Plant liposome nano-carrier enhanced drug targeting cancer treatment method

Abstract

The invention provides a method for packaging and targeted delivery of a tetravalent platinum drug by using an innovative plant-derived lipidosome nano-carrier (PDENs), and aims to improve the efficiency and selectivity of cancer treatment. A novel PDENs is developed by using a biosynthesis pathway of a specific plant, and the carrier has unique surface modification and internal structure and can efficiently package a tetravalent platinum drug. The core of the method is that fixed-point and fixed-time activation of the drug in cancer cells is realized through a red light response molecular switch, so that the targeting property and the treatment effect of the drug are greatly improved. In addition, targeting ligand modification on the surfaces of the PDENs further enhances the specific recognition of the drug on tumor cells. Compared with traditional chemotherapeutic drugs, the methods of the present invention exhibit higher therapeutic efficiency, lower side effects and better patient compliance. A sustained-release and controlled-release system is constructed through a double-layer membrane structure of the PDENs, so that the circulation time of a drug in a body is prolonged, and the drug administration frequency is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and particularly to a method for enhancing the targeting of tetravalent platinum drugs in cancer treatment, improving the therapeutic effect and reducing the damage to normal cells by using plant-derived liposome nanoparticles (PDENs) as a novel drug delivery system. Background Art

[0002] In the field of cancer treatment, traditional chemotherapy drugs often cause serious damage to normal cells while killing tumor cells due to the lack of targeting, resulting in significant side effects for patients. In recent years, nano-drug delivery systems (NDDS) have received extensive attention because they can improve the pharmacokinetic properties of drugs, enhance the tumor targeting of drugs, and reduce the toxic side effects of drugs in the body. In particular, liposome nanoparticles have become a research hotspot due to their good biocompatibility, simple preparation, and the ability to encapsulate hydrophilic and lipophilic drugs.

[0003] However, the existing liposome nanoparticles still need to be improved in terms of permeability in tumor tissues, drug controlled-release ability, and targeting. In addition, how to achieve the site-specific activation of drugs in tumor cells to further improve the therapeutic effect and selectivity of drugs is the key point of current research. As a new type of anti-cancer drug, tetravalent platinum drugs show great therapeutic potential due to their unique chemical properties and high activity. However, the poor water solubility, low stability, and difficulty in achieving efficient activation in tumor cells of tetravalent platinum drugs limit their application in clinical treatment.

[0004] To address the above problems, the present invention provides a method for enhancing the targeted cancer treatment of tetravalent platinum drugs by using plant-derived liposome nanoparticles (PDENs). By modifying the surface of PDENs with targeting ligands and photosensitizers, specific recognition of tumor cells and light-controlled release of drugs are achieved, thereby improving the therapeutic effect and targeting of tetravalent platinum drugs and providing a new strategy for cancer treatment. Summary of the Invention

[0005] The present invention relates to a method for enhancing the targeted cancer treatment of tetravalent platinum drugs by using plant-derived liposome nanoparticles (PDENs), which is achieved through the following specific and rigorous technical steps: S1. Preparation of PDENs: Extract and purify lipids with a vinblastine content of 5-10% from Catharanthus plants, and prepare PDENs using the reverse evaporation technique. By adjusting the ratio of organic solvent to aqueous phase and the ultrasonic treatment time, precisely control the particle size distribution of PDENs to ensure that it is in the range of 50-200 nanometers.

[0006] S2. Drug encapsulation: Mix the tetravalent platinum prodrug with lipids, remove the organic solvent in a rotary evaporator to form a thin film, then add buffer solution for hydration, separate the PDENs with uniform particle size by size exclusion chromatography (SEC), and determine the encapsulation efficiency by ultraviolet-visible spectroscopy to ensure it is not less than 90%.

[0007] S3. Surface modification: Use 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as activators to react folic acid molecules with the free carboxyl groups on the surface of PDENs to form stable covalent bonds, and determine the folic acid modification density by fluorescence labeling and flow cytometry analysis.

[0008] S4. Photosensitizer binding: Select porphyrin photosensitizers with a maximum absorption peak at 650 nm, fix them on the surface of PDENs through thiol-maleimide reaction, and determine the introduced amount by high performance liquid chromatography (HPLC) to ensure that the photoactivation efficiency of the photosensitizer reaches more than 80%.

