Targeted tumor area-based functionalized nano drug loading method and platform

Abstract

The invention discloses a targeted tumor area-based functionalized nano drug loading method and platform, the method can enhance the FUAS curative effect and realize the efficient nano platform of accurate drug targeting, and the method solves the problems that the drug is difficult to target the tumor area in the breast cancer treatment process and the tumor residue exists in the focused ultrasound ablation operation. Precise targeted delivery and controlled release of the drug and a synergistic anti-tumor effect of the drug and FUAS are realized, so that the treatment effect of the breast cancer is remarkably improved. Through the targeting ability of the BF to a tumor hypoxic region, precise delivery of the nano platform is realized, and non-specific distribution of drugs in healthy tissues is effectively reduced. Drug release is triggered through the FUAS, the tumor ablation effect is enhanced, the treatment effect is remarkably improved, and the risk of residual tumors is reduced. PEG and PEI are adopted for modification, so that the biocompatibility of the nanoparticles is improved, and the systematic toxicity of drugs is reduced through a controlled release mechanism of TD.

Description

Technical Field

[0001] The invention relates to the technical field of targeted drug-carrying materials, in particular to a functionalized nano drug-carrying method based on targeting tumor areas. Background Art

[0002] Breast cancer is one of the most common malignant tumors in the world. Although the treatment methods are constantly improving, it still faces many challenges. Currently, the commonly used treatment methods include chemotherapy, radiotherapy and surgery, but chemotherapy has the limitations of drug diffusion throughout the body, large toxic side effects, and difficulty in targeting the tumor area. In addition, focused ultrasound ablation surgery (FUAS), as an emerging non-invasive treatment technology, has shown good prospects in the treatment of breast cancer, but it still has the shortcomings of incomplete ablation, tumor residue and damage to the tissues around the acoustic pathway.

[0003] Therefore, it is of great significance to develop an efficient nanoplatform that can enhance the efficacy of FUAS and achieve precise drug targeting. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a functionalized nano-drug delivery method based on targeting the tumor area, which can enhance the efficacy of FUAS and achieve precise drug targeting.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The functionalized nano drug delivery method based on targeting tumor areas provided by the present invention comprises the following steps: S1. Prepare drug nanoplatforms using mesoporous silica nanoparticles MSNs, and functionalize the mesoporous silica nanoparticles MSNs into polyethylene glycol PEG and polyethyleneimine PEI; S2. Setting up drug loading of astragaloside AS and controlled release mechanism thermosensitive gate molecules on mesoporous silica nanoparticles MSNs: S3. Develop a tumor targeting strategy using electrostatic adsorption to deliver nanoparticles to the target tumor area; S4. Use focused ultrasound ablation surgery FUAS to release drugs to achieve synergistic treatment.

[0006] Furthermore, in step S1, the mesoporous silica nanoparticles MSNs are functionalized to polyethylene glycol PEG and polyethyleneimine PEI, and the specific process is as follows: S11 Synthesis of mesoporous silica nanoparticles MSNs; S12 Preparation of polyethylene glycol PEG modified 3PEG-NHS; Covalent coupling of S13 with mesoporous silica nanoparticles MSNs; S14 preparing a modification solution of polyethyleneimine PEI; S15 The PEG-modified MSNs were mixed with the PEI-modified solution through electrostatic adsorption and covalent bonding; S16 Cleaning and purification: After the reaction is completed, the functionalized modified MSNs are collected by centrifugation and washed with deionized water for multiple times to remove unbound PEI and reaction byproducts, and finally, PEG and PEI dual-modified MSNs are obtained.

[0007] Furthermore, the synthesis of mesoporous silica nanoparticles MSNs in step S11 is carried out in the following manner: mesoporous silica nanoparticles MSNs are prepared by a sol-gel method, with tetraethyl orthosilicate TEOS as a silicon source and cetyltrimethylammonium bromide CTAB as a template, and a nanostructure is formed by hydrolysis under alkaline conditions. After the preparation is completed, the CTAB template is removed by washing with ethanol and calcining at high temperature to obtain MSNs with uniform pore size and high specific surface area.

[0008] Furthermore, in step S2, astragaloside AS is further loaded on the nano-platform, and 1-tetradecanol TD is used as a thermosensitive gate-controlled molecule. The specific process is as follows: The amino-modified PEG-PEI-MSN was dispersed in methanol, subjected to ultrasonic treatment, and then astragaloside IV was added and stirred to obtain a mixture; Then, 1-tetradecanol TD was added, and hot water was added after stirring; then, centrifugation was immediately performed and the supernatant was removed; Finally, TD / astragaloside AS-loaded PEG-PEI-MSNs were dispersed in ice-cold ultrapure water and subjected to a centrifugation-washing cycle to remove all unbound astragaloside AS, and then dispersed in ice-cold ultrapure water under ultrasonication.

[0009] Furthermore, in step S3, a tumor targeting strategy is formulated by electrostatic adsorption to deliver the nanoparticles to the target tumor area. The specific process is as follows: The positively charged PEI-modified nanoparticles are combined with negatively charged drugs through electrostatic adsorption, and the specific targeting ability of the drugs to the hypoxic areas of the tumor is utilized to achieve the precise delivery of the nanoparticles to the tumor area.

[0010] Furthermore, the drug uses astragaloside IV (AS) or Bifidobacterium BF.

[0011] Furthermore, in step S4, the focused ultrasound ablation surgery FUAS is used to release drugs to achieve synergistic treatment, and the specific process is as follows: 1-Tetradecanol TD was used as a thermosensitive gate molecule to achieve controlled release of the drug under FUAS triggering.

