A functionalized nano-drug delivery method and platform based on targeting tumor regions

Abstract

This invention discloses a functionalized nanoparticle drug delivery method and platform based on targeted tumor regions. This method enhances the efficacy of FUAS (Fusion-Assisted Assay) and achieves a highly efficient nanoplatform for precise drug targeting. This method addresses the challenges of drug targeting of tumor regions during breast cancer treatment and the tumor residue issues associated with focused ultrasound ablation surgery. It achieves precise targeted drug delivery, controlled release, and synergistic anti-tumor effects with FUAS, thereby significantly improving the treatment efficacy for breast cancer. The precise delivery of the nanoplatform is achieved through the targeting ability of BF (Body-Focused Flow) to hypoxic tumor regions, effectively reducing non-specific drug distribution in healthy tissues. FUAS triggers drug release and enhances the tumor ablation effect, significantly improving treatment efficacy and reducing the risk of residual tumor. PEG and PEI modification improves the biocompatibility of the nanoparticles, and the controlled-release mechanism of TD (Digital Transmission Therapy) reduces systemic drug toxicity.

Description

Technical Field

[0001] This invention relates to the field of targeted drug delivery materials technology, and in particular to a functionalized nano-drug delivery method based on targeting tumor regions. Background Technology

[0002] Breast cancer, one of the most common malignant tumors worldwide, faces numerous challenges despite continuous advancements in treatment methods. Currently used treatments include chemotherapy, radiotherapy, and surgery. However, chemotherapy has limitations such as systemic drug diffusion, significant toxic side effects, and difficulty in targeting specific tumor areas. Furthermore, focused ultrasound ablation surgery (FUAS), as an emerging non-invasive treatment technique, has shown promising promise in breast cancer treatment, but it still suffers from incomplete ablation, tumor residue, and damage to surrounding tissues.

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

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

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The functionalized nano-drug delivery method based on targeted tumor regions provided by this invention includes the following steps:

[0007] S1. A drug nanoplatform was prepared using mesoporous silica nanoparticles (MSNs). The mesoporous silica nanoparticles (MSNs) were functionalized and modified into polyethylene glycol (PEG) and polyethyleneimine (PEI).

[0008] S2. Astragaloside A (AS) and a thermosensitive gated molecule for controlled release are loaded onto mesoporous silica nanoparticles (MSNs):

[0009] S3. A tumor-targeting strategy is developed using electrostatic adsorption to deliver nanoparticles to the target tumor region;

[0010] S4. Utilize focused ultrasound ablation surgery to release drugs via FUAS for synergistic treatment.

[0011] Furthermore, in step S1, the mesoporous silica nanoparticles (MSNs) are functionalized with polyethylene glycol (PEG) and polyethyleneimine (PEI). The specific process is as follows:

[0012] S11 Synthesis of Mesoporous Silica Nanoparticles (MSNs);

[0013] S12 Preparation of modified 3PEG-NHS for polyethylene glycol PEG;

[0014] Covalent coupling of S13 with mesoporous silica nanoparticles (MSNs);

[0015] S14 Preparation of a modified solution of polyethyleneimine (PEI);

[0016] S15 mixes PEG-modified MSNs with PEI-modified solutions via electrostatic adsorption and covalent bonding.

[0017] S16 Cleaning and Purification: After the reaction, the functionalized MSNs were collected by centrifugation and washed repeatedly with deionized water to remove unbound PEI and reaction byproducts. Finally, MSNs modified with both PEG and PEI were obtained.

[0018] Furthermore, the synthesis of mesoporous silica nanoparticles (MSNs) in step S11 is carried out as follows: mesoporous silica nanoparticles (MSNs) are prepared using the sol-gel method, with tetraethyl orthosilicate (TEOS) as the silicon source and hexadecyltrimethylammonium bromide (CTAB) as the template agent. The nanostructure is formed by hydrolysis under alkaline conditions. After preparation, the nanostructure is washed with ethanol and calcined at high temperature to remove the CTAB template, thereby obtaining MSNs with uniform pore size and high specific surface area.

[0019] Furthermore, in step S2, astragaloside A (AS) is further loaded onto the nanoplatform, and 1-tetradecanoic acid (TD) is used as a thermosensitive gate molecule. The specific process is as follows:

[0020] Amino-modified PEG-PEI-MSN was dispersed in methanol, sonicated, and then astragaloside A was added and stirred to obtain a mixture.

[0021] Then add 1-tetradecyl alcohol TD, stir, and then add hot water; subsequently, immediately centrifuge and remove the supernatant;

[0022] Finally, the TD / astragaloside A AS-loaded PEG-PEI-MSN was dispersed in ice-cold ultrapure water, and all unbound astragaloside A AS was removed by centrifugation-washing cycle, followed by ultrasonication to disperse it in ice-cold ultrapure water.

[0023] Furthermore, in step S3, an electrostatic adsorption method is used to formulate a tumor-targeting strategy to deliver nanoparticles to the target tumor region. The specific process is as follows:

[0024] By combining positively charged PEI-modified nanoparticles with negatively charged drugs through electrostatic adsorption, the precise delivery of nanoparticles to the tumor region can be achieved by utilizing the drug's specific targeting ability to the hypoxic area of ​​the tumor.

[0025] Furthermore, the drug is astragaloside A (AS) or Bifidobacterium BF.

[0026] Furthermore, in step S4, focused ultrasound ablation surgery is used to release drugs via FUAS to achieve synergistic treatment. The specific process is as follows:

[0027] Using 1-tetradecanool TD as a thermosensitive gated molecule, controlled drug release is achieved under FUAS triggering.

