-responsive polymer (DS-PLGA): prepared by chemical synthesis method, or purchased from related commercial products.

[0100] 4. M2 TAMs binding peptide (M2pep): liver cancer related peptides, such as GPC-3, EpCAM, CXCR4, etc., synthesized or purchased on demand.

[0101] 5. PEG modified materials: used to improve the biocompatibility and stability of nanoparticles.

[0102] instrument:

[0103] 1. Transmission electron microscopy (TEM): used to characterize the size and morphology of nanoparticles.

[0104] 2. Conventional laboratory equipment such as rotary evaporator, ultrafiltration equipment, ultrasonic cleaner, etc.

[0105] Example 1: Preparation method of nanoparticles

[0106] In this example, DS-PLGA polymer was used to load FSP1 inhibitor (viFSP1) and sorafenib (SF), and surface modification was performed to improve drug targeting.

[0107] Step 1: DS-PLGA dissolution and Sv addition

[0108] 1. DS-PLGA dissolution: Take DS-PLGA polymer and dissolve it in ethyl acetate and dichloromethane (volume ratio is 3:7). Prepare a solution with appropriate concentration, usually 10 mg / mL solution.

[0109] 2. Sv was added to the solution: Sv (wSF / wvF=1.4 / 1) dissolved in dichloromethane was added to the above solution. Different mass ratios of DS-PLGA to Sv were tested, including 1:1, 2.5:1, 5:1, 7.5:1 and 10:1.

[0110] Step 2: Sonication and emulsification

[0111] The obtained solution was placed in an ice water bath and subjected to ultrasonic treatment. The operating conditions were an ultrasonic power of 180 W, a duty cycle of 3 s on / 1 s off, and a duration of 5 minutes to form a uniform emulsion.

[0112] Step 3: Mix with PVA solution

[0113] 1. Slowly add the obtained emulsion into the 3% PVA solution and continue ultrasonic treatment to ensure that the emulsion and PVA solution are fully mixed.

[0114] 2. Use a rotary evaporator to remove the organic solvent at low temperature to remove the dichloromethane in the emulsion and form a nanoparticle suspension.

[0115] Step 4: Centrifugation and collection of precipitate

[0116] 1. Collect the precipitate by centrifugation (12,000 g, 40 min, 4°C).

[0117] 2. The precipitate (labeled as Sv@P) was washed twice with PBS to remove the unreacted surfactant.

[0118] 3. Finally, resuspend the washed precipitate in PBS for later use.

[0119] Step 5: Surface modification of nanoparticles

[0120] 1. Preparation and modification of HCC membrane:

[0121] The nanoparticles were surface modified with M2-type TAMs binding peptide (M2pep) to improve the targeting of tumor cells. The specific method was as follows: 100 μg of DSPE-PEG-M2pep was mixed with 1 mg of HCC membrane and incubated at 4°C for 30 minutes. The mixture was extruded through a 0.2 μm polycarbonate membrane to obtain M2pep-modified HCC membrane (labeled as M-M2p).

[0122] 2. Formation of nanoparticles: M-M2p and Sv@P were mixed in different mass ratios (e.g., 1:1, 2.5:1, 5:1, 7.5:1, 10:1). The mixture was shaken in a vortex oscillator for 5 minutes and then sonicated for 3 minutes. Finally, the mixture was co-extruded through a Miniextruder using polycarbonate membranes of different thicknesses (400nm, 200nm, and 100nm) for 15 cycles to obtain M2pep-targeted Sv-loaded DS-PLGA nanoparticles (labeled as Sv@PM-M2p).

[0123] 3. Preparation of control group: No DSPE-PEG-M2pep was added, and Sv@PM was prepared as the control group.

[0124] Step 6: Characterization and Analysis

[0125] 1. Use transmission electron microscopy (TEM) to observe the morphology of the obtained nanoparticles and confirm the size and uniformity of the nanoparticles.

[0126] 2. Use dynamic light scattering (DLS) to measure the particle size distribution and Zeta potential of the nanoparticles to ensure that the particle size is between 10-150nm and has good stability.

[0127] Example 2: Combination therapy of in vitro hepatoma cell lines

[0128] (I) Experimental steps:

[0129] 1. Cell culture: HepG2 or Huh7 liver cancer cell lines were cultured at 37°C, 5% CO, and DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) was used.

[0130] 2. Drug preparation and treatment: Nanoparticles loaded with FSP1 inhibitor (viFSP1) and sorafenib (SF) were used for drug delivery according to the preparation method. Different treatment groups were set up, including single treatment group (viFSP1 or SF) and combined treatment group (viFSP1+SF). The cell treatment time was 24 hours, and each group of cells was treated 3 times in total.

[0131] 3. Cell proliferation detection: The treated cells were tested for cell proliferation and toxicity using CCK-8 reagent. According to the kit instructions, the absorbance (OD value) of each group of cells was measured to calculate the cell survival rate.

[0132] 4. Ferroptosis detection: Use flow cytometry and immunofluorescence staining to detect intracellular ferroptosis markers (such as changes in MDA and GSH levels). The effect of combined treatment can be determined based on the changes in intracellular markers.

[0133] (II) Experimental results:

[0134] CCK-8 assay showed that the combined treatment group showed significant inhibitory effect on cell proliferation, and the inhibitory effect of the combined group was more significant than that of the treatment group using viFSP1 or SF alone.

