Nanoreactor for jointly blocking autophagy and macropinocytosis as well as preparation method and application of nanoreactor

Abstract

The invention provides a nano-reactor for jointly blocking autophagy and macropinocytosis, the nano-reactor comprises a first nano-particle CPZIs synthesized by integrating copper peroxide (CP), Zn < 2 + > ions and 2-methylimidazole (2-MIM), and an NRF2 inhibitor is loaded in a pore channel of the CPZIs to form a second nano-particle CPZI-Ms. The invention further provides a preparation method of the nanoreactor for jointly blocking the autophagy and macropinocytosis effects. The nanoreactor provided by the invention enhances the killing effect on tumor cells by blocking autophagy and macropinocytosis at the same time.

Description

Technical Field

[0001] The present invention relates to the field of nanotechnology, and in particular to a nanoreactor for jointly blocking autophagy and macropinocytosis, and a preparation method and application thereof. Background Art

[0002] Autophagy is an endogenous protective mechanism that can sequester and degrade harmful components, thereby alleviating potential cellular damage, while recovering essential nutrients from the degraded substances. However, for cancer cells, this self-protection process can make them more resistant to anti-tumor interventions, significantly reducing the therapeutic effect. Therefore, inhibiting autophagy in tumor cells has become a promising strategy to improve the efficacy of anti-tumor treatment. Lysosomes play a key role in cellular autophagy, promoting the fusion of autophagosomes at the late stage of autophagy, thereby mediating the degradation of subsequent substances. Reactive oxygen species (ROS) can induce lysosomal lipid peroxidation, leading to increased lysosomal membrane permeability, ultimately leading to lysosomal dysfunction and autophagy blockade. Nanomaterials have been widely demonstrated to exist in lysosomes for a long time. Therefore, utilizing the intrinsic ROS generation ability of nanoplatforms to preferentially induce elevated ROS levels in lysosomes is a promising autophagy blocking strategy.

[0003] However, studies have shown that inhibiting autophagy in tumors will prompt tumor cells to compensatorily upregulate macropinocytosis, allowing cells to take up nutrients such as extracellular fluid and macromolecules, thereby reducing the therapeutic effect, among which nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role. When autophagy is blocked, NRF2 expression is upregulated, allowing tumor cells to use macropinocytosis to take up extracellular nutrients and maintain intracellular homeostasis, and support the biosynthesis of intracellular amino acid pools and central carbon metabolites, thereby maintaining the proliferation ability of tumor cells, ultimately reducing the therapeutic effect of autophagy inhibition and enhancing resistance to tumor treatment. Summary of the invention

[0004] The present invention aims to design a nanoreactor to simultaneously block autophagy and macropinocytosis and enhance the killing effect on tumor cells in order to solve the problem that the existing inhibition of autophagy causes the upregulation of macropinocytosis and weakens the therapeutic effect of tumors.

[0005] According to a first aspect of the present invention, a nanoreactor is provided for jointly blocking autophagy and macropinocytosis, wherein the nanoreactor comprises a nanoreactor comprising: ... 2+ The first nanoparticle CPZIs is synthesized by ions and 2-methylimidazole (2-MIM), and the NRF2 inhibitor is loaded in the pores of the CPZIs to constitute the second nanoparticle CPZI-Ms.

[0006] As an optional embodiment, the NRF2 inhibitor includes ML385.

[0007] As an optional embodiment, the loading rate of the NRF2 inhibitor is greater than 50%.

[0008] As an optional embodiment, the particle size of the second nanoparticle CPZI-Ms is 170 nm to 200 nm, and the Zeta potential is +5 mV to +10 mV.

