Ruthenium-based diagnosis and treatment nano material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano materials, in particular to a ruthenium-based diagnosis and treatment nano material and a preparation method and application thereof. The ruthenium-based diagnosis and treatment nano material structurally comprises a copper-zinc compound (CZO) core formed by ZnO2 and copper salt; the [Ru (dcbpy) 3] Cl2 layer ((at) R) is coated on the surface of the CZO core; and the arginine-hyaluronic acid compound ((at) AH) is modified on the outermost layer. The CZO (at) R (at) AH material disclosed by the invention not only has excellent drug delivery efficiency, but also shows relatively strong anti-tumor activity. In a cytotoxicity experiment, the material effectively inhibits the growth of melanoma cells B16F10 and A375, and has relatively low toxicity to normal skin cells HaCaT. Accurate striking on tumor cells is achieved, and systemic toxic and side effects caused by traditional chemotherapy are avoided. Meanwhile, due to the red fluorescence of CZO-R-AH, the purpose of diagnosis and treatment integration can be achieved in the whole treatment process, the treatment time of a patient is saved, and the treatment cycle efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a ruthenium-based therapeutic nanomaterial, its preparation method, and its application. Background Technology

[0002] Cancer treatment primarily includes surgical resection, chemotherapy, and radiotherapy. When choosing a treatment plan, factors such as the type, location, and stage of the tumor are typically considered to determine the most suitable approach. The ultimate goal of these treatments is to achieve complete tumor removal as much as possible. However, in advanced stages of cancer, even if a tumor is successfully removed through surgical resection, the possibility of cancer invasion and metastasis cannot be ruled out. Therefore, for malignant tumors such as melanoma, a comprehensive systemic diagnosis and treatment are necessary. While chemotherapy, as a systemic treatment, can act on cancer cells throughout the body, the accumulation of most chemotherapy drugs in tumor tissue is not ideal. To improve treatment success rates, multiple high-dose administrations are often required. However, this high-dose administration can lead to significant drug accumulation in healthy organs and tissues, triggering a series of serious side effects. These side effects have a significant impact on the patient's overall health and are a problem that must be carefully considered and addressed during treatment.

[0003] Currently, most drugs or probes are delivered to the tumor area via injection or blood flow, relying on their inherent properties to image the tumor or elicit responsive changes, thereby achieving diagnostic and monitoring functions. These methods include photoacoustic imaging (PAI), photothermal imaging (PTI), ultrasound imaging (USI), and fluorescence imaging (FLI). Existing tumor diagnosis and treatment research largely utilizes near-infrared light irradiation of small-molecule photosensitizers or nanoparticles to achieve imaging diagnosis, photothermal therapy, or to enhance treatment efficacy through co-delivery of chemotherapy drugs. However, these drugs or probes either only possess imaging capabilities without therapeutic effects, or require large, sophisticated instruments to achieve therapeutic functions.

[0004] To reduce the toxicity to normal tissues and improve drug utilization, researchers are actively developing targeted or responsive drugs. Tumor-targeted drugs can accumulate at the tumor site, improving utilization efficiency. Drug targeting can be passive or active. Passive targeting of nanomedicines utilizes their small size and the high permeability of tumor blood vessels to enhance drug penetration and retention in the tumor area, but there are significant differences among patients. Active targeting leverages the special structures of cancer cells or the tumor microenvironment (TME), such as overexpressed receptors, to allow nanomedicines to actively accumulate in the tumor area, increasing contact and uptake by tumor cells. Response drugs, in tumor uptake or within the TME, utilize characteristics such as low pH, high GSH, and high H2O2 to initiate drug delivery or orthogonally catalyze the production of toxic substances, reducing the toxic side effects of systemic administration. Therefore, developing targeted and responsive drugs against tumors and the TME is of great significance.