[0009] S5. PEGylation: Modify PEG molecules with a molecular weight of 5000 Da onto the surface of PDENs through the reaction of amino groups and carboxyl groups, and determine the degree of PEGylation by dynamic light scattering (DLS) and potential measurement to balance the long-circulation characteristics of PDENs and the exposure of targeting ligands.

[0010] S6. Drug release kinetics: In phosphate buffer (PBS) at 37°C and pH 7.4, determine the drug release kinetics by dialysis bag method to simulate the in vivo environment and ensure that the cumulative release rate reaches 60 - 80% within 72 hours.

[0011] S7. Auxiliary component optimization: During the preparation of PDENs, add 0.1% (w / v) of Tween 80 as a surfactant, 1% (w / v) of cholesterol to enhance membrane stability, and 0.5% (w / v) of PEGylated molecules to improve stability and long-circulation characteristics.

[0012] S8. Personalized treatment plan: Utilize the folic acid receptor expression level in tumor tissues, patient body weight, and tumor volume to customize the dose of PDENs and the red light irradiation conditions (wavelength 650 nm, power 7 mW / cm²) through a computational model for personalized treatment.

[0013] S9. Treatment effect evaluation: Evaluate the inhibitory effect of PDENs on tumor cells through in vitro MTT experiments, evaluate the in vivo anti-tumor effect through a nude mouse model of tumor cells with overexpressed folic acid receptors, and evaluate the tumor cell apoptosis rate through immunohistochemical staining.

[0014] S10. Data collection and analysis: Collect data such as drug concentration, tumor volume change, body weight change, and immunohistochemistry results, and use statistical software for t-tests and analysis of variance to verify the treatment effect and optimize the treatment plan.

[0015] The innovation of the present invention lies in that by precisely controlling the preparation conditions of PDENs, drug encapsulation efficiency, surface modification density, photosensitizer introduction amount, and PEGylation treatment, high-efficiency encapsulation, specific targeting, and controlled release of tetravalent platinum drugs are achieved, significantly improving the efficacy and selectivity of the drugs, while reducing the damage to normal cells. In addition, the present invention also includes the formulation of personalized treatment plans and the comprehensive evaluation of treatment effects, providing a brand-new strategy for cancer treatment.

[0016] The two main technical problems addressed by the present invention are as follows: First, traditional tetravalent platinum drugs are difficult to achieve efficient enrichment and activation in tumor cells due to poor water solubility and lack of targeting, which limits their application in clinical treatment. Second, existing liposome nanocarriers have insufficient deep penetration in tumor tissues and are difficult to achieve site-specific controlled release of drugs in tumor cells, resulting in poor efficacy and possible systemic side effects. The present invention uses liposome nanocarriers (PDENs) derived from plants and integrates folic acid molecules as targeting ligands and porphyrin-based photosensitizers as red light-responsive molecular switches, successfully achieving specific targeting of tumor cells and controlled release of drugs in the tumor microenvironment, effectively solving the above problems and improving the treatment effect and application potential of tetravalent platinum drugs.

[0017] The key protection points of the present invention lie in two key innovations: First, the use of liposome nanocarriers (PDENs) derived from Catharanthus plants as a drug delivery platform, which not only has excellent biocompatibility and biodegradability, but also achieves high-efficiency encapsulation and stable carriage of tetravalent platinum drugs through specific lipid compositions; Second, the innovative application of surface modification strategies, including covalent bonding of folic acid molecules to enhance targeting to tumor cells, and the introduction of porphyrin derivatives of photosensitizers to achieve drug release under red light control, ensuring site-specific activation and rapid release of drugs in tumor cells. These innovation points together constitute the core protected content of the present invention, providing a new high-efficiency and low-toxic treatment plan for cancer treatment.

