A multifunctional bimetallic nanozyme for treating oxaliplatin-resistant colorectal cancer, its preparation method and application

Abstract

The present invention provides a multifunctional bimetallic nanozyme for treating oxaliplatin-resistant colorectal cancer, its preparation method and application, belonging to the field of nano biomedicine. It includes a copper-manganese oxygen vacancy nanoflower core prepared by chemical doping method, and a soybean phospholipid modified shell after oxaliplatin loading, constructing a biodegradable metal-based oxide nanozyme rich in Cu-Mn bimetallic active sites with oxygen vacancies. The prepared core-shell nanoparticles can efficiently deliver oxaliplatin to colorectal cancer tissues, in-situ consume nicotinamide adenine dinucleotide (NAD+) in tumor cells, and simultaneously supply oxygen in-situ to break the tumor redox balance and reverse oxaliplatin resistance. The preparation method of the oxygen vacancy nanozyme in this scheme is simple, with high tumor retention rate, high catalytic activity, good persistence, high drug delivery efficiency and good biocompatibility. It can quickly reduce NAD+ in tumor cells to block cell metabolism while delivering chemotherapy drugs to reverse tumor treatment resistance.

Description

Technical Field

[0001] The present invention belongs to the field of nano biomedicine, and particularly relates to a multifunctional bimetallic nanozyme for treating oxaliplatin-resistant colorectal cancer, a preparation method thereof and an application thereof. Background Art

[0002] Colorectal cancer chemotherapy resistance usually occurs in a nicotinamide adenine dinucleotide (NAD + ) - dependent manner. NAD + is a key cofactor necessary for maintaining cellular energy metabolism, participates in tumor energy reprogramming, such as glycolysis, oxidative phosphorylation, fatty acid oxidation and tricarboxylic acid cycle (TCA) cycle, and maintains cellular gene stability, participating in DNA damage repair. At the same time, NAD + also acts as a second messenger necessary for cell survival, and has important functions such as participating in maintaining mitochondrial function, antioxidant, promoting gene expression, inhibiting cell senescence, etc. However, the NAD + inhibitors currently used in clinical practice mainly rely on inhibiting the expression of the NAD + synthesis rate - limiting enzyme NAMPT to produce effects. Due to the problems of primary or acquired tumor resistance, no objective tumor remission was observed in phase I / II clinical trials, and various systemic side effects occurred, resulting in the interruption of the research. Therefore, developing new drugs with better efficacy, reduced systemic toxicity, targeting tumor cells, and rapidly depleting NAD + in tumor cells has great clinical application prospects. Summary of the Invention

[0003] To solve the above - mentioned technical problems, the present invention provides a multifunctional bimetallic nanozyme for treating oxaliplatin - resistant colorectal cancer, a preparation method thereof and an application thereof, including a copper - manganese oxygen - vacancy nanoflower core prepared by a chemical doping method, and after loading oxaliplatin (Oxa), a surface polymer - modified shell is constructed, and an oxygen - vacancy (OV) biodegradable metal - based oxide nanozyme rich in Cu - Mn bimetallic active sites is constructed (referred to as CuMnO x-V @Oxa@SP). The prepared core - shell nanoparticles can efficiently deliver oxaliplatin to colorectal cancer tissues, in - situ consume nicotinamide adenine dinucleotide (NAD + ) in tumor cells, reduce tumor metabolic activity, and at the same time supply oxygen in - situ to break the tumor redox balance and reverse oxaliplatin resistance.

[0004] To achieve the above object, the present solution provides a multifunctional bimetallic nanozyme for treating oxaliplatin - resistant colorectal cancer, and the multifunctional bimetallic nanozyme includes a copper - manganese oxygen - vacancy nanoflower core, and a soybean phospholipid - modified shell after the copper - manganese oxygen - vacancy nanoflower core is loaded with oxaliplatin.

