A supramolecular carrier-free self-delivery system, a preparation method thereof and application thereof in tumor treatment

Abstract

This invention discloses a supramolecular carrier-free self-delivery system, its preparation method, and its application in tumor treatment, belonging to the field of biomedical technology. The supramolecular carrier-free self-delivery system is prepared by non-covalent self-assembly of mitoxantrone, dihydroporphyrin (Ce6), and ultrasmall iron oxide nanoparticles in an aqueous phase. This invention utilizes the co-assembly of mitoxantrone (MTX), dihydroporphyrin (Ce6), and ultrasmall iron oxide nanoparticles (IONP) to form a supramolecular carrier-free self-delivery system CMIOs. This system is simple to prepare, stable in performance, and highly biosafety. It can synergistically inhibit cancer through multiple mechanisms, achieving highly efficient inhibition of breast cancer, providing a new strategy for tumor treatment, and has broad application prospects in solving clinical problems.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a supramolecular carrier-free self-delivery system, its preparation method, and its application in tumor treatment. Background Technology

[0002] Cancer, due to its heterogeneity, diversity, high recurrence rate, and metastasis rate, has become one of the major obstacles to increasing human lifespan. To overcome the limitations of traditional treatments (such as chemotherapy, surgery, and radiotherapy), drug delivery systems (DDSs) based on nanocarriers have emerged. These systems (including micelles, dendritic macromolecules, liposomes, and polymer vesicles) are generally constructed through covalent bonds or supramolecular weak interactions, primarily benefiting from their inherent nanoscale properties, such as prolonged circulation time, enhanced tumor site accumulation, and improved retention, leading to more effective tumor treatment. However, these nanocarrier-based nanomedicines face challenges such as limited drug loading capacity, poor encapsulation stability, complex preparation processes, and potential immunogenicity and toxicity induced by the carrier, limiting their clinical application. Recent studies have shown that the median drug dose ultimately reaching solid tumors is only 0.7%. Furthermore, the high difficulty in reproducibly preparing DDSs also hinders their clinical application.

[0003] To address the aforementioned challenges, a novel strategy for drug self-delivery systems (DSDSs) has been proposed in recent years. These systems primarily consist of small-molecule drugs and require no nanocarriers. Typically, nanostructures are assembled through intermolecular or intramolecular interactions between different drug molecules. Of particular note are these self-delivered supramolecular nanomedicines, which have attracted widespread research interest in combination drug delivery and therapy due to their customized nanostructures, simple preparation methods, high drug loading efficiency, excellent biocompatibility, and strong tumor targeting capabilities. For example, chemotherapeutic drugs, peptides, prodrugs, or metal ions have all been reported as assembly units for constructing self-delivery systems. Despite significant progress in this field, achieving a balance between assembly performance and synergistic effects among different components remains challenging. Maintaining supramolecular interactions to ensure assembly stability in the complex biological environment of living organisms is also a challenge. Therefore, there is an urgent need to develop a carrier-free self-delivery system that can simultaneously achieve both high assembly performance and therapeutic efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a supramolecular carrier-free self-delivery system, its preparation method, and its application in tumor therapy, thereby addressing the problems existing in the prior art. This invention utilizes mitoxantrone (MTX), dihydroporphyrin (Ce6), and ultrasmall iron oxide nanoparticles (IONP) to co-assemble a supramolecular carrier-free self-delivery system, CMIOs. This system is simple to prepare, stable in performance, and highly biosafety. It can synergistically inhibit cancer through multiple mechanisms, achieving highly efficient inhibition of breast cancer, providing a new strategy for tumor therapy, and has broad application prospects in solving clinical problems.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a supramolecular carrier-free self-delivery system, which is prepared by non-covalent self-assembly of mitoxantrone, dihydroporphyrin, and ultrasmall iron oxide nanoparticles in an aqueous phase.

[0006] The present invention also provides a method for preparing the above-mentioned supramolecular carrier-free self-delivery system, wherein the mitoxantrone, dihydroporphyrin and ultrasmall iron oxide nanoparticles are dissolved in water and allowed to react statically to obtain the supramolecular carrier-free self-delivery system.

[0007] Furthermore, the final concentration ratio of mitoxanone, dihydroporphyrin, and ultrasmall iron oxide nanoparticles is 1:3:1.

[0008] Furthermore, the settling time is 20-40 minutes.

[0009] This invention also provides the application of the above-mentioned supramolecular carrier-free self-delivery system in the preparation of drugs for treating tumors.

