Method for enhancing deep penetration of tumor based on NO gas nano-drug

Abstract

The invention belongs to the technical field of nano-drugs, and discloses a method capable of enhancing deep penetration of tumors based on NO gas nano-drugs, and the method comprises the following specific steps: step 1, selection and pretreatment of a starting material for preparing a nano-drug carrier: selecting Boc-NH-PEG-NH2 as a starting material of a PEG chain; according to the present invention, with the accurately prepared nano-drug carrier, the NO donor can be efficiently loaded and the responsive release can be achieved, the effective accumulation of the NO gas at the tumor site can be ensured so as to provide the anti-tumor effect, the release of the NO gas can trigger the expression and the activity enhancement of the matrix metalloproteinase (MMPs), and the tumor matrix collagen can be degraded so as to achieve the anti-tumor effect; compared with the prior art, the nano-drug provided by the invention has the advantages that the penetration barrier of tumor tissues is obviously reduced, favorable conditions are provided for the deep penetration of the nano-drug, in addition, the deep penetration of the nano-drug is further promoted by the responsive disintegration of the nano-drug in a tumor microenvironment, the anti-tumor effect is enhanced, and finally, the nano-drug has a selective toxic effect and is relatively low in toxicity to normal cells.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicine, and in particular is a method for enhancing deep penetration of tumors based on NO gas nanomedicine. Background Art

[0002] A tumor is a new growth formed when a cell in a local tissue loses normal growth regulation at the genetic level under the influence of various carcinogenic factors, resulting in its clonal abnormal proliferation. These new growths usually appear as lumps, which may cause symptoms such as compression and obstruction to the human body, and even endanger life in severe cases. Tumors are divided into two categories: benign tumors and malignant tumors. Malignant tumors, namely cancer, are invasive and metastatic, posing a great threat to human health.

[0003] Tumor treatment has always been an important challenge in the medical field, especially for the penetration and treatment of deep tumor cells. The penetration of traditional chemotherapy drugs in tumor tissues is often affected by the complexity of the tumor microenvironment, such as dense matrix collagen, abnormal vascular structure and high-pressure interstitial fluid. These factors limit the deep penetration and therapeutic effect of drugs. Therefore, the development of new nano-drug carriers and the improvement of the deep tumor penetration ability of drugs have become the hot spots of current tumor treatment research. Among them, poor tumor penetration is the main dilemma of nano-drugs that limits the anti-cancer efficacy. The dense extracellular matrix (ECM) in tumors is one of the main obstacles to the deep penetration of nano-drugs. Existing nano-drugs are The specific pathological microenvironment, after accumulating in the tumor, is usually confined to the perivascular area and is difficult to penetrate into the deep area of ​​the tumor. The tumor penetration efficiency is low, which inevitably leads to insufficient drug infiltration and incomplete eradication of cancer cells. NO, as a bioactive gas molecule, has a wide range of physiological and pathological regulatory effects in vivo, including anti-inflammatory, anti-angiogenic and inducing cell apoptosis. In recent years, NO gas therapy has shown great potential in the field of tumors, especially in promoting tumor vascular normalization, inhibiting tumor growth and metastasis. However, how to effectively deliver NO to the tumor site and achieve deep penetration in tumor tissue is a key issue facing NO gas therapy. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for enhancing deep tumor penetration based on NO gas nanomedicine, which has the advantage of increasing the depth of tumor penetration.