[0012] The present invention also provides a functionalized nano-drug delivery platform based on targeting tumor areas, comprising mesoporous silica nanoparticles MSNs whose surfaces are modified with polyethylene glycol PEG and polyethyleneimine PEI, and drugs loaded by the mesoporous silica nanoparticles MSNs; the mesoporous silica nanoparticles MSNs are loaded with 1-tetradecanol TD, and the 1-tetradecanol TD serves as a thermosensitive gate-controlled molecule.

[0013] Furthermore, the drug is any one of astragaloside IV (AS) and Bifidobacterium BF.

[0014] Furthermore, the PEI-modified nanoparticles are positively charged, and the drug is a drug with a negative charge.

[0015] The beneficial effects of the present invention are: The present invention provides a functionalized nano-drug delivery method based on targeting the tumor area, and an efficient nano-platform that can enhance the efficacy of FUAS and achieve precise drug targeting. This method solves the problems of difficulty in targeting drugs to the tumor area during the treatment of breast cancer and tumor residues in focused ultrasound ablation surgery, and achieves precise targeted delivery and controlled release of drugs as well as synergistic anti-tumor effects with FUAS, thereby significantly improving the therapeutic effect of breast cancer.

[0016] Precise targeting: Through the targeting ability of BF to the hypoxic area of ​​the tumor, the precise delivery of the nanoplatform is achieved, effectively reducing the nonspecific distribution of drugs in healthy tissues. Synergistic treatment: By triggering drug release and enhancing the tumor ablation effect through FUAS, the treatment effect is significantly improved and the risk of residual tumors is reduced. High safety: PEG and PEI modification are used to improve the biocompatibility of nanoparticles, and the controlled release mechanism of TD is used to reduce the systemic toxicity of drugs. Multifunctional design: Combining drug delivery, controlled release and acoustic enhancement characteristics, it provides a safe and efficient comprehensive treatment strategy for breast cancer.

[0017] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for explanation.

[0019] Figure 1 It is a functionalized nano-drug delivery method based on targeting the tumor area.

[0020] Figure 2It is a functionalized nano-drug delivery platform based on targeting tumor areas.

[0021] Figure 3 This is a diagram showing the results of an experiment on mice. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0023] Example 1 like Figure 1 As shown, this embodiment provides a functionalized nano drug delivery method based on targeted tumor area. The nano drug delivery platform can achieve precise targeted delivery and controlled release of drugs and synergistic anti-tumor effects with FUAS, thereby significantly improving the therapeutic effect of breast cancer, including the following steps: S1. Preparation of drug nanoplatforms using mesoporous silica nanoparticles (MSNs); In this embodiment, mesoporous silica nanoparticles (MSNs) are functionalized and modified into polyethylene glycol (PEG) and polyethyleneimine (PEI) based on the excellent drug loading capacity and high acoustic impedance characteristics of mesoporous silica nanoparticles (MSNs) to enhance their biocompatibility and surface activity; S11 Synthesis of mesoporous silica nanoparticles (MSNs): Mesoporous silica nanoparticles (MSNs) were prepared by a sol-gel method, with tetraethyl orthosilicate (TEOS) as the silicon source and cetyltrimethylammonium bromide (CTAB) as the template. The nanostructures were formed by hydrolysis under alkaline conditions. After the preparation, the MSNs were washed with ethanol and calcined at high temperature to remove the CTAB template, and obtained MSNs with uniform pore size and high specific surface area. S12 Modification of polyethylene glycol PEG Preparation of 3PEG-NHS: Take a certain amount of polyethylene glycol (PEG) and react it with N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to form terminal activated NHS ester (PEG-NHS); Covalent coupling of S13 with mesoporous silica nanoparticles MSNs: PEG-NHS was added to a MSNs solution dispersed in anhydrous dimethyl sulfoxide (DMSO) and stirred for 12 h. PEG was covalently coupled to the surface hydroxyl groups of MSNs via esterification reaction to form PEG-modified MSNs (PEG-MSNs). S14 Preparation of polyethyleneimine PEI modification solution: Dissolve polyethyleneimine (PEI) in anhydrous DMSO and adjust the pH to neutral.

[0024] S15 Electrostatic adsorption and covalent bonding: PEG-modified MSNs (PEG-MSNs) were mixed with PEI solution and stirred at room temperature for 24 hours. Since PEI molecules have abundant amino groups, they can be adsorbed to the surface of MSNs through electrostatic action. At the same time, glutaraldehyde (GA) was used as a cross-linking agent to promote the covalent bonding between PEI and the surface of MSNs.

[0025] S16 Cleaning and purification: After the reaction, the functionalized MSNs were collected by centrifugation and washed with deionized water for multiple times to remove unbound PEI and reaction byproducts. Finally, PEG and PEI double-modified MSNs (PEG / PEI-MSNs) were obtained.

[0026] Polyethylene glycol (PEG) is added in this embodiment, which has the following characteristics: 1. Shielding immune recognition PEG can form a hydrophobic protective layer to reduce the interaction between the surface of nanoparticles and serum proteins, thereby avoiding rapid clearance by the mononuclear macrophage system (MPS). This feature can significantly prolong the half-life of nanoparticles in the blood circulation.

[0027] 2. Increase solubility and stability The hydrophilic molecular structure of PEG can reduce the aggregation risk of nanoparticles in physiological environments and improve the stability of nanoparticles in complex biological fluids such as blood. This is crucial to ensure the functional integrity of nanoparticles during targeted delivery.

[0028] 3. Reduce toxicity and side effects PEG modification can effectively reduce the nonspecific effects of naked nanoparticles on normal tissues, thereby reducing side effects caused by immune responses or nonspecific adsorption and enhancing the safety of drug treatment.

