Preparation method of nano composite material based on zinc-based cystine coordination polymer and application of nano composite material in anti-tumor treatment

Abstract

The invention discloses a construction method of a nano composite material based on a zinc-based cystine coordination polymer and application of the nano composite material in anti-tumor treatment, and belongs to the technical field of composite medical materials. The preparation method comprises the following steps: preparing polydopamine coated curcumin-gelatin drug nanocrystals (Cur-coated PDA DNCs) so as to enhance the stability and modifiability of the polydopamine coated curcumin-gelatin drug nanocrystals; furthermore, the Cur (at) PDA DNCs, glucose oxidase (GOx) and a metal amino acid coordination polymer (Zn-Cys) are mixed, and the Cur (at) PDA (at) GOx / Zn-Cys composite nanomaterial (CPGZC NPs) is prepared through an in-situ growth method. The design of the whole composite material treatment system gives full play to the synergistic effect of all parts of the material, and effective compounding of multiple components is achieved. The nano composite material disclosed by the invention has the advantages that the cancer cell killing effect is obviously improved by activating cancer cell disulfide death and cell apoptosis pathways in vitro; furthermore, the compound also has an excellent anti-tumor effect in a mouse body, and has relatively high biological safety.

Description

Technical Field The present invention belongs to the technical field of composite pharmaceutical materials, and particularly relates to a preparation method of a nanocomposite material based on a zinc-based cysteine coordination polymer and its application in anti-tumor treatment. Background Art

[0001] Cell death, as a common phenomenon of degradation and damage, is an indispensable part of the development and pathophysiological processes of organisms. Among them, regulated cell death (RCD) is a spontaneous and programmed cell death process, involving a signal cascade reaction participated by specific effector molecules, and this signal pathway can be intervened and regulated by drug or gene molecules to achieve selective targeted induction of cancer cell death without harming normal cells. Therefore, it has always received extensive attention from people (Signal Transduct Target Ther., 2022, 7, 286). With the continuous development and in-depth study of various RCD modes, some key pathway factors have gradually become powerful targets for developing new cancer treatment methods. However, the complexity and diversity of cancer are constantly evolving, and the discovered RCD cell death modes still have their respective limitations in tumor treatment. In particular, cancer cells can escape from certain mode paths and develop drug resistance, while different cell death modes will help to enrich the target factors and signal pathways for activating cell death, reduce the probability of cancer cell escape, and fundamentally improve the tumor treatment effect. Therefore, the exploration and utilization of new RCD modes have always been a research hotspot.

[0002] Inspired by natural metalloproteins, the self-assembly strategy driven by coordination and non-covalent interactions between metal ions and organic cofactors (Coord. Chem. Rev., 2019, 398, 113009) has increasingly become a new means for constructing multifunctional nano-biological anti-cancer agents. Among these biological agents based on nano-coordination polymers (NCPs), metal-amino acid-mediated self-assembly has received particular attention due to its significant advantages such as simple processing, fine tunability of nanostructures, and good biological properties. In particular, these NCPs combine metal ions (e.g., Zn 2+ 、Fe 3+ 、Cu 2+) and the excellent properties of amino acid ligands, and can also co-assemble a variety of functional small molecules (e.g., drugs, fluorophores), thus achieving efficient drug loading and multifunctionalization (Adv. Sci., 2023, 10, e2303818). In most related studies, amino acids can coordinate and self-assemble with metal ions through multiple binding sites, which can enhance stability, improve biocompatibility and reduce immunogenicity, etc., thus reducing the potential side effects of nanoassemblies (Angew. Chem. Int. Ed., 2018, 57, 17084). However, there are few reports on the pharmacological effects of amino acids in promoting cancer treatment in such nanoassemblies. In addition, metal-amino acid coordination mainly focuses on constructing self-assembled nanoparticles (e.g., nanospheres, nanoshuttles). Further exploration is still needed for the work of assembling metal-amino acid coordination networks on the surface of functional nanoparticles (e.g., organic or inorganic nanoparticles) for cancer treatment, which will lay an important foundation for the development of more efficient multifunctional anti-cancer nanoformulations.