[0009] According to a second aspect of the present invention, there is provided a method for preparing the aforementioned nanoreactor for jointly blocking autophagy and macropinocytosis, comprising the following steps:

[0010] dissolving PVP in an aqueous solution containing CuCl2·2H2O to form a first solution;

[0011] Adding NaOH, 2-MIM, H2O2 and zinc acetate to the first solution in sequence to form a second solution, ensuring that the second solution is stirred and reacted at room temperature under strong alkaline conditions, and centrifuging and washing with water after the reaction is completed to obtain the first nanoparticle CPZIs;

[0012] CPZIs and NRF2 inhibitors are mixed, stirred, and centrifuged in methanol to load the NRF2 inhibitors into the pores of CPZIs to obtain the second nanoparticle CPZI-Ms.

[0013] As an optional embodiment, the concentration of the CuCl2·2H2O aqueous solution is 0.005M to 0.015M.

[0014] As an optional embodiment, in the first solution, the mass ratio of PVP to CuCl2·2H2O is (0.04-0.06):1.

[0015] As an optional embodiment, the pH of the second solution is >11, and in the second solution, the mass ratio of 2-MIM, H2O2, zinc acetate and CuCl2·2H2O is 1:(0.12-0.15):(0.02-0.04):(0.02-0.03).

[0016] As an optional embodiment, the mass ratio of CPZIs to NRF2 inhibitor is 1:(0.5-2), and the NRF2 inhibitor includes ML385.

[0017] According to a third aspect of the present invention, there is provided a use of the aforementioned nanoreactor for jointly blocking autophagy and macropinocytosis in the preparation of autophagy blocking tumor drugs.

[0018] It can be seen from the above technical solutions of the present invention that the nanoreactor proposed by the present invention for jointly blocking autophagy and macropinocytosis is internalized into the lysosome and utilizes the acidic lysosomal environment of tumor cells for degradation, releasing copper ions and hydrogen peroxide, generating excessive oxidative stress to trigger lysosomal membrane rupture, inducing lysosomal dysfunction, and effectively inhibiting the autophagy process through the lysosomal pathway; subsequently, the NRF2 inhibitor ML385 is released to downregulate biomarkers (NHE1 and SDC1) related to macropinocytosis, blocking the compensatory macropinocytosis reaction activated by autophagy inhibition, hindering the uptake of extracellular nutrients, and also providing a relatively stable environment for copper ions to continue to play a role, making the oxidative damage environment caused by copper ions less likely to be improved, so that copper ions can better play their role in triggering cell apoptosis; in this way, through combined blocking, the killing effect on tumor cells is enhanced, and the therapeutic effect of tumors is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 1A is a transmission electron microscope image of CPZIs and CPZI-Ms in the examples of the present invention; wherein 1A is CPZIs and 1B is CPZI-Ms.

[0020] Figure 2 This is the element mapping diagram of CPZIs in the example of the present invention.

[0021] Figure 3 Characterization diagram of CPZIs before and after drug loading in the examples of the present invention; wherein 3A is the hydrated particle size, 3B is the Zeta potential, 3C is the FTIR spectrum, and 3D is the high performance liquid chromatogram of ML385 (where the inset is the linear relationship between the integral of the chromatographic peak area with a retention time of 3.335 minutes and the ML385 concentration).

[0022] Figure 4 4A is a transmission electron microscopy image, and 4B is a copper ion release image.

[0023] Figure 5 This is a release curve of ML385 of CPZI-Ms in the example of the present invention under different pH conditions.

[0024] Figure 6 It is the ability of CPZI-Ms in the example of the present invention to release active oxygen; wherein, 6A is the ability of CPZI-Ms to release peroxy groups, 6B is the ability of CPZI-Ms to generate hydroxyl radicals at different concentrations, and 6C is the ability of CPZI-Ms to generate hydroxyl radicals that changes with time at a concentration of 60 μg / mL.

[0025] Figure 7A is the spatiotemporal distribution diagram of CPZI-Ms in the example of the present invention within the cell.

[0026] Figure 7 B is a detection diagram of the generation of reactive oxygen species by CPZIs and CPZI-Ms in cells in the examples of the present invention.