[0005] Based on a general inventive concept, this solution also provides a method for preparing a multifunctional bimetallic nanozyme, including the following steps:

[0006] S1. Prepare the copper-manganese oxygen vacancy nanoflower core: Mix Cu(acac)2, Mn(acac)3, and triethylene glycol under water bath ultrasonic treatment, then slowly heat to reflux in a N2 environment. After cooling, add ethyl acetate to the suspension and mix under magnetic stirring. Centrifuge to collect the nanoparticles, wash with ethyl acetate, and then dry in vacuum to obtain the copper-manganese oxygen vacancy nanoflower core CuMnO x-V ;

[0007] S2. Load oxaliplatin (Oxa) onto the copper-manganese oxygen vacancy nanoflower core: Dissolve the CuMnO prepared in step S1 x-V in water, add a dimethylformamide solution containing oxaliplatin, stir in the dark at room temperature, centrifuge, and then purify the nanoparticles to remove the unloaded free oxaliplatin to obtain CuMnO x-V @Oxa;

[0008] S3. Modify CuMnO x-V @Oxa with soy lecithin (SP): Disperse the CuMnO x-V @Oxa prepared in S2 in ethanol, add a dichloromethane solution containing soy lecithin, stir overnight, centrifuge to collect the product, wash with dichloromethane, and then wash with phosphate buffered saline to remove the free soy lecithin, obtaining the multifunctional bimetallic nanozyme CuMnO x-V @Oxa@SP.

[0009] Preferably, in step S1, the molar ratio of Cu(acac)2 to Mn(acac)3 is 1:1; the heating time is 3 h; the centrifugation speed is 8000 rpm, and the time is 10 min.

[0010] Preferably, in step S2, the concentration of oxaliplatin is 1 mg / ml; the stirring time is 36 h; the centrifugation speed is 10000 rpm, and the time is 10 min.

[0011] Preferably, in step S3, the concentration of soy lecithin is 10 mg / ml; the stirring temperature is ۲۵ °C; the centrifugation speed is 11000 rpm, and the time is 10 min.

[0012] Preferably, in step S3, the particle size of the multifunctional bimetallic nanozyme is 50 - 100 nm.

[0013] Based on a general inventive concept, this solution also provides an application of the multifunctional bimetallic nanozyme in the preparation of a drug for treating oxaliplatin-resistant colorectal cancer.

[0014] The mechanism of the nanozyme prepared by this scheme for treating oxaliplatin-resistant colorectal cancer is as follows:

[0015] In this study, soybean phospholipid (SP) surface modification and oxaliplatin (Oxa) loading (termed CuMnO x-V @Oxa@SP) were used to construct a biodegradable metal-based oxide nanozyme rich in oxygen vacancies (OVs) for the treatment of oxaliplatin-resistant human colorectal cancer. In addition to peroxidase (POD) and oxidase (OXD) activities related to ROS generation, CuMnO x-V @Oxa@SP also has NAD + -enzyme mimicking activity. On the one hand, the NADH oxidase mimicking catalytic activity of CuMnO x-V @Oxa@SP can effectively disrupt the NADH / NAD + balance in colorectal cancer cells, leading to mitochondrial dysfunction and inhibiting ATP production in the tricarboxylic acid cycle (TCA) pathway. CuMnO x-V @Oxa@SP can further oxidize NADH to ADP-ribose radicals and nicotinamide, thus effectively interfering with metabolic plasticity and restoring chemosensitivity. Therefore, the sensitivity of colorectal cancer cells to Oxa is further enhanced, resulting in enhanced DNA co-damage and induction of apoptosis. On the other hand, the thermal decomposition process induces the formation of oxygen vacancies (OVs), which elongate and weaken the O-O bond of H2O2, thus enhancing ROS generation. It should be noted that oxygen vacancies are both electron donors and electron acceptors, which capture surrounding O2 to generate superoxide anions (·O2 - ). These anions are further oxidized by holes to generate singlet oxygen (1O2), which has strong cytotoxicity to cancer cells. Subsequently, these nanozymes enter colorectal cancer cells through endocytosis, and release Cu 2+ and Mn 4+ in the acidic tumor microenvironment (TME), and are reduced by intracellular H2O2 to Cu + and Mn 2 + , showing strong hydroxyl radical (-OH) generation ability. This study proposed a direct and highly enzyme-powered OV engineering bimetallic nanozyme, which is expected to synergistically treat drug-resistant human colorectal cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The preparation method of the oxygen vacancy nanozyme of the present invention is simple, with a particle size of 50-100 nm, high tumor retention rate, high catalytic activity, good persistence, high drug delivery efficiency, and good pH response rate, good biocompatibility, and can efficiently catalyze NAD +nanomaterials can rapidly reduce NAD in tumor cells + While blocking cell metabolism, it delivers chemotherapeutic drugs, reverses tumor treatment resistance, and has broad application prospects in the treatment of refractory tumors.

