Brain glioma targeted multifunctional nano-enzyme and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and discloses a brain glioma targeted multifunctional nano-enzyme and a preparation method thereof.The multifunctional nano-enzyme is prepared by the steps that copper sulfide nanoparticles wrapped by polydopamine serve as an inner core, folic acid and glucose oxidase are modified to the surfaces of the nanoparticles to form nano-enzyme, and then the nano-enzyme is prepared into the brain glioma targeted multifunctional nano-enzyme. Lactoferrin is further modified on the surface of the nano-enzyme through electrostatic adsorption, and the brain glioma targeted multifunctional nano-enzyme is constructed.

Description

A multifunctional nanozyme targeting glioma and its preparation method Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically relating to a multifunctional nanozyme targeting glioma and its preparation method. Background Technology

[0002] Gliomas are the most common malignant tumors of the central nervous system, characterized by high invasiveness, high mortality, and difficult prognosis, with a median survival of only about 15 months. Currently, clinical treatments include traditional methods such as surgical resection, chemotherapy, and radiotherapy, as well as subsequent adjuvant therapies. However, the blood-brain barrier, characterized by tight junctions between endothelial cells, severely restricts the delivery of antitumor drugs to brain tumors. With the development of nanoparticle formulations, some nanocarriers, due to their excellent biocompatibility and flexible modification properties, offer new solutions for crossing the blood-brain barrier unique to the central nervous system. Therefore, it is necessary to design and construct a nanomedicine delivery system that can effectively penetrate the blood-brain barrier and precisely target gliomas after penetrating the brain.

[0003] Nanozymes are a class of nanomaterials with enzymatic catalytic properties. Compared with natural enzymes, nanozymes have good stability, lower production costs, and simpler preparation methods. Nanozymes utilize their physicochemical properties to regulate redox homeostasis in the tumor microenvironment (TME) and induce oxidative damage in tumor cells. Nanozymes are mainly classified into four types: peroxidase (POD), catalase, oxidase, and superoxide dismutase. Among them, POD is a class of transition metal-based nanozymes such as Cu, Fe, and Mn. It is a nanomaterial that consumes peroxidase activity and can catalyze the Fenton / Fenton-like reaction of hydrogen peroxide (H2O2) to generate highly cytotoxic hydroxyl radicals (·OH), inducing tumor cell apoptosis.

[0004] Chemokinetic therapy (CDT) is a novel approach to cancer treatment. Compared to traditional cancer therapies, CDT offers higher tumor specificity and selectivity, as well as lower toxicity. CDT primarily relies on the Fenton reaction activated by nanomedicines within a tumor microenvironment (TME), consuming H₂O₂ to generate highly oxidizing ·OH radicals. These free radicals are among the most active reactive oxygen species (ROS) and can specifically attack tumor cells, inducing apoptosis. However, CDT alone offers limited efficacy in anti-tumor therapy, mainly due to the complexity of the TME. The endogenous H₂O₂ in the TME is insufficient to generate enough ·OH radicals to achieve the desired therapeutic effect, and the Fenton reaction occurs under strongly acidic conditions (pH 2-4), while the weak acidity of the TME limits the efficiency of CDT. Introducing glucose oxidase (GOx) can address these issues. GOx catalyzes the oxidation of glucose in the TME to generate exogenous H₂O₂ while simultaneously lowering the local pH. This dual action provides sufficient substrate for the Fenton reaction and regulates the acidity of the reaction system, effectively solving the problem of slow reaction rates caused by insufficient substrate and limited reaction conditions in traditional Fenton reactions.

[0005] Gas therapy is widely used in cancer treatment due to its therapeutic efficacy and good biocompatibility. Hydrogen sulfide (H2S), carbon monoxide, nitric oxide, and hydrogen act as gaseous messengers, playing different roles in cancer treatment. H2S is a key gas affecting intracellular signaling pathways, possessing anti-tumor, anti-inflammatory, and angiogenesis-promoting effects. H2S induces apoptosis by inhibiting the activation of signal transducers and activator of transcription 3 and by downregulating NF-κB, triggering a series of immune responses. Furthermore, H2S can inhibit the activity of high-level ROS-scavenging enzymes in the tumor microenvironment (TME), such as by inhibiting catalase and reducing thioredoxin reductase activity, leading to the accumulation of more ROS in the TME to kill tumor cells.