[0018] The present invention encapsulates a tetravalent platinum drug using a liposome nanocarrier (PDENs) derived from Catharanthus plants, and combines a folic acid molecular targeting ligand and a porphyrin-based photosensitizer, achieving the following beneficial effects: First, the biocompatibility and biodegradability of PDENs provide a stable and safe delivery environment for the drug, significantly improving the stability and circulation time of the drug in vivo; Second, the modification of the folic acid molecular targeting ligand significantly enhances the specific recognition ability of the drug for tumor cells, reducing damage to normal cells; Third, the introduction of the porphyrin-based photosensitizer enables light-controlled release of the drug in the tumor microenvironment, improving the therapeutic index and selectivity of the drug; Finally, by precisely controlling the particle size, surface modification density, and drug release kinetics of PDENs, the present invention provides a new strategy for cancer treatment that is efficient, low-toxic, has good targeting and controlled release characteristics, and is expected to significantly improve the treatment effect and quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is a flowchart of the method for enhancing drug-targeted cancer treatment using the plant liposome nanocarrier of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the attached Figure 1 drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1: S1. Preparation of PDENs: Lipids are extracted from Catharanthus plants, and the lipids containing vinblastine are purified using high-performance liquid chromatography (HPLC). PDENs are prepared by the reverse evaporation technique, controlling the ratio of organic solvent to aqueous phase to be 3:1 and the sonication time to be 30 minutes to form PDENs with uniform particle size.

[0022] S2. Encapsulation of the tetravalent platinum drug: The tetravalent platinum prodrug is mixed with the lipid, and the organic solvent is removed in a rotary evaporator to form a film. Phosphate buffer (PBS) with a pH of 7.4 is added for hydration, and PDENs with uniform particle size are separated by SEC. The encapsulation efficiency is determined by ultraviolet-visible spectroscopy, and the results show that the encapsulation efficiency reaches 92%.

[0023] S3. Surface modification and fixation of the targeting ligand: Using EDC and NHS as activators, the folic acid molecule is reacted with the free carboxyl groups on the surface of PDENs to form stable covalent bonds. The folic acid modification density is determined by fluorescence labeling and flow cytometry analysis, and the results show that the molar ratio of folic acid molecule to PDENs is 1:100.

[0024] S4. Introduction of photosensitizer and control of drug release: Select porphyrin photosensitizers with a maximum absorption peak at 650 nm and immobilize them on the surface of PDENs through thiol-maleimide reaction. The introduction amount is determined by HPLC to ensure that the photoactivation efficiency of the photosensitizer reaches over 85%.

[0025] S5. PEGylation treatment: Modify PEG molecules with a molecular weight of 5000 Da onto the surface of PDENs through the reaction of amino groups and carboxyl groups. The degree of PEGylation is determined by DLS and ζ-potential measurement to ensure the long-circulation characteristics of PDENs and the exposure of targeting ligands.

[0026] S6. Drug release kinetics: In PBS at 37°C and pH 7.4, the drug release kinetics is measured by the dialysis bag method. The results show that the cumulative release rate reaches 65% within 72 hours, conforming to the Higuchi model.

[0027] S7. Optimization of auxiliary components: During the preparation of PDENs, add 0.1% (w / v) of Tween 80 as a surfactant and 1% (w / v) of cholesterol to enhance the membrane stability. Add 0.5% (w / v) of PEGylated molecules to improve the stability and long-circulation characteristics.

[0028] S8. Personalized treatment plan: Utilize the folate receptor expression level in tumor tissues, patient weight, and tumor volume to customize the dose of PDENs and the red light irradiation conditions through a computational model.

[0029] S9. Evaluation of treatment effect: Evaluate the inhibitory effect of PDENs on tumor cells through in vitro MTT experiments. The results show that compared with unmodified PDENs, the inhibitory rate of modified PDENs on tumor cells is increased by about 60%. Evaluate the in vivo anti-tumor effect through a nude mouse model of tumor cells with overexpressed folate receptors. The results show that the growth rate of tumor volume in the treatment group is significantly lower than that in the control group.

[0030] S10. Data collection and analysis: Collect data such as drug concentration, changes in tumor volume, changes in body weight, and immunohistochemical results. Use statistical software for t-tests and analysis of variance. A P-value less than 0.05 indicates that the results are statistically significant.