[0005] To achieve the above purpose, the present invention provides the following technical solution: a method for enhancing deep tumor penetration based on NO gas nanomedicine, the specific steps are as follows: Step 1: Preparation of nano drug carriers Selection and pretreatment of starting materials: Select Boc-NH-PEG-NH2 as the starting material for the PEG chain and ensure its purity and reactivity. Prepare the required functional groups such as phenylboronic acid (BA) and lactobionic acid (LA), as well as reagents such as Fmoc-protected lysine for subsequent steps; Modification of PEG chain: The Boc protecting group of Boc-NH-PEG-NH2 is removed by using trifluoroacetic acid (TFA) under appropriate solvents and conditions to expose the amino group. Subsequently, the Fmoc-protected lysine is sequentially coupled to the PEG chain through amide reaction to form PEG derivatives with multiple Fmoc-protected amino groups. The reaction conditions, such as temperature, time and pH value, need to be precisely controlled to ensure the coupling efficiency and purity of the product. Introduction of functional groups: Phenylboronic acid (BA) or lactobionic acid (LA) is coupled to the end of the PEG chain through an amide reaction to form dendritic macromolecules BACA (containing phenylboronic acid) and LACA (containing lactobionic acid) with specific functional groups. Self-assembled nanomicelles: BACA and LACA dendrimers were mixed in a certain ratio in a PBS solution with a pH value of 7.4 and self-assembled into nanomicelles by solvent evaporation. During this process, borate ester bonds were formed between phenylboronic acid and lactobionic acid, thereby achieving cross-linking of the nanomicelles. These nanomicelles can be used as the basic carrier of nanomedicines and can be loaded with anticancer drugs and NO donors; Step 2: Loading anti-cancer drugs Loading anticancer drugs: The chemotherapy drug sorafenib tosylate (SOR) and nanomicelles were dissolved in chloroform organic solvent, and then the drug-loaded nanodrug carrier (SOR@BLN) was obtained by solvent evaporation method; Step 3: Loading anticancer drugs and NO donor Loading NO donor: Similarly, NO donor (PSF) and nanomicelles are dissolved in an organic solvent. By precisely controlling the amount of drug molecules added and the reaction conditions, efficient drug loading and uniform distribution can be achieved, and nanodrugs loaded with NO donors (NO@BLN) are prepared by solvent evaporation. Co-loading of drugs and NO donors: In order to combine the dual advantages of chemotherapy and gas therapy, anticancer drugs and NO donors can be simultaneously loaded into nanomicelles to prepare SOR-NO@BLN nanomedicines; Step 4: Verify the properties of the nanomedicine Characterization and optimization: Scanning electron microscopy, transmission electron microscopy, and dynamic light scattering are used to observe the prepared nanomedicines for characterization of morphology, structure, and particle size. At the same time, fluorescence labeling and fluorescence spectrophotometry are used to evaluate the cross-linking degree and drug loading efficiency of the nanomedicines, and optimization and adjustment are performed as needed; Stability test: Under simulated physiological conditions, such as PBS solution at pH 7.4, the stability of nanomedicines is evaluated, including particle size changes, drug leakage, etc., to ensure that the nanomedicines can remain stable during blood circulation and avoid premature drug release or nanostructure disintegration; Responsiveness test: Evaluate the responsiveness of nanomedicines under conditions that simulate tumor microenvironment, such as acidic pH and high concentration of glutathione (GSH). By observing the changes in the particle size of nanomedicines, drug release, and the amount of NO produced, verify that nanomedicines can respond to microenvironmental changes inside tumors and achieve controlled drug release and NO gas generation. Through a series of characterization methods, the morphology, structure and particle size changes of nanomedicines can be verified; using fluorescent probes and NO gas detection kits, the ability of nanomedicines to release NO in cells and their effects on cells can be evaluated; Step 5: NO gas release and tumor matrix collagen degradation Release of NO gas: When the nanomedicine is internalized by tumor cells, the PSF loaded inside reacts with the high concentration of GSH in the cells to release NO gas. This process is responsive, that is, it will only occur under specific conditions, such as acidic tumor microenvironment and high GSH concentration in cells, thus ensuring the effective release of NO gas at the tumor site. NO-mediated expression of matrix metalloproteinases (MMPs): The released NO gas can enhance the expression and activity of matrix metalloproteinases, such as MMP-1 and MMP-2, through the NO→ONOO-→MMPs signaling pathway. These matrix metalloproteinases can degrade the collagen components in the tumor matrix, thereby further reducing the permeability barrier of tumor tissue and paving the way for deep penetration of nanomedicines. As a biological signal molecule, NO can regulate a variety of physiological and pathological processes, including inflammatory response, angiogenesis and cell apoptosis. NO reacts with superoxide radical anions (·O2 - ) reacts to form peroxynitrite anion (ONOO - ), which in turn triggers the expression and activation of MMPs. MMPs are a class of enzymes that can degrade the extracellular matrix (ECM), including collagenase, gelatinase and matrix lysin. Their expression and activity have an important impact on tumor growth, invasion and metastasis; Matrix collagen degradation: Activated matrix metalloproteinases (MMPs) further degrade tumor matrix collagen. Tumor matrix collagen is an important component of the tumor microenvironment. Its dense structure often hinders the deep penetration of nanomedicines. By degrading matrix collagen, the barrier to tumor penetration can be reduced, and the permeability and therapeutic effect of nanomedicines can be improved. Through NO-mediated MMPs activation and matrix collagen degradation, nanomedicines can more effectively penetrate tumor tissues and reach deeper tumor cells. This process not only enhances the anti-tumor effect of nanomedicines, but also provides new ideas and methods for the precise treatment of tumors. Step 6: Deep penetration of nanomedicine Responsive disintegration: When SOR-NO@BLN nanodrugs enter tumor tissues, the boronate bonds in the nanodrugs will hydrolyze due to the acidity of the tumor microenvironment, causing the nanodrugs to disintegrate into smaller nanomicelles. This disintegration behavior allows the nanodrugs to penetrate deeper into tumor tissues. This change in particle size allows the nanodrugs to more easily penetrate the dense intercellular spaces and matrix collagen of tumor tissues, laying the foundation for subsequent deep penetration. Deep penetration: Enhanced MMPs activity can degrade tumor matrix (such as collagen fibers), thus paving the way for deep penetration of nanomedicines. The reduction of particle size and degradation of matrix collagen work together to form a positive feedback loop, which greatly promotes the deep penetration of nanomedicines in tumors. Step 7: Efficacy evaluation and optimization Evaluation of permeability: In vitro tumor spheroid experiments, tumor spheroids with a diameter of about 200µm were constructed using Hepa1-6 cells to simulate the environment of solid tumors. These tumor spheroids were placed in PBS with different pH values, and Cy5-labeled SOR-NO@BLN nanodrugs were added to observe the permeation of the drug at different time points. In vivo experiments, the permeability and therapeutic effects of nanodrugs were evaluated through animal experiments. SOR-NO@BLN nanodrugs were injected into the tumor site in the animal body, and advanced imaging techniques such as fluorescence imaging and MRI were used to observe the distribution and permeation of the drug in the tumor tissue. The experimental results showed that nanodrugs can achieve good penetration in tumor tissues and significantly inhibit tumor growth, confirming the potential of nanodrugs in tumor treatment. According to the experimental results, the preparation conditions, loading amount, release rate and other parameters of nanodrugs were optimized. By adjusting these parameters, the deep tumor penetration ability and therapeutic effect of nanodrugs were improved, laying a solid foundation for their clinical application. Safety assessment: Toxicity test, using the CCK-8 method to determine the toxicity of nanomedicines to mouse (Hepa1-6) and human (HepG2) liver cancer cells. The experimental results show that within the appropriate dosage range, nanomedicines have low toxicity to normal cells and high toxicity to cancer cells, proving the selective toxicity of nanomedicines; in vivo metabolism and excretion monitoring, conducting in vivo animal pharmacokinetic experiments, by monitoring changes in drug concentrations in the blood and the distribution of drugs in major organs, understanding the metabolic pathways and excretion pathways of nanomedicines in the body, and determining that nanomedicines have good stability in the body and can be metabolized and excreted through organs such as the liver and kidneys.