[0006] Currently, in the treatment of gliomas, nanoparticles are often used in combination with photothermal therapy. Patent announcement number CN119684318A discloses a near-infrared photothermal / photodynamic / chemotherapy combined nanoplatform for glioblastoma treatment and its preparation method. The nanoparticles, modified from macrophage membranes and loaded with curcumin and the near-infrared aggregation-induced emission molecule BDTA, can reduce the expression of the heat shock protein HSP-70 through curcumin, thereby enhancing the sensitivity of photothermal therapy. Through the Foster resonance energy transfer mechanism, it effectively improves the photothermal conversion efficiency (87.6%) and singlet oxygen generation (1.7 times higher than the control group). This nanoplatform utilizes photothermal therapy, photodynamic therapy, and chemotherapy to treat gliomas. However, Figure 6 of the patent shows that this nanoplatform has a low efficiency of 1 W / cm². 2Under near-infrared light irradiation, the temperature of the highest concentration of nanoparticles reaches over 55°C, a temperature that can induce irreversible damage such as neuronal apoptosis and blood-brain barrier disruption, hindering further clinical translation. In contrast, this invention innovatively proposes a multifunctional nanozyme with a core combined mechanism of chemokinetic therapy and gas therapy, which can completely avoid the risk of brain damage associated with thermotherapy, providing an efficient and safe innovative treatment strategy for the precision treatment of gliomas.

[0007] In summary, current nanotherapy approaches for gliomas still have some shortcomings: (1) Existing formulations mostly rely on single-target ligand-mediated nanodelivery systems, which suffer from low targeting efficiency and insufficient blood-brain barrier penetration, making it difficult to achieve precise treatment. (2) Traditional chemokinetic therapy requires H2O2 and strongly acidic conditions, but the low H2O2 concentration and neutral pH of TME limit the Fenton reaction rate and result in insufficient ROS generation. (3) Existing chemokinetic therapies are mostly combined with photothermal therapy, which requires exogenous light stimulation. Excessive temperature can easily damage brain tissue, making further clinical translation difficult. Therefore, there is an urgent need for a new nanoplatform to achieve precise and safe treatment of gliomas. Summary of the Invention

[0008] The purpose of this invention is to address the problems in existing glioma treatment technologies, such as poor targeting of nanotherapy systems, difficulty in penetrating the blood-brain barrier, single treatment mechanism, insufficient responsiveness of the tumor microenvironment, and limited treatment efficiency. The invention proposes a multifunctional nanozyme with glioma targeting capability, combined with CDT combined with gas therapy, and its preparation method.

[0009] Based on the aforementioned prior art, this invention provides a multifunctional nanozyme targeting gliomas and its preparation method. This invention first prepares copper sulfide nanoparticles coated with polydopamine using a hydrothermal method. Utilizing the abundant functional groups on the surface of polydopamine, folic acid and glucose oxidase are co-loaded onto the polydopamine-coated copper sulfide nanoparticles via Michael addition reaction and covalent coupling. Finally, lactoferrin is further modified onto the nanozyme surface through electrostatic adsorption, constructing a multifunctional nanozyme targeting gliomas. After intravenous injection, this multifunctional nanozyme, under the action of folic acid and lactoferrin, can cross the blood-brain barrier to further target gliomas. In the weakly acidic environment of the tumor, polydopamine degrades, rapidly releasing glucose oxidase and copper sulfide nanoparticles. The released glucose oxidase can then react with… Glucose in the tumor microenvironment undergoes a catalytic reaction; on the one hand, it provides additional hydrogen peroxide to replenish the substrate required for the Fenton reaction; on the other hand, it further reduces the pH value of the tumor microenvironment, breaking the original weakly acidic environment's limitation on the Fenton reaction, significantly improving the efficiency of the Fenton reaction, and thus enhancing the efficacy of chemokinetics; in addition, the reduction in the pH value of the tumor microenvironment can simultaneously trigger the release of copper ions and hydrogen sulfide gas from copper sulfide nanoparticles, realizing gas therapy. The released copper ions consume H2O2 to generate ROS, and the released H2S gas can also inhibit the activity of endogenous ROS enzymes, accelerating the cascade generation of ROS, ultimately forming a tumor treatment mode of chemokinetics combined with gas therapy.

[0010] This invention successfully prepared a multifunctional nanozyme targeting glioma, and the prepared nanozyme was uniformly dispersed and had a uniform particle size.