[0028] The present invention also provides a functionalized nano-drug delivery platform based on targeting tumor regions, comprising mesoporous silica nanoparticles (MSNs) with surfaces modified by polyethylene glycol (PEG) and polyethyleneimine (PEI), and a drug loaded on the mesoporous silica nanoparticles (MSNs); wherein the mesoporous silica nanoparticles (MSNs) are loaded with 1-tetradecanoic acid TD, and the 1-tetradecanoic acid TD serves as a thermosensitive gate molecule.

[0029] Furthermore, the drug is either astragaloside A (AS) or Bifidobacterium BF.

[0030] Furthermore, the PEI-modified nanoparticles are positively charged, and the drug is a negatively charged drug.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention provides a functionalized nano-drug delivery method based on targeting tumor regions, and a highly efficient nanoplatform that can enhance the efficacy of FUAS and achieve precise drug targeting. This method solves the problems of difficulty in targeting tumor regions with drugs during breast cancer treatment and tumor residue in focused ultrasound ablation surgery. It achieves precise targeted delivery and controlled release of drugs, as well as synergistic anti-tumor effects with FUAS, thereby significantly improving the treatment effect of breast cancer.

[0033] Precise Targeting: Utilizing the targeting capability of BF (Body-Fluid Fiber) to hypoxic tumor regions, the nanoplatform achieves precise delivery, effectively reducing non-specific drug distribution in healthy tissues. Synergistic Therapy: FUAS (Fusion-Ablation-Induced Synthesis) triggers drug release and enhances tumor ablation effects, significantly improving treatment efficacy and reducing the risk of residual tumor. High Safety: PEG and PEI modification improves the biocompatibility of the nanoparticles, and the controlled-release mechanism of TD (Digital Transmission Device) reduces systemic drug toxicity. Multifunctional Design: Combining drug delivery, controlled release, and acoustic enhancement properties, this provides a safe and efficient comprehensive treatment strategy for breast cancer.

[0034] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0035] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration.

[0036] Figure 1 This is a functionalized nano-drug delivery method based on targeting tumor regions.

[0037] Figure 2 This is a functionalized nano-drug delivery platform based on targeting tumor regions.

[0038] Figure 3 This is a diagram showing the results of an experiment using mice. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0040] Example 1

[0041] like Figure 1 As shown in this embodiment, a functionalized nano-drug delivery method based on targeting tumor regions is provided. This nano-drug delivery platform can achieve precise targeted delivery, controlled release, and synergistic anti-tumor effects with FUAS, thereby significantly improving the treatment effect of breast cancer. The method includes the following steps:

[0042] S1. Preparation of drug nanoplatform using mesoporous silica nanoparticles (MSNs);

[0043] In this embodiment, the excellent drug loading capacity and high acoustic impedance of mesoporous silica nanoparticles (MSNs) are utilized to functionalize MSNs into polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance their biocompatibility and surface activity.

[0044] Synthesis of S11 mesoporous silica nanoparticles (MSNs): Mesoporous silica nanoparticles (MSNs) were prepared using the sol-gel method. Tetraethyl orthosilicate (TEOS) was used as the silicon source, and hexadecyltrimethylammonium bromide (CTAB) was used as the template agent. The nanostructures were formed by hydrolysis under alkaline conditions. After preparation, the nanoparticles were washed with ethanol and calcined at high temperature to remove the CTAB template, resulting in MSNs with uniform pore size and high specific surface area.

[0045] Preparation of S12 polyethylene glycol (PEG) modified 3PEG-NHS: A certain amount of polyethylene glycol (PEG) is reacted with N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to form terminally activated NHS ester (PEG-NHS).

[0046] Covalent coupling of S13 with mesoporous silica nanoparticles (MSNs): PEG-NHS was added to an MSNs solution dispersed in anhydrous dimethyl sulfoxide (DMSO), and the reaction was stirred for 12 hours. PEG was covalently coupled with the hydroxyl groups on the surface of MSNs through esterification reaction to form PEG-modified MSNs (PEG-MSNs).

[0047] Preparation of S14 modified solution of polyethyleneimine (PEI): Polyethyleneimine (PEI) was dissolved in anhydrous DMSO and the pH was adjusted to neutral.

[0048] S15 Electrostatic Adsorption and Covalent Bonding: PEG-modified MSNs (PEG-MSNs) were mixed with a PEI solution and reacted at room temperature with stirring for 24 hours. Because PEI molecules are rich in amino groups, they can be adsorbed onto the MSN surface via electrostatic interactions. Simultaneously, glutaraldehyde (GA) was used as a cross-linking agent to promote the covalent bonding between PEI and the MSN surface.

[0049] S16 Washing and Purification: After the reaction, the functionalized MSNs were collected by centrifugation and washed repeatedly with deionized water to remove unbound PEI and reaction byproducts. Finally, MSNs dual-modified with PEG and PEI (PEG / PEI-MSNs) were obtained.

[0050] The addition of polyethylene glycol (PEG) in this embodiment has the following characteristics:

[0051] 1. PEG can shield immune recognition by forming a hydrophobic protective layer, reducing the interaction between the nanoparticle surface and serum proteins, thereby preventing rapid clearance by the mononuclear macrophage system (MPS). This property can significantly prolong the half-life of nanoparticles in the bloodstream.

[0052] 2. Enhancing Solubility and Stability: The hydrophilic molecular structure of PEG can reduce the risk of nanoparticle aggregation in physiological environments and improve the stability of nanoparticles in complex biological fluids such as blood. This is crucial for ensuring the functional integrity of nanoparticles during targeted delivery.

[0053] 3. Reduce toxicity and side effects: PEG modification can effectively reduce the non-specific effects of exposed nanoparticles on normal tissues, thereby reducing side effects caused by immune responses or non-specific adsorption and enhancing the safety of drug therapy.