[0031] Through the above specific implementation methods, the present invention has successfully achieved the efficient encapsulation of tetravalent platinum drugs, specific targeting of tumor cells, and controlled release, significantly improving the efficacy and selectivity of the drugs, and providing a new high-efficiency and low-toxic treatment plan for cancer treatment.

[0032] Example 2: This example details a method for encapsulating a tetravalent platinum drug using plant-derived lipid nanoparticle carriers (PDENs), which is optimized for its application in breast cancer treatment.

[0033] S1. Preparation of PDENs: Phosphatidylcholine (PC) was extracted from soybean lecithin, and PDENs were prepared by the thin-film hydration method. PC was dissolved in ethanol in a rotary evaporator to form a film, and then PBS preheated to 37°C was added for hydration. PDENs with a particle size of 150 nm were obtained through gentle sonication.

[0034] S2. Encapsulation of the tetravalent platinum drug: The tetravalent platinum prodrug was dissolved in PBS, mixed with PDENs, and sonicated with a probe for 20 seconds to ensure that the drug molecules were encapsulated inside the PDENs. The mixed solution was centrifuged using an ultrafiltration device to remove the unencapsulated drug. The encapsulation efficiency was measured at 280 nm using a UV spectrophotometer and reached 93%.

[0035] S3. Surface modification: Folate labeled with 5-fluorescein isothiocyanate was added to the PDENs, and folate was immobilized on the surface of the PDENs through an EDC / NHS-mediated amide coupling reaction to achieve targeted modification of tumor cells. After modification, the amount of folate immobilized was detected using a flow cytometer to ensure that at least one folate molecule was modified on the surface of each PDEN.

[0036] S4. Binding of the photosensitizer: The photosensitizer silicon phthalocyanine with near-infrared light absorption characteristics was added to the PDENs and immobilized on the surface of the PDENs through a thiol-maleimide reaction. The amount of the introduced photosensitizer was determined by fluorescence spectroscopy to ensure effective catalysis of drug release under near-infrared light irradiation.

[0037] S5. PEGylation treatment: mPEG-5000 was reacted with the carboxyl groups on the surface of the PDENs to form PEGylated PDENs to improve their long-circulation characteristics in the blood circulation. The degree of PEGylation was measured by DLS to ensure that the average particle size of the PDENs increased by no more than 10 nanometers.

[0038] S6. Drug release kinetics: The PEGylated PDENs were suspended in a release medium simulating the in vivo environment, and a constant temperature condition of 37°C was set. The in vivo drug release was simulated by the dialysis bag method. The change in drug concentration was analyzed by HPLC to obtain the drug release curve, and the results showed that the cumulative release rate reached 70% within 72 hours.

[0039] S7. Addition of auxiliary components: During the preparation of the PDENs, 0.1% (w / v) of Tween 80 was added as a surfactant, and 1% (w / v) of cholesterol was added to enhance the stability of the liposome membrane.

[0040] S8. Personalized treatment plan: According to the tumor characteristics and folate receptor expression levels of breast cancer patients, the dose and light irradiation conditions of PDENs are customized through a computational model to ensure the treatment effect.

[0041] S9. Evaluation of treatment effect: The inhibitory effect of PDENs on breast cancer cells is evaluated using in vitro MTT assay, and the proliferation inhibition rate of the drug on tumor cells is determined by CCK-8 assay. In the established animal model of breast cancer tumors, the in vivo distribution of the drug and tumor growth are monitored by fluorescence imaging technology.

[0042] S10. Data analysis: Data such as tumor volume, body weight changes, and drug concentration before and after treatment are collected, and statistical software is used for data analysis to evaluate the treatment effect.

[0043] Through the above specific embodiments, the present invention provides a tetravalent platinum drug targeted treatment method for breast cancer treatment. By optimizing the preparation of PDENs, drug encapsulation, surface modification, photosensitizer binding, PEGylation treatment, and personalized treatment plan, efficient drug encapsulation, specific targeting of tumor cells, and controlled release are achieved, significantly improving the treatment effect and providing a new strategy for breast cancer treatment. Specific Embodiment 3: This embodiment details a method for encapsulating tetravalent platinum drugs using plant-derived liposome nanoparticles (PDENs), which is optimized specifically for the treatment of non-small cell lung cancer (NSCLC).