[0011] To achieve this objective, the present invention adopts the following technical solution:

[0012] A multifunctional nanozyme targeting glioma is characterized in that the multifunctional nanozyme uses copper sulfide nanoparticles encapsulated with polydopamine as the core, and modifies the surface of the nanoparticles with folic acid and glucose oxidase to form a nanozyme. Then, lactoferrin is further modified on the surface of the nanozyme through electrostatic adsorption, thus constructing a multifunctional nanozyme targeting glioma.

[0013] Furthermore, the preparation method of the glioma-targeting multifunctional nanozyme of the present invention includes the following specific steps:

[0014] (1) Dissolve CuCl2·2H2O in an appropriate amount of deionized water to form 1mM Cu 2+In a solution prepared by adding 10 mg of sodium citrate to the above solution in an ice-water bath and stirring for 5 min, 20 mg of dopamine was added and stirred until homogeneous. 1 mL of Na₂S·9H₂O (100 mM) solution was added dropwise to the above solution, and the mixture was sonicated for 15 min. The mixture was then transferred to a 90 °C water bath and reacted for 1 h. After the reaction was complete, the product was collected by centrifugation, washed three times with deionized water, and lyophilized to obtain polydopamine-encapsulated copper sulfide nanoparticles (PDA-CuSNPs).

[0015] (2) Weigh 20 mg of folic acid and dissolve it in 2 mL of Tris solution (pH 8.5, 50 mM). Add 20 mg of EDC while stirring and react for 10 min. Then add 20 mg of NHS to activate for 30 min. Dissolve 40 mg of PDA-CuS NPs in a certain amount of Tris solution. Add the activated folic acid solution dropwise to the above solution and stir for 12 h. Centrifuge the obtained mixture, collect the precipitate, and freeze-dry it to obtain folic acid modified PDA-CuS NPs (FP-CuSNPs).

[0016] (3) Dissolve 2 mg of GOx in a certain amount of pure water, add EDC and NHS catalysts and activate at room temperature for 1 h. Then add GOx:FP-CuS NPs at a mass ratio of 2:1 to react, stir for 1 h, centrifuge to obtain the precipitate, freeze dry to obtain GOx-loaded FP-CuS NPs (FP-CuS@GOx NPs).

[0017] (4) Resuspend FP-CuS@GOx NPs in Tris solution, add 1 mL of lactoferrin aqueous solution (0.5 mg / mL), stir at room temperature for 4 h, and centrifuge to obtain lactoferrin modified FP-CuS@GOx NPs (LFP-CuS@GOxNPs).

[0018] This invention provides the application of a multifunctional nanozyme targeting glioma in the preparation of anti-glioma products.

[0019] Compared with existing technologies, the present invention has the following advantages:

[0020] (1) To address the challenge of the blood-brain barrier severely restricting the delivery of antitumor drugs to brain tumors, thus preventing drug accumulation in the tumor, this invention employs a "folic acid (FA)-lactoferrin (LF)" dual-targeting system to solve the problems of "easy off-target effects in blood circulation and insufficient blood-brain barrier penetration" in traditional targeted formulations. This multifunctional nanozyme (LFP-CuS@GOxNPs) can effectively cross the blood-brain barrier through the penetrating effect of LF. At the same time, FA and LF can bind to both FA receptors and LRP-1 receptors on the surface of gliomas, thereby mediating greater accumulation of the multifunctional nanozyme at the tumor site and enabling the formulation to exert better therapeutic effects.

[0021] (2) Addressing the challenges of limited treatment mechanisms and efficiency, this invention employs CDT combined with gas therapy to treat gliomas. On one hand, GOx catalyzes the generation of H2O2 from glucose in the tumor microenvironment, providing ample substrate for CDT. Simultaneously, the generated gluconic acid lowers the pH of the tumor microenvironment, accelerating the copper-mediated Fenton reaction rate. On the other hand, lowering the pH within tumor cells promotes the release of copper ions and H2S gas from CuS, resulting in the release of Cu... 2+ The consumption of H2O2 to generate ROS, along with the released H2S gas, can inhibit the activity of endogenous ROS enzymes, reducing the tolerance of tumor cells to ROS. By combining CDT with gas therapy to enhance the cascade production of ROS, the issue of the efficacy of single-therapy treatment for gliomas is resolved.

[0022] (3) Currently, chemokinetic therapy is often combined with photothermal therapy to treat tumors. Although the local thermal effect mediated by photothermal therapy can enhance the catalytic effect of chemokinetics, even slight photothermal stimulation can cause irreversible damage such as neuronal apoptosis and blood-brain barrier disruption in gliomas, which are malignant tumors of the central nervous system. This invention combines chemokinetic therapy with gas therapy, which can avoid the associated risks of brain damage and provides a new approach for the safe treatment of gliomas.