[0054] 4. Enhancing Tumor Targeting: PEG-modified nanoparticles more easily enter tumor tissue through the "enhanced penetration and retention effect" (EPR effect). The protective effect of PEG allows nanoparticles to cross the vascular barrier more efficiently and accumulate in tumor tissue.

[0055] The addition of polyethyleneimine (PEI) in this embodiment has the following characteristics:

[0056] 1. Positively Charged Targeting and Cellular Uptake: PEI is a cationic polymer with a high density of amino groups and a positively charged surface. These positive charges can interact with the negative charges on the cell membrane, promoting the adsorption and internalization of nanoparticles into cells. The positively charged nature of PEI enhances the cell membrane permeability of the nanocarrier, enabling it to enter cells more efficiently, especially tumor cells.

[0057] 2. Improved Drug Delivery Efficiency: Due to the cationic nature of PEI, the nanoparticles bind more tightly to drug molecules, thereby increasing drug loading capacity. After cellular uptake, PEI helps drugs be effectively released into the cell or organelles, increasing drug bioavailability and optimizing therapeutic effects.

[0058] 3. Increasing the stability of nanoparticles in blood: PEI can reduce the aggregation of nanoparticles in vivo through charge shielding effect, thereby increasing their stability in blood. In this way, nanoparticles can maintain a stable state in vivo for a longer period of time, avoiding the impact on their biodistribution due to aggregation.

[0059] 4. Enhancing the efficacy of targeted therapy: PEIs can also enhance tumor-specific delivery by binding to receptors on the surface of tumor cells. For example, the positive charge of PEIs can bind to anion receptors or gene targets on the surface of tumor cells, thereby improving the precision of targeted therapy.

[0060] 5. Promoting Intracellular Release and Antitumor Effects: PEI, through its cationic properties, facilitates the release of drug-loaded nanoparticles from endosomes into the cytoplasm. This process is enhanced by the "protonation" effect of PEI, thereby improving the biological efficacy of the drug.

[0061] In this embodiment, mesoporous silica nanoparticles (MSNs) are used as the core carrier, combined with surface modification of polyethylene glycol (PEG) and polyethyleneimine (PEI), to achieve excellent drug loading capacity, biocompatibility and stability.

[0062] The nanoplatform in this embodiment is designed with biocompatibility and systemic safety in mind. It reduces toxic side effects through PEG modification and drug controlled release mechanisms, providing a safer treatment option for breast cancer patients.

[0063] S2. Assembling drug-loaded astragaloside A (AS) and a thermosensitive gated molecule for controlled release on mesoporous silica nanoparticles (MSNs):

[0064] Astragaloside A (AS) was further loaded onto the nanoplatform, and 1-tetradecyl alcohol (TD) was used as a thermosensitive gate molecule to achieve drug encapsulation and controlled release triggered by FUAS, ensuring efficient drug release at the tumor site and avoiding side effects caused by early release.

[0065] The process of loading astragaloside A (AS) onto the nanoplatform in this embodiment is as follows:

[0066] 6 mg of amino-modified PEG-PEI-MSN was dispersed in 3 mL of methanol and sonicated. Then, 1.2 mg of astragaloside A (AS, dissolved in 3 mL of methanol) was added. The mixture was gently stirred at 68°C for 5 hours to allow the methanol to evaporate completely.

[0067] Then add 2 mg of 1-tetradecyl alcohol (TD), stir for 1 hour, add 5 mL of hot water (70°C); then immediately centrifuge (1200 rpm, 10 minutes) at 50°C and remove the supernatant;

[0068] Finally, the TD / astragaloside A (AS) loaded PEG-PEI-MSN was dispersed in ice-cold ultrapure water and all unbound astragaloside A (AS) was removed by centrifugation-washing cycles at least six times, and then dispersed in ice-cold ultrapure water under sonication (30 seconds).

[0069] In this embodiment, unlike the modifications with PEG and PEI, the loading of astragaloside A (AS) during the drug loading process mainly occurs within the pores of MSNs or on their surface. PEG improves the biocompatibility of the nanoparticles, enabling them to remain in vivo for a longer period; PEI enhances their affinity for cell membranes by imparting a positive charge to the nanoparticle surface. AS loading primarily relies on the interaction between the pore structure of the MSNs and the surface functionalized groups. The loading of AS is mainly due to physical adsorption, while the PEG / PEI modification is a chemical modification.

[0070] Meanwhile, the controlled release process and mechanism of the drug are as follows:

[0071] The role of the thermosensitive gated molecule TD: 1-Tetradecanol (TD) is a thermosensitive gated molecule capable of undergoing a phase transition with temperature changes. Its principle is based on the fact that TD alters its molecular structure at specific temperatures, thereby affecting drug release. During FUAS treatment, the local temperature in the tumor area rises, causing TD to undergo a phase transition, opening the drug's "gated" mechanism and allowing the release of the loaded astragaloside A (AS). The specific process is as follows:

[0072] At room temperature: At room temperature, TD acts as a gated molecule to firmly seal the drug AS within the channels of MSNs, preventing premature drug release.

[0073] 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 changes in the molecular structure of TD, forming pores or channels, thereby allowing the blocked drug to be released.

[0074] Drug release: With the phase transition of TD, AS detaches from the nanoparticles and enters the tumor tissue for local treatment. At this point, the release rate and amount of drug can be precisely controlled by the thermal effect of FUAS.

[0075] The controlled-release mechanism of the drug utilizes a thermal effect to drive release: FUAS triggers a local temperature increase, promoting the TD phase transition and simultaneously facilitating the release of astragaloside A (AS). Because AS release is temperature-controlled, the drug can achieve more concentrated treatment at the tumor site, avoiding the side effects associated with systemic release.