[0045] S1. Preparation of PDENs: Phospholipids are extracted from sunflower seed oil, and PDENs are prepared by the thin-film hydration method. The phospholipids are dissolved in chloroform in a rotary evaporator to form a thin film, and then preheated PBS at 40°C is added for hydration. PDENs with a particle size of 120 nm are obtained through gentle ultrasonic treatment.

[0046] S2. Encapsulation of tetravalent platinum drugs: The tetravalent platinum prodrug is dissolved in PBS, mixed with PDENs, and sonicated with a probe for 30 seconds to ensure that drug molecules are encapsulated inside PDENs. The mixed solution is centrifuged using an ultrafiltration device to remove unencapsulated drugs. The encapsulation efficiency is measured by a UV spectrophotometer at λmax = 300 nm and reaches 91%.

[0047] S3. Surface modification: FITC (fluorescein isothiocyanate)-labeled folic acid is added to PDENs, and folic acid is immobilized on the surface of PDENs through an EDC / NHS-mediated amide coupling reaction to achieve targeted modification of tumor cells. After modification, the amount of immobilized folic acid is detected using a flow cytometer to ensure that at least one folic acid molecule is modified on the surface of each PDEN.

[0048] S4. Binding of photosensitizer: A porphyrin photosensitizer with an absorption peak at 650 nm was added to the PDENs and immobilized on the surface of the PDENs through a thiol-maleimide reaction. The amount of the introduced photosensitizer was determined by fluorescence spectroscopy to ensure effective catalysis of drug release under red light irradiation.

[0049] S5. PEGylation treatment: mPEG-5000 was reacted with the carboxyl groups on the surface of the PDENs to form PEGylated PDENs, so as to improve their long-circulation characteristics in the blood circulation. The degree of PEGylation was measured by DLS to ensure that the average particle size of the PDENs increased by no more than 5 nanometers.

[0050] S6. Drug release kinetics: The PEGylated PDENs were suspended in a release medium simulating the in vivo environment, and a constant temperature condition of 37 °C was set. The in vivo drug release was simulated by the dialysis bag method. The change in drug concentration was analyzed by HPLC to obtain the drug release curve, and the results showed that the cumulative release rate reached 68% within 72 hours.

[0051] S7. Addition of auxiliary components: During the preparation of the PDENs, 0.05% (w / v) of Tween 80 was added as a surfactant, and 0.5% (w / v) of cholesterol was added to enhance the stability of the liposome membrane.

[0052] S8. Personalized treatment plan: According to the tumor characteristics and folate receptor expression levels of non-small cell lung cancer patients, the dose and light irradiation conditions of the PDENs were customized through a computational model to ensure the treatment effect.

[0053] S9. Evaluation of treatment effect: The inhibitory effect of the PDENs on non-small cell lung cancer cells was evaluated by an in vitro MTT assay, and the proliferation inhibition rate of the drug on tumor cells was determined by a CCK-8 assay.

[0054] In the established non-small cell lung cancer tumor animal model, the in vivo distribution of the drug and the tumor growth were monitored through biodistribution experiments.

[0055] S10. Data analysis: Data such as tumor volume, body weight change, and drug concentration before and after treatment were collected, and data analysis was performed using statistical software to evaluate the treatment effect.

[0056] Through the above specific embodiments, the present invention provides a tetravalent platinum drug targeted treatment method for the treatment of non-small cell lung cancer. By optimizing the preparation of PDENs, drug encapsulation, surface modification, photosensitizer binding, PEGylation treatment, and personalized treatment plan, efficient drug encapsulation, specific targeting of tumor cells, and controlled release have been achieved, significantly improving the treatment effect and providing a new strategy for the treatment of non-small cell lung cancer.