[0023] (4) To address the shortcomings of insufficient responsiveness of formulations in the tumor microenvironment and difficulty in achieving stable circulation in vivo, this invention introduces polydopamine (PDA) to solve these problems. On one hand, PDA has the advantage of good biocompatibility. PDA is structurally stable in normal tissues, preventing premature leakage of CuS and GOx. It rapidly decomposes under the weakly acidic conditions of the tumor, reducing the probability of the multifunctional nanozyme being recognized and cleared by the immune system, prolonging its circulation time in vivo, and ensuring that the multifunctional nanozyme can effectively reach the tumor site to exert its therapeutic effect, thus improving the safety and efficacy of treatment. On the other hand, the surface of PDA contains abundant functional groups, facilitating the modification of targeting molecules, drugs, etc., to enhance the targeting effect.

[0024] (5) The synthesis process of nanozymes is relatively simple, the production cost is low, and it is capable of mass production. These unique advantages make nanozymes of great significance in the treatment of cancer. Attached Figure Description

[0025] Figure 1 shows the infrared images of PDA-CuS NPs, FP-CuS NPs, FP-CuS@GOx NPs and LFP-CuS@GOx NPs;

[0026] Figure 2 shows the H2S gas release of LFP-CuS@GOx NPs under different conditions;

[0027] Figure 3 shows the cascade catalytic performance of LFP-CuS@GOx NPs;

[0028] Figure 4 shows the particle size change of LFP-CuS@GOx NPs after 12 hours of incubation with plasma;

[0029] Figure 5 shows the cytotoxicity assay performed using the MTT assay.

[0030] Figure 6 shows images of reactive oxygen species generated by GL261 cells after incubation for 4 hours with different concentrations of FP-CuS@GOx NPs and LFP-CuS@GOx NPs using an inverted fluorescence microscope.

[0031] Figure 7 is a schematic diagram of the therapeutic mechanism of multifunctional nanozymes. Detailed Implementation

[0032] To better understand the present invention, the following embodiments are provided to further illustrate the invention, but the invention is not limited to the following examples.

[0033] Example 1

[0034] Preparation of PDA-CuS NPs

[0035] (1) Dissolve CuCl2·H2O in deionized water to form 1mM Cu 2+ In a solution prepared by ice bath, 10 mg of sodium citrate was added to the above solution and stirred for 5 min. Then, 20 mg of DA was added and stirred until homogeneous. 1 mL of Na2S·H2O (100 mM) solution was added dropwise to the above solution, and the mixture was sonicated for 15 min. The mixture was then transferred to a 90 °C water bath and reacted for 1 h.

[0036] (2) After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 10 min, washed three times with deionized water, and the particle size was measured using a ZetaPALS potentiometer and particle size analyzer.

[0037] Example 2

[0038] Preparation of FP-CuS NPs

[0039] (1) Weigh 20 mg of FA and dissolve it in 2 mL of Tris solution (pH 8.5, 50 mM). Add 20 mg of EDC while stirring and react for 10 min. Then add 20 mg of NHS to activate for 30 min.

[0040] (2) Dissolve 40 mg PDA-CuS NPs in 2 mL of Tris solution, add the activated FA solution dropwise to the above solution, and stir for 12 h;

[0041] (3) After the reaction was completed, the product was collected by centrifugation at 12,000 rpm for 10 min, washed three times with deionized water, and the particle size was measured using a ZetaPALS potentiometer and particle size analyzer.

[0042] Example 3

[0043] Preparation of FP-CuS@GOx NPs

[0044] (1) Dissolve 2 mg GOx in 2 mL of deionized water, add 35 mg EDC and 35 mg NHS and activate at room temperature for 1 h;

[0045] (2) Add GOx:FP-CuS NPs to the reaction at a mass ratio of 2:1 and stir for 2 hours;

[0046] (3) After the reaction was completed, the product was collected by centrifugation at 12,000 rpm for 10 min, washed with deionized water 3 times, and the particle size was measured using a ZetaPALS potentiometer and particle size analyzer.