[0005] Therefore, combining nanomaterial imaging with chemokinetics utilizing the tumor microenvironment (TME) to construct an integrated diagnostic and therapeutic strategy is expected to predict treatment response based on real-time monitoring and achieve selective clearance of cancer cells. Summary of the Invention

[0006] The purpose of this invention is to provide a ruthenium-based therapeutic nanomaterial, its preparation method, and its application. The ruthenium-based therapeutic nanomaterial is a CZO@R@AH nanomaterial with a copper-zinc complex as the core and a surface modified with [Ru(dcbpy)3]Cl2 and an arginine-hyaluronic acid layer. It releases copper / zinc ions in response to acid to kill B16F10 / A375 melanoma cells, and achieves targeted imaging using red fluorescence.

[0007] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0008] A CZO@R@AH nanomaterial has the following structure: a copper-zinc complex (CZO) core formed by ZnO2 and copper salt; a [Ru(dcbpy)3]Cl2 layer (@R) covering the surface of the CZO core; and an arginine-hyaluronic acid complex (@AH) modified on the outermost layer.

[0009] Furthermore, the nanomaterials release copper / zinc ions in a tumor microenvironment with a pH < 7.4.

[0010] Furthermore, the nanomaterial targets the CD44 receptor overexpressed in melanoma cells via hyaluronic acid.

[0011] Furthermore, the nanomaterial uses the red fluorescence of the @R layer to locate drug distribution and monitor the therapeutic effect.

[0012] Furthermore, the CZO core is prepared by the following steps: dispersing ZnO2 in ethanol, adding CuCl2·2H2O and stirring, and centrifuging and washing to obtain the core; wherein ZnO2 is prepared by reacting zinc acetate, PVP K30 and H2O2 in water.

[0013] Furthermore, the @R layer modification method includes: reacting CZO with APTES in anhydrous ethanol, centrifuging and washing to obtain aminated CZO; and connecting the aminated CZO with EDC / NHS activated [Ru(dcbpy)2]Cl2.

[0014] Furthermore, the @AH layer modification method includes: reacting EDC / NHS activated hyaluronic acid with L-arginine, and obtaining Arg-HA after dialysis; ultrasonically mixing Arg-HA aqueous solution with CZO@R aqueous solution, and forming it through electrostatic adsorption.

[0015] On the other hand, the present invention proposes a method for preparing the above-mentioned nanomaterials, comprising the following steps:

[0016] S1: Preparation of ZnO2: Dissolve zinc acetate and PVP K30 in ultrapure water, add H2O2 and stir, centrifuge, wash and dry;

[0017] S2: Preparation of CZO: Disperse ZnO2 in ethanol, add CuCl2·2H2O and stir, centrifuge at 8000 r / min for 5 min, wash with ethanol and dry;

[0018] S3: Preparation of CZO@R: CZO and APTES were reacted in an oil bath in anhydrous ethanol, centrifuged and washed, and then connected with EDC / NHS activated [Ru(dcbpy)3]Cl2. After centrifugation at 10000 r / min for 5 min, the mixture was washed with water / ethanol and dried.

[0019] S4: Preparation of Arg-HA: Hyaluronic acid is activated by EDC / NHS and then reacted with L-arginine, followed by lyophilization via dialyzation.

[0020] S5: Preparation of CZO@R@AH: Sonically mix Arg-HA aqueous solution and CZO@R aqueous solution, stir, centrifuge at 10000 r / min for 5 min and wash.

[0021] On the other hand, this invention proposes the application of the above-mentioned nanomaterials in the diagnosis and treatment of melanoma, including: hyaluronic acid-mediated targeted uptake by melanoma cells; acidic tumor microenvironment triggering the release of copper / zinc ions, inducing tumor cell death; and ruthenium complex layer providing fluorescence imaging function to monitor drug distribution in real time.

[0022] The beneficial effects of this invention are:

[0023] This invention, the CZO@R@AH nanomaterial, achieves highly efficient melanoma cell targeting through its hyaluronic acid (HA) layer, addressing the lack of targeted therapy in most current chemotherapy drugs. Hyaluronic acid specifically binds to the overexpressed CD44 receptor on the surface of tumor cells, not only improving drug uptake but also ensuring drug concentration near tumor cells, thereby effectively enhancing drug utilization. After binding to the high receptor expression in tumor tissue, hyaluronic acid promotes the accumulation of the nanomaterial at the tumor site, significantly reducing drug accumulation in normal cells and minimizing potential side effects.