[0003] Drug nanocrystals (DNCs) have been applied to the treatment of infectious and immune diseases, with advantages such as long-lasting efficacy, high drug loading, easy clinical production and low cost. Compared with other drug delivery systems, such as liposomes or polymer nanoparticles, drug nanocrystals are composed of self-assembled drugs without carrier excipients, achieving a drug loading efficiency of nearly 100% (J. Biomed. Nanotechnol., 2022, 18, 939). However, the key challenge is to achieve precise control of the surface modification of DNCs to optimize drug transport and therapeutic effects. Currently, in the developed strategies, the surface of DNCs is mostly functionalized with polymers, small molecules or biomolecules to improve their stability or targeting, but these coatings may introduce conflicting interactions, potentially leading to premature drug release or poor biodistribution. In addition, few studies have focused on achieving synergistic effects by combining multiple active ingredients on DNCs, especially in regulating the cell death mechanism. Therefore, research on the above key issues is crucial for overcoming these obstacles and obtaining more controllable and effective cancer treatment effects.

[0004] Therefore, combining the construction of NCP composites with the latest development direction of RCD pathway regulation, and carrying out the self-assembly preparation of NCP-based biological agents to promote their research and utilization in regulating the cell death mechanism will surely lead to the rapid development of this field. Summary of the Invention

[0001] The present invention belongs to the technical field of composite medical materials, and relates to a preparation method of a composite material of a poly-dopamine-curcumin drug nanocrystal coated with a zinc-based cystine coordination polymer, and its application research on mediating the synergistic regulation of multiple cell death mechanisms for efficient cancer treatment.

[0002] Curcumin-gelatin drug nanocrystals were prepared by the anti-solvent method and then coated with polydopamine to obtain a drug nanocrystal material of curcumin composite polydopamine (Cur@PDADNCs), enhancing its stability and modifiability.

[0003] Cur@PDADNCs, glucose oxidase (GOx), and metal amino acid coordination polymer (Zn-Cys) were mixed into the reaction mother liquor, and Cur@PDA@GOx / Zn-Cys nanocomposite (CPGZC NPs) was prepared by in-situ growth method to achieve effective multi-component composite.

[0004] The nanocomposite can synergistically promote cancer cell death by activating the mechanisms of cell disulfidptosis and apoptosis in vitro, and has significant anti-tumor effects in vivo with high in vivo safety.

[0005] In the construction of zinc-based cysteine coordination polymer nanocomposite, Zn-Cys and GOx grew on the surface of Cur@PDADNCs together by in-situ growth.

[0006] The specific preparation method is as follows:

[0007] The preparation of Cur DNCs was obtained by dropping a curcumin acetone solution into a gelatin solution and centrifuging at 10000 rpm.

[0008] The concentration of the curcumin acetone solution was 10 mg / mL -1 ; the concentration of the gelatin solution was 5 mg / mL -1 .

[0009] When preparing Cur@PDADNCs, every 2 mg of Cur DNCs was dispersed in a 4 mM dopamine hydrochloride solution with a pH of 8.0 - 8.5, stirred uniformly, and obtained after centrifugation.

[0010] To prepare CPGZC NPs, Cur@PDA was dispersed in a GOx solution of 0.2 mg / mL -1 , and the zinc acetate mother liquor and cystine mother liquor were added dropwise and reacted in sequence, and finally obtained after centrifugation.

[0011] The concentration of the zinc acetate mother liquor was 11 mg / mL -1 , and the concentration of the cystine mother liquor was 12 mg / mL -1 .

[0012] The coordination polymer nanocomposite provided by the present invention has a particle size of 150 - 200 nm.

[0013] The ζ potential of the coordination polymer nanocomposite provided by the present invention in PBS solution with pH = 7.4 is -23.1 mV.

[0014] This coordination polymer nanocomposite can be applied to the preparation of liver cancer treatment drugs.

[0015] A cancer treatment drug contains the coordination polymer nanocomposite described in the present invention, and the present invention combines the construction of NCPs composite with the regulation of RCD pathway to explore its application in synergistically activating a new ferroptosis mechanism and the classical apoptosis mechanism.