[0047] Example 4

[0048] Preparation of LFP-CuS@GOx NPs

[0049] (1) Resuspend FP-CuS@GOxNPs in 2 mL Tris solution, add 1 mL LF aqueous solution (0.5 mg / mL), and stir at room temperature for 4 h;

[0050] (2) After the reaction was completed, the product was collected by centrifugation at 12,000 rpm for 10 min, washed three times with deionized water, and the particle size was measured using a ZetaPALS potentiometer and particle size analyzer.

[0051] Example 5

[0052] Referring to Table 1, the particle size, PDI, and potential of PDA-CuS NPs, FP-CuS NPs, FP-CuS@GOx NPs, and LFP-CuS@GOx NPs samples in aqueous solution were measured using a Zeta PALS potentiometer and particle size analyzer. The PDI data in Table 1 shows that the four nanoparticles were uniformly dispersed in the aqueous solution, with a maximum particle size not exceeding 220 nm. The prepared FP-CuS@GOx NPs had a particle size of 178.23 ± 1.67 nm, a PDI of 0.224 ± 0.024, and a Zeta potential of -34.2 ± 1.21 mV. To enhance the blood-brain barrier penetration ability, targeting ability, and stability of FP-CuS@GOx NPs after brain penetration, LF was adsorbed onto the surface of FP-CuS@GOx NPs via electrostatic interaction, thus preparing LFP-CuS@GOx NPs. The results showed that the LFP-CuS@GOx NP particle size increased slightly to 212.94±0.97 nm, the PDI decreased to 0.146±0.025, and the particle size distribution became more uniform. Since LF is a cationic protein, its positive surface charge interacts electrostatically with the negative surface charge of FP-CuS@GOxNPs, resulting in an increase in potential to -29.2±0.93 mV.

[0053]

[0054] Table 1. Water content, particle size, PDI, and potential of PDA-CuS NPs, FP-CuS NPs, FP-CuS@GOx NPs, and LFP-CuS@GOx NPs

[0055] Example 6

[0056] Referring to Figure 1, weigh 10 mg of the four freeze-dried samples into a mortar, add an appropriate amount of KBr powder, grind thoroughly to ensure uniform dispersion, and then press into thin sheets. Press at 400-4000 cm⁻¹ -1 The spectra were measured and recorded using an infrared spectrometer.

[0057] FT-IR spectroscopy confirmed the successful preparation of PDA-CuS NPs, FP-CuS NPs, FP-CuS@GOx NPs, and LFP-CuS@GOx NPs. As shown in Figure 1, the PDA-CuS NPs contain the characteristic phenolic hydroxyl and amino groups of PDA, with a wavelength of 3349.5 cm⁻¹. -1 and 1510.7cm -1 The peaks that appear are mainly attributed to the stretching vibrations of OH and the shear vibrations of NH, and the peak at 1630.0 cm⁻¹ is... -1 The stretching vibrations of the aromatic ring C=C skeleton belonging to PDA indicate that PDA has been successfully modified onto the CuS surface. After coupling with FA, the thickness is 1641.3 cm⁻¹. -1and 1235.2cm -1 The peaks at these locations are attributed to the bending vibrations of C=O and CO, respectively, indicating the presence of carboxyl groups. These results demonstrate that FA is coupled to the surface of PDA-CuSNPs. GOx is a water-soluble protein containing abundant amide bonds. The amide I band (1647.0 cm⁻¹) -1 This is mainly attributed to the C=O stretching vibration of the peptide bond, specifically the amide II band (1547.7 cm⁻¹). -1 This is mainly attributed to NH bending vibration, with a relatively weaker intensity of 1301.9 cm. -1 and 1287.0cm -1 The results, primarily attributed to the stretching vibrations of CC and CN, indicate the successful synthesis of FP-CuS@GOx NPs. Furthermore, the infrared spectra of FP-CuS@GOx NPs and LFP-CuS@GOx NPs are largely identical to those of PDA-CuS NPs, suggesting that the introduction of FA and LF does not affect the chemical composition of PDA-CuS NPs.

[0058] Example 7

[0059] H2S release behavior detection

[0060] Referring to Figure 2, accurately transfer an appropriate amount of LFP-CuS@GOx NPs solution into a dialysis bag (MWCO = 10000 Da). Release media are equal volumes of PBS buffer and Glu solution, respectively, with the dialysis bag placed within each. Gently stir the release buffer solution in a 37°C water bath. Accurately transfer 1 mL of the release solution at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h, replacing the solution with fresh solution. Take 1 mL of the sample and thoroughly mix it with 100 μL of Zn(Ac)₂, then add 200 μL of LMPD and 200 μL of FFeCl₃. Incubate at room temperature in the dark for 30 min to form methylene blue. Measure the absorbance at 665 nm using UV light.