[0024] The responsive ion release properties exhibited by CZO@R@AH in the acidic tumor microenvironment of this invention further enhance the efficacy of treatment, addressing the problem that most current drugs lack controllable release capabilities and have high systemic toxicity. The tumor microenvironment is typically characterized by a low pH value. Based on its peroxide properties, the nanomaterial of this invention can accelerate the release of zinc and copper ions in this environment. This selective ion release effectively promotes tumor cell death by inducing intracellular oxidative stress and DNA damage. Due to the core structure of the copper-zinc complex (CZO), its stability decreases upon entering the tumor microenvironment, thereby accelerating the ion release rate. Because of the unique acidic microenvironment of tumor cells, normal cells are not significantly affected at neutral pH, demonstrating the selectivity and safety of the treatment.

[0025] This invention, the CZO@R@AH nanomaterial, endows the material with excellent fluorescence imaging capabilities through a ruthenium complex layer (@R), enabling simultaneous visualization and monitoring of drugs during treatment. This overcomes the limitation of current treatment methods that require separate diagnosis and treatment steps, saving medical resources and reducing patient waiting time. Due to its strong fluorescence emission properties, the ruthenium complex provides real-time imaging feedback during drug delivery and targeting, facilitating the tracking of drug distribution and release dynamics within the body. This ensures real-time observation of the drug's location and extent of action during treatment, providing timely feedback to physicians, assisting in decision-making, and further evaluating treatment efficacy. The enhanced tumor uptake mechanism through drug targeting also makes imaging more reliable. This integrated imaging and treatment feature eliminates the need for large, sophisticated instruments, enabling precise tumor treatment and monitoring.

[0026] The CZO@R@AH material of this invention exhibits a strong nitric oxide release capacity by encapsulating arginine in a hyaluronic acid layer (@AH). Through the ROS effect induced by copper-zinc ions in tumor cells, it induces a ROS / RNS cascade to produce ONOO. − This enhances the tumor-killing effect. Under the premise of pH response, it greatly improves the treatment efficiency.

[0027] The CZO@R@AH material of this invention not only possesses excellent drug delivery efficiency but also exhibits strong anti-tumor activity. In cytotoxicity experiments, this material effectively inhibited the growth of B16F10 and A375 melanoma cells, while showing low toxicity to normal skin cells (HaCaT). This achieves precise targeting of tumor cells while avoiding the systemic toxic side effects of traditional chemotherapy.

[0028] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of 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.

[0030] Figure 1 Schematic diagram of the cytotoxic effects of CZO@R@AH nanomaterials; A: Cytotoxic effect on B16F10 mouse melanoma cells at a concentration of 0–40 μg / mL; B: Cytotoxic effect on A375 human malignant melanoma cells at a concentration of 0–40 μg / mL; C: Cytotoxic effect on HaCaT human immortalized keratinocytes at a concentration of 0–40 μg / mL.

[0031] Figure 2 A schematic diagram characterizing the cellular mechanism of action of CZO@R@AH; A: Staining results of live and dead cells; B: Semi-quantitative analysis of live and dead cell staining; C: Subcellular localization confocal microscopy images; D: Cell-targeted imaging comparison.

[0032] Figure 3 The diagram shows the ion release characteristics of CZO@R@AH; A: Zinc ion release curves at pH 5.0 vs pH 7.4; B: Copper ion release curves at pH 5.0 vs pH 7.4. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] The preparation method of the arginine-hyaluronic acid complex (Arg-HA) as described in this embodiment includes: dissolving 0.2 g of hyaluronic acid in ultrapure water, adding EDC and NHS to activate HA, and adding L-Arg and stirring after activation. After stirring, dialyzing the product overnight using a dialysis bag, and then freeze-drying to obtain a solid product.

[0036] Example 2

[0037] The preparation method of CZO@R@AH nanomaterials as described in this embodiment includes the following steps:

[0038] Preparation of ZnO2: Zinc acetate and PVP K30 were dissolved in 50 ml of ultrapure water. Then, hydrogen peroxide was added, and the mixture was stirred rapidly at room temperature to obtain a white product. The product was then centrifuged and washed three times with ethanol, and dried in a vacuum drying oven.