[0016] Compared with the prior art, the beneficial effects or advantages of the present invention:

[0017] The present invention provides a multifunctional zinc-based cysteine coordination polymer nanocomposite with the ability to induce apoptosis and ferroptosis, which shows excellent effects as an antitumor agent; the present invention provides a multifunctional zinc-based cysteine coordination polymer nanocomposite with the ability to induce apoptosis and ferroptosis, which shows good biosafety. The present invention develops an antitumor agent based on zinc-based cysteine coordination polymer nanocomposite, demonstrating a potentially feasible treatment paradigm for synergistically treating cancer by inducing apoptosis and ferroptosis, with the potential for clinical translation. This also indicates that metal amino acid coordination polymers have great prospects in translational nanomedicine and treating human diseases. Description of the Drawings

[0018] Figure 1 a is the TEM image of Cur@PDA DNCs, and its size is about 150.5 nm; Figure 1 b is the TEM image of the prepared CPGZC NPs, and its size is about 171.6 nm. The enlarged TEM image shows that there is indeed a Zn-Cys shell in the CPGZC NPs prepared in Example 2 and the shell thickness is about 10 nm; Figure 1 c is the SEM image of CPGZC NPs; Figure 1 d is the elemental distribution of CPGZC NPs.

[0019] Figure 2 are the structural characteristics of CPGZC NPs; Figure 2 a is the XRD pattern of pure Cur drug, Cur@PDADNCs (CP NPs), CPGZCNPs, and pure Zn-Cys material; Figure 2 b is the Zn 2p XPS spectrum of CPGZC NPs; Figure 2 c is the S2p XPS spectrum of CPGZC NPs.

[0020] Figure 3are the enzyme activity and release characteristics of CPGZC NPs; Figure 3 a is the infrared spectra of GOx, pure Cur drug, and CPGZC NPs; Figure 3 b is the change in the content of H2O2 in the solution after CPGZC NPs were incubated with different concentrations of glucose solution for 5 h; Figure 3 c is the drug release curve of CPGZC NPs under different pH conditions.

[0021] Figure 4 is the in vitro toxicity test of CPGZC NPs, Figure 4 a is the activity detection of HUVEC and HepG2 cells after treatment with CPGZC NPs; Figure 4 b is the activity detection of HepG2 cells after treatment with pure Cur drug, CP NPs, and CPGZC NPs.

[0022] Figure 5 is the induction of cell disulfidptosis by CPGZC NPs; Figure 5 a is the Drebrin protein immunoblotting of HepG2 cells under reducing and non-reducing conditions after treatment with CPGZC NPs; Figure 5 b is the ATP content of HepG2 cells after treatment with pure Cur drug, CP NPs, and CPGZC NPs; Figure 5 c is the NADP+ / NADPH content of HepG2 cells after treatment with Cur, CP NPs, and CPGZC NPs. P values were calculated by t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0023] Figure 6 is the detection chart of the apoptosis degree of HepG2 cells after treatment with Cur, CP NPs, and CPGZC NPs.

[0024] Figure 7 is the in vivo antitumor activity of CPGZC NPs; a is the photo of the subcutaneous transplanted tumor collected at the end of the experiment; b is the change in tumor volume size (n = 5); c is the change in tumor weight (n = 5). P values were calculated by t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0025] Figure 8 are the histological HE staining images of the heart, liver, spleen, lung, and kidney of mice under different treatments (scale bar: 100 μm). Specific implementation manners

[0026] The present invention can be further described by the following examples. The examples are intended to illustrate the present invention rather than to limit the present invention, and the protection scope of the present invention is not limited thereto.

[0027] Example 1

[0028] Synthesis of Cur@PDADNCs (CP NPs)

[0029] Dissolve the Cur drug in acetone to prepare a concentration of 10 mg mL -1 Then, 200 μL of this solution was gradually added dropwise to a 5% (W / V) gelatin solution. The mixture was centrifuged at 10,000 rpm / min to obtain gelatin-stabilized Cur DNCs. A concentration of 0.6 mg mL -1 The dopamine solution was then stirred with Cur DNCs for 30 min and subsequently centrifuged to obtain stable polydopamine (PDA)-coated curcumin nanoparticles, i.e., CP NPs.

[0030] Example 2

[0031] Synthesis of Cur@PDA@GOx / Zn-Cys nanoparticles (CPGZC NPs)

[0032] CP NPs were mixed with a concentration of 0.2 mg mL -1 The GOx solution was mixed and stirred at room temperature in the dark for 30 minutes. Then, 200 μL of zinc acetate solution (11 mg mL -1 ), and the reaction was continued for another 30 minutes. Subsequently, an equal volume of cystine (Cys) solution (12 mg mL-1) was added dropwise. After stirring for 30 minutes, the mixture was centrifuged and washed three times to obtain CPGZCNPs.