[0061] The results showed that LFP-CuS@GOx NPs continuously released H2S under acidic conditions, while almost no H2S was detected under neutral conditions. Under the same acidic conditions, the H2S release from LFP-CuS@GOx NPs in the presence of glucose was 4.25 times that in the glucose-free system. This may be attributed to GOx catalyzing the reaction of glucose to gluconic acid, leading to a continuous increase in acidity in the system. This is consistent with the pH detection results in Example 8, indicating that LFP-CuS@GOx NPs can continuously release high concentrations of H2S. Under neutral conditions with added glucose, the amount of H2S released remained in equilibrium with the acidic conditions without added glucose. These experiments demonstrate that GOx can effectively catalyze the continuous release of H2S from LFP-CuS@GOx NPs under acidic conditions.

[0062] Example 8

[0063] Cascade catalytic performance

[0064] Referring to Figure 3a, GOx can react with glucose in aqueous solution to produce gluconic acid and H2O2. The gluconic acid produced can cause changes in the pH of the system. The pH changes of LFP-CuS@GOx NPs and GOx solution in glucose solutions of different concentrations (0mM, 5mM, 10mM) were detected by pH meter.

[0065] The results showed that, in the absence of Glu, the pH values ​​of free GOx and LFP-CuS@GOx NPs remained stable at around 7.30. However, after the addition of 5 mM Glu solution, the pH values ​​of both free GOx and LFP-CuS@GOx NPs decreased significantly over time, eventually stabilizing at around 3.80. Furthermore, this formulation exhibited Glu-dependent behavior; the higher the Glu concentration, the faster the pH decreased, consistent with the trend observed in the free GOx solution. This indicates that the formulation possesses good GOx catalytic activity, capable of catalyzing Glu to gluconic acid and lowering the pH of the reaction system.

[0066] Referring to Figure 3b, the activity of POD enzyme was investigated using the TMB method. PBS buffer (pH 4.0) was mixed with 200 μL LFP-CuS@GOxNPs solution, and then 200 μL LTMB solution (10 mM) and 200 μL H2O2 solution (10 mM) were added. This constituted the experimental group. The LFP-CuS@GOx+TMB group and the H2O2+TMB group served as controls. Oxidized TMB appeared blue; photographs were taken and the UV-Vis absorption spectra were recorded.

[0067] The results showed that neither the H2O2+TMB group nor the LFP-CuS@GOx+TMB group solution turned significantly blue. The former indicates that the above reaction could not occur due to the lack of peroxidase; the latter indicates that the preparation can release Cu under acidic conditions. 2+ However, it cannot catalyze the formation of ·OH without additional H2O2. Only the LFP-CuS@GOx+TMB+H2O2 group solution turned blue, which confirms that LFP-CuS@GOx exhibits POD enzyme activity under H2O2 conditions, consistent with the UV full scan spectrum.

[0068] Referring to Figure 3c, to investigate the H2O2 produced in the catalytic system, ammonium oxalate was used as a probe. 1 mL of LFP-CuS@GOx solution was added to 1 mL of glucose solution (10 mM). After different time periods, 200 μL of ammonium oxalate solution (20 mM) was added to obtain a yellow suspension. After reacting for 8 min, the absorbance at 385 nm was measured using a UV-Vis spectrophotometer.

[0069] The results showed that after the addition of glucose solution, the concentration of H2O2 increased with time in the first 20 minutes, indicating that GOx in LFP-CuS@GOx could react with glucose to produce H2O2 during this period. However, after 20 minutes, the H2O2 content decreased with time, possibly because the reaction of GOx with glucose also lowered the pH of the system. A decrease in pH leads to the decomposition of PDA, thereby releasing Cu from CuS. 2+ Cu 2+ This process consumes the H2O2 produced in the system, causing the H2O2 content to decrease over time and eventually stabilize. This study demonstrates that LFP-CuS@GOx can effectively convert glucose into H2O2, providing additional H2O2 to the system.

[0070] Example 9

[0071] Plasma stability of nanoparticles

[0072] Referring to Figure 4, FP-CuS@GOx solution and LFP-CuS@GOx solution were mixed with rat plasma filtered through a 0.22 μm microporous membrane and incubated in a 37℃ constant temperature water bath for 12 h. The particle size of the incubated sample solution was measured at different time points using a Zeta PALS particle size analyzer to investigate the stability of the formulation in plasma.