[0006] Preferably, in step 1, the Boc protecting group of Boc-NH-PEG-NH2 is removed using trifluoroacetic acid in an organic solvent (DMF) at a temperature between 3°C and 5°C above room temperature and a pH of 7.

[0007] Preferably, in the introduction of the functional group in step 1, when coupling phenylboronic acid or lactobionic acid through an amide reaction, the reaction conditions need to be strictly controlled and necessary purification steps need to be performed to obtain high-purity BACA and LACA dendrimers.

[0008] Preferably, the nanomicelles described in step 1 have a larger initial particle size of 150 nm, which is beneficial to stability in blood circulation. This cross-linked structure can responsively dissociate in the acidic tumor microenvironment to release smaller nanomicelles of 25 nm.

[0009] Preferably, the Cy5-labeled SOR-NO@BLN nanodrug described in step 7 is prepared by replacing 5% of LACA with LACA-Cy5 in order to facilitate tracking of the nanodrug in vitro and in vivo.

[0010] Preferably, by adjusting the ratio of BACA and LACA in step 1, the cross-linking degree and particle size of the nanomicelles can be regulated, and Alizarin Red S can react with boric acid to emit strong fluorescence, thereby evaluating the cross-linking degree of nanoparticles of different formulations.

[0011] Preferably, while the deep penetration described in step 6 occurs, the SOR and NO gas in the nanomedicine exert a synergistic therapeutic effect inside the tumor, and SOR, as a chemotherapeutic drug, can directly inhibit the growth and division of tumor cells.

[0012] Preferably, when the pH value of the nanodrug in step 7 is 6.5 in an acidic environment, the nanodrug can dissociate into smaller nanomicelles more quickly, thereby penetrating the tumor spheroids more effectively, and the penetration ability is significantly improved compared to that under pH 7.4 conditions.

[0013] Preferably, the acidity of the tumor microenvironment in step six refers to the situation when the pH value of the tumor microenvironment is 6.5, and the borate ester bonds in the nanodrug will be hydrolyzed, causing the nanodrug to disintegrate into smaller nanomicelles.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Through the precisely prepared nano-drug carrier, NO donors can be efficiently loaded and responsively released, ensuring the effective accumulation of NO gas at the tumor site, thereby exerting its anti-tumor effect. Secondly, the release of NO gas can trigger the expression and activity enhancement of matrix metalloproteinases (MMPs), degrade tumor matrix collagen, and significantly reduce the permeability barrier of tumor tissue, providing favorable conditions for the deep penetration of nano-drugs. In addition, the responsive disintegration of nano-drugs in the tumor microenvironment further promotes its deep penetration and enhances the anti-tumor effect. Finally, nano-drugs have selective toxic effects, low toxicity to normal cells, and good stability in the body. They can be metabolized and excreted through organs such as the liver and kidneys, providing new ideas and methods for the clinical treatment of tumors. DETAILED DESCRIPTION