[0076] Improved treatment efficiency: Through a controlled release mechanism, AS can act continuously at the tumor site, increasing local drug concentration and thus enhancing the anti-tumor effect of the drug.

[0077] Prolonged treatment effect: Because drug release is controlled, the treatment effect can be prolonged, which helps reduce the risk of tumor recurrence.

[0078] S3. A tumor-targeting strategy is developed using electrostatic adsorption to deliver nanoparticles to the target tumor region;

[0079] Positively charged PEI-modified nanoparticles are combined with negatively charged probiotics **Bifidobacterium bifidum (BF)** via electrostatic adsorption. By utilizing BF's specific targeting ability to hypoxic areas of tumors, the nanoparticles are precisely delivered to the tumor region.

[0080] Bifidobacterium (BF) injection and enrichment in tumor areas;

[0081] 1. Bifidobacterium injection: To ensure the accumulation of Bifidobacterium in the tumor area, 200 µL of Bifidobacterium suspension (concentration of 1×10⁻⁶) was injected via tail vein. 6 Bifidobacteria (CFU / mL) were injected into the experimental animals. This injection protocol was performed 3 days in advance to ensure that Bifidobacteria could proliferate and accumulate in the tumor area.

[0082] 2. Enrichment Process Validation: Tissue homogenization experiments were used to verify the enrichment of Bifidobacteria in the tumor region. The results showed that Bifidobacteria can accumulate in large quantities in the tumor region, especially in the hypoxic areas. This characteristic enables BF to serve as a targeted delivery system, precisely delivering subsequent nanoparticles to tumor tissue.

[0083] 3. Injection of PEI-modified nanoparticles and their electrostatic adsorption with BF;

[0084] 4. PEI-modified nanoparticle injection: On the third day after Bifidobacterium injection, 200 µL of nanoparticle suspension (concentration 200 µg / mL) was injected via the tail vein, allowing the nanoparticles to enter the body through blood circulation. The PEI-modified nanoparticles have a positively charged surface, thus enabling them to bind to the negatively charged Bifidobacterium previously enriched at the tumor site through electrostatic adsorption.

[0085] 5. Electrostatic Adsorption Binding: Because the cell wall of Bifidobacteria contains abundant negatively charged groups (such as fatty acid and phosphate groups), while PEI-modified nanoparticles carry a positive charge, the two are tightly bound together through electrostatic interactions. This binding ensures that the drug carrier can accurately locate and enter the tumor region, thereby achieving precise drug delivery.

[0086] The validation of targeted delivery is as follows:

[0087] 1. In vivo fluorescence imaging: In vivo fluorescence imaging technology was used to verify the binding of nanoparticles to Bifidobacteria and their enrichment in the tumor region. The results showed that the injected nanoparticles effectively bound to Bifidobacteria enriched in the tumor site, and the nanoparticles could accurately target the tumor region.

[0088] 2. Frozen Section Analysis: Further observation using frozen sections and fluorescence microscopy verified the aggregation of nanoparticles in the tumor region. The intensity of the fluorescence signal indicated that the nanoparticles could effectively accumulate in tumor tissue, and the binding with Bifidobacteria improved the efficiency of targeted delivery.

[0089] S4. Utilize focused ultrasound ablation surgery (FUAS) to release drugs for synergistic treatment;

[0090] Under the thermal effect triggering of focused ultrasound ablation surgery (FUAS), 1-tetradecanool (TD) undergoes a phase transition to release the drug. At the same time, the acoustic impedance properties of nanoparticles are used to improve the tumor ablation efficiency of FUAS, thereby significantly enhancing the overall effect of breast cancer treatment. By introducing 1-tetradecanool (TD) as a thermosensitive gated molecule, controlled drug release is achieved under FUAS triggering, avoiding early drug leakage and ensuring the drug's efficient action at the tumor site.

[0091] The thermosensitive phase transition principle of TD in this embodiment is as follows: 1-Tetradecanol (TD) is a molecule with thermally responsive properties, capable of undergoing a phase transition at a specific temperature. At room temperature, TD molecules are in a liquid state and can seal the pores or surface of drug-loaded nanoparticles, preventing premature drug release. When the local temperature rises to the phase transition temperature of TD (typically between 40°C and 45°C), the TD molecules undergo a phase transition, thereby changing their molecular structure, forming pores or channels, and promoting the release of drug-loaded molecules.

[0092] In this embodiment, the FUAS thermal effect triggers drug release. FUAS heating effect: Focused ultrasound ablation surgery (FUAS) heats the tumor area using focused ultrasound energy. During treatment, the local temperature of the tumor rises rapidly to the range of 40°C to 45°C, a temperature sufficient to trigger the phase transition of TD molecules.

[0093] In this embodiment, drug release follows the TD phase transition: when the TD molecules undergo a phase transition, the previously closed nanoparticle pores are opened, and drugs such as astragaloside A (AS) begin to be released. The release rate and amount can be precisely controlled based on the treatment time of FUAS, temperature changes, and the phase transition characteristics of TD. Drug release is controlled, preventing early leakage and ensuring maximum efficacy of the drug at the tumor site.

[0094] To prevent early drug leakage, the gate control effect of TD is utilized: 1-Tetradecanol (TD) acts as a thermosensitive gate control molecule, ensuring that the drug does not distribute nonspecifically or leak prematurely in the body before treatment. The drug is precisely released only when the local temperature of the tumor area rises to the trigger temperature. This mechanism greatly improves the safety and efficiency of treatment and avoids side effects on normal tissues.