[0073] The results showed that during plasma incubation, FP-CuS@GOx NPs and LFP-CuS@GOx NPs exhibited protein adsorption on their nanoparticle surfaces, leading to an increase in particle size. The particle size of LFP-CuS@GOx NPs decreased slightly after 0-1 h and FP-CuS@GOx NPs after 1-2 h. This may be because the loaded GOx may partially unfold or fold its enzyme structure in plasma, altering its binding mode with the nanoparticles. Conformational adjustments of the enzyme molecules in the plasma ionic environment may reduce the space they occupy, thus slightly decreasing the composite particle size. Analysis of the overall dynamic trends showed that all formulations stabilized after a particle size increase of approximately 30-40 nm within 12 h. This phenomenon is attributed to the dynamic equilibrium process of protein adsorption-desorption, indicating that FP-CuS@GOx NPs and LFP-CuS@GOx NPs have good stability in blood circulation.

[0074] Example 10

[0075] Nanozyme cytotoxicity assay for GL261 cells

[0076] Referring to Figure 5, GL261 cells in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates and incubated for 24 h. The culture medium was then removed, and 200 μL / well of gradient concentrations (NPs concentration 2.5-20 μg / mL) of FP-CuS@GOx and LFP-CuS@GOx solutions (diluted with serum-free DMEM) were added to each well. The plates were incubated for another 24 h, and the drug-containing medium was discarded. Cells were washed three times with PBS buffer. 100 μL of MTT solution (0.5 mg / mL, dissolved in pH 7-4 PBS) was added to each well, and the plates were incubated for 4 h. The supernatant was removed, and 100 μL of DMSO was added to each well, with gentle shaking to dissolve any crystals. The absorbance of each well was measured at 595 nm using a microplate reader. Cells incubated with serum-free DMEM under the same conditions served as a blank control (100% viability). Each sample was processed in triplicate.

[0077] The results showed that both FP-CuS@GOx NPs and LFP-CuS@GOx NPs exhibited concentration-dependent cytotoxicity against GL261 cells. As the concentration of the formulation increased, the survival rate of GL261 cells decreased. Furthermore, LFP-CuS@GOx NPs, modified with both LF and FA, showed stronger cytotoxicity against GL261 cells than FP-CuS@GOx NPs modified with FA alone. It is possible that the dual targeting effect of LF and FA specifically enhances the binding affinity of the formulation to the LRP-1 and FA receptors on the surface of GL261 cells, promoting the accumulation of the formulation within tumor cells and thus increasing the effective intracellular concentration.

[0078] Example 11

[0079] Intracellular reactive oxygen species generation characterization using inverted fluorescence microscopy

[0080] Referring to Figure 6, GL261 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Seeds were placed in 24-well plates and incubated for 24 h. The culture medium was then removed, and 400 μL of a gradient concentration (NPs concentration 6.25-50 μg / mL) of FP-CuS@GOx and LFP-CuS@GOx solution (diluted with serum-free DMEM) was added to each well. The plates were then returned to the incubator for another 4 h. The drug-containing culture medium was discarded, and the plates were washed three times with PBS buffer. 100 μL of 5 μM DCFH-DA solution was added to each well, and the plates were incubated for 30 min. The supernatant was then removed, and each well was washed three times with PBS. Fluorescence was observed under an inverted fluorescence microscope. Each well was replicated three times.

[0081] The results showed that the induction of intracellular ROS by FP-CuS@GOx NPs and LFP-CuS@GOx NPs was concentration-dependent. With increasing concentrations of FP-CuS@GOx NPs and LFP-CuS@GOx NPs, the intensity of the green fluorescence labeled with the ROS-specific DCFH-DA probe increased, demonstrating that FP-CuS@GOx NPs and LFP-CuS@GOx NPs could effectively induce the accumulation of large amounts of ROS through cascade catalysis. Furthermore, under the same concentration conditions, the green fluorescence intensity of the LFP-CuS@GOx NPs group was higher than that of the FP-CuS@GOx NPs group, proving that the dual targeting effect of LF and FA can enhance the recognition and binding ability of nano-formulations to specific receptors on the surface of tumor cells. Compared with a single targeting modification strategy, this approach can better promote the endocytic uptake efficiency of nano-formulations by tumor cells, resulting in the generation of more ROS in the TME, which is ultimately reflected in the enhanced fluorescence signal.