[0027] Figure 8 These are expression analysis diagrams of autophagy markers after cells were treated with CPZIs and CPZI-Ms in the examples of the present invention; wherein, 8A is a Western blot protein expression diagram, 8B and 8C are corresponding quantitative analysis diagrams, and 8D and 8E are fluorescence staining diagrams of LC3B and P62, respectively.

[0028] Fig. 9 CPZI-Ms in the example of the present invention inhibits macropinocytosis; wherein, 9A is a Western blot analysis of the expression of cellular NRF2 protein, 9B is a fluorescent image of cellular NRF2 immunostaining, 9C is a fluorescent image of TMR-dextran uptake by cells after different treatments, 9D is a quantification of TMR-dextran uptake, 9E is a Western blot analysis of the expression of NHE1 and SDC1 proteins in cells, and 9F is the intracellular ATP level.

[0029] Fig.10 The figures show the therapeutic effects of CPIZs and CPZI-Ms on tumor cells at different concentrations in the examples of the present invention.

[0030] Fig.11 The therapeutic effects of CPIZs and CPZI-Ms on mouse tumors in the examples of the present invention; wherein 11A is the growth curve of the tumor, 11B is the weight of the tumor after the treatment, and 11C is the immunohistochemical staining images of different groups after treatment. DETAILED DESCRIPTION

[0031] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0032] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0033] In an exemplary embodiment of the present invention, a nanoreactor for jointly blocking autophagy and macropinocytosis is provided, wherein the nanoreactor comprises a nanoreactor comprising: ... 2+The first nanoparticle CPZIs is synthesized by ions and 2-methylimidazole (2-MIM), and the NRF2 inhibitor is loaded in the pores of the CPZIs to constitute the second nanoparticle CPZI-Ms.

[0034] As an optional example, the NRF2 inhibitor includes ML385.

[0035] As an optional embodiment, the loading rate of the NRF2 inhibitor is greater than 50%, and particularly preferably 54.6%.

[0036] As an optional example, the particle size of the second nanoparticle CPZI-Ms is 170 nm to 200 nm, and is particularly preferably 187 nm.

[0037] As an optional example, the Zeta potential of the second nanoparticle CPZI-Ms is +5 mV to +10 mV, and is particularly preferably +8.83 mV.

[0038] As an optional example, both the first nanoparticle CPZIs and the second nanoparticle CPZI-Ms have a flower-like morphology.

[0039] In another exemplary embodiment of the present invention, a method for preparing the aforementioned nanoreactor for jointly blocking autophagy and macropinocytosis is also provided, comprising the following steps:

[0040] PVP was dissolved in an aqueous solution containing CuCl2·2H2O, and then NaOH, 2-MIM, H2O2 and zinc acetate were added in sequence, ensuring that the second solution was under strong alkaline conditions, and stirred at room temperature for reaction. After the reaction was completed, the solution was centrifuged and washed with water to obtain the first nanoparticle CPZIs;

[0041] CPZIs and NRF2 inhibitors are mixed, stirred, and centrifuged in methanol to load the NRF2 inhibitors into the pores of CPZIs to obtain the second nanoparticle CPZI-Ms.

[0042] As an optional example, the concentration of the CuCl2·2H2O aqueous solution is 0.005M to 0.015M.

[0043] As an optional example, in the first solution, the mass ratio of PVP to CuCl2·2H2O is (0.04-0.06):1.

[0044] As an optional example, the pH of the second solution is >11, and in the second solution, the mass ratio of 2-MIM, H2O2, zinc acetate and CuCl2·2H2O is 1:(0.12-0.15):(0.02-0.04):(0.02-0.03).

[0045] As an optional example, the mass ratio of CPZIs to NRF2 inhibitor is 1:(0.5-2), and the NRF2 inhibitor is ML385.