[0018] (2) The CuMnO x-V @Oxa@SP's NADH oxidase-mimicking catalytic activity can effectively disrupt the NADH / NAD + balance in colorectal cancer cells, leading to mitochondrial dysfunction and inhibiting the production of ATP in the tricarboxylic acid cycle (TCA) pathway. CuMnO x-V @Oxa@SP can further oxidize NADH to ADP ribose radicals and nicotinamide, thus effectively interfering with metabolic plasticity and restoring chemosensitivity.

[0019] (3) The thermal decomposition process induces the formation of oxygen vacancies (OVs). OVs elongate and weaken the O-O bond of H2O2, thereby enhancing the generation of ROS. It is worth noting that oxygen vacancies are both electron donors and electron acceptors. They capture surrounding O2 to generate superoxide anions, which are further oxidized by holes to generate singlet oxygen, which has strong cytotoxicity to cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 For Experimental Example 1, the structure and properties of CuMnO x-V @Oxa@SP NFs Figure 1 A is the synthesis process and structural schematic diagram of CuMnO x-V @Oxa@SP NFs Figure 1 B is the TEM image of CuMnO x-V @Oxa@SP NFs, Figure 1 C is the HRTEM image, Figure 1 D is the HAADF scanning TEM image, Figure 1 E is the corresponding elemental mapping, Figure 1 F is the PXRD pattern of CuMnO x-V NFs, Figure 1 G is the XPS survey spectrum of CuMnO x-V NFs, Figure 1 H is the high-resolution Cu 2p, Figure 1I is Mn 2p, Figure 1 J is the O1s spectrum, Figure 1 K is CuMnO x-V the ESR spectrum of NFs, Figure 1 L is the measurement of CuMnO in water x-V NFs, CuMnO x-V @Oxa and CuMnO x-V the hydrodynamic diameter of @Oxa@SPNFs, Figure 1 M is the Zeta potential;

[0022] Figure 2 is CuMnO in Experimental Example 2 x-V the multi-enzyme activity of @Oxa@SPNFs, Figure 2 A is CuMnO x-V the ultraviolet-visible absorption spectrum of NADH changing with time within 45 minutes in the presence of @Oxa@SPNFs, Figure 2 B is CuMnO x-V the comparison of NAD + and NADH production under the treatment of the @Oxa@SP NFs + H2O2 group;

[0023] Figure 3 is CuMnO in Experimental Example 3 x-V the schematic diagram of the OXD simulation catalytic process of @Oxa@SPNFs, 3A is the detection of CuMnO for degrading DPBF x-V the ROS generated by the @Oxa@SPNFs + H2O2 group, 3B is the comparison of DPBF, DPBF + H2O2, DPBF + CuMnO x-V @Oxa@SP and DPBF + H2O2 + CuMnO x-V the oxidation of @Oxa@SP, 3C is the ESR of CuMnO captured by DMPO x-V the ESR of @Oxa@SPNFs;

[0024] Figure 4 is CuMnO in Experimental Example 3 x-V the characteristics of @Oxa@SP NFs for degrading ABDA, 4A is the detection of CuMnO x-V the 1 O2 generated by the @Oxa@SPNFs + H2O2 group through the degradation of ABDA, 4B is ABDA, ABDA + H2O2, ABDA + CuMnO x-V @Oxa@SP and ABDA + H2O2 + CuMnO x-V of @Oxa@SP 1 the O2 production comparison, 4C is the ESR of CuMnO captured by TEMP x-V the ESR of @Oxa@SPNFs;