[0029] 4. Promote tumor targeting effect PEG-modified nanoparticles are more likely to enter tumor tissues through the "enhanced permeability and retention effect" (EPR effect). The protective effect of PEG enables nanoparticles to more efficiently pass through the vascular barrier and accumulate in tumor tissues. ); Polyethyleneimine (PEI) is added in this embodiment, which has the following characteristics: 1 Positive charge guides targeting and cell uptake PEI is a cationic polymer with a high density of amino groups and a positive charge on the surface. These positive charges can interact with the negative charges on the cell membrane to promote the cell adsorption and internalization of nanoparticles. The positive charge property of PEI enhances the cell membrane permeability of nanocarriers, enabling them to enter cells, especially tumor cells, more efficiently.

[0030] 2 Improve drug delivery efficiency Due to the cationic properties of PEI, nanoparticles bind more tightly to drug molecules, thereby improving drug loading capacity. After cell uptake, PEI can help drugs be effectively released into cells or organelles, increase drug bioavailability, and optimize therapeutic effects.

[0031] 3. Increase the stability of nanoparticles in the blood PEI can reduce the aggregation of nanoparticles in the body through the charge shielding effect and increase their stability in the blood. In this way, nanoparticles can maintain a stable state in the body for a longer period of time, avoiding the impact of aggregation on their biodistribution.

[0032] 4 Enhanced targeted therapy effect PEI can also enhance tumor-specific delivery by binding to receptors on the surface of tumor cells. For example, the positive charge of PEI can bind to anion receptors or gene targets on the surface of tumor cells, thereby improving the accuracy of targeted therapy.

[0033] 5 Promote intracellular release and anti-tumor effect PEI, through its cationic properties, helps release drug-loaded nanoparticles from endosomes into the cytoplasm. This process is enhanced by the "protonation" effect of PEI, thereby improving the biological effect of the drug. ); This example uses mesoporous silica nanoparticles (MSNs) as the core carrier, combined with surface modification of polyethylene glycol (PEG) and polyethyleneimine (PEI), to achieve excellent drug loading capacity, biocompatibility and stability; The nanoplatform of this embodiment focuses on biocompatibility and systemic safety in its design, reduces toxic side effects through PEG modification and drug controlled release mechanism, and provides a safer treatment option for breast cancer patients; S2. Drug loading of astragaloside (AS) and thermosensitive gate molecules with controlled release mechanism on mesoporous silica nanoparticles (MSNs): Astragaloside (AS) was further loaded on the nanoplatform, and 1-tetradecanol (TD) was used as a thermosensitive gate molecule to achieve drug encapsulation and controlled release under FUAS triggering, ensuring efficient drug release at the tumor site and avoiding side effects caused by early release. The process of loading astragaloside IV (AS) on the nanoplatform in this embodiment is as follows: 6 mg of amino-modified PEG-PEI-MSN was dispersed in 3 mL of methanol and sonicated, then 1.2 mg of astragaloside IV (AS, dissolved in 3 mL of methanol) was added, and the mixture was gently stirred at 68 °C for 5 h to completely evaporate the methanol. Then, 2 mg of 1-tetradecanol (TD) was added, stirred for another 1 h, and 5 mL of hot water (70 °C) was added; subsequently, centrifugation was immediately performed (1200 rpm, 10 min) at 50 °C, and the supernatant was removed; Finally, TD / AS-loaded PEG-PEI-MSNs were dispersed in ice-cold ultrapure water and subjected to at least six centrifugation-washing cycles to remove all unbound AS, followed by dispersion in ice-cold ultrapure water under sonication (30 s).

[0034] In this embodiment, during the loading process of astragaloside (AS), unlike the modification of PEG and PEI, drug loading mainly occurs in the pores or on the surface of MSNs. The role of PEG is to improve the biocompatibility of nanoparticles so that they can remain in the body for a long time; PEI enhances their affinity with cell membranes by giving the surface of nanoparticles positive charges. The loading of AS mainly depends on the interaction between the pore structure of MSNs and the surface functional groups. The loading of drug AS is mainly physical adsorption, while the modification of PEG / PEI is chemical modification.

[0035] At the same time, the controlled release process and mechanism of the drug are as follows: The role of the thermosensitive gate-controlled molecule TD: 1-Tetradecanol (TD) is a thermosensitive gate-controlled molecule that can undergo phase change when the temperature changes. The principle is based on the fact that TD changes its molecular structure at a specific temperature, thereby affecting the release of the drug. During FUAS treatment, the local temperature of the tumor area will rise, TD will undergo a phase change, and the "gate control" mechanism of the drug will be turned on, allowing the loaded astragaloside (AS) to be released. The specific process is as follows: Room temperature state: At room temperature, TD acts as a gate molecule to firmly seal the drug AS in the pores of MSNs to prevent premature release of the drug.

[0036] FUAS triggering: When the thermal effect of FUAS acts on the tumor area, the local temperature rises to the phase transition temperature of TD, causing the TD molecular structure to change and form pores or channels, thereby allowing the enclosed drugs to be released.

[0037] Drug release: With the phase change of TD, AS detaches from the nanoparticles and enters the tumor tissue for local treatment. At this time, the release rate and amount of the drug can be precisely controlled by the thermal effect of FUAS.

[0038] The controlled release mechanism of the drug uses thermal effects to drive release: FUAS triggers a local temperature increase, which promotes the TD phase transition and the release of astragaloside (AS). Since the release of AS is controlled by temperature, the drug can be treated more concentratedly at the tumor site, avoiding the side effects of systemic release.

[0039] Improve treatment efficiency: Through the controlled release mechanism, AS can continue to act at the tumor site, increase local drug concentration, and thus enhance the anti-tumor effect of the drug.

[0040] Prolonged therapeutic effect: Due to the controlled drug release, the therapeutic effect can be prolonged, helping to reduce the risk of tumor recurrence.