[0010] In the supramolecular carrier-free self-delivery system of this invention, mitoxantrone (MTX) acts as a chemotherapeutic drug, and dihydroporphyrin (Ce6) acts as a photosensitizer, which can generate singlet oxygen under light conditions. 1 In the presence of highly expressed hydrogen peroxide (H2O2) in the tumor microenvironment, ultrasmall iron oxide nanoparticles (IONP) can undergo the Fenton reaction to generate hydroxyl radicals (•OH). The three assembly units work together to exert tumor-killing functions and achieve significant inhibition of tumors.

[0011] Optionally, the tumor includes breast cancer.

[0012] Optionally, the drug includes a photodynamic therapy drug; the photodynamic therapy drug, combined with light irradiation, achieves the effect of treating tumors.

[0013] The present invention also provides a medicament for treating tumors, wherein the active ingredient of the medicament comprises the above-mentioned supramolecular carrier-free self-delivery system.

[0014] Optionally, the tumor includes breast cancer.

[0015] Optionally, the drug includes a photodynamic therapy drug; the photodynamic therapy drug, combined with light irradiation, achieves the effect of treating tumors.

[0016] The present invention discloses the following technical effects: This invention reveals that the supramolecular carrier-free self-delivery system CMIOs, co-assembled from mitoxanone (MTX), dihydroporphyrin (Ce6), and ultrafine iron oxide nanoparticles (IONP), possesses advantages such as regular morphology, uniform dispersion, good reproducibility, high stability, and good biocompatibility. Utilizing the chemotherapeutic properties of MTX, the photosensitizing properties of Ce6, and the Fenton reaction properties of IONP, combined therapy against tumors can be achieved. Cellular and animal experiments demonstrate that this system exhibits strong killing ability against breast cancer cells (4T1) and significantly reduces the toxicity of MTX to normal cells; it can also significantly inhibit tumor growth in mice, with a tumor inhibition rate as high as 80.4%.

[0017] The supramolecular carrier-free self-delivery system CMIOs of this invention is simple to prepare, has stable performance, and high biosafety. It can synergistically inhibit cancer through multiple mechanisms, achieving highly efficient inhibition of breast cancer. It provides a new strategy for tumor treatment and has broad application prospects in solving clinical problems. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The structural formulas and schematic diagrams of mitoxantrone (MTX), dihydroporphyrin (Ce6), and ultrasmall iron oxide nanoparticles (IONP) are shown. Figure 2 TEM images of the morphology of CMIOs, a supramolecular carrier-free self-delivery system; Figure 3 The particle size distribution of CMIOs, a supramolecular carrier-free self-delivery system; Figure 4 The surface potential distribution of CMIOs, a supramolecular carrier-free self-delivery system; Figure 5 The particle size distribution of the supramolecular carrier-free self-delivery system CMIOs after 7 days in phosphate buffer solution; Figure 6The graphs show the release curves of Ce6 drug from the supramolecular carrier-free self-delivery system CMIOs in solutions with different pH values. Figure 7 To investigate the in vitro cytotoxic effects of different concentrations of the drug on 4T1 breast cancer cells; Figure 8 To investigate the in vitro cytotoxic effects of different concentrations of the drug on normal human kidney 293T cells; Figure 9 The graph shows the changes in body weight of mice in different groups; Figure 10 A statistical graph showing the tumor inhibition rate of mice in different groups; Figure 11 The graph shows the results of the main indicators of liver and kidney function in mice from different groups; where a is the ALT test result, b is the AST test result, c is the BUN test result, and d is the Crea test result. Figure 12 H&E staining images of major internal organs of mice in different groups. Detailed Implementation

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

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

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

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

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

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0026] Mitoxantrone (MTX) and dihydroporphyrin (Ce6) involved in the following examples were purchased from Shanghai Titan Technology Co., Ltd. and Shanghai Aladdin Biochemical Technology Co., Ltd., respectively. Ultrasmall iron oxide nanoparticles (IONPs) were synthesized according to the literature (Kim BH, Lee N, Kim H, et al. Large-Scale Synthesis of Uniform and Extremely Small-Sized Iron Oxide Nanoparticles for High-Resolution T1 Magnetic Resonance Imaging Contrast Agents[J].), and their structural formula and schematic diagram are shown below. Figure 1 As shown.