Claims

1. A method for enhancing the targeted cancer therapy of tetravalent platinum drugs using plant-derived liposomal nanoparticles (PDENs), characterized in that, The method includes the following steps: a. Extract and purify the lipid containing vinblastine from Catharanthus plants to form PDENs, wherein the concentration of vinblastine is 5-10% of the total weight of PDENs; b. Encapsulate the tetravalent platinum drug into the PDENs by thin-film hydration method and size exclusion chromatography to form a drug-carrier complex, wherein the particle size of PDENs is 50-200 nanometers and the drug encapsulation efficiency reaches more than 90%; c. Fix folic acid molecules on the surface of PDENs by covalent bonding technology, and the molar ratio of folic acid molecules to PDENs is 1:50 to 1:200 to improve the targeting of the drug to tumor cells with overexpressed folate receptors; d. Introduce photosensitizers such as porphyrin derivatives on the surface of PDENs, and activate drug release by red light irradiation (650 nm wavelength, 7 mW / cm²) to enhance the therapeutic effect and reduce damage to normal cells, wherein the photoactivation efficiency of the photosensitizer reaches more than 80%.

2. The method according to claim 1, characterized in that, In the stability test of the tetravalent platinum drug in a simulated in vivo environment, the drug release rate does not exceed 10% within 24 hours, and the cumulative release rate reaches 60-80% within 72 hours at 37°C.

3. The method according to claim 1, characterized in that, The surface modification of the PDENs also includes polyethylene glycol (PEG) molecules, and the molar ratio of PEG molecules to PDENs is 1:10 to 1:50 to enhance the long-circulation characteristics of PDENs in the blood circulation and reduce the clearance of the reticuloendothelial system (RES).

4. The method according to claim 1, wherein The red light activation mechanism includes but is not limited to photosensitizer-mediated drug release or light-responsive liposome membrane design, wherein the photoactivation efficiency of the photosensitizer reaches more than 80%, and under red light irradiation, the drug release rate exceeds 95% within 5 minutes.

5. A tetravalent platinum pharmaceutical composition encapsulated in plant-derived liposomal nanocarriers (PDENs) prepared by any of the methods of claims 1 to 4, characterized in that, The controlled release characteristics of the tetravalent platinum drug in the composition conform to the Higuchi model or the first-order kinetic model, and the cumulative release rate in a simulated in vivo environment reaches 60-80% within 72 hours.

6. The composition according to claim 5, wherein The composition also includes auxiliary components such as surfactants such as Tween 80, stabilizers such as cholesterol, or PEGylated molecules to enhance the stability and bioavailability of the drug, wherein the total weight of the auxiliary components does not exceed 10% of the total weight of the composition.

7. Use of any of the methods or compositions of claims 1 to 6 for cancer treatment, characterized in that, The cancers include but are not limited to breast cancer, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, etc., and the treatment method also includes the following steps: a. Evaluate the size and location of the tumor by imaging techniques such as CT, MRI or PET-CT to determine the folate receptor expression level of the tumor; b. Customize a personalized treatment plan according to the tumor characteristics and patient conditions, including drug dosage, light irradiation conditions and treatment cycles, wherein the drug dosage is calculated according to the patient's weight and tumor size, and the light irradiation conditions are adjusted according to the tumor location and depth.

8. A plant-derived liposomal nanocarrier (PDENs) for cancer treatment, characterized in that, The PDENs are nanocarriers prepared by the method of claims 1 to 4, and the surface of the PDENs is modified with a red light-responsive molecular switch and a targeting ligand for tumor cell-specific receptors, wherein the total weight of the surface-modified molecular switch and targeting ligand does not exceed 20% of the total weight of the PDENs.

9. A method for detecting and evaluating the efficacy of any one of the methods or compositions according to claims 1 to 8 in cancer treatment, characterized in that, The detection and evaluation method includes the following steps: a. Evaluating the inhibitory effect of the drug on tumor cells through in vitro cell experiments, such as CCK-8 proliferation experiments or colony formation experiments, where the experimental settings include drug treatment groups with different concentrations and an untreated control group; b. Evaluating the in vivo anti-tumor effect and safety of the drug through in vivo animal models, such as nude mouse models established with tumor cells overexpressing folate receptors, where the evaluation indicators include tumor volume, body weight changes, and histopathological changes of major organs; c. Evaluating the impact of the drug on the tumor microenvironment, such as the apoptosis rate of tumor cells, angiogenesis, and immune cell infiltration, through immunohistochemical staining or flow cytometry.

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