[0041] S3. Develop a tumor targeting strategy using electrostatic adsorption to deliver nanoparticles to the target tumor area; The positively charged PEI-modified nanoparticles are combined with the negatively charged probiotics **Bifidobacterium bifidum (BF)** by electrostatic adsorption, and the specific targeting ability of BF to the hypoxic area of ​​the tumor is used to achieve the precise delivery of nanoparticles to the tumor area; Bifidobacterium (BF) injection and enrichment in tumor areas; 1 Bifidobacterium injection: To ensure that bifidobacteria can be enriched in the tumor area, 200 μL of bifidobacterium suspension (concentration of 1×10 6 CFU / mL) was injected into the experimental animals. This injection was performed 3 days in advance to ensure that Bifidobacterium could proliferate and accumulate in the tumor area.

[0042] 2 Enrichment process verification: Through tissue homogenization experiments, the enrichment of bifidobacteria in the tumor area was verified. The results showed that bifidobacteria can accumulate in large quantities in the tumor area, especially in the hypoxic area of ​​the tumor. This feature enables BF to be used as a targeted delivery system to accurately deliver subsequent nanoparticles to tumor tissues.

[0043] 3 Injection of PEI-modified nanoparticles and electrostatic adsorption with BF; 4 PEI-modified nanoparticle injection: On the third day after the bifidobacterium injection, 200 µL of nanoparticle suspension (concentration of 200 µg / mL) was injected through the tail vein, allowing the nanoparticles to enter the body through the blood circulation. The surface of PEI-modified nanoparticles has a positive charge, so they can bind to the negatively charged bifidobacteria previously enriched in the tumor site through electrostatic adsorption.

[0044] 5 Electrostatic adsorption binding: Since the cell wall of bifidobacteria contains abundant negatively charged groups (such as fatty acids and phosphate groups), and the PEI-modified nanoparticles are positively charged, the two are tightly bound by electrostatic action. This combination ensures that the drug carrier can accurately locate and enter the tumor area, thereby achieving precise drug delivery.

[0045] Targeted delivery is validated as follows: 1 In vivo fluorescence imaging: The combination of nanoparticles and bifidobacteria and their enrichment in the tumor area were verified through in vivo fluorescence imaging technology. The results showed that the injected nanoparticles effectively combined with bifidobacteria enriched in the tumor site, and the nanoparticles were able to accurately target the tumor area.

[0046] 2 Frozen section analysis: The aggregation of nanoparticles in the tumor area was further verified by frozen section and fluorescence microscopy observation. The intensity of the fluorescence signal showed that the nanoparticles could effectively accumulate in the tumor tissue, and the combination with bifidobacteria improved the efficiency of targeted delivery. S4. Utilize focused ultrasound ablation surgery (FUAS) to release drugs for synergistic therapy; Triggered by the thermal effect of focused ultrasound ablation surgery (FUAS), 1-tetradecanol (TD) undergoes a phase change to release drugs, while the acoustic impedance characteristics of nanoparticles are used to improve the tumor ablation efficiency of FUAS, thereby significantly enhancing the comprehensive effect of breast cancer treatment. By introducing 1-tetradecanol (TD) as a thermosensitive gate-controlled molecule, controlled release of drugs is achieved under FUAS triggering, avoiding early leakage of drugs and ensuring efficient action of drugs at the tumor site.

[0047] Thermosensitive phase change principle of TD in this embodiment: 1-Tetradecanol (TD) is a molecule with thermal response characteristics, which can undergo phase change at a specific temperature. At room temperature, TD molecules are in a liquid state and can block the pores or surfaces of drug-loaded nanoparticles to prevent premature release of drugs. When the local temperature rises to the phase change temperature of TD (usually between 40°C and 45°C), the TD molecules undergo a phase change, thereby changing their molecular structure, forming pores or channels, and promoting the release of drug-loaded molecules.

[0048] In this embodiment, the FUAS thermal effect triggers drug release FUAS heating effect: Focused ultrasound ablation surgery (FUAS) heats the tumor area through focused ultrasonic energy. During the treatment process, the local temperature of the tumor will rise rapidly to a range of 40°C to 45°C, which is high enough to trigger the phase change of TD molecules.

[0049] In this embodiment, the drug is released after the TD phase change: when the TD molecule undergoes a phase change, the originally closed nanoparticle pores are opened, and drugs such as astragaloside (AS) begin to be released. The speed and amount of release can be precisely controlled according to the treatment time of FUAS, the change in temperature, and the phase change characteristics of TD. The release of the drug is controlled, and early leakage will not occur, ensuring that the drug is maximized at the tumor site.

[0050] Among them, the gate control effect of TD is used to avoid early leakage of drugs: 1-tetradecanol (TD) is used as a thermosensitive gate control molecule to ensure that the drug will not be non-specifically distributed in the body or leak in advance before treatment. Only when the local temperature of the tumor area rises to the trigger temperature will the drug be accurately released. This mechanism greatly improves the safety and efficiency of treatment and avoids the side effects of drugs on normal tissues.

[0051] This embodiment utilizes the specific targeting ability of Bifidobacterium (BF) to the hypoxic area of ​​the tumor, and accurately delivers the nanoparticles to the tumor site through electrostatic adsorption, which greatly improves the accuracy of treatment and avoids the nonspecific distribution of the drug; and achieves accurate targeting of the tumor area; This embodiment uses this method to perform FUAS-based collaborative treatment, combining focused ultrasound ablation surgery (FUAS) with nano-drug delivery, triggering drug release through the thermal effect of FUAS, enhancing the tumor ablation effect, and achieving controlled release of drugs at the same time, effectively solving the shortcomings of FUAS single treatment mode; The method provided in this embodiment can realize multi-modal treatment integration, and combines the nano-platform with tumor targeting, drug controlled release and acoustic enhancement functions, which significantly improves the comprehensive effect of breast cancer treatment and provides a safe, efficient and multifunctional treatment strategy; it has the characteristics of high biosafety and has broad clinical potential. It is not only suitable for the treatment of breast cancer, but also provides technical support for the combined treatment of other solid tumors, and has broad application prospects.