[0025] Figure 5 For Experimental Example 4, CuMnO x-V @Oxa@SPNFs' POD-like activity, Figure 5 A is for detecting CuMnO by degrading MB x-V ROS generated by @Oxa@SP NFs + H2O2 group, Figure 5 B is for comparing MB, MB + H2O2, MB + CuMnO x-V @Oxa@SP and MB + H2O2 + CuMnO x-V The oxidation of MB in @Oxa@SP, Figure 5 C is the ESR of CuMnO x-V @Oxa@SPNFs captured by DMPO;

[0026] Figure 6 For Experimental Example 5, CuMnO x-V Study on the fusion process of @ICG@SP with oxaliplatin-resistant colorectal cancer cell line (HCT116 / L cells). 6A is the confocal microscope image showing the morphology and fluorescence pattern of HCT116 / L cells after being labeled with ICG-labeled CuMnO x-V @ICG@SP from 0 to 48 hours, Figure 6 B is the correlation between incubation time and ICG fluorescence intensity;

[0027] Figure 7 For Experimental Example 6, CuMnO x-V The chemosensitivity of @Oxa@SPNFs. 7A is the effect of different concentrations of CuMnO x-V @SP and oxaliplatin on cell viability. 7B is the two-dimensional heat map of the zero interaction potency (ZIP) synergy score in the study of the combination of CuMnO x-V @SP and oxaliplatin. 7C is the anti-tumor efficacy of different concentrations of CuMnO x-V @Oxa@SP on HCT116L cells;

[0028] Figure 8 For Experimental Example 7, the H&E staining of various organs of HCT116L xenograft tumor-bearing mice after 14 days of treatment with different methods;

[0029] Figure 9 For Experimental Example 7, the treatment response in HCT116L tumor-bearing mice, Figure 9 A is the tumor volume after 14 days of treatment, 9B is the body weight fluctuation, 9C is the tumor weight, and 9D is the survival curve of mice after treatment (n = 3). Detailed implementation method

[0030] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail in conjunction with the accompanying drawings and specific embodiments.

[0031] The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps, or conditions of the present invention belongs to the scope of the present invention.

[0032] If not specifically specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art; if not specifically specified, the reagents used in the embodiments are all commercially available.

[0033] Example 1 CuMnO x-V Design, Synthesis, and Characterization of @Oxa@SP

[0034] (1) Preparation of Copper-Manganese Oxygen Vacancy Nanoflower Core (CuMnO x-V )

[0035] Mix 20.261 g (1.0 mmol) of Cu(acac)2, 0.253 g (1.0 mmol) of Mn(acac)3, and 100 mL of triethylene glycol (TEG) with the help of bath ultrasound, and then slowly heat for 3 hours to reflux under a nitrogen atmosphere. After cooling, add 20 mL of ethyl acetate (EA) to the homogeneous suspension and mix under magnetic stirring. Centrifuge at 8000 rpm for 10 minutes, collect the nanoparticles, wash them 5 times with EA, and then dry them under vacuum to obtain 0.15 g of solid.

[0036] (2) Preparation of Copper-Manganese Oxygen Vacancy Nanoflower Core Loaded with Oxaliplatin (CuMnO x-V @Oxa)

[0037] Add 5 mL of a dimethylformamide (DMF) solution containing 1.0 mg / ml of oxaliplatin (Oxa) to the CuMnO x-V solution, and stir in the dark at room temperature for 36 hours. Finally, purify the nanoparticles by centrifuging at 10000 rpm for 10 minutes to remove the unloaded free Oxa, and the Oxa loading content is calculated by ICP-MS.

[0038] (3) Preparation of Multifunctional Bimetallic Nanozyme (CuMnO x-V @Oxa@SP)

[0039] To improve the biocompatibility of CuMnO x-V @Oxa, the researchers used soy phosphatidylcholine (SP) to modify the surface of CuMnO x-V @Oxa. Briefly, disperse 10 mg of CuMnO in 10 mL of ethanol.x-V @Oxa was added to a 10 mL dichloromethane (DCM) solution containing 10 mg / mL - 1 SP. The mixture was stirred overnight at 25 °C and then the product was collected by centrifugation at 11000 rpm for 10 min. Finally, CuMnO x-V @Oxa@SP was washed 5 times with DCM and then 3 times with phosphate buffered saline (PBS) to remove free SP. Finally, the collected CuMnO x-V @Oxa@SP had better solubility, stability and biocompatibility.