[0095] This embodiment utilizes the specific targeting ability of Bifidobacterium (BF) to hypoxic areas of tumors, and precisely delivers nanoparticles to the tumor site through electrostatic adsorption, which greatly improves the accuracy of treatment and avoids non-specific drug distribution; thus achieving precise targeting of the tumor area.

[0096] This embodiment utilizes the method to perform FUAS-based synergistic therapy, combining focused ultrasound ablation surgery (FUAS) with nanomedicine delivery. The thermal effect of FUAS triggers drug release, enhancing the tumor ablation effect and simultaneously achieving controlled drug release therapy, effectively addressing the shortcomings of FUAS as a single treatment modality.

[0097] The method provided in this embodiment can achieve multimodal treatment integration. By combining a nanoplatform with tumor targeting, controlled drug release, and acoustic enhancement functions, it significantly improves the overall 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 applicable to the treatment of breast cancer, but also provides technical support for the combined treatment of other solid tumors, and has broad application prospects.

[0098] Example 2

[0099] This embodiment provides a functionalized nano-drug delivery platform based on targeting tumor regions, comprising mesoporous silica nanoparticles (MSNs) with surfaces modified by polyethylene glycol (PEG) and polyethyleneimine (PEI) and a drug; the mesoporous silica nanoparticles (MSNs) are loaded with 1-tetradecanoic acid TD, which serves as a thermosensitive gated molecule.

[0100] The drug is either astragaloside A (AS) or Bifidobacterium BF;

[0101] The drug provided in this embodiment has anti-tumor effects: Astragaloside A (AS) has significant anti-tumor activity. Studies have shown that astragaloside A can inhibit the growth, migration, and invasion of tumor cells through multiple mechanisms, including inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting tumor development by regulating the tumor microenvironment.

[0102] Immunomodulatory Effects: Astragaloside A exhibits strong immunomodulatory effects, enhancing the body's immune function. It strengthens the immune system's recognition and elimination of tumor cells by promoting the function of immune cells such as macrophages and T cells. Furthermore, astragaloside A can also enhance the body's immune response against tumor cells by regulating cytokine secretion.

[0103] Antioxidant activity: Astragaloside A possesses antioxidant activity, which can scavenge free radicals in the body and slow down oxidative damage to cells. This property helps protect healthy cells from oxidative stress and may play an important role in anti-tumor therapy.

[0104] Anti-inflammatory effects: Astragaloside A exhibits positive anti-inflammatory properties. It can reduce chronic inflammatory responses 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 A helps improve treatment efficacy and reduce side effects.

[0105] Studies on improving treatment tolerance have found that astragaloside A can improve patient tolerance to treatment and prolong the effective period of treatment by reducing the side effects caused by chemotherapy or radiotherapy (such as immunosuppression and bone marrow suppression). It can also enhance the repair capacity of healthy cells in the body and promote anti-tumor immune responses.

[0106] Astragaloside A also has an inhibitory effect on tumor angiogenesis, inhibiting tumor growth and metastasis by interfering with tumor blood vessel formation. This effect helps reduce the nutrient supply to the tumor and promotes tumor cell death.

[0107] Pharmacokinetics: Astragaloside A exhibits high bioavailability, effectively absorbed via oral or injection routes, entering the bloodstream and rapidly distributing throughout the body. Due to its water and lipid solubility, astragaloside A rapidly reaches effective concentrations in vivo and exerts its pharmacological effects. Modification with nanocarriers can further enhance the targeting and bioavailability of astragaloside A.

[0108] The mesoporous silica nanoparticles have the following characteristics:

[0109] High specific surface area and large pore volume: Mesoporous silica nanoparticles (MSNs) possess significantly high specific surface areas (typically between 700–1000 m² / g) and large pore volumes. This allows MSNs to provide ample space for drug loading, making them particularly suitable for loading large molecular drugs (such as astragaloside A). By adjusting the synthesis conditions, the pore size (typically in the range of 2–10 nm) can be controlled, resulting in high drug loading capacity.

[0110] Tunable Pore Size and Functionalized Surfaces: MSNs possess tunable pore sizes, typically in the 2–10 nm range, allowing for optimized loading based on drug molecular size. Surface functionalization enhances the biocompatibility, stability, and targeting of nanoparticles. For example, modification with polyethylene glycol (PEG) and polyethyleneimine (PEI) enables MSNs to maintain long-term stability in vivo and increases their interaction with target cells. For loading drugs such as astragaloside A (AS), the pore size and surface functionalization properties of MSNs offer unique advantages for precise drug delivery and controlled release.

[0111] High drug loading capacity: Due to their high specific surface area and tunable pore size, MSNs offer high drug loading capacity. Through optimized porosity and functionalization modifications, MSNs can effectively load drugs, making them particularly suitable for natural drugs like astragaloside A (AS), which have a large molecular weight and poor water solubility. MSNs can improve the bioavailability of astragaloside A, ensuring its effectiveness in treatment.

[0112] Size requirements: For the astragaloside A (AS) drug delivery system in this study, the particle size of MSNs used should be between 100 nm and 150 nm. This size can maintain good blood circulation time and effectively target the tumor site.

[0113] Biocompatibility and Low Toxicity: MSNs are inherently highly biocompatible, reducing immune and toxic responses. Surface modification can further enhance their compatibility with the biological environment. For example, PEG-modified MSNs can avoid rapid clearance by the mononuclear macrophage system (MPS), prolonging their circulation time in vivo. For the delivery of astragaloside A, the biocompatibility of MSNs ensures drug safety in vivo and reduces side effects.