[0027] Example 1: Preparation and Characterization of Supramolecular Carrier-Free Self-Delivery System CMIOs A method for preparing a supramolecular carrier-free self-delivery system CMIOs includes the following steps: A concentrated solution of MTX, Ce6, and IONP at a certain concentration was diluted in a certain volume of deionized water, so that the concentrations of MTX, Ce6, and IONP in the mixed solution were 50 μM, 150 μM, and 50 μM, respectively, in a concentration ratio of 1:3:1. After standing for 30 minutes, the solution was tested using a transmission electron microscope. The results are as follows: Figure 2 As shown, CMIOs with uniform particle size and good dispersibility were obtained. The average particle size of the CMIOs assembly system is approximately 35 nm.

[0028] Example 2: Performance Characterization and Stability Study of CMIOs Take 1 mL of the CMIOs solution prepared in Example 1, and use a dynamic light scattering particle size analyzer to test the particle size distribution and surface potential of the CMIOs. The results are as follows: Figure 3 As shown, the average particle size of CMIOs is 40 nm, consistent with the electron microscopy characterization results; Figure 4 As shown, the surface potential of CMIOs is negative, with a value of -28.7 mV, which is conducive to the uptake of assemblies by cells.

[0029] The solvent for the CMIOs solution was changed from pure water to an equal volume of phosphate buffer solution (pH=7.4, incubated at 37℃, with particle size changes monitored every day). The results are as follows: Figure 5 As shown, the particle size of CMIOs remained essentially unchanged over 5 days, indicating that CMIOs have good physiological stability, laying a solid foundation for subsequent biological testing.

[0030] The solvent for the CMIOs solution was changed from pure water to an equal volume of phosphate buffer solutions with different pH values, and the solution was placed at 37°C. The release of Ce6 was monitored in real time. The results are as follows: Figure 6 As shown, under acidic conditions (pH = 5.5), CMIOs disassemble, releasing the drug Ce6. Compared to the physiological environment at pH = 7.4 (11.2%), the total release of Ce6 within 10 hours was 40.6%, indicating that CMIOs have acid-responsive properties, and the microacidic environment of the tumor can induce the disassembly of CMIOs, thereby releasing the drug.

[0031] Example 3: Evaluation of the in vitro antitumor activity of CMIOs Using breast cancer cells 4T1 or normal cells 293T as experimental subjects, five experimental groups were set up: Ce6 group, Ce6+L group, MTX group, CMIOs group, and CMIOs+L group. Among them, the Ce6+L group and the CMIOs+L group were exposed to light (660 nm, 0.5 W / cm²) after the drug was mixed with the cells. 2 (5 minutes). Each of the above experimental groups was further divided into 5 concentration groups, as follows: Ce6 group: 2.5μM, 5μM, 10μM, 12μM, 15μM; Ce6+L group: 2.5μM, 5μM, 10μM, 12μM, 15μM; MTX group: 0.8μM, 1.7μM, 3.3μM, 4μM, 5μM; CMIOs group: ([MTX] = [IONP] = 0.8 μM, [Ce6] = 2.5 μM), ([MTX] = [IONP] = 1.7 μM, [Ce6] = 5 μM), ([MTX] = [IONP] = 3.3 μM, [Ce6] = 10 μM), ([MTX] = [IONP] = 4 μM, [Ce6] = 12 μM), ([MTX] = [IONP] = 5 μM, [Ce6] = 15 μM); the preparation method of CMIOs is the same as in Example 1; CMIOs+L group: ([MTX] = [IONP] = 0.8 μM, [Ce6] = 2.5 μM), ([MTX] = [IONP] = 1.7 μM, [Ce6] = 5 μM), ([MTX] = [IONP] = 3.3 μM, [Ce6] = 10 μM), ([MTX] = [IONP] = 4 μM, [Ce6] = 12 μM), ([MTX] = [IONP] = 5 μM, [Ce6] = 15 μM); the preparation method of CMIOs is the same as in Example 1.

[0032] Following the above grouping, each drug was co-incubated with the cells for 24 hours. After incubation, the absorbance of the cells was measured using a CCK8 assay kit, and their viability was calculated. Results are as follows: Figure 7 As shown, under illumination conditions (660 nm, 0.5 W / cm²), 2 (After 5 minutes), the CMIOs+L group showed the most significant inhibitory effect on 4T1 cell activity, achieving the best anti-tumor effect, which proves that the drug components in CMIOs have a synergistic anti-cancer effect. Figure 8 As shown, the CMIOs group also significantly reduced the toxicity of MTX to 293T cells, indicating that the CMIOs assembly has a certain selectivity in killing tumor cells and normal cells, and can reduce toxicity to normal tissues and improve biosafety.