[0033] Figure 1 The TEM data of a shows that the size of CP NPs prepared in Example 1 is 150.5 nm; Figure 1 The TEM data in b show that the size of CPGZC NPs prepared in Example 2 is about 171.6 nm, and the TEM magnified image shows that the Zn-Cys shell layer does exist in the CPGZC NPs prepared in Example 2 and the thickness is about 10 nm; Figure 1 The SEM data of c shows that the CPGZC NPs prepared in Example 2 exhibit a spherical structure with uniform particle size and good dispersion; Figure 1 The element distribution in d proves that the constituent elements such as C, N, O, S and Zn are uniformly distributed throughout the nanostructure. In summary, CPGZC NPs meet the size requirements of nanoparticles for in vivo drug delivery therapy.

[0034] Through the XRD pattern ( Figure 2 a) Analyze the crystallinity of the nanoparticles. The CP NPs are amorphous. The new XRD peaks of the CPGZC NPs match with Zn-cys, confirming that the Zn-Cys network is successfully coated on the surface of CPGZC; The XPS Zn 2p binding energy confirms that there is a stable and clear coordination interaction between the Zn(II) metal nodes and the organic functional groups in CPGZC. The analysis of the S2p binding energy confirms that there are S-S and C-S bonds derived from Cys in the CPGZC NPs. In summary, the CPGZC NPs are composite nanomaterials based on Zn-Cys coordination polymers.

[0035] The positive effects of the present invention are shown by the following specific experimental examples:

[0036] Example 3

[0037] This example provides tests on the enzyme activity and in vitro release performance of CPGZC NPs.

[0038] The infrared spectrum confirms the effective loading of GOx into the CPGZC NPs ( Figure 3 a); Co-incubate the CPGZC NPs with the glucose solution, and it is observed that as the glucose concentration in the system gradually increases, the production amount of H2O2 also increases significantly ( Figure 3 b); In the phosphate buffer solution environment with pH values of 7.4 and 5.5, the CPGZC NPs exhibit good acid-responsive release performance ( Figure 3 c).

[0039] Example 4

[0040] This example provides tests on the in vitro induction of cell disulfidptosis and apoptosis by CPGZC NPs.

[0041] To accurately evaluate the treatment effects of different experimental groups, we carefully incubated the HepG2 cells with each different group. As Figure 4 shown, after the CPGZC NPs act on the HepG2 cells for 12 h, they exhibit extremely strong cytotoxicity. When the concentration reaches 20 μg mL -1 , the viability of the HepG2 cells decreases significantly, down to only 17.6%. In sharp contrast, when the concentration of the CP NPs is 20 μg mL -1 , the cell viability remains at about 50% level.

[0042] The first thing to be satisfied in preparing a new type of nano-medical material is that the material itself has no obvious toxic and side effects on normal cells, Figure 4a demonstrated the low toxicity of CPGZC NPs to normal cells; in addition, by comparing the MTT assay results of pure Cur drug, CP NPs, and CPGZC NPs on HepG2 cells, it was shown that the multi-component composite nanosystem in the present invention had a better therapeutic effect than single-component ones, such as Figure 4 b.

[0043] Furthermore, the mechanism of CPGZC NPs-induced cell bisulfide death was studied. The actin-binding protein drebrin was detected by Western blotting, and the accumulation of disulfide bonds in HepG2 cells treated with CPGZC NPs was confirmed ( Figure 5 a); at the same time, the contents of ATP and NADPH in HepG2 cells treated with CPGZC NPs also decreased significantly ( Figure 5 b,c).

[0044] To characterize the apoptosis phenomenon induced by different treatment methods, we used the double staining method of Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI), and analyzed it by flow cytometry, as Figure 6 shown. In this experimental system, the apoptosis rate of the control group was extremely low, only 3.3%, which reflected that cells maintained a good physiological state under normal culture conditions and the apoptosis process was at a relatively low level. Although the apoptosis rate of the pure Cur drug group increased to 17.2%, overall, its effect of inducing cell apoptosis was still relatively limited. When HepG2 cells were incubated with CPGZC NPs, the apoptosis rate increased significantly, up to 63.8%. The apoptosis rate of the CP NPs group was 30.8%, which was significantly lower than that of the CPGZC NPs group.