[0040] Experimental Example 1 Characterization of CuMnO x-V @Oxa@SP

[0041] The synthesis process of the multifunctional bimetallic nanozyme and the structure of copper-manganese oxygen vacancy nanoflowers (CuMnO x-V ) are as Figure 1 shown in A.

[0042] To improve the physiological stability of CuMnO x-V nanoflowers and have no obvious toxicity in vitro and in vivo, the surface of CuMnO x-V nanoflowers was modified with soy phosphatidylcholine (SP). The morphology of CuMnO x-V @Oxa@SPNFs was observed by transmission electron microscopy (TEM).

[0043] The results are as Figure 1 shown in B. CuMnO x-V presented a "flower"-like nanostructure (CuMnO x-V NFs).

[0044] Figure 1 C is a high-resolution transmission electron microscopy (HRTEM) image, and the defects (dislocations marked by circles) can be clearly seen. Figure 1 D and E are X-ray energy dispersive spectroscopy (EDS) elemental spectra, showing the uniform distribution of copper, manganese, platinum and phosphorus elements, confirming the successful synthesis of CuMnO x-V @Oxa@SPNFs.

[0045] Figure 1 G is X-ray photoelectron spectroscopy (XPS) characterizing the surface chemical state, elemental composition and oxygen vacancies of CuMnO x-V . The results show that four prominent peaks were observed at 285.5, 532.1, 642.3 and 934.8 eV, corresponding to the elemental species of C, O, Mn and Cu respectively.

[0046] Figure 1H is the high-resolution spectrum of Cu 2p. The peaks at 934.2, 941.4, 944.1, 953.9, and 962.3 eV correspond to Cu2p3Cu(I)-O, Cu2p3sat., Cu2p1Cu(II)-O, and Cu2p1sat., respectively.

[0047] Figure 1 I is the high-resolution spectrum of Mn2p. The maximum value of the Mn2p3 peak is in the range of 641.3 - 643.0 eV, the peak at 648.0 eV is attributed to Mn2p3sat., and the maximum value of the Mn2p1 peak is in the range of 652.9 - 654.2 eV.

[0048] Figure 1 J is the high-resolution spectrum of O1s. The three main peaks at 530.1, 531.6, and 533.14 eV come from lattice oxygen, oxygen vacancies, and surface oxygen, respectively. In the O1s spectrum, the peaks at 530.1 eV and 533.1 eV correspond to lattice oxygen and surface oxygen in the Cu-O / Mn-O bonds, respectively. The characteristic peak at 531.6 eV can be attributed to oxygen vacancies. More importantly, Figure 1 K is the electron spin resonance (ESR) spectrum, which further verifies the formation of oxygen vacancies. An obvious signal centered at a g value of 2.003 is observed, which is a typical feature of oxygen vacancies, confirming the successful preparation of OV-engineered CuMnO x-V NFs. The presence of oxygen vacancies will promote the electron-hole separation of CuMnO x-V during the reaction process and improve its enzyme catalytic performance.

[0049] Figure 1 L is the average hydrodynamic size of CuMnO x-V 、CuMnO x-V @Oxa and CuMnO x-V @Oxa@SP, which are 88.6 ± 6.5 nm, 85.3 ± 7.4 nm, and 108.9 ± 2.8 nm, respectively.

[0050] Figure 1 M shows that after adding oxaliplatin (Oxa), the ZETA potential increases from -10.5 ± 3.8 mV (CuMnO x-V @Oxa) to -7.7 ± 2.6 mV (CuMnO x-V @Oxa). The zeta potential of CuMnO x-V @Oxa@SP is -27.7 ± 6.6 mV because the free phosphate groups of SP are exposed on the surface of CuMnO x-V @Oxa.

[0051] Experimental Example 2 examines CuMnO x-VMultienzyme activities of @Oxa@SPNFs

[0052] Considering the special performance of catalytic biomaterials with enzyme mimetic activity in cancer treatment, CuMnO x-V The multi-enzyme mimetic activity of @Oxa@SPNFs was studied. NADH and NAD + As an important cofactor of intracellular redox metabolism, it participates in the tricarboxylic acid cycle (TCA) pathway. x-V The NADH oxidase mimetic activity of @Oxa@SPNFs was evaluated by analyzing NADH consumption.