[0052] Example 2 The present embodiment provides a functionalized nano-drug delivery platform based on targeting tumor areas, comprising mesoporous silica nanoparticles (MSNs) whose surfaces are modified by polyethylene glycol (PEG) and polyethyleneimine (PEI) and drugs; the mesoporous silica nanoparticles MSNs are loaded with 1-tetradecanol TD, and the 1-tetradecanol TD serves as a thermosensitive gating molecule.

[0053] The drug is any one of astragaloside IV (AS) and Bifidobacterium BF; The drug provided in this embodiment has an anti-tumor effect: Astragaloside IV (AS) has significant anti-tumor activity. Studies have shown that astragaloside IV can inhibit the growth, migration and invasion of tumor cells through multiple mechanisms, including inhibiting the proliferation of tumor cells, inducing cell apoptosis, and inhibiting the development of tumors by regulating the tumor microenvironment.

[0054] Immunomodulatory effect Astragaloside IV has a strong immunomodulatory effect and can enhance the body's immune function. It enhances the immune system's recognition and clearance of tumor cells by promoting the functions of immune cells such as macrophages and T cells. In addition, astragaloside IV can also enhance the body's immune response to tumor cells by regulating the secretion of cytokines.

[0055] Antioxidant Effect Astragaloside IV has antioxidant activity, which can scavenge free radicals in the body and slow down cellular oxidative damage. This property helps protect healthy cells from oxidative stress and may play an important role in anti-tumor therapy.

[0056] Anti-inflammatory effect Astragaloside IV shows a positive effect in anti-inflammatory aspects. It can reduce chronic inflammatory response and alleviate tissue damage caused by tumors or other diseases by inhibiting the release of inflammatory factors. By reducing the level of inflammation in the tumor microenvironment, astragaloside IV helps to improve the therapeutic effect and reduce side effects.

[0057] Improving treatment tolerance Studies have found that astragaloside IV can improve patients' tolerance to treatment and prolong the effective period of treatment by reducing the side effects caused by chemotherapy or radiotherapy (such as immunosuppression, bone marrow suppression, etc.). It can enhance the repair ability of healthy cells in the body and promote anti-tumor immune response.

[0058] Angiogenesis inhibition effect Astragaloside IV also has the effect of inhibiting tumor angiogenesis, which can inhibit tumor growth and metastasis by interfering with the formation of tumor blood vessels. This effect helps to reduce the nutrient supply of the tumor and promote the death of tumor cells.

[0059] Pharmacokinetic properties Astragaloside IV has a high bioavailability and can be effectively absorbed by oral administration or injection, enter the blood circulation and quickly distribute throughout the body. Astragaloside IV has certain water solubility and fat solubility, it can quickly reach an effective concentration in the body and exert its pharmacological effects. The targeting and bioavailability of Astragaloside IV can be further improved by modifying the nanocarrier.

[0060] The mesoporous silica nanoparticles have the following characteristics: High specific surface area and large pore volume: Mesoporous silica nanoparticles have a significantly high specific surface area (usually between 700–1000 m² / g) and large pore volume. This enables MSNs to provide a large amount of space to load drugs, especially suitable for loading macromolecular drugs (such as astragaloside IV). By adjusting the synthesis conditions, the pore size can be controlled (usually in the range of 2-10 nm), giving it a high drug loading capacity.

[0061] Adjustable pore size and functionalized surface: MSNs have adjustable pore size, usually in the range of 2–10 nm, which can optimize the loading according to the molecular size of the drug. Through surface functionalization, the biocompatibility, stability and targeting of nanoparticles can be improved. For example, through polyethylene glycol (PEG) and polyethyleneimine (PEI) modification, MSNs can remain stable in vivo for a long time and increase the interaction with target cells. For the loading of drugs such as astragaloside (AS), the pore size and surface functionalization characteristics of MSNs give it unique advantages in the precise delivery and controlled release of drugs.

[0062] High drug loading capacity: Due to the high specific surface area and adjustable pore size, MSNs have a high drug loading capacity. Through optimized porosity and functional modification, MSNs can effectively load drugs, especially for natural drugs such as astragaloside (AS), which has a large molecular weight and poor water solubility. MSNs can improve the bioavailability of astragaloside IV and ensure its effectiveness during treatment.

[0063] Size requirements: For the astragaloside (AS) drug delivery system in this study, the particle size of the MSNs used should be between 100nm and 150nm. This size can maintain a good blood circulation time and effectively target the tumor site.

[0064] Biocompatibility and low toxicity: MSNs are highly biocompatible and can reduce immune and toxic reactions. Through surface modification, MSNs can further improve their compatibility with the biological environment. For example, PEG-modified MSNs can avoid rapid clearance by the mononuclear macrophage system (MPS) and prolong their circulation time in the body. For the delivery of astragaloside IV, the biocompatibility of MSNs ensures the safety of the drug in the body and reduces side effects.

[0065] Controlled drug release: MSNs achieve controlled drug release through surface functionalization, such as the thermosensitive gate molecule 1-tetradecanol (TD). This feature is particularly important for the delivery of astragaloside (AS) because it ensures the precise release of the drug at the tumor site and avoids side effects caused by early release. The combination of MSNs and TD allows precise control of drug release, effectively releasing astragaloside (AS) under the triggering of the thermal effect of FUAS (focused ultrasound ablation surgery).