[0039] Preparation of CZO: ZnO2 was dispersed in anhydrous ethanol, CuCl2∙2H2O was added, and the mixture was stirred and centrifuged (8000 r / min, 5 min). The mixture was washed three times with ethanol and dried in a vacuum drying oven.

[0040] Preparation of CZO@R: CZO and APTES were dispersed in anhydrous ethanol, centrifuged after an oil bath, washed three times with ethanol, and dried in a vacuum drying oven to obtain powder. [Ru(dcbpy)3]Cl2, EDC, and NHS were activated in water by stirring, and the above powder was added. After stirring, the mixture was centrifuged (10000 r / min, 5 min), washed three times each with H2O and anhydrous ethanol, and then dried in a vacuum drying oven.

[0041] Preparation of CZO@R@AH: Arg-HA was dispersed in ultrapure water to prepare solution A. CZO@R was dispersed in water to prepare solution B. Solution B was slowly added to solution A under ultrasonication, followed by stirring. The product was then centrifuged (10000 r / min, 5 min) and washed.

[0042] Example 3

[0043] Cell viability assay: The cytotoxicity of the materials was assessed using B16F10 mouse skin melanoma cells and A375 human malignant melanoma cells to evaluate antitumor efficacy, while HaCaT human immortalized keratinocytes were used to assess biosafety. Cells were first seeded at appropriate density in 96-well plates and pre-cultured overnight. Then, a series of concentration gradients of the materials were added and co-incubated with the cells for 24 h. Finally, CCK-8 solution was added according to the kit instructions, and after color development, the absorbance was measured at 450 nm using a microplate reader. A control group was used, and the cytotoxicity of the materials was determined by comparing the absorbance values ​​of the experimental and control groups. (n=3)

[0044] Live and dead cell staining: After incubating CZO@R@AH with cells for 24 h, the cells were incubated with Calcein AM / PI for 30 min, and the live and dead cell status was observed under a microscope (Calcein AM: Ex=494 nm, Em=500–530 nm; PI: Ex=535 nm, Em=600–630 nm; red fluorescence indicates dead cells, and green fluorescence indicates live cells).

[0045] Cell uptake: Cells were seeded on culture dishes and incubated with the material for 4 h before being stained with different organelle dyes. The colocalization of fluorescence in the material and organelles was observed using laser confocal microscopy.

[0046] Cell targeting: Three cell lines (B16F10, A375, and HaCaT) were seeded on culture dishes and incubated with the material for 4 hours. After washing, the fluorescence of the material and the colocalization of cells were observed by laser confocal microscopy.

[0047] Figure 1 In samples A and B, CZO@R@AH was observed to have a strong anti-melanoma effect. At 40 μg / mL, it showed at least 50% cytotoxicity against B16-F10 and A375 cells, while exhibiting good biocompatibility against normal human skin tissue cells (HaCaT).

[0048] Figure 2 In study A, it was observed that CZO@R@AH had a better tumor cell lethality effect compared to the control group. Figure 2 Semi-quantitative analysis in B showed that CZO@R@AH exhibited significant tumor cell cytotoxicity. Figure 2 CZO@R@AH in C exhibited good cell imaging and subcellular localization (mitochondria and lysosomes) through its own red fluorescence. Figure 2 In D, comparing B16-F10 and A375 melanoma cells with HaCaT normal human skin cells, CZO@R@AH showed better targeted imaging performance. B16-F10 and A375 cells showed more obvious red fluorescence, while HaCaT showed almost no fluorescence after washing.

[0049] Figure 3 In study A, it was observed that within 24 hours, CZO@R@AH released approximately 26% of zinc ions and approximately 18% of copper ions under acidic conditions, significantly higher than the release effect under neutral conditions. This effect of releasing large amounts of copper and zinc ions in response to the specific acidic pH of the tumor microenvironment, combined with its own targeting action, enables a more specific effect on tumor cells and greatly reduces the toxicity to normal tissue cells.