[0053] Figure 2 A, 2B are UV-visible absorption spectra monitoring the oxidation process of NADH and NAD under the treatment of H2O2 group. + Compared with the generation of NADH, as the reaction time (0-35 minutes) increases, the characteristic absorption peak of NADH at 340nm wavelength decreases significantly, while NAD + The absorption peak at 260 nm wavelength increases significantly. This change in NADH absorption is due to the conversion of NADH to NAD + The conversion of CuMnO x-V @Oxa@SP NFs can induce NADH oxidation in the presence of H2O2. As the reaction time increases, NADH is almost completely degraded to NAD. + , which confirms that CuMnO x-V @Oxa@SPNFs converts NADH into NAD + With the extension of reaction time, NAD + The characteristic absorption peak at 260 nm decreased significantly with the increase of reaction time (35-45 min), confirming that CuMnO x-V @Oxa@SPNFs has catalytic activity similar to that of nano-NAD. + This change in absorption is related to the CuMnO x-V The oxygen vacancies of @Oxa@SPNFs are closely related.

[0054] Experimental Example 3: Investigation of CuMnO x-V Enzyme-like catalytic oxidation performance of @Oxa@SPNFs

[0055] 1,3-Diphenylisobenzofuran (DPBF), a colorless yellow probe that reacts with ROS, was used to evaluate the CuMnO x-V Enzyme-like catalytic oxidation performance of @Oxa@SPNFs.

[0056] The results are as follows Figure 3As shown in A, in CuMnO x-V In the presence of @Oxa@SP+H2O2, the characteristic peak of DPBF gradually decreases with the extension of reaction time, indicating that CuMnO x-V @Oxa@SP can generate ROS in the presence of H2O2.

[0057] Figure 3 B shows that in CuMnO x-V In the @Oxa@SP+H2O2 group, this characteristic peak of DPBF gradually weakened with time and decreased to 20% of the initial value after 30 minutes.

[0058] In order to further determine the O2 - The presence of species was investigated by electron spin resonance (ESR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in methanol as a free radical scavenger. Figure 3 As shown in C, it further shows that O2 - The typical formation mode is CuMnO x-V The electron donor of @Oxa@SP NFs donates one electron to O2, thus x-V The @Oxa@SP+H2O2 group produces more O2 - , while there is no signal in the pure H2O2 group.

[0059] Subsequently, the singlet oxygen probe 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used to detect OV-mediated singlet oxygen ( 1 O2) is generated, and the result is as follows Figure 4 As shown in A and 4B, consistent with the above results, only CuMnO x-V The @Oxa@SP+H2O2 group showed obvious ABDA degradation. Figure 4 C shows that in CuMnO x-V The spectrum of @Oxa@SP+H2O2 group was observed to be similar to that of 1 The obvious characteristic signal corresponding to O2, rather than CuMnO x-V The @Oxa@SP group (pure H2O2) did not show any signal.

[0060] The above results show that oxygen vacancies are both electron donors and electron acceptors, which can - Reaction, play H + The role of generating 1 O2.

[0061] oxygen vacancies(OV ·+ )+O2→OV ++ + O2 -(1)

[0062] oxygen vacancies(OV ·+ )+·O2 - →OV ·· + 1 O2 (2)

[0063] Experimental Example 4 investigated the POD-like activity of CuMnO x-V @Oxa@SPNFs

[0064] Due to the different valence states of copper ions and manganese ions, it was investigated whether CuMnO x-V @Oxa@SPNFs, as a POD-like nanozyme, could convert H2O2 into highly toxic -OH through the classical Fenton reaction. Methylene blue (MB) was used as a probe to detect the generation of -OH. Under weakly acidic conditions (pH = 5.2), MB can be oxidized by highly active -OH to form colorless MB-OH, and DMPO was used as an -OH scavenger.

[0065] Figure 5 A shows the generation of -OH evaluated by ESR spectroscopy. A quartet resonance peak with a signal intensity ratio of 1:2:2:1 can be clearly observed, which is a characteristic DMPO--OH adduct, indicating that CuMnO x-V @Oxa@SP can convert H2O2 into highly toxic -OH.