[0046] In other exemplary embodiments of the present invention, there is also provided an application of the aforementioned nanoreactor for jointly blocking autophagy and macropinocytosis in the preparation of autophagy blocking tumor drugs. After the nanoreactor enters the lysosome, on the one hand, due to impaired autophagic clearance, damaged proteins and cytotoxic components accumulate; on the other hand, by inhibiting macropinocytosis, the uptake of extracellular nutrients is hindered, leading to tumor cell death, and providing a relatively stable environment for copper ions to continue to exert their effects, making the oxidative damage environment caused by copper ions less likely to be improved, thereby allowing copper ions to better exert their role in triggering cell apoptosis.

[0047] The autophagy blocking strategy provided by the present invention is efficient, economical and easy to operate, and simultaneously inhibits autophagy blocking and the macropinocytosis upregulation effect caused by it. This combined blocking effect can enhance the effect of treating tumors.

[0048] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0049] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0050] Example 1

[0051] [Synthesis and characterization of CPZI-Ms nanoreactor]

[0052] PVP (1.5 g) was dissolved in an aqueous solution containing CuCl2·2H2O (15 mL, 0.01 M), followed by the addition of NaOH (15 mL, 0.015 M), 2-MIM (8 mL, 114.94 mg / mL), H2O2 (300 μL, 30%) and zinc acetate (8 mL, 3.69 mg / mL), stirred at room temperature for 2 h, and centrifuged to obtain CPZIs. Then 1 mg CPZIs was mixed with 1 mg ML385 in methanol, stirred for 12 h, and centrifuged to obtain the final product, which was the CPZI-Ms nanoreactor.

[0053] Figure 1 is a transmission electron microscopy (TEM) image, showing that the obtained CPZIs and CPZI-Ms have flower-like morphology; Figure 2 This is the element distribution diagram. The results show that CPZIs contain characteristic elements such as Cu, O, N, Zn and C, indicating that CPZIs were successfully synthesized.

[0054] Figure 3A shows that the hydrated particle size of CPZI-Ms increased from 171 nm to 187 nm after loading with NRF inhibitor. Figure 3 B shows that the zeta potential dropped from +14.19mV to +8.83mV. Figure 3 Fourier transform infrared spectroscopy showed that CPZI-Ms was at 1039 cm -1 (=COC stretching vibration) shows the characteristic stretching vibration belonging to ML385. The above results can prove that CPZIs are successfully loaded with ML385.

[0055] Figure 3 D shows that the loading efficiency of ML385 was 54.6% as measured by high performance liquid chromatography (HPLC) at a wavelength of 254 nm.

[0056] Therefore, it can be proved that the present invention successfully synthesized the CPZI-Ms nanoreactor.

[0057] Example 2

[0058] [pH-responsive disintegration of CPZI-Ms]

[0059] The obtained CPZI-Ms were dispersed in PBS solutions with different pH values ​​(7.6 and 5.0) and gently shaken at 100 rpm at room temperature. After 30 min, the solution was treated by centrifugation, the residue was imaged by transmission electron microscopy (TEM), and the UV-vis spectrum of the solution was measured. In addition, the hydrodynamic size of the residue was measured at predetermined time points (0h, 2h, 4h, etc.), and the solution was centrifuged at different times, 4mL of the supernatant was taken and replaced with the same volume of fresh PBS solution, and the released Cu was detected by ICP-OES. 2+ .

[0060] Figure 4 A is a transmission electron microscopy image at different times. Under the condition of pH 7.6, CPZIs maintained a regular framework structure and did not show obvious degradation. In contrast, after being treated with acidic PBS solution for 30 minutes, CPZIs showed obvious degradation and no intact nanoparticles were seen.

[0061] Figure 4 B shows copper ions (Cu 2+ ) showed similar responsive release behavior under acidic conditions. At pH 5.0, which simulated the acidic environment in tumor cell lysosomes, the release rate of copper ions was 57.4%, while at pH 7.6 and pH 6.5, the release of copper ions was very slow and the release amount was extremely small.