[0066] Synthesis and functionalization of nanoparticles in this example: mesoporous silica nanoparticles were prepared and modified with PEG and PEI to give them good biocompatibility and surface positive properties; 1 Polyethylene glycol (PEG) modification - imparts good biocompatibility; **Polyethylene glycol (PEG)** is a commonly used surface modification molecule that can improve the biocompatibility of nanoparticles, reduce the recognition and clearance of the immune system, and prolong the circulation time of nanoparticles. The specific modification process is as follows: Synthesis and activation of PEG: PEG molecules can be activated through amino (–NH2) or carboxyl (–COOH) groups. PEG-NHS (PEG terminal activation) or PEG-COOH is usually used, where PEG-NHS can form a covalent bond through esterification reaction with the hydroxyl (–OH) on the surface of MSNs.

[0067] PEG modification process: PEG-NHS is dissolved in a suitable solvent (such as deionized water or DMSO), and then added to the MSNs solution for about 12 hours. PEG forms a stable covalent bond by esterification with the siloxy groups (–SiOH) on the surface of MSNs, thereby successfully connecting the PEG molecules to the surface of MSNs. PEG molecules form a hydrophilic protective layer on the surface of nanoparticles, effectively avoiding nonspecific adsorption and immune recognition of nanoparticles in the body.

[0068] Functions and effects: Enhanced biocompatibility: After PEG modification, the surface of MSNs carries hydrophilic PEG chains, which can improve the stability of nanoparticles and reduce their aggregation in the blood.

[0069] Reduce immune response: PEG-modified nanoparticles can avoid being recognized and cleared by the mononuclear macrophage system (MPS), prolonging their circulation time in the body, thereby improving the effectiveness of the drug.

[0070] 2. Polyethyleneimine (PEI) modification - giving the surface positive electrical properties **Polyethyleneimine (PEI)** is a cationic polymer with rich amino groups, which can give the surface of nanoparticles positive charges. The positive charge enhances the interaction between nanoparticles and cell membranes or negatively charged biomolecules, promoting the cellular uptake of nanoparticles. The specific modification process is as follows: PEI dissolution and preparation: Dissolve a certain amount of PEI in deionized water or an appropriate solvent. The solution concentration is generally 0.1–1 mg / mL. Ensure that the solution is uniform.

[0071] PEI modification process: Add PEI solution to the PEG-modified MSNs solution and stir for 24 hours to allow PEI to form a stable bond with the surface of the nanoparticles through electrostatic adsorption. Since PEI molecules carry a large number of amino groups (–NH2), these amino groups can adsorb with negatively charged groups (such as Si–O–) on the surface of the nanoparticles to form a positively charged surface.

[0072] Cross-linking agent assistance: In order to enhance the binding force between PEI and nanoparticles, a cross-linking agent such as glutaraldehyde (GA) can be added to further promote the covalent binding between PEI and the surface of MSNs.

[0073] Functions and effects: Imparting positive charge to the surface: After PEI modification, the surface of the nanoparticles carries a large amount of positive charge, which helps to have a strong electrostatic interaction with the negatively charged area of ​​the cell membrane and promotes cell endocytosis. Especially in targeted delivery, positively charged nanoparticles can bind to tumor cells or receptors more effectively.

[0074] Enhanced cell uptake and targeting: PEI modification with positive surface charge can increase the affinity of nanoparticles to tumor cells and promote their entry into cells through endocytosis of the cell membrane, thereby enhancing the effect of targeted therapy.

[0075] 3. Advantages of combined modification of PEG and PEI By combining PEG and PEI for surface modification, MSNs gain the advantages of both: The hydrophilic protective layer of PEG can effectively improve the biocompatibility and stability of MSNs and prolong their circulation time in the body.

[0076] The positive charge of PEI enhances the cell affinity and endocytosis ability of nanoparticles, improving the efficiency of targeted drug delivery.

[0077] This dual modification not only enhances the drug loading capacity and targeting of MSNs, but also avoids the problems of uneven drug distribution and side effects caused by the lack of targeting in traditional nanocarriers.

[0078] Drug loading and gate-controlled encapsulation in this embodiment: AS is loaded into MSNs through physical adsorption and chemical modification technology, and TD is used as a thermosensitive gate-controlled molecule for encapsulation; The drug loading and gate-controlled packaging of this embodiment are as follows: Drug loading: loading of astragaloside IV (AS), which is an active ingredient extracted from the traditional Chinese medicine Astragalus, has significant anti-tumor, immunomodulatory, antioxidant and other multiple biological effects. In this example, AS is loaded into mesoporous silica nanoparticles (MSNs) as a therapeutic drug, and the specific loading process is as follows: Dissolving astragaloside IV (AS): First, a certain amount of astragaloside IV (AS) is dissolved in an appropriate solvent (e.g., deionized water or PBS buffer). For example, 4 mg of AS can be dissolved in 4 mL of deionized water to obtain an AS solution with a suitable concentration. By adjusting the concentration of AS, the drug loading can be optimized.

[0079] Preparation of nanoparticles: Prepare surface-modified mesoporous silica nanoparticles (MSNs), which usually have a pore size range of 2–10 nm and a specific surface area of ​​700–1000 m² / g, providing sufficient space to load drugs. MSNs can be synthesized by the sol-gel method and modified with PEG and PEI to ensure good biocompatibility and surface positive properties.

[0080] Drug loading process: Add the dissolved AS solution to the MSNs solution and mix them in a certain ratio (for example, the mass ratio of AS to MSNs is 1:5). Through stirring and ultrasonic treatment, astragaloside (AS) enters the pores of MSNs by physical adsorption. This process usually takes 12 hours to ensure that the drug molecules can fully enter the pores of the nanoparticles and interact with the surface functional groups of the nanoparticles. The loading efficiency can be optimized by adjusting the solution concentration, stirring time, temperature and other conditions.

[0081] Loading effect verification: HPLC detection; The specific process of drug loading and gate-controlled packaging is as follows: Preparation and dispersion of PEG-PEI-MSN: Take 6 mg of amino-modified PEG-PEI-MSN and disperse it in 3 mL of methanol. Ultrasonic treatment is used to ensure that the nanoparticles are fully dispersed and a stable solution is formed.