[0114] Controlled drug release: MSNs achieve controlled drug release through surface functionalization (such as the thermosensitive gated molecule 1-tetradecanool (TD)). This property is particularly important for the delivery of astragaloside A (AS), as it ensures precise drug release at the tumor site and avoids side effects caused by early release. The combination of MSNs and TDs allows for precise control of drug release, effectively releasing astragaloside A (AS) under the triggering thermal effect of FUAS (Focused Ultrasound Ablation Surgery).

[0115] The synthesis and functionalization of nanoparticles in this embodiment: mesoporous silica nanoparticles were prepared and modified with PEG and PEI to endow them with good biocompatibility and positive surface electrical properties;

[0116] 1. Polyethylene glycol (PEG) modification – imparts good biocompatibility;

[0117] Polyethylene glycol (PEG) is a commonly used surface-modifying molecule that can improve the biocompatibility of nanoparticles, reduce their recognition and clearance by the immune system, and prolong their cycling time. The specific modification process is as follows:

[0118] Synthesis and activation of PEG: PEG molecules can be activated through either amino (–NH2) or carboxyl (–COOH) groups. PEG-NHS (PEG-terminated activation) or PEG-COOH is commonly used. PEG-NHS can form covalent bonds by esterification with the hydroxyl (–OH) groups on the surface of MSNs.

[0119] PEG modification process: PEG-NHS is dissolved in a suitable solvent (such as deionized water or DMSO) and then added to the MSNs solution. The reaction is allowed to proceed for approximately 12 hours. PEG successfully attaches to the MSNs surface by undergoing esterification with the siloxy groups (–SiOH) on the MSNs surface, forming stable covalent bonds. The PEG molecules form a hydrophilic protective layer on the nanoparticle surface, effectively preventing non-specific adsorption and immune recognition of the nanoparticles in vivo.

[0120] Functions and effects:

[0121] Enhanced biocompatibility: After PEG modification, MSNs have hydrophilic PEG chains on their surface, which can improve the stability of nanoparticles and reduce their aggregation in blood.

[0122] Reduced immune response: PEG-modified nanoparticles can avoid being recognized and cleared by the mononuclear macrophage system (MPS), prolonging their circulation time in the body and thus improving drug efficacy.

[0123] 2. Polyethyleneimine (PEI) modification – imparting positive electrical properties to the surface

[0124] Polyethyleneimine (PEI) is a cationic polymer rich in amino groups, which can impart a positive charge to the surface of nanoparticles. This positive charge enhances the interaction between the nanoparticles and cell membranes or negatively charged biomolecules, promoting cellular uptake of the nanoparticles. The specific modification process is as follows:

[0125] PEI Dissolution and Preparation: Dissolve a certain amount of PEI in deionized water or a suitable solvent. The solution concentration is generally 0.1–1 mg / mL. Ensure that the solution is homogeneous.

[0126] PEI modification process: PEI solution was added to the PEG-modified MSNs solution, and the mixture was stirred for 24 hours to allow PEI to form a stable bond with the nanoparticle surface through electrostatic adsorption. Since PEI molecules contain a large number of amino groups (–NH2), these amino groups can adsorb onto negatively charged groups (such as Si–O–) on the nanoparticle surface, forming a positively charged surface.

[0127] Crosslinking agent assistance: In order to enhance the binding force between PEI and nanoparticles, crosslinking agents such as glutaraldehyde (GA) can be added to further promote the covalent bonding between PEI and MSNs surface.

[0128] Functions and effects:

[0129] Imparting a positive surface charge: After PEI modification, the nanoparticles have a large number of positive charges on their surface, which helps to generate strong electrostatic interactions with the negatively charged regions of the cell membrane, promoting endocytosis. Especially in targeted delivery, positively charged nanoparticles can bind more effectively to tumor cells or receptors.

[0130] Enhanced cellular uptake and targeting: PEI modification with a positive surface charge can improve the affinity of nanoparticles for tumor cells, promote their entry into cells through endocytosis via the cell membrane, thereby enhancing the effect of targeted therapy.

[0131] 3. Advantages of co-modification with PEG and PEI

[0132] By combining PEG and PEI for surface modification, MSNs achieve the advantages of both:

[0133] The hydrophilic protective layer of PEG can effectively improve the biocompatibility and stability of MSNs and prolong their circulation time in vivo.

[0134] The positive charge of PEI enhances the cell affinity and endocytic capacity of nanoparticles, thereby improving the efficiency of targeted drug delivery.

[0135] 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.

[0136] Drug loading and gated encapsulation in this embodiment: As is loaded into MSNs through physical adsorption and chemical modification techniques, and TD is used as a thermosensitive gated molecule for encapsulation;

[0137] The drug loading and gate-controlled packaging in this embodiment are detailed as follows:

[0138] Drug Loading: Astragaloside A (AS) loading. Astragaloside A (AS) is an active ingredient extracted from the traditional Chinese medicine Astragalus membranaceus, possessing significant anti-tumor, immunomodulatory, and antioxidant effects. In this embodiment, AS is loaded as a therapeutic drug into mesoporous silica nanoparticles (MSNs). The specific loading process is as follows:

[0139] Dissolving Astragaloside A (AS): First, dissolve a certain amount of astragaloside A (AS) in a suitable 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 of appropriate concentration. By adjusting the concentration of AS, the drug loading can be optimized.

[0140] Nanoparticle preparation: Prepare surface-modified mesoporous silica nanoparticles (MSNs) with pore sizes typically ranging from 2 to 10 nm and specific surface areas reaching 700–1000 m² / g, providing sufficient space for drug loading. MSNs can be synthesized via the sol-gel method and modified with PEG and PEI to ensure good biocompatibility and positive surface charge properties.