[0062] Figure 5is the release curve of ML385, which shows that under the condition of pH 5.0, 39.8% of ML385 was released from CPZI-Ms within 30 min, while the release rate was slow under the conditions of pH 7.6 and pH 6.5.

[0063] The above results show that under neutral or slightly acidic conditions, Cu 2+ and ML385 were able to remain stable but were effectively released in an acidic environment.

[0064] Therefore, it can be proved that the CPZI-Ms of the present invention utilizes the acidic lysosomal environment of tumor cells to be degraded.

[0065] Example 3

[0066] [pH responsiveness and OH generation of CPZI-Ms]

[0067] Figure 6 A is the result of KMnO4 colorimetric experiment, which shows that under acidic conditions, CPZI-Ms can fade the KMnO4 solution, confirming the release of peroxide groups in CPZI-Ms.

[0068] Then, different concentrations of CPZI-Ms (0, 7.5, 15, 30, 60, 120 μg / mL) were mixed with an acetate buffer solution (pH 5.0) containing TMB (40 μg / mL). After centrifugation, the supernatant was collected and the generation of ·OH was detected by the absorption peak at 650 nm.

[0069] To test the time-dependent generation of ·OH radicals, CPZI-Ms were mixed with acetate buffer solution (pH 5.0) containing TMB (40 μg / mL) at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h). After centrifugation, the supernatant was collected and the generation of ·OH was detected by the absorption peak at 650 nm.

[0070] Figure 6 B shows that the generated amount gradually increases with the increase of CPZI-Ms concentration; Figure 6 C shows that the amount of ·OH generated gradually increases with time. These results indicate that CPZI-Ms can generate a large amount of ·OH under acidic conditions (pH 5.0) in a concentration- and time-dependent manner.

[0071] Example 4

[0072] [Spatial and temporal distribution of CPZI-Ms in cells and detection of reactive oxygen species generated]

[0073] In order to detect the spatiotemporal distribution of nanoreactors in tumor cells, Panc02 cells were cultured in laser confocal culture dishes and fresh CPZIs / CPZI-Ms suspension (FITC labeled) was added. Lyso-TrackerRed dye (50nM) and Hoechst dye solution were added at different time points (0h, 1h, 2h, 4h, 8h) to mark lysosomes and cell nuclei. The phagocytosis of CPZIs / CPZI-Ms and their localization in lysosomes were observed by laser confocal microscopy, and the data were analyzed by ImageJ software.

[0074] In order to determine the generation of reactive oxygen species (·OH) in cells, DCFH-DA was used as a detection reagent. Panc02 cells were cultured in a laser confocal culture dish and fresh CPZIs / CPZI-Ms suspension (FITC labeled) was added. Lyso-TrackerRed dye (50nM) and Hoechst dye solution were added at different time points (4h, 8h) to mark lysosomes and cell nuclei. After washing with PBS three times, the fluorescence of DCFH-DA and the localization of ·OH in lysosomes were observed by laser confocal microscopy, and the data were analyzed by ImageJ software.

[0075] Figure 7 A shows that after incubation with Panc02 cancer cells, CPZI-Ms first entered the lysosomal region and reached peak accumulation in the lysosome at 4 h. After 4 h, the co-localization of the nanoreactor and the lysosome gradually weakened, and even the fluorescence signals of the nanoreactor and the lysosome decreased, which may be due to the disintegration of the nanoreactor in the lysosome and the damage of the lysosome caused by the simultaneous generation of ·OH free radicals.

[0076] Figure 7 As shown in B, after 4 h of incubation with CPZIs and CPZI-Ms, the fluorescence signal of 2′,7′-dichlorodifluorofluorescein ester (DCFH-DA), an ROS indicator, was mainly co-localized with lysosomes, while there was almost no fluorescence signal in the lysosomes of the control group