[0082] Dissolution and addition of astragaloside IV (AS): 1.2 mg of astragaloside IV (AS) was dissolved in 3 mL of methanol to prepare AS solution. Then, the AS solution was added to the PEG-PEI-MSN solution and mixed thoroughly. Ultrasonic treatment helped the AS molecules to better enter the pores or surface of the nanoparticles and be loaded onto the nanoparticles by physical adsorption.

[0083] Solvent evaporation and drug loading: The above mixture was gently stirred at 68°C for 5 hours to allow the methanol to evaporate completely. During this process, astragaloside (AS) was firmly loaded on the surface or in the pores of PEG-PEI-MSN by physical adsorption.

[0084] Addition and encapsulation of 1-tetradecanol (TD): Add 2 mg of 1-tetradecanol (TD) to the drug-loaded PEG-PEI-MSN solution and stir again for 1 hour. TD, as a thermosensitive gate-controlled molecule, can undergo phase change at a specific temperature to control the release of the drug. At this point, the TD molecule will be encapsulated on the surface or pores of the nanoparticles to ensure the stable encapsulation of astragaloside (AS).

[0085] Hot water addition and centrifugal separation: 5 mL of hot water (70°C) was added to the mixed solution to help TD further bind to the nanoparticles and promote the formation of a stable encapsulation structure between the drug and TD. Subsequently, centrifugation was immediately performed (1200 rpm, 10 minutes) at a temperature of 50°C, and the supernatant was removed to obtain TD / astragaloside (AS)-loaded PEG-PEI-MSN complexes.

[0086] Washing and purification: The centrifuged PEG-PEI-MSN complex was dispersed in ice-cold ultrapure water and centrifuged and washed at least six times to remove unbound astragaloside (AS). This process ensures that there is no excess free drug in the complex and maintains the purity of the nanoparticles. After each wash, the supernatant was removed by centrifugation to ensure that the drug was completely loaded in the nanoparticles.

[0087] Ultrasonic treatment and dispersion: Finally, the purified TD / astragaloside (AS)-loaded PEG-PEI-MSNs were dispersed in ice-cold ultrapure water and ultrasonicated (30 s) to ensure complete dispersion of the nanoparticles and prevent particle aggregation to obtain a uniformly dispersed nanoparticle suspension.

[0088] Advantages of Drug Loading and Gate-Controlled Packaging Improving drug loading capacity: Through the combination of physical adsorption and chemical modification, MSNs can effectively load astragaloside (AS) and ensure the stability and controlled release of the drug through TD encapsulation.

[0089] Prolong drug release time: The gate control effect of TD ensures that the drug can be continuously released in the tumor area, increasing the intensity of the drug's effect on tumor cells during the treatment process.

[0090] Reduced side effects: Since the drug can be precisely released at the tumor site, systemic side effects and non-specific drug exposure are greatly reduced, thereby improving the safety of treatment. Targeted binding experiment in this example: functionalized nanoparticles were mixed with BF and bound by electrostatic adsorption to confirm their binding efficiency and stability; The method for confirming the binding efficiency and stability in this example is as follows: The efficiency and stability of the binding between the nanoparticles and BF are confirmed mainly by the following two experimental methods: laser confocal microscopy imaging and flow cytometry (FCM) detection.

[0091] Confocal laser scanning microscopy imaging: To observe the binding of NPs to BF, fluorescently labeled NPs and BF were used for observation by confocal microscopy (CLSM): DII-labeled NPs: First, the NPs were labeled with DII dye, which enabled them to emit red fluorescence under confocal laser scanning microscopy. FITC-labeled BF: Bifidobacterium (BF) was labeled with FITC dye, which enabled them to emit green fluorescence under confocal laser scanning microscopy.

[0092] Confocal microscopy observation: The complex of the labeled nanoparticles and bifidobacteria is observed under a laser confocal microscope. The overlap of fluorescence signals is used to observe whether the nanoparticles effectively bind to the bifidobacteria. If the complex successfully binds, the red fluorescent nanoparticles and the green fluorescent BF will overlap and appear on the cell surface.

[0093] Flow cytometry (FCM): In order to quantitatively detect the binding efficiency of nanoparticles and BF, flow cytometry can be used for analysis: Flow cytometry pretreatment: After the labeled and mixed complexes are resuspended in PBS buffer, flow cytometry is performed. The fluorescence intensity of FITC-labeled BF and DII-labeled nanoparticles is detected by flow cytometry.

[0094] Binding efficiency analysis: Based on the overlap of fluorescence signals, flow cytometry can analyze the efficiency of nanoparticle binding to BF. Binding efficiency is quantitatively analyzed by calculating the proportion of cells with both red and green fluorescence on the surface of BF cells.

[0095] Stability test: Flow cytometry can also be used to test the stability of the complex. By sampling at regular intervals, observe whether the binding of nanoparticles to BF is stable, especially the distribution changes after the binding of nanoparticles to BF, and whether there is dissociation or other instability. Analysis of binding efficiency and stability results Binding efficiency: The results obtained by flow cytometry can show the binding efficiency of the complex, which is usually determined by calculating the ratio of cells with red and green fluorescence. If the complex is stable and successfully bound, the overlapping area of ​​red and green will be more obvious.

[0096] Stability analysis: The stability of the combination of nanoparticles and BF can be evaluated by dynamically monitoring the changes in the complex (such as the distribution inside and outside the cell after binding, changes in the intensity of the fluorescence signal, etc.). If the fluorescence intensity of the complex does not change significantly within a certain period of time, it means that the combination of nanoparticles and BF is relatively stable.