[0033] Example 4: In vivo antitumor activity assessment of CMIOs The in vivo antitumor activity of CMIOs was investigated using a 4T1 tumor-bearing mouse model. BALB / c mice (purchased from Shanghai Regent Technology Co., Ltd.) were randomly divided into four groups: control group (Control), model group (CM), model light-illuminated group (CM+L), Ce6+L group, MTX+L group, CMIOs group, and CMIOs+L group. The CM, CM+L, Ce6+L, MTX+L, CMIOs, and CMIOs+L groups were used to establish a 4T1 tumor-bearing mouse model via subcutaneous injection of 4T1 cells. The tumor size was determined by... 3The medication was administered via tail vein injection. The administration methods for each group are as follows: Control group: An equal volume of normal saline was injected into the tail vein; CM group: One intravenous injection of a mixture of Ce6 (4 mg / kg body weight) and MTX (1 mg / kg body weight); CM+L group: A single intravenous injection of a mixture of Ce6 (4 mg / kg body weight) and MTX (1 mg / kg body weight) was administered via tail vein; phototherapy (660 nm, 0.5 W / cm²) was administered every two days. 2 (5 minutes), a total of 3 times; Ce6+L group: Ce6 (4 mg / kg body weight) was injected once via tail vein; light exposure was given every 2 days (660 nm, 0.5 W / cm²). 2 (5 minutes), a total of 3 times; MTX+L group: MTX (1 mg / kg body weight) was injected once via tail vein; light exposure was given every 2 days (660 nm, 0.5 W / cm²). 2 (5 minutes), a total of 3 times; CMIOs group: CMIOs ([Ce6] = 4 mg / kg body weight) were injected once via the tail vein; CMIOs+L group: CMIOs ([Ce6] = 4 mg / kg body weight) were injected once via tail vein; light exposure was given every 2 days (660nm, 0.5W / cm²). 2 (5 minutes), a total of 3 times.

[0034] Body weight and tumor volume of mice in each group were continuously monitored for 12 days. Results are as follows: Figure 9 As shown, the mice exhibited a steady increase in body weight, indicating that the drugs and CMIOs did not have significant side effects on mouse growth. Figure 10 As shown, compared with the MTX + L group (23.9%), Ce6 + L group (32.2%) and CM + L group (54.0%), the CMIOs + L group significantly inhibited the growth rate of 4T1 tumors, with a tumor inhibition rate as high as 80.4%, indicating that the CMIOs + L group has superior anti-tumor activity due to its tumor targeting and synergistic effects through multiple mechanisms.

[0035] Blood and major internal organs of mice in each group were collected on day 12 after drug administration for serum biochemical indicators and H&E staining. Results are as follows: Figure 11 As shown, the liver and kidney function indicators of mice in each group were within the normal range, indicating that CMIOs have high biocompatibility. Figure 12 As shown, compared with the blank group, no significant abnormalities were observed in the H&E staining of the viscera of mice treated with CMIOs, indicating that this combination therapy has excellent biocompatibility.

[0036] All the above results indicate that the CMIOs materials prepared in this invention have high biocompatibility.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A supramolecular carrier-free self-delivery system, characterized in that, The supramolecular carrier-free self-delivery system was prepared by non-covalent self-assembly of mitoxantrone, dihydroporphyrin, and ultrasmall iron oxide nanoparticles in an aqueous phase.

2. A method for preparing the supramolecular carrier-free self-delivery system according to claim 1, characterized in that, The mitoxantrone, dihydroporphyrin, and ultrafine iron oxide nanoparticles were dissolved in water and allowed to react statically to obtain the supramolecular carrier-free self-delivery system.

3. The preparation method according to claim 2, characterized in that, The final concentration ratio of mitoxanone, dihydroporphyrin, and ultrafine iron oxide nanoparticles is 1:3:

1.

4. The preparation method according to claim 2, characterized in that, The static reaction time is 20-40 minutes.

5. The use of the supramolecular carrier-free self-delivery system according to claim 1 in the preparation of drugs for treating tumors.

6. The application according to claim 5, characterized in that, The tumors include breast cancer.

7. The application according to claim 5, characterized in that, The drug includes a photodynamic therapy drug; the photodynamic therapy drug, combined with light irradiation, achieves the effect of treating tumors.

8. A drug for treating tumors, characterized in that, The active ingredient of the drug includes the supramolecular carrier-free self-delivery system as described in claim 1.

9. The medicament according to claim 8, characterized in that, The tumors include breast cancer.

10. The medicament according to claim 8, characterized in that, The drug includes a photodynamic therapy drug; the photodynamic therapy drug, combined with light irradiation, achieves the effect of treating tumors.

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