[0015] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0016] The embodiment of the present invention provides a method for enhancing deep tumor penetration based on NO gas nanomedicine, and the specific steps are as follows: Step 1: Preparation of nano drug carriers Selection and pretreatment of starting materials: Select Boc-NH-PEG-NH2 as the starting material for the PEG chain and ensure its purity and reactivity. Prepare the required functional groups such as phenylboronic acid (BA) and lactobionic acid (LA), as well as reagents such as Fmoc-protected lysine for subsequent steps; Modification of PEG chain: The Boc protecting group of Boc-NH-PEG-NH2 is removed by using trifluoroacetic acid (TFA) under appropriate solvents and conditions to expose the amino group. Subsequently, the Fmoc-protected lysine is sequentially coupled to the PEG chain through amide reaction to form PEG derivatives with multiple Fmoc-protected amino groups. The reaction conditions, such as temperature, time and pH value, need to be precisely controlled to ensure the coupling efficiency and purity of the product. Introduction of functional groups: Phenylboronic acid (BA) or lactobionic acid (LA) is coupled to the end of the PEG chain through an amide reaction to form dendritic macromolecules BACA (containing phenylboronic acid) and LACA (containing lactobionic acid) with specific functional groups. Self-assembled nanomicelles: BACA and LACA dendrimers were mixed in a certain ratio in a PBS solution with a pH value of 7.4 and self-assembled into nanomicelles by solvent evaporation. During this process, borate ester bonds were formed between phenylboronic acid and lactobionic acid, thereby achieving cross-linking of the nanomicelles. These nanomicelles can be used as the basic carrier of nanomedicines and can be loaded with anticancer drugs and NO donors; Step 2: Loading anti-cancer drugs Loading anticancer drugs: The chemotherapy drug sorafenib tosylate (SOR) and nanomicelles were dissolved in chloroform organic solvent, and then the drug-loaded nanodrug carrier (SOR@BLN) was obtained by solvent evaporation method; Step 3: Loading anticancer drugs and NO donor Loading NO donor: Similarly, NO donor (PSF) and nanomicelles are dissolved in an organic solvent. By precisely controlling the amount of drug molecules added and the reaction conditions, efficient drug loading and uniform distribution can be achieved, and nanodrugs loaded with NO donors (NO@BLN) are prepared by solvent evaporation. Co-loading of drugs and NO donors: In order to combine the dual advantages of chemotherapy and gas therapy, anticancer drugs and NO donors can be simultaneously loaded into nanomicelles to prepare SOR-NO@BLN nanomedicines; Step 4: Verify the properties of the nanomedicine Characterization and optimization: Scanning electron microscopy, transmission electron microscopy, and dynamic light scattering are used to observe the prepared nanomedicines for characterization of morphology, structure, and particle size. At the same time, fluorescence labeling and fluorescence spectrophotometry are used to evaluate the cross-linking degree and drug loading efficiency of the nanomedicines, and optimization and adjustment are performed as needed; Stability test: Under simulated physiological conditions, such as PBS solution at pH 7.4, the stability of nanomedicines is evaluated, including particle size changes, drug leakage, etc., to ensure that the nanomedicines can remain stable during blood circulation and avoid premature drug release or nanostructure disintegration; Responsiveness test: Evaluate the responsiveness of nanomedicines under conditions that simulate tumor microenvironment, such as acidic pH and high concentration of glutathione. By observing the changes in the particle size of nanomedicines, drug release, and the amount of NO produced, verify that nanomedicines can respond to microenvironmental changes inside the tumor and achieve controlled drug release and NO production. Through a series of characterization methods, the morphology, structure and particle size changes of nanomedicines can be verified; using fluorescent probes and NO detection kits, the ability of nanomedicines to release NO in cells and their effects on cells can be evaluated; Step 5: NO gas release and tumor matrix collagen degradation Release of NO gas: When the nanomedicine is internalized by tumor cells, the PSF loaded inside reacts with the high concentration of glutathione (GSH) in the cells to release NO gas. This process is responsive, that is, it only occurs under specific conditions, such as acidic tumor microenvironment and high intracellular GSH concentration, thus ensuring the effective release of NO gas at the