Iron-based nanomedicine integrating chemotherapy / chemodynamic therapy, and preparation method and application thereof

Abstract

The application discloses an iron-based nano drug integrating chemotherapy and chemical power therapy, and a preparation method and application thereof, and belongs to the technical field of nanomedicine. The iron-based nano drug comprises a core and a shell. The core comprises an active ingredient and ferrous ions, and the shell is hyaluronic acid. The hyaluronic acid shell prolongs the circulation time of the nano particles in blood. Through HA / CD44 specific interaction, cells effectively phagocytize the nano particles. ROS generated through the Fenton reaction induced by the iron ions can cause the redox balance imbalance in tumor cells, thereby improving the sensitivity of the tumor cells to the active ingredient of the drug and enhancing the chemotherapy effect.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to an iron-based nanomedicine integrating chemotherapy / chemodynamic therapy, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common malignant tumor worldwide. It is a primary liver cancer with a high mortality rate, poor prognosis, a still high recurrence rate, and a significantly low survival rate after cancer metastasis.

[0003] Chemotherapy remains one of the first-line clinical treatment strategies for HCC, but insufficient targeted drug delivery and the tumor immunosuppressive microenvironment affect chemotherapeutic drug resistance and hinder its effectiveness. Therefore, there is an urgent need to find a new treatment strategy. Nanomedicines possess superior physicochemical properties, such as targeted drug delivery and responsive drug release in the tumor microenvironment. These systems have shown potential in the treatment of HCC by facilitating the synergistic delivery of various therapeutic agents.

[0004] In recent years, Fenton-like reactions based on iron-based materials have attracted widespread attention. The active surface of iron-based nanoparticles and the iron ions released from tumors can increase the production of reactive oxygen species (ROS) and promote the conversion to highly reactive ROS free radicals in tumor cells through Fenton-like reactions, leading to increased intracellular oxidation levels, DNA necrosis, protein inactivation, lipid oxidation, and ultimately inducing apoptosis in cancer cells.

[0005] Tumor immunotherapy has achieved remarkable progress over the past decade. In-depth research in tumor immunotherapy has confirmed the crucial role of the immune system in regulating tumorigenesis, development, metastasis, and drug resistance. Compared to other organs, the liver possesses rich immune tolerance mechanisms, protecting it from damage. However, the resulting immunosuppressive microenvironment has become a major cause of the disruption of the inhibitory effects of chemotherapeutic drugs on tumor cells. Chemokines are considered the most influential mediators, playing a vital regulatory role in the immune system's control of immune cell recruitment, angiogenesis, cell migration, and metastasis. CXC receptor 4 (CXCR4) is one of the most widely expressed chemokines in human cancers, exhibiting a clear interaction with CXC ligand 12 (CXCL12). The CXCL12 / CXCR4 axis can induce the recruitment of immunosuppressive cells to tumors, thereby enhancing the immunosuppressive microenvironment. Numerous studies have demonstrated that blocking CXCR4 or CXCL12 (e.g., using AMD3100) can significantly improve anti-tumor efficacy in various tumor models, which is related to its reversal of tumor immunosuppression.

[0006] Single cancer therapies such as chemotherapy, immunotherapy, and chemokinetics can only moderately modulate tumor suppression, but their treatment efficiency and survival rates after cancer metastasis remain low. Therefore, finding a new treatment strategy is an urgent problem to be solved. Summary of the Invention

[0007] The purpose of this invention is to provide an iron-based nanomedicine integrating chemotherapy / chemodynamic therapy, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] One of the technical solutions of this invention is a nanomedicine delivery system, comprising a core and a shell; the core includes an active ingredient and Fe. 2+ The outer shell is hyaluronic acid.

[0010] The second technical solution of the present invention, the preparation method of the nanomedicine delivery system, involves adding the active ingredient containing Fe 2+ The solution is stirred and reacted, and then the reacted solution is dispersed in a hyaluronic acid solution for further reaction. Centrifugation yields the nanomedicine delivery system.

[0011] The third technical solution of the present invention is the application of the nanomedicine delivery system in the preparation of drugs for treating hepatocellular carcinoma.

[0012] The fourth technical solution of the present invention is a nanomedicine for treating hepatocellular carcinoma, comprising the nanomedicine delivery system.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] This invention provides an iron-based nanomedicine integrating chemotherapy / chemodynamic therapy, its preparation method, and its application. The hyaluronic acid (HA) structure contains numerous carboxyl groups, giving the entire molecule a negative charge. The HA coating on the surface can conceal the positive charge on the surface of the targeted nanocore. When the nanoparticles reach the tumor microenvironment, overexpressed hyaluronidase in the tumor promotes the degradation of the outer shell, causing the surface hyaluronic acid to detach and reverse the charge, providing conditions for the nanocore to target and accumulate inside the cell. Furthermore, hyaluronic acid promotes the cellular uptake and internalization of nanoparticles through specific interaction with the CD44 receptor on the cell surface. The HA shell prolongs the circulation time of the nanoparticles in the bloodstream and enables cells to effectively engulf the nanoparticles through the specific HA / CD44 interaction. The ROS generated by the iron-induced Fenton-like reaction leads to an imbalance of redox balance within tumor cells, thereby increasing the sensitivity of tumor cells to the active pharmaceutical ingredient and enhancing the efficacy of chemotherapy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the FDAH structure;

[0017] Figure 2 The diagram shows the grouping of mice; where PBS is the control group, FDH is the control group with plexafor (FDAH removed), and FDP is the control group with HA in FDAH replaced by PVP.