[0045] In summary, these results indicated that CPGZC NPs could not only effectively induce the accumulation of cellular disulfides to cause cell bisulfide death, but also induce apoptosis, and achieve cancer treatment by synergistically inducing two cell death mechanisms.

[0046] Example 5

[0047] This example provided an in vivo tumor inhibition effect test of CPGZC NPs.

[0048] SPF-grade adult male BALB / c nude mice aged 5 - 6 weeks (weighing 17 - 22 g) were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd. During the cultivation period, the mice could eat and drink freely and were kept in pathogen-free cages, maintaining a 12 h light / dark cycle, and the breeding temperature was controlled at 20 - 25 °C. All animal experiments and research protocols were approved by the Animal Protection and Use Committee of Qingdao University of Science and Technology (Approval No.: QKDLL-2024-51). In addition, the animal experiments in this study followed the ARRIVE guidelines and were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and related guidelines.

[0049] 5×10 6 cells / 100 μL of HepG2 cells were inoculated into the axilla of nude mice. When the tumor grew to 50 mm 3 , the mice were randomly divided into five groups. Subsequently, different drug preparations, including normal saline, pure Cur drug, CPNPs, and CPGZC NPs (curcumin dose was 5 mg / kg body weight), were administered by intravenous injection every two days for 14 days. The tumor volume was calculated according to the following formula: V = π / 6 × L × W 2 .

[0050] To study the therapeutic effect of CPGZC NPs in vivo, a tumor-bearing BALB / c nude mouse model was established by subcutaneous implantation of HepG2 cells. Seven days after implantation, the mice were treated with normal saline, pure Cur drug, CP NPs, and CPGZC NPs respectively. As Figure 7 shown in a - c, compared with other groups, the tumor volume and weight in the CPGZC NPs group were significantly reduced. According to the calculation of the change in tumor volume, the tumor inhibition rates of the pure Cur drug, CP NPs, and CPGZC NPs were 36.5%, 53.4%, and 84.8% respectively.

[0051] In summary, in vivo experiments confirmed that CPGZC NPs could effectively inhibit tumor growth.

[0052] Example 6

[0053] This example provides an in vivo safety evaluation test of CPGZC NPs.

[0054] To further evaluate whether CPGZC NPs have adverse effects on the body's normal tissues, H&E staining analysis was performed on the main organs such as the mouse heart, liver, spleen, lung, and kidney. The staining results showed that the tissue structures of each main organ were clear, the morphology was normal, and no obvious inflammation, necrosis, or other pathological changes were observed ( Figure 8 ).

[0055] As described above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A preparation method of a zinc-based cystine coordination polymer nanocomposite (CPGZC NPs), characterized in that The composite material uses curcumin - gelatin nanocrystals coated with polydopamine (Cur@PDA DNCs) as the core. Through the in - situ growth method, zinc - based cysteine coordination polymer (Zn - Cys) and glucose oxidase (GOx) are coated on the surface of Cur@PDA together to construct this composite material.

2. The nanocomposite material according to claim 1 is characterized as follows: 1) For the preparation of the Cur@PDA DNCs, the curcumin nanocrystals (Cur DNCs) prepared by the anti - solvent method are dispersed at 2 mg each in a 4 mM dopamine hydrochloride solution with a pH of 8.0 - 8.5, and a polydopamine coating layer is formed by stirring. 2) The preparation of the CPGZC NPs requires dispersing Cur@PDA DNCs in a GOx solution of 0.2 mg mL -1 . Subsequently, 0.2 mL of a zinc acetate mother liquor of 11 mg mL -1 is added and reacted for 30 minutes. Then, 0.2 mL of a cystine mother liquor of 12 mg mL -1 is added and reacted for 30 minutes. The product is obtained by centrifugation and collection, where the molar ratio of zinc ions to cystine is 1:1.

64.

3. The coordination polymer nanocomposite according to claim 1, wherein The particle size of the coordination polymer nanocomposite material is 150 - 200 nm.

4. Use of the coordination polymer nanocomposite according to claim 1 in the preparation of a liver cancer therapeutic drug, characterized in that, This nanocomposite material exerts an anti - tumor effect by inducing the mechanism of disulfidptosis and apoptosis pathways in liver cancer cells.

5. A pharmaceutical composition for treating liver cancer, characterized in that, A zinc - based cysteine coordination polymer nanocomposite material prepared by the preparation method described in claims 1 and 2 is included as an active ingredient.

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