[0135] Analysis of ferroptosis markers showed that the combined treatment could significantly induce cellular ferroptosis and enhance the therapeutic effect.

[0136] Example 3: In vivo treatment of liver cancer in situ

[0137] (I) Experimental steps:

[0138] 1. Mouse model establishment: Huh7 liver cancer cells were inoculated into the pancreas of nude mice, and treatment was started after the tumor grew to a certain size. Tumor size was measured before treatment to ensure that the initial volume of the tumor was consistent.

[0139] 2. Drug treatment: The prepared FSP1 inhibitor (viFSP1) and sorafenib-loaded nanoparticles were injected into mice. Each mouse received a drug dose of 5 mg per kg of body weight for 7 consecutive days. The treatment group, blank group and control group were set up.

[0140] 3. Tumor monitoring and evaluation: During the treatment period, the size of the mouse tumor was measured every 2 days, and the tumor volume was calculated (formula: volume = length × width 2 / 2). After the treatment, the mice were euthanized and the tumors were removed for weight measurement.

[0141] (II) Experimental results:

[0142] The growth of tumor volume and weight of mice in the treatment group were significantly slowed down. Compared with the control group, the tumor volume and weight were reduced by about 60%.

[0143] The in vivo therapeutic effects showed that the combined therapeutic nanoparticle system could effectively inhibit tumor growth and enhance the therapeutic effect.

[0144] Through the above examples, the FSP1 inhibitor (viFSP1) and sorafenib combined with M2pep targeted nanoparticle system provided by the present invention can effectively enhance the effect of liver cancer treatment. The system not only improves the accumulation of drugs in the tumor area through precise drug delivery, but also significantly enhances the durability and safety of treatment.

[0145] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. The viFSP1 combined with sorafenib ferroptosis-enhanced liver cancer treatment system based on bionic nanoparticles is characterized by: The liver cancer treatment system is a targeted nanoparticle loaded with viFSP1 and sorafenib. The nanoparticle is loaded with viFSP1 and sorafenib by a responsive release factor, and is prepared by using a delivery factor as a delivery system and modifying a targeted polypeptide on its surface.

2. The liver cancer treatment system according to claim 1, characterized in that: The responsive release factor is a GSH-responsive polymer DS-PLGA, the delivery factor is a bionic nanoparticle; and the targeting polypeptide is an M2 TAMs binding peptide.

3. The method for preparing the liver cancer treatment system according to claim 2, characterized in that: The following steps are involved: Step 1, dissolution of DS-PLGA polymer and addition of drugs: using a mixture of ethyl acetate and dichloroethane to dissolve DS-PLGA to form a DS-PLGA solution; mixing a Sv mixture dissolved in dichloromethane with the DS-PLGA solution; the Sv refers to viFSP1 and sorafenib; Step 2, ultrasonic treatment and emulsification: placing the solution obtained in step 1 in an ice water bath for ultrasonic treatment to form a uniform emulsion; Step 3: Mixing with PVA solution: slowly add the emulsion obtained in step 2 into the 3% PVA solution, and continue to perform ultrasonic treatment to ensure that the emulsion and PVA solution are fully mixed; The organic solvent was removed using a rotary evaporator at low temperature to form a nanoparticle suspension; Step 4, centrifugation and precipitate collection: collect the precipitate Sv@P by low-temperature centrifugation, and resuspend the precipitate in PBS after washing for later use; Step 5: Surface modification of nanoparticles: (1) Preparation and modification of HCC membranes: The nanoparticles were surface-modified with M2-type TAMs-binding peptides to obtain M2pep-modified HCC membranes, namely M-M2p. (2) Forming nanoparticles: mixing M-M2p with Sv@P; (3) The mixture was co-extruded to obtain M2pep-targeted Sv-loaded DS-PLGA nanoparticles, namely Sv@PM-M2p.

4. The preparation method according to claim 3, characterized in that: In step 1, the volume ratio of ethyl acetate and dichloromethane in the DS-PLGA solution is 3:7; the concentration of the DS-PLGA solution is 10 mg / mL; the mass ratio of sorafenib and viFSP1 is 1.4:1; the mass ratio of DS-PLGA to Sv is 1:1, 2.5:1, 5:1, 7.5:1 or 10:

1.

5. The preparation method according to claim 3, characterized in that: In step 2, the operating conditions of the ultrasonic treatment are: 180 W ultrasonic power, 3s on / 1s off duty cycle, and 5 minutes of duration.

6. The preparation method according to claim 3, characterized in that: In step 5, the specific method for preparing and modifying the HCC membrane is as follows: 100 μg of DSPE-PEG-M2pep is mixed with 1 mg of HCC membrane, and the mixture is incubated at 4° C. for 30 minutes; and the mixture is extruded through a 0.2 μm polycarbonate membrane.

7. The preparation method according to claim 3, characterized in that: In step five, the mixing mass ratio of the M-M2p and Sv@P is 1:1, 2.5:1, 5:1, 7.5:1, and 10:

1.

8. Use of the liver cancer treatment system according to any one of claims 1-2 or the liver cancer treatment system prepared by the preparation method according to any one of claims 3-7 in preparing liver cancer treatment drugs.

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