[0018] Figure 3 Tumor growth curves (A) and tumor inhibition rates (B) after treatment of different groups;

[0019] Figure 4 Bar charts showing ex vivo tumor tissue after treatment in different groups (A) and tumor weight after treatment in different groups (B); where # indicates comparison with the control group and PBS group; * indicates comparison between the two groups at the ends of the horizontal line; * table P<0.05, ** table P<0.01, *** table P<0.001, **** table P<0.0001; # table P<0.05, ## table P<0.05, ### table P<0.001, #### table P<0.0001. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0026] This invention provides a nanomedicine delivery system comprising a core and a shell; the core includes an active ingredient and Fe. 2+ The outer shell is hyaluronic acid.

[0027] This invention also provides a method for preparing the nanomedicine delivery system, wherein the active ingredient is added to a container containing Fe 2+ The solution is stirred and reacted, and then the reacted solution is dispersed in a hyaluronic acid solution for further reaction. Centrifugation yields the nanomedicine delivery system.

[0028] In some specific implementations, the Fe 2+ The molar ratio of the active ingredient to the active ingredient is approximately 25:(20-25).

[0029] In some specific implementations, the Fe 2+ The mass ratio of hyaluronic acid to hyaluronic acid is 1:10.

[0030] In some specific implementations, the stirring reaction is carried out under dark conditions with stirring at 600 rpm for 2 hours.

[0031] In some specific implementations, the condition for continuing the reaction is stirring at 600 rpm for 24 hours in the dark.

[0032] In some specific implementations, the Fe-containing 2+ The solution is formed by uniformly dispersing ferrous chloride tetrahydrate FeCl2·4H2O (10 mg) in DMSO (2 mL).

[0033] In some specific implementations, triethylamine (3 μL) is added before the stirring reaction (to desalt DOX-HCl).

[0034] In some specific implementations, the hyaluronic acid solution is a solution formed by uniformly dispersing hyaluronic acid (50 mg) in deionized water (5 mL).

[0035] In some specific implementations, the centrifugation conditions are centrifugation at a speed of 10,000 rpm for 10 minutes.

[0036] This invention also provides the application of the nanomedicine delivery system in the preparation of drugs for treating hepatocellular carcinoma.

[0037] This invention also provides a nanomedicine for treating hepatocellular carcinoma, including the nanomedicine delivery system.

[0038] In some specific embodiments, the active ingredient is selected from at least one of doxorubicin or praxavir (AMD3100).

[0039] Example 1

[0040] This embodiment provides an iron-based nanoparticle comprising a core and a shell. Two clinical drugs, doxorubicin (DOX) and plerixafor (AMD3100), work together with ferrous ions to form the core of the nanoparticle FDAH, while hyaluronic acid (HA) serves as the shell.

[0041] Preparation method:

[0042] First, ferrous chloride tetrahydrate (FeCl₂·4H₂O) (10 mg) was uniformly dispersed in DMSO (2 mL). Then, doxorubicin hydrochloride (DOX-HCl) (12 mg) and prazosofol (AMD3100) (10 mg) were added to the FeCl₂·4H₂O solution, along with triethylamine (3 μL). The mixture was stirred at 600 rpm in the dark for 2 hours. Hyaluronic acid (HA, 50 mg) was uniformly dispersed in deionized water (5 mL). The Fe-DOX-AMD3100 solution was then slowly added dropwise to the uniformly dispersed HA solution. The mixture was stirred at 600 rpm in the dark for 24 hours. Finally, the mixture was centrifuged at 10,000 rpm for 10 minutes to obtain FDAH nanoparticles.

[0043] Experimental Example 1

[0044] After co-culturing FDAH with HepG2 liver cancer cells for 12 hours, the nanomedicine was taken up by the cancer cells and remained within lysosomes. It then slowly leaked from the lysosomes into the cytoplasm by altering the permeability of the lysosomal membrane. This may lead to the release of hydrolytic enzymes from the lysosomes into the cytoplasm, thereby promoting and accelerating the induction of apoptosis. Furthermore, cytotoxicity assays showed that FDAH combination therapy exhibited the best inhibitory effect on HepG2 cells compared to single-therapy (chemotherapy, chemokinetic therapy).

[0045] Experimental Example 2

[0046] Hepatocellular carcinoma modeling in mice and its grouping intervention

[0047] Given that the metabolic enzymes in HepG2 cells do not change with excessive passage number in drug-related studies, and that, as a human hepatocellular carcinoma cell line, its biotransformation and metabolic enzymes are homologous to those in normal human hepatocytes, it is often used as an ideal cell line for in vitro hepatocellular metabolism or genotoxicity assays. However, this human hepatocellular carcinoma cell line does not induce tumors in immunosuppressed mice; therefore, female BALB / c nude mice were selected as experimental subjects.