[0097] like Figure 3 As shown, Figure 3The following is a graph showing the effect of an experiment on mice. The results of the functionalized nano-drug delivery platform based on targeted tumor areas provided in this embodiment are as follows: If strong fluorescence overlap between nanoparticles and BF is observed under a laser confocal microscope, and flow cytometry analysis shows that the binding efficiency reaches the expected value (e.g., more than 80%), it can be confirmed that the binding of nanoparticles and BF is successful. In terms of stability, if the complex does not dissociate after a period of time and the results in flow cytometry are stable, it indicates that it has good stability in the body and can provide a guarantee for subsequent drug delivery.

[0098] In vitro release experiment of this example: simulate FUAS triggering conditions to verify the phase change release behavior and drug controlled release effect of TD under thermal effect; in vivo experiment: inject functionalized nanoparticles into mouse breast cancer model, combined with FUAS for treatment, observe tumor ablation rate and drug distribution. Tumor inhibition of simple FUAS group and FUAS+Bifidobacterium+nanoparticle group.

[0099] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A functionalized nano drug delivery method based on targeting tumor areas, characterized in that: The following steps are involved: S1. Prepare drug nanoplatforms using mesoporous silica nanoparticles MSNs, and functionalize the mesoporous silica nanoparticles MSNs into polyethylene glycol PEG and polyethyleneimine PEI; S2. Setting up drug loading of astragaloside AS and controlled release mechanism thermosensitive gate molecules on mesoporous silica nanoparticles MSNs: S3. Develop a tumor targeting strategy using electrostatic adsorption to deliver nanoparticles to the target tumor area; S4. Use focused ultrasound ablation surgery FUAS to release drugs to achieve synergistic treatment.

2. The functionalized nano drug delivery method based on targeting tumor area according to claim 1, characterized in that: In step S1, mesoporous silica nanoparticles MSNs are functionalized to polyethylene glycol PEG and polyethyleneimine PEI, and the specific process is as follows: S11 Synthesis of mesoporous silica nanoparticles MSNs; S12 Preparation of polyethylene glycol PEG modified 3PEG-NHS; Covalent coupling of S13 with mesoporous silica nanoparticles MSNs; S14 preparing a modification solution of polyethyleneimine PEI; S15 The PEG-modified MSNs were mixed with the PEI-modified solution through electrostatic adsorption and covalent bonding; S16 Cleaning and purification: After the reaction is completed, the functionalized modified MSNs are collected by centrifugation and washed with deionized water for multiple times to remove unbound PEI and reaction byproducts, and finally, PEG and PEI dual-modified MSNs are obtained.

3. The functionalized nano drug delivery method based on targeting tumor area according to claim 2, characterized in that: The synthesis of mesoporous silica nanoparticles MSNs in step S11 is carried out in the following manner: mesoporous silica nanoparticles MSNs are prepared by a sol-gel method, tetraethyl orthosilicate TEOS is used as a silicon source, hexadecyltrimethylammonium bromide CTAB is used as a template, and a nanostructure is formed by hydrolysis under alkaline conditions. After the preparation is completed, ethanol washing and high-temperature calcination are used to remove the CTAB template to obtain MSNs with uniform pore size and high specific surface area.

4. The functionalized nano drug delivery method based on tumor region targeting according to claim 1, characterized in that: In step S2, astragaloside AS is further loaded on the nano-platform, and 1-tetradecanol TD is used as a thermosensitive gate-control molecule. The specific process is as follows: The amino-modified PEG-PEI-MSN was dispersed in methanol, subjected to ultrasonic treatment, and then astragaloside IV was added and stirred to obtain a mixture; Then, 1-tetradecanol TD was added, and hot water was added after stirring; then, centrifugation was immediately performed and the supernatant was removed; Finally, TD / astragaloside AS-loaded PEG-PEI-MSNs were dispersed in ice-cold ultrapure water and subjected to a centrifugation-washing cycle to remove all unbound astragaloside AS, and then dispersed in ice-cold ultrapure water under ultrasonication.

5. The functionalized nano drug delivery method based on targeting tumor area according to claim 1, characterized in that: In step S3, a tumor targeting strategy is formulated by electrostatic adsorption to deliver the nanoparticles to the target tumor area. The specific process is as follows: The positively charged PEI-modified nanoparticles are combined with negatively charged drugs through electrostatic adsorption, and the specific targeting ability of the drugs to the hypoxic areas of the tumor is utilized to achieve the precise delivery of the nanoparticles to the tumor area.

6. The functionalized nano drug delivery method based on targeting tumor area according to claim 5, characterized in that: The medicine adopts astragaloside AS or bifidobacterium BF.

7. The functionalized nano drug delivery method based on targeting tumor area according to claim 1, characterized in that: In step S4, focused ultrasound ablation surgery FUAS is used to release drugs to achieve synergistic treatment, and the specific process is as follows: 1-Tetradecanol TD was used as a thermosensitive gating molecule to achieve controlled release of drugs under the triggering of focused ultrasound ablation surgery FUAS.

8. A functionalized nano drug delivery platform based on targeting tumor areas, characterized in that: The invention comprises mesoporous silica nanoparticles MSNs whose surfaces are modified by polyethylene glycol PEG and polyethyleneimine PEI, and drugs loaded by the mesoporous silica nanoparticles MSNs; the mesoporous silica nanoparticles MSNs are loaded with 1-tetradecanol TD, and the 1-tetradecanol TD is used as a thermosensitive gate control molecule.

9. The functionalized nano drug delivery platform based on tumor region targeting according to claim 8, characterized in that: The drug is any one of astragaloside AS and Bifidobacterium BF.

10. The functionalized nano drug delivery platform based on tumor region targeting according to claim 8, characterized in that: The PEI modified nanoparticles are positively charged, and the drug is a drug with a negative charge.

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