tumor site. NO mediates the expression of matrix metalloproteinases (MMPs): the released NO gas is converted into ONOO via NO→ONOO - →MMPs signaling pathway can enhance the expression and activity of matrix metalloproteinases, such as MMP-1 and MMP-2. These matrix metalloproteinases can degrade the collagen components in the tumor matrix, thereby further reducing the permeability barrier of tumor tissue and paving the way for deep penetration of nanomedicines. NO, as a biological signal molecule, can regulate a variety of physiological and pathological processes, including inflammatory response, angiogenesis and cell apoptosis. NO gas reacts with oxygen free radicals (·O2 - ) reacts to form peroxynitrite anion (ONOO - ), which in turn triggers the expression and activation of MMPs. MMPs are a class of enzymes that can degrade the extracellular matrix (ECM), including collagenase, gelatinase and matrix lysin. Their expression and activity have an important impact on tumor growth, invasion and metastasis; Matrix collagen degradation: Activated matrix metalloproteinases (MMPs) further degrade tumor matrix collagen. Tumor matrix collagen is an important component of the tumor microenvironment. Its dense structure often hinders the deep penetration of nanomedicines. By degrading matrix collagen, the barrier to tumor penetration can be reduced, and the permeability and therapeutic effect of nanomedicines can be improved. Through NO-mediated MMPs activation and matrix collagen degradation, nanomedicines can more effectively penetrate tumor tissues and reach deeper tumor cells. This process not only enhances the anti-tumor effect of nanomedicines, but also provides new ideas and methods for the precise treatment of tumors. Step 6: Deep penetration of nanomedicine Responsive disintegration: When SOR-NO@BLN nanodrugs enter tumor tissues, the boronate bonds in the nanodrugs will hydrolyze due to the acidity of the tumor microenvironment, causing the nanodrugs to disintegrate into smaller nanomicelles. This disintegration behavior allows the nanodrugs to penetrate tumor tissues more deeply. This change in particle size allows the nanodrugs to more easily pass through the dense intercellular spaces and matrix collagen of tumor tissues, laying the foundation for subsequent deep penetration. Deep penetration: Enhanced MMPs activity can degrade tumor matrix (such as collagen fibers), thus paving the way for deep penetration of nanomedicines. The reduction of particle size and degradation of matrix collagen work together to form a positive feedback loop, which greatly promotes the deep penetration of nanomedicines in tumors. Step 7: Efficacy evaluation and optimization Evaluation of permeability: In vitro tumor spheroid experiments, tumor spheroids with a diameter of about 200µm were constructed using Hepa1-6 cells to simulate the environment of solid tumors. These tumor spheroids were placed in PBS with different pH values, and Cy5-labeled SOR-NO@BLN nanodrugs were added to observe the permeation of the drug at different time points. In vivo experiments, the permeability and therapeutic effects of nanodrugs were evaluated through animal experiments. SOR-NO@BLN nanodrugs were injected into the tumor site in the animal body, and advanced imaging techniques such as fluorescence imaging and MRI were used to observe the distribution and permeation of the drug in the tumor tissue. The experimental results showed that nanodrugs can achieve good penetration in tumor tissues and significantly inhibit tumor growth, confirming the potential of nanodrugs in tumor treatment. According to the experimental results, the preparation conditions, loading amount, release rate and other parameters of nanodrugs were optimized. By adjusting these parameters, the deep tumor penetration ability and therapeutic effect of nanodrugs were improved, laying a solid foundation for their clinical application. Safety assessment: Toxicity test, using the CCK-8 method to determine the toxicity of nanomedicines to mouse (Hepa1-6) and human (HepG2) liver cancer cells. The experimental results show that within the appropriate dosage range, nanomedicines have low toxicity to normal cells and high toxicity to cancer cells, proving the selective toxicity of nanomedicines; in vivo metabolism and excretion monitoring, conducting in vivo animal pharmacokinetic experiments, by monitoring changes in drug concentrations in the blood and the distribution of drugs in major organs, understanding the metabolic pathways and excretion pathways of nanomedicines in the body, and determining that nanomedicines have good stability in the body and can be metabolized and excreted through organs such as the liver and kidneys.