[0048] Twenty healthy female BALB / c nude mice were acclimatized in a 25°C constant-temperature animal room with free access to water and food. Light was artificially controlled for 12 hours a day, and bedding was changed three times a week. After one week of acclimatization, they were randomly divided into three groups of four mice each based on their weight: PBS group (normal control group), FDH group, FDP group, FDAH group, and DOX group.

[0049] Modeling: 1×10⁻⁶ mmol / L was subcutaneously injected into the right leg of 4-5 week old female BALB / c nude mice. 7 One HepG2 cell. When the tumor volume is approximately 50 mm. 3 Mice were randomly divided into five groups: PBS, DOX, FDH, FDP, and FDAH.

[0050] Intervention: When the tumor volume is approximately 50 mm 3Subsequently, the PBS group was intravenously injected with PBS (pH=7.4, 200 μl) every two days; the DOX group was intravenously injected with DOX-PBS solution (pH=7.4, 10 mg / kg, 200 μl) every two days; the FDH group was intravenously injected with FDH-NaH2PO4 solution (pH=7.4, DOX concentration 10 mg / kg, 200 μl) every two days; the FDP group was intravenously injected with FDP-PBS solution (pH=7.4, DOX concentration 10 mg / kg, 200 μl) every two days; and the FDAH group was intravenously injected with FDAH-PBS solution (pH=7.4, DOX concentration 10 mg / kg, 200 μl) every two days. Tumor volume and body weight were recorded every two days.

[0051] Animal in vivo experiments were conducted by constructing a mouse model, and the results were as follows: Figure 4 As shown, the results of tumor size and weight after treatment indicate that the FDAH treatment group had the best anti-tumor therapeutic effect. Furthermore, chemotherapy alone (DOX group) caused significant liver damage. This may be because AMD3100 can reduce or avoid the liver damage caused by chemotherapy drugs, while also inhibiting liver cancer metastasis. This phenomenon also reflects that the nanomedicine FDAH has better biocompatibility than free DOX, and can protect normal tissues from the toxicity of chemotherapy drugs alone.

[0052] In summary, two clinical drugs, doxorubicin (DOX) and plerixafor (AMD3100), were used in combination with ferrous ions to form the core of the nanoparticle FDAH. This core was then encapsulated with a hyaluronic acid (HA) shell to prepare the FDAH nanoparticles. Hyaluronic acid contains numerous carboxyl groups, giving the entire molecule a negative charge. The hyaluronic acid coating can mask the positive charge on the surface of the targeted nanoparticle core. When the nanoparticles reach the tumor microenvironment, the overexpressed hyaluronidase (HAase) in the tumor promotes the degradation of the shell, causing the hyaluronic acid layer to detach and reverse the charge, providing conditions for the nanoparticle core to target and accumulate within the cell. Furthermore, hyaluronic acid, through its specific interaction with the CD44 receptor on the cell surface, can promote the cellular uptake and internalization of the nanoparticles. Therefore, hyaluronic acid can serve as a relatively ideal shell material for targeted nanoparticles.

[0053] Following intravenous injection, the HA shell prolongs the circulation time of nanoparticles in the bloodstream and enables cells to effectively engulf the nanoparticles through the HA / CD44-specific interaction. ROS generated through the iron-induced Fenton reaction leads to an imbalance of redox balance within tumor cells, thereby increasing tumor cell sensitivity to DOX and enhancing chemotherapy efficacy. Furthermore, AMD3100 can also intervene in the CXCL12 / CXCR4 axis, reducing tumor immunosuppression and promoting DOX chemotherapy in tumor cells.

[0054] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a nanomedicine delivery system for treating hepatocellular carcinoma, characterized in that, This includes a nanomedicine delivery system comprising a core and a shell; the core includes an active ingredient and Fe. 2+ The outer shell is hyaluronic acid; the active ingredients are doxorubicin and plexafor; the Fe... 2+ The molar ratio of the active ingredient to the active ingredient is 25:(20-25); The preparation method is as follows: adding the active ingredient to a substance containing Fe... 2+ The solution is stirred and reacted, and then the reacted solution is dispersed in a hyaluronic acid solution for further reaction. Centrifugation yields the nanomedicine delivery system.

2. The preparation method according to claim 1, characterized in that, The Fe 2+ The mass ratio of hyaluronic acid to hyaluronic acid is 1:

10.

3. The preparation method according to claim 1, characterized in that, The stirring reaction was carried out under dark conditions at 600 rpm for 2 hours.

4. The preparation method according to claim 1, characterized in that, The condition for continuing the reaction was stirring at 600 rpm for 24 hours in the dark.

5. A nanomedicine delivery system for treating hepatocellular carcinoma, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

6. Use of the nanomedicine delivery system of claim 5 in the preparation of a medicament for treating hepatocellular carcinoma.

7. A nanomedicine for treating hepatocellular carcinoma, characterized in that, Includes the nanomedicine delivery system of claim 5.

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