[0141] Drug loading process: The dissolved astragaloside (AS) solution is added to the MSNs solution at a specific ratio (e.g., AS to MSNs mass ratio of 1:5). Astragaloside A (AS) is then physically adsorbed into the pores of the MSNs through stirring and sonication. This process typically takes 12 hours to ensure sufficient drug molecule entry into the nanoparticle pores and interaction with the surface functional groups of the nanoparticles. Loading efficiency can be optimized by adjusting conditions such as solution concentration, stirring time, and temperature.

[0142] Validation of loading effect: HPLC detection;

[0143] The specific process of drug loading and gate-controlled packaging is as follows:

[0144] Preparation and dispersion of PEG-PEI-MSN: Take 6 mg of amino-modified PEG-PEI-MSN and disperse it in 3 mL of methanol. Use sonication to ensure that the nanoparticles are fully dispersed and form a stable solution.

[0145] Dissolution and addition of astragaloside A (AS): 1.2 mg of astragaloside A (AS) was dissolved in 3 mL of methanol to prepare an AS solution. Then, the AS solution was added to the PEG-PEI-MSN solution and mixed thoroughly. Ultrasonic treatment was used to help AS molecules better enter the pores or surface of the nanoparticles and be loaded onto the nanoparticles through physical adsorption.

[0146] Solvent evaporation and drug loading: The above mixture was gently stirred at 68°C for 5 hours to allow complete methanol evaporation. During this process, astragaloside A (AS) was firmly loaded onto the surface or pores of PEG-PEI-MSN through physical adsorption.

[0147] Addition and encapsulation of 1-Tetradecyl alcohol (TD): 2 mg of 1-tetradecyl alcohol (TD) was added to the drug-loaded PEG-PEI-MSN solution, and the mixture was stirred again for 1 hour. TD, as a thermosensitive gated molecule, can undergo a phase transition at a specific temperature, thereby controlling drug release. At this point, the TD molecules are encapsulated on the surface or within the pores of the nanoparticles, ensuring stable encapsulation of astragaloside A (AS).

[0148] Hot water addition and centrifugation: 5 mL of hot water (70°C) was added to the mixed solution. The addition of hot water helped TD to further bind with the nanoparticles and promoted the formation of a stable encapsulation structure between the drug and TD. Subsequently, centrifugation was performed immediately (1200 rpm, 10 min) while maintaining the temperature at 50°C. The supernatant was removed to obtain the TD / astragaloside A (AS)-loaded PEG-PEI-MSN complex.

[0149] Washing and purification: The centrifuged PEG-PEI-MSN complex was dispersed in ice-cold ultrapure water and subjected to at least six centrifugation-wash cycles to remove unbound astragaloside A (AS). This process ensured that there was no excess free drug in the complex, maintaining the purity of the nanoparticles. After each wash, the supernatant was removed by centrifugation to ensure that the drug was completely loaded into the nanoparticles.

[0150] Ultrasonic treatment and dispersion: Finally, the purified TD / astragaloside A (AS) loaded PEG-PEI-MSN was dispersed in ice-cold ultrapure water and ultrasonically treated (30 seconds) to ensure complete dispersion of nanoparticles, prevent particle aggregation, and obtain a uniformly dispersed nanoparticle suspension.

[0151] Advantages of drug loading and gate-controlled packaging

[0152] Enhancing drug loading capacity: Through a combination of physical adsorption and chemical modification, MSNs can effectively load astragaloside A (AS), and the encapsulation of TD ensures the stability and controlled release of the drug.

[0153] Prolonged drug release time: The barrier effect of TD ensures that the drug can be continuously released in the tumor area, thereby increasing the intensity of the drug's effect on tumor cells during treatment.

[0154] Reduced side effects: Because the drug can be precisely released at the tumor site, systemic side effects and non-specific drug exposure are greatly reduced, thereby improving treatment safety.

[0155] The targeted binding experiment in this embodiment: functionalized nanoparticles were mixed with BF and bound by electrostatic adsorption to confirm their binding efficiency and stability.

[0156] The methods for confirming the binding efficiency and stability in this embodiment are as follows: The efficiency and stability of the binding between nanoparticles and BF are confirmed mainly through the following two experimental methods: laser confocal microscopy imaging and flow cytometry (FCM) detection.

[0157] Laser confocal microscopy imaging: To observe the binding of nanoparticles to Bifidobacterium fibrillation (BF), fluorescently labeled nanoparticles and BF were used for observation via confocal microscopy (CLSM): DII-labeled nanoparticles: First, the nanoparticles were labeled with DII dye, enabling them to fluoresce red under a laser confocal microscope. FITC-labeled BF: Bifidobacterium fibrillation (BF) was labeled with FITC dye, enabling it to fluoresce green under a laser confocal microscope.

[0158] Confocal microscopy observation: The complex of labeled nanoparticles and Bifidobacterium was observed using laser confocal microscopy. The effectiveness of the nanoparticles binding to Bifidobacterium was observed by observing the overlap of fluorescence signals. If the complex successfully bound, the red fluorescent nanoparticles and the green fluorescent BF would overlap and appear on the cell surface.

[0159] Flow cytometry (FCM): To quantitatively detect the binding efficiency of nanoparticles to BF, flow cytometry can be used for analysis. Pretreatment for flow cytometry: The labeled and mixed complex is resuspended in PBS buffer before flow cytometry detection. The fluorescence intensity of FITC-labeled BF and DII-labeled nanoparticles is detected using a flow cytometer.

[0160] Binding efficiency analysis: Based on the overlap of fluorescence signals, flow cytometry can analyze the binding efficiency of nanoparticles to BF. Binding efficiency is quantitatively analyzed by calculating the proportion of BF cell surfaces simultaneously exhibiting red and green fluorescence.