[0017] First, functionalized PEG derivatives were prepared and self-assembled into nanomicelles, which were then loaded with the anticancer drug sorafenib and NO donor to form SOR-NO@BLN nanodrugs. The drug was stable under simulated physiological conditions and responsively released NO in the tumor microenvironment, enhancing the expression of matrix metalloproteinases, degrading tumor matrix collagen, and lowering the permeability barrier. At the same time, the acidic environment triggered the disintegration of the nanodrugs, reducing the particle size and promoting deep penetration. The permeability and therapeutic effects were evaluated through in vitro and in vivo experiments, and the preparation conditions were optimized to ensure that the drug had low selective toxicity, was stable in vivo, and had good metabolic excretion. This provided new ideas and methods for the precision treatment of tumors and significantly improved the therapeutic effects.

[0018] In step 1, the Boc protecting group of Boc-NH-PEG-NH2 is removed using trifluoroacetic acid in an organic solvent (DMF) at a temperature between 3 and 5 degrees Celsius above room temperature and a pH of 7.

[0019] N,N-dimethylformamide is a commonly used organic solvent and is widely used as a dissolving and reaction medium in chemical synthesis. It has good solubility and can dissolve many organic substances, including PEG chains and reaction reagents. In the modification process of PEG chains, DMF as a solvent can provide a good reaction environment and promote the removal of Boc protecting groups and subsequent amide reactions.

[0020] Among them, in the introduction of functional groups in step 1, when coupling phenylboronic acid or lactobionic acid through amide reaction, it is necessary to strictly control the reaction conditions and perform necessary purification steps to obtain high-purity BACA and LACA dendrimers.

[0021] By utilizing the principle that small molecules can pass through but large molecules cannot, different molecules with large molecular weight differences can be separated, thereby removing small molecule impurities. Through dialysis, the product can be effectively separated from these impurities, thereby improving the purity of BACA and LACA dendrimers.

[0022] Among them, the nanomicelles in step one have a larger initial particle size of 150nm, which is beneficial to stability in blood circulation. This cross-linked structure can responsively dissociate in the acidic tumor microenvironment to release smaller nanomicelles of 25nm.

[0023] When the nanodrug enters the acidic tumor microenvironment with a pH of 6.5, the borate ester bond between phenylboronic acid and lactic acid breaks, leading to the dissociation of the nanomicelles and a reduction in particle size, which facilitates their deep penetration into tumor tissue.

[0024] Among them, the Cy5-labeled SOR-NO@BLN nanodrug in step seven was prepared by replacing 5% of LACA with LACA-Cy5 in order to facilitate the tracking of nanodrugs in vitro and in vivo.

[0025] The synthesis method of LACA-Cy5 is as follows: 150 mg of LACA and 20 mg of Cy5 were dissolved in 5 mL of DMF, and then 10 mg of DMAP and 80 mg of EDC were added. After the reaction solution was stirred at room temperature for 48 h, it was dialyzed with deionized water to remove small molecules, and LACA-Cy5 was obtained by lyophilization.

[0026] Among them, in step 1, by adjusting the ratio of BACA and LACA, the cross-linking degree and particle size of the nanomicelles can be controlled. By using Alizarin Red S, it can react with boric acid to emit strong fluorescence, thereby evaluating the cross-linking degree of nanoparticles with different formulations.

[0027] When the molar ratio of BACA to LACA reached 1:6, the fluorescence signal was stable, indicating the equilibrium point of assembly / cross-linking, therefore, the BACA / LACA ratio was optimized to 1:6 to prepare BLN nanomedicine.

[0028] Among them, in step six, while deeply penetrating, the SOR and NO gas in the nanomedicine exert a synergistic therapeutic effect inside the tumor. As a chemotherapy drug, SOR can directly inhibit the growth and division of tumor cells.

[0029] NO gas further enhances the therapeutic effect by regulating the tumor microenvironment and inducing tumor cell apoptosis. This synergistic therapeutic effect not only improves the therapeutic effect, but also reduces the dosage of a single drug, thereby reducing potential side effects.

[0030] Among them, in step seven, when the pH value of the nanodrug is 6.5 in an acidic environment, the nanodrug can dissociate into smaller nanomicelles more quickly, thereby penetrating the tumor spheroids more effectively, and the penetration ability is significantly improved compared to that under pH 7.4 conditions.

[0031] This result demonstrated that the acid-responsive dissociation mechanism of nanodrugs played an important role in deep penetration.

[0032] Among them, the acidity of the tumor microenvironment in step six refers to the situation when the pH value of the tumor microenvironment is 6.5, and the borate ester bonds in the nanodrug will be hydrolyzed, causing the nanodrug to disintegrate into smaller nanomicelles.

[0033] This responsive disintegration enables the nanomicelles to take effect in a timely manner when they reach the tumor microenvironment and avoid disintegration before reaching the vicinity of the tumor.