[0066] Figure 5 B and 5C show that in the MB + H2O2 + CuMnO x-V @Oxa@SP group, the characteristic peak of MB significantly weakened within 5 minutes, highlighting its excellent -OH generation ability and verifying the POD-like activity of CuMnO x-V @Oxa@SP.

[0067] Experimental Example 5 investigated the endocytosis of CuMnO x-V @Oxa@SPNFs

[0068] Efficient cellular uptake is crucial for the tumor accumulation of nanozymes. To study the endocytosis of nanoparticles, we cultured CuMnO x-V @ICG@SP nanozyme labeled with ICG (indocyanine green) with human colorectal cancer oxaliplatin-resistant cell line (HCT116L) for a period of time.

[0069] Figure 6 A shows CuMnO monitored by confocal laser scanning microscopy (CLSM) x-VThe intracellular distribution of @ICG@SP in HCT116L cells. The results showed that the fluorescence intensity was continuously increasing in HCT116L cells over time, indicating that the nanozyme was effectively internalized. Figure 6 B showed its absorption over time. These results confirmed that CuMnO x-V @SP could be effectively absorbed by HCT116L cells to carry out effective catalytic reactions to eliminate cancer cells.

[0070] Experimental Example 6 investigated the chemosensitivity of CuMnO x-V @Oxa@SPNFs

[0071] To further explore whether the nanocatalyst could improve the chemosensitivity of oxaliplatin, HCT116L cells were treated with different concentrations of oxaliplatin and CuMnO x-V @SP. The cytotoxicity to HCT116L was detected using the standard MTT assay.

[0072] The results were as Figure 7 shown in A. The comparison of IC50 values of CuMnO x-V @SP (0, 50, 100, 200 μg / mL) combined with oxaliplatin showed that the respective IC50 values were 28.20, 7.26, 1.51, and 0.41 μg / mL, showing a gradient-enhanced sensitivity to oxaliplatin.

[0073] Figure 7 B was for further evaluation using the SynergyFinder web application. The results showed a strong synergistic effect between CuMnO x-V @SP and oxaliplatin. When the concentration of CuMnO x-V @SP was 50 - 200 μg / mL and the concentration of oxaliplatin was 6.2 - 25 μg / mL, the zero interaction potency (ZIP) synergy score exceeded 10.

[0074] Finally, as Figure 7 shown in C, the IC50 of treating HCT116L cells with CuMnO x-V @Oxa@SP was 28.15 / 5.63 μg / mL. The results showed that compared with the combination therapy of CuMnO x-V @SP and oxaliplatin, CuMnO x-V @Oxa@SP had a stronger antitumor effect.

[0075] Experimental Example 7 investigated the in vivo antitumor efficacy of CuMnO x-V @Oxa@SPNFs

[0076] Treatment started on the 12th day after tumor inoculation when the tumor volume was approximately 100 mm3. HCT116L tumor-bearing mice were randomly divided into five groups and received the following treatments respectively: G1) PBS; G2) Oxa 5 mg / kg; G3) Oxa 10 mg / kg; G4) CuMnO x-V @SP; G5) CuMnO x-V @Oxa@SP, which were administered via tail vein injection on days 0, 2, 4, and 6 respectively. The tumor volume and mouse body weight were monitored twice a day, and the survival time was recorded.

[0077] During the observation period, Figure 8 Figure 9 shows the HE staining of various organs of tumor-bearing mice 14 days later. The results showed that although the H&E images of the major organs of mice treated with Oxa 5 mg / kg and Oxa 10 mg / kg did not show obvious abnormalities, Figure 9 Figures 9B and 9C showed that these two groups of mice had varying degrees of anorexia and weight loss during the treatment, resulting in shortened survival time. In contrast, the CuMnO x-V @SP group and the CuMnO x-V @Oxa@SP group of mice maintained stable body weights, and no histopathological changes in the major organs were observed in the H&E stained sections after treatment, indicating that the adverse effects of CuMnO x-V @SP and its Oxa delivery on metabolism were negligible.

[0078] In addition, there was no significant difference in the change of tumor volume between the Oxa 5 mg / kg group and the PBS control group, indicating that the conventional dose of Oxa had limited effect on the growth of HCT116L xenograft tumors. In contrast, the CuMnO x-V @SP group showed moderate tumor suppression, which might be due to the depletion of NAD + disrupting the metabolism of tumor cells and the enhanced oxidative stress caused by accelerating the Fenton reaction. Notably, the CuMnO x-V @Oxa@SP group had stronger tumor suppression and significantly prolonged survival, which was attributed to the combined action of the anticancer activity of CuMnO x-V @SP and the oxaliplatin additive. The CuMnO x-V @Oxa@SP complex significantly reduced the level of NAD + and reversed the oxaliplatin resistance observed in HCT116L cells. When this complex was used in combination with oxaliplatin, it also showed an obvious synergistic antitumor effect. In addition, Figure 9 Figures 9A-C showed that administering oxaliplatin at a dose of 10 mg / kg could moderately inhibit tumor growth; however, its associated neurotoxicity induced anorexia, resulting in a rapid decrease in the body weight of mice.

[0079] The above are only the preferred embodiments of the present invention patent, and the protection scope of the present invention patent is not limited to the above embodiments. For those skilled in the art, the improvements and transformations obtained without departing from the technical concept of the present invention patent should also be regarded as the protection scope of the present invention patent.

Claims

1. A multifunctional bimetallic nanozyme for treating oxaliplatin-resistant colorectal cancer, characterized in that: The multifunctional bimetallic nanozyme comprises a copper-manganese-oxygen vacancy nanoflower core, a copper-manganese-oxygen vacancy nanoflower core loaded with oxaliplatin and a soybean lecithin-modified shell; The preparation method of the multifunctional bimetallic nanozyme comprises the following steps: S1. Preparation of copper manganese oxygen vacancy nanoflower core: Cu(acac)2, Mn(acac)3, and triethylene glycol were mixed under water bath ultrasound, then slowly heated to reflux under N2 environment. After cooling, ethyl acetate was added to the suspension and mixed under magnetic stirring. Nanoparticles were collected by centrifugation, washed with ethyl acetate, and vacuum dried to obtain copper manganese oxygen vacancy nanoflower core CuMnO x-V ; S2, Copper manganese oxygen vacancy nanoflower core loaded with oxaliplatin Oxa: CuMnO prepared in step S1 x-V Dissolve in water, add dimethylformamide solution containing oxaliplatin, stir in the dark at room temperature, and purify the nanoparticles after centrifugation to remove the unloaded free oxaliplatin to obtain CuMnO x-V @Oxa; S3, soybean lecithin SP modified CuMnO x-V @Oxa: CuMnO prepared by S2 x-V @Oxa was dispersed in ethanol, and a dichloromethane solution containing soybean lecithin was added. The mixture was stirred overnight, and the product was collected by centrifugation. The product was washed with dichloromethane and then with phosphate-buffered saline to remove free soybean lecithin, thereby obtaining the multifunctional bimetallic nanozyme CuMnO. x-V @Oxa@SP.

2. The multifunctional bimetallic nanozyme according to claim 1, characterized in that In the step S1, the molar ratio of Cu(acac)2 to Mn(acac)3 is 1:1; the heating time is 3 hours; and the centrifugal speed is 8000 rpm for 10 minutes.

3. The multifunctional bimetallic nanozyme according to claim 1, characterized in that In step S2, the concentration of oxaliplatin is 1 mg / ml; the stirring time is 36 h; the centrifugal speed is 10,000 rpm, and the time is 10 min.

4. The multifunctional bimetallic nanozyme according to claim 1, characterized in that In the step S3, the concentration of soybean lecithin is 10 mg / ml; the stirring temperature is 25° C.; the centrifugal speed is 11000 rpm, and the time is 10 min.

5. The multifunctional bimetallic nanozyme according to claim 1, characterized in that The particle size of the multifunctional bimetallic nanozyme in step S3 is 50-100 nm.

6. Use of the multifunctional bimetallic nanozyme according to any one of claims 1 to 5 in the preparation of a drug for treating oxaliplatin-resistant colorectal cancer.

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