[0050] This invention develops a responsive, self-catalyzed copper-zinc-ruthenium trimetallic nanomaterial (CZO@R@AH) that enables targeted therapy for melanoma. In the material synthesis, arginine is grafted onto hyaluronic acid and [Ru(dcbpy)3]Cl2 to endow the material with targeting and acid-responsive properties against melanoma. By targeting tumor cells, the material exhibits significant fluorescence imaging capabilities after uptake by tumor cells. Furthermore, the low pH of the tumor microenvironment causes acid-responsive decomposition, releasing large amounts of copper and zinc ions, inducing various cell death mechanisms, and achieving a killing effect on tumor cells. In cytotoxicity assays, 40 μg / mL of CZO@R@AH caused approximately 87% toxicity to mouse melanoma cells and approximately 50% toxicity to human melanoma cells, while showing almost no toxicity to normal human skin cells at a concentration of 40 μg / mL.

[0051] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A CZO@R@AH nanomaterial, characterized in that, The structure comprises: a copper-zinc complex core formed by ZnO2 and copper salt; a [Ru(dcbpy)3]Cl2 layer coated on the surface of the CZO core; and an arginine-hyaluronic acid complex modified on the outermost layer.

2. The CZO@R@AH nanomaterial of claim 1, wherein: The nanomaterial releases copper / zinc ions in a tumor microenvironment with pH < 7.

4.

3. The CZO@R@AH nanomaterial of claim 1, wherein: The nanomaterial targets CD44 receptors overexpressed by melanoma cells through hyaluronic acid.

4. The CZO@R@AH nanomaterial of claim 1, wherein: The nanomaterial locates drug distribution and monitors treatment effect through red fluorescence of the R layer.

5. The CZO@R@AH nanomaterial as described in claim 1, characterized in that: The CZO core is prepared by dispersing ZnO2 in ethanol, adding CuCl2·2H2O and stirring, and centrifuging and washing to obtain; wherein the ZnO2 is prepared by reacting zinc acetate, PVP K30 and H2O2 in water.

6. The CZO@R@AH nanomaterial of claim 1, wherein: The R layer modification method comprises: reacting CZO with APTES in anhydrous ethanol, centrifuging and washing to obtain aminated CZO; and connecting the aminated CZO with EDC / NHS activated [Ru(dcbpy)2]Cl2.

7. The CZO@R@AH nanomaterial of claim 1, wherein: The AH layer modification method comprises: reacting EDC / NHS activated hyaluronic acid with L-arginine, and dialyzing to obtain Arg-HA; and ultrasonically mixing an Arg-HA aqueous solution with a CZO@R aqueous solution to form by electrostatic adsorption.

8. The method of claim 1-7, wherein: The method comprises the following steps: S1: preparing ZnO2: dissolving zinc acetate and PVP K30 in ultrapure water, adding H2O2 and stirring, centrifuging and drying; S2: preparing CZO: dispersing ZnO2 in ethanol, adding CuCl2·2H2O and stirring, centrifuging at 8000 r / min for 5 min, and washing with ethanol and drying; S3: preparing CZO@R: oil bath reacting CZO and APTES in anhydrous ethanol, connecting with EDC / NHS activated [Ru(dcbpy)3]Cl2 after centrifuging and washing, centrifuging at 10000 r / min for 5 min, and washing with water / ethanol and drying; S4: preparing Arg-HA: reacting hyaluronic acid after EDC / NHS activation with L-arginine, dialyzing and freeze-drying; S5: preparing CZO@R@AH: ultrasonically mixing an Arg-HA aqueous solution with a CZO@R aqueous solution, stirring, centrifuging at 10000 r / min for 5 min and washing.

9. The nanomaterial for use in the diagnosis and treatment of melanoma according to any one of claims 1 to 7, characterized in that: The method comprises: Hyaluronic acid-mediated melanoma cell targeting uptake; acid tumor microenvironment triggers copper / zinc ion release, inducing tumor cell death; and a ruthenium complex layer provides fluorescence imaging function, real-time monitoring of drug distribution.

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