[0034] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0035] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for enhancing deep tumor penetration based on NO gas nanomedicine, characterized in that: The specific steps are as follows: Step 1: Preparation of nano drug carriers Selection and pretreatment of starting materials: Select Boc-NH-PEG-NH2 as the starting material for the PEG chain and ensure its purity and reactivity. Prepare the required functional groups such as phenylboronic acid (BA) and lactobionic acid (LA), as well as reagents such as Fmoc-protected lysine for subsequent steps; Modification of PEG chain: The Boc protecting group of Boc-NH-PEG-NH2 is removed by using trifluoroacetic acid (TFA) under appropriate solvents and conditions to expose the amino group. Subsequently, the Fmoc-protected lysine is sequentially coupled to the PEG chain through amide reaction to form PEG derivatives with multiple Fmoc-protected amino groups. The reaction conditions, such as temperature, time and pH value, need to be precisely controlled to ensure the coupling efficiency and purity of the product. Introduction of functional groups: Phenylboronic acid (BA) or lactobionic acid (LA) is coupled to the end of the PEG chain through an amide reaction to form dendritic macromolecules BACA (containing phenylboronic acid) and LACA (containing lactobionic acid) with specific functional groups. Self-assembled nanomicelles: BACA and LACA dendrimers were mixed in a certain ratio in a PBS solution with a pH value of 7.4 and self-assembled into nanomicelles by solvent evaporation. During this process, borate ester bonds were formed between phenylboronic acid and lactobionic acid, thereby achieving cross-linking of the nanomicelles. These nanomicelles can be used as the basic carrier of nanomedicines and can be loaded with anticancer drugs and NO donors; Step 2: Loading anti-cancer drugs Loading anticancer drugs: The chemotherapy drug sorafenib tosylate (SOR) and nanomicelles were dissolved in chloroform organic solvent, and then the drug-loaded nanodrug carrier (SOR@BLN) was obtained by solvent evaporation method; Step 3: Loading anticancer drugs and NO donor Loading NO donor: Similarly, NO donor (PSF) and nanomicelles are dissolved in an organic solvent. By precisely controlling the amount of drug molecules added and the reaction conditions, efficient drug loading and uniform distribution can be achieved, and nanodrugs loaded with NO donors (NO@BLN) are prepared by solvent evaporation. Co-loading of anticancer drugs and NO donors: In order to combine the dual advantages of chemotherapy and gas therapy, anticancer drugs and NO donors can be simultaneously loaded into nanomicelles to prepare SOR-NO@BLN nanomedicines; Step 4: Verify the properties of the nanomedicine Characterization and optimization: Scanning electron microscopy, transmission electron microscopy, and dynamic light scattering are used to characterize the morphology, structure, and particle size of the prepared nanomedicines. At the same time, the cross-linking degree and drug loading efficiency of the nanomedicines are evaluated through fluorescent labeling and fluorescence spectrophotometry, and optimization and adjustment are performed as needed; Stability test: Under simulated physiological conditions, such as PBS solution at pH 7.4, the stability of nanomedicines is evaluated, including particle size changes, drug leakage, etc., to ensure that the nanomedicines can remain stable during blood circulation and avoid premature drug release or nanostructure disintegration; Responsiveness test: Under simulated tumor microenvironment conditions, such as acidic pH and high concentration of glutathione (GSH), the responsiveness of nanomedicines is evaluated. By observing the changes in the particle size of nanomedicines, drug release, and the amount of NO gas produced, it is verified that nanomedicines can respond to microenvironmental changes inside the tumor and achieve controlled drug release and NO gas generation; Through a series of characterization methods, the morphology, structure and particle size changes of nanomedicines can be verified; using fluorescent probes and NO gas detection kits, the ability of nanomedicines to release NO in cells and their effects on cells can be evaluated; Step 5: NO gas release and tumor matrix collagen degradation Release of NO gas: When the nanomedicine is internalized by tumor cells, the PSF loaded inside reacts with the high concentration of GSH in the cells to release NO gas. This process is responsive, that is, it will only occur under specific conditions, such as acidic tumor microenvironment and high GSH concentration in cells, thus ensuring the effective release of NO gas at the tumor site. NO-mediated expression of matrix metalloproteinases (MMPs): The released NO gas can enhance the expression and activity of matrix metalloproteinases, such as MMP-1 and MMP-2, through the NO→ONOO-→MMPs signaling pathway. These matrix metalloproteinases can degrade the collagen components in the tumor matrix, thereby further reducing the permeability barrier of tumor tissue and paving the way for deep penetration of nanomedicines. As a biological signal molecule, NO can regulate a variety of physiological and pathological processes, including inflammatory response, angiogenesis and cell apoptosis. NO reacts with superoxide radical anions (·O2 - ) to generate peroxynitrite anion (ONOO - ), which in turn triggers the expression and activation of MMPs. MMPs are a class of enzymes that can degrade the extracellular matrix (ECM), including collagenase, gelatinase and matrix lysin. Their expression and activity have an important impact on tumor growth, invasion and metastasis; Matrix collagen degradation: Activated matrix metalloproteinases (MMPs) further degrade tumor matrix collagen. Tumor matrix collagen is an important component of the tumor microenvironment. Its dense structure often hinders the deep penetration of nanomedicines. By degrading matrix collagen, the barrier to tumor penetration can be reduced, and the permeability and therapeutic effect of nanomedicines can be improved. Through NO-mediated MMPs activation and matrix collagen degradation, nanomedicines can more effectively penetrate tumor tissues and reach deeper tumor cells. This process not only enhances the anti-tumor effect of nanomedicines, but also provides new ideas and methods for the precise treatment of tumors. Step 6: Deep penetration of nanomedicine Responsive disintegration: When SOR-NO@BLN nanodrugs enter tumor tissues, the boronate bonds in the nanodrugs will hydrolyze due to the acidity of the tumor microenvironment, causing the nanodrugs to disintegrate into smaller nanomicelles. This disintegration behavior allows the nanodrugs to penetrate deeper into tumor tissues. This change in particle size allows the nanodrugs to more easily penetrate the dense intercellular spaces and matrix collagen of tumor tissues, laying the foundation for subsequent deep penetration. Deep penetration: Enhanced MMPs activity can degrade tumor matrix (such as collagen fibers), thus paving the way for deep penetration of nanomedicines. The reduction of particle size and degradation of matrix collagen work together to form a positive feedback loop, which greatly promotes the deep penetration of nanomedicines in tumors. Step 7: Efficacy evaluation and optimization Evaluation of permeability: In vitro tumor spheroid experiments were conducted using Hepa1-6 cells to construct tumor spheroids with a diameter of approximately 200 µm to simulate the environment of solid tumors. These tumor spheroids were placed in PBS with different pH values ​​and Cy5-labeled SOR-NO@BLN nanomedicines were added to observe the permeability of the drug at different time points. In vivo experiments were conducted using animals to evaluate the permeability and therapeutic effects of nanomedicines. SOR-NO@BLN nanomedicines were injected into the tumor site in the animal body, and the distribution and permeation of the drug in the tumor tissue were observed using fluorescence imaging technology. The experimental results showed that nanomedicines were able to achieve good permeation in tumor tissues and significantly inhibit tumor growth, confirming the potential of nanomedicines in tumor treatment. Based on the experimental results, the preparation conditions, loading amount, release rate and other parameters of nanomedicines were optimized. By adjusting these parameters, the deep tumor penetration ability and therapeutic effect of nanomedicines were improved, laying a solid foundation for their clinical application. Safety assessment: Toxicity test, using the CCK-8 method to determine the toxicity of nanomedicines to mouse (Hepa1-6) and human (HepG2) liver cancer cells. The experimental results show that within the appropriate dosage range, nanomedicines have low toxicity to normal cells and high toxicity to cancer cells, proving the selective toxicity of nanomedicines; in vivo metabolism and excretion monitoring, conducting in vivo animal pharmacokinetic experiments, by monitoring changes in drug concentrations in the blood and the distribution of drugs in major organs, understanding the metabolic pathways and excretion pathways of nanomedicines in the body, and determining that nanomedicines have good stability in the body and can be metabolized and excreted through organs such as the liver and kidneys.

2. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: In step 1, the Boc protecting group of Boc-NH-PEG-NH2 is removed using trifluoroacetic acid in an organic solvent (DMF) at a temperature between 3 and 5 degrees Celsius above room temperature and a pH of 7.

3. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: In the introduction of the functional group in step 1, when phenylboronic acid or lactobionic acid is coupled via an amide reaction, the reaction conditions need to be strictly controlled and necessary purification steps need to be performed to obtain high-purity BACA and LACA dendrimers.

4. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: The nanomicelles described in step 1 have a larger initial particle size of 150 nm, which is beneficial to stability in blood circulation. This cross-linked structure can responsively dissociate in the acidic tumor microenvironment to release smaller nanomicelles of 25 nm.

5. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: In the Cy5-labeled SOR-NO@BLN nanodrug described in step 7, LACA-Cy5 was used to replace 5% of LACA to prepare the nanodrug in order to facilitate the tracking of the nanodrug in vitro and in vivo.

6. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: As described in step 1, by adjusting the ratio of BACA and LACA, the cross-linking degree and particle size of the nanomicelles can be regulated. By using Alizarin Red S, which can react with boric acid to emit strong fluorescence, the cross-linking degree of nanoparticles of different formulations can be evaluated.

7. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: As described in step six, while penetrating deeply, the SOR and NO gas in the nanomedicine exert a synergistic therapeutic effect inside the tumor. As a chemotherapeutic drug, SOR can directly inhibit the growth and division of tumor cells.

8. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: When the nanomedicine described in step 7 is in an acidic environment with a pH value of 6.5, the nanomedicine can dissociate into smaller nanomicelles more quickly, thereby penetrating the tumor spheroids more effectively, and the penetration ability is significantly improved compared to that under pH 7.4 conditions.

9. The method for enhancing deep tumor penetration based on NO gas nanomedicine according to claim 1, characterized in that: The acidity of the tumor microenvironment described in step six refers to the situation when the pH value of the tumor microenvironment is 6.5, and the borate ester bonds in the nanodrug will be hydrolyzed, causing the nanodrug to disintegrate into smaller nanomicelles.