[0161] Stability testing: Flow cytometry can also be used to test the stability of the complex. By sampling at regular intervals, the stability of the binding between nanoparticles and BF is observed, especially the distribution changes of the nanoparticles after binding with BF, and whether dissociation or other unstable phenomena occur. Analysis of binding efficiency and stability results.

[0162] Binding efficiency: Flow cytometry results can show the binding efficiency of the complex, typically determined by calculating the proportion of cells exhibiting red and green fluorescence. If the complex is stable and binds successfully, the overlapping area of ​​red and green fluorescence will be quite noticeable.

[0163] Stability analysis: The stability of the binding between nanoparticles and BF can be assessed by dynamically monitoring changes in the complex (such as intracellular and extracellular distribution after binding, changes in fluorescence signal intensity, etc.). If the fluorescence intensity of the complex does not change significantly within a certain period of time, it indicates that the binding between nanoparticles and BF is relatively stable.

[0164] like Figure 3 As shown, Figure 3To illustrate the results using mice as an example, the functionalized nanoparticle drug delivery platform based on tumor-targeting technology provided in this embodiment yielded the following results: If strong fluorescence overlap between the 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., above 80%), then the binding of the nanoparticles and BF can be confirmed as successful. Regarding stability, if the complex does not dissociate after a period of time and the flow cytometry results are stable, it indicates good in vivo stability, which can provide assurance for subsequent drug delivery.

[0165] In vitro release experiments in this embodiment: simulating FUAS triggering conditions to verify the phase transition release behavior of TD under thermal effects and the controlled drug release effect; In vivo experiments: functionalized nanoparticles were injected into a mouse breast cancer model and treated with FUAS to observe tumor ablation rate and drug distribution. Tumor inhibition was compared between the FUAS-only group and the FUAS + Bifidobacterium + nanoparticle group.

[0166] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing a functionalized nano-drug delivery platform based on targeting tumor regions, characterized in that: Includes the following steps: S1. A drug nanoplatform was prepared using mesoporous silica nanoparticles (MSNs), and the mesoporous silica nanoparticles (MSNs) were functionalized using polyethylene glycol (PEG) and polyethyleneimine (PEI). S2. Drug-loaded astragaloside A (AS) and the thermosensitive gate molecule 1-tetradecyl alcohol (TD) with controlled release mechanism are set on mesoporous silica nanoparticles (MSNs); S3. Positively charged PEI-modified nanoparticles are combined with negatively charged Bifidobacterium BF via electrostatic adsorption to obtain a functionalized nano-drug delivery platform.

2. The method for preparing a functionalized nano-drug delivery platform based on targeting tumor regions as described in claim 1, characterized in that: In step S1, mesoporous silica nanoparticles (MSNs) are functionalized with polyethylene glycol (PEG) and polyethyleneimine (PEI). The specific process is as follows: S11 Synthesis of Mesoporous Silica Nanoparticles (MSNs); S12 Preparation of modified 3PEG-NHS for polyethylene glycol PEG; Covalent coupling of S13 with mesoporous silica nanoparticles (MSNs); S14 Preparation of a modified solution of polyethyleneimine (PEI); S15 mixes PEG-modified MSNs with PEI-modified solutions via electrostatic adsorption and covalent bonding. S16 Cleaning and Purification: After the reaction, the functionalized MSNs were collected by centrifugation and washed repeatedly with deionized water to remove unbound PEI and reaction byproducts. Finally, MSNs modified with both PEG and PEI were obtained.

3. The preparation method of the functionalized nano-drug delivery platform based on targeted tumor regions as described in claim 2, characterized in that: The synthesis of mesoporous silica nanoparticles (MSNs) in step S11 is carried out as follows: mesoporous silica nanoparticles (MSNs) are prepared using the sol-gel method, with tetraethyl orthosilicate (TEOS) as the silicon source and hexadecyltrimethylammonium bromide (CTAB) as the template agent. The nanostructure is formed by hydrolysis under alkaline conditions. After preparation, the nanostructure is washed with ethanol and calcined at high temperature to remove the CTAB template, thereby obtaining MSNs with uniform pore size and high specific surface area.

4. The method for preparing a functionalized nano-drug delivery platform based on targeting tumor regions as described in claim 1, characterized in that: In step S2, astragaloside A (AS) is further loaded onto the nanoplatform, and 1-tetradecanool (TD) is used as a thermosensitive gate molecule. The specific process is as follows: PEG-PEI-MSN was dispersed in methanol, ultrasonicated, and then astragaloside A was added and stirred to obtain a mixture. Then add 1-tetradecyl alcohol TD, stir, and then add hot water; subsequently, immediately centrifuge and remove the supernatant; Finally, the TD / astragaloside A AS-loaded PEG-PEI-MSN was dispersed in ice-cold ultrapure water, and all unbound astragaloside A AS was removed by centrifugation-washing cycle, followed by ultrasonication to disperse it in ice-cold ultrapure water.

5. The method for preparing a functionalized nano-drug delivery platform based on targeting tumor regions as described in claim 1, characterized in that: Using 1-tetradecanool TD as a thermosensitive gated molecule, controlled drug release is achieved under FUAS triggering during focused ultrasound ablation surgery.

6. A functionalized nanomedicine delivery platform based on targeting tumor regions, characterized in that: The invention comprises mesoporous silica nanoparticles (MSNs) with surfaces modified by polyethylene glycol (PEG) and polyethyleneimine (PEI), and drugs loaded on the mesoporous silica nanoparticles (MSNs); the mesoporous silica nanoparticles (MSNs) are loaded with 1-tetradecanoic acid (TD), which serves as a thermosensitive gated molecule; the drugs are astragaloside A (AS) and Bifidobacterium BF.

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