Claims

1. A multifunctional nanozyme targeting glioma, characterized in that, The multifunctional nanozyme described herein uses copper sulfide nanoparticles encapsulated with polydopamine as the core, and modifies the surface of the nanoparticles with folic acid and glucose oxidase to form a nanozyme. Then, lactoferrin is further modified on the surface of the nanozyme through electrostatic adsorption, thus constructing a multifunctional nanozyme for targeting glioma.

2. The multifunctional nanozyme for glioma targeting according to claim 1, characterized in that, The multifunctional nanozyme was first prepared via a hydrothermal method using copper sulfide nanoparticles encapsulated with polydopamine. Utilizing the abundant functional groups on the polydopamine surface, folic acid and glucose oxidase were co-loaded onto the polydopamine-encapsulated copper sulfide nanoparticles through Michael addition and covalent coupling. Finally, lactoferrin was further modified onto the nanozyme surface via electrostatic adsorption, constructing a multifunctional nanozyme targeting gliomas. After intravenous injection, this multifunctional nanozyme, under the influence of folic acid and lactoferrin, can cross the blood-brain barrier to further target gliomas. In the weakly acidic environment of the tumor, polydopamine degrades, rapidly releasing glucose oxidase and copper sulfide nanoparticles. The released glucose oxidase can... It catalyzes a reaction with glucose in the tumor microenvironment; on the one hand, it provides additional hydrogen peroxide to supplement the substrate required for the Fenton reaction; on the other hand, it further reduces the pH value of the tumor microenvironment, breaking the original weakly acidic environment's limitation on the Fenton reaction, significantly improving the efficiency of the Fenton reaction, and thus enhancing the efficacy of chemokinetics; in addition, the reduction of the pH value of the tumor microenvironment can simultaneously trigger the release of copper ions and hydrogen sulfide gas from copper sulfide nanoparticles, realizing gas therapy. The released copper ions consume H2O2 to generate ROS, and the released H2S gas can also inhibit the activity of endogenous ROS enzymes, accelerating the cascade generation of ROS, ultimately forming a tumor treatment mode with synergistic effects of chemokinetics combined with gas therapy.

3. The method for preparing a multifunctional nanozyme targeting glioma according to claim 1, characterized in that, The method includes the following specific steps: (1) Dissolving copper chloride dihydrate (CuCl2·2H2O) in an appropriate amount of deionized water to form 1 mM Cu 2+ In an ice-water bath, 10 mg of sodium citrate was added to the above solution and stirred for 5 min. Then, 20 mg of dopamine was added and stirred until homogeneous. 1 mL of sodium sulfide nonahydrate (Na2S·9H2O, 100 mM) solution was added dropwise to the above solution. After sonication for 15 min, the solution was transferred to a 90 °C water bath and reacted for 1 h. After the reaction was completed, the product was collected by centrifugation, washed three times with deionized water, and lyophilized to obtain polydopamine-encapsulated copper sulfide nanoparticles (PDA-CuS NPs). (2) 20 mg of folic acid was weighed and dissolved in 2 mL of Tris solution (pH 8.5, 50 mM). 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) was added under stirring and reacted for 10 min. Then, 20 mg of N-hydroxysuccinamide imine (NHS) was added and activated for 30 min. 40 mg of PDA-CuS NPs were dissolved in a certain amount of Tris solution. The activated folic acid solution was added dropwise to the above solution and stirred for 12 h. The resulting mixture was centrifuged, the precipitate was collected, and lyophilized to obtain folic acid-modified PDA-CuS NPs (FP-CuS NPs). (3) 2 mg of glucose oxidase (GOx) was dissolved in a certain amount of pure water and activated at room temperature for 1 h with EDC and NHS catalysts. Then, GOx:FP-CuS NPs were added to the reaction at a mass ratio of 2:1 and stirred for 1 h. The precipitate was obtained by centrifugation and lyophilized to obtain GOx-loaded FP-CuS NPs (FP-CuS@GOx NPs). (4) FP-CuS@GOx NPs were resuspended in Tris solution and 1 mL of lactoferrin aqueous solution (0.5 mg / mL) was added. The mixture was stirred at room temperature for 4 h and centrifuged to obtain lactoferrin-modified FP-CuS@GOx NPs (LFP-CuS@GOx NPs).

4. The application of the glioma-targeting multifunctional nanozyme according to claim 1 in the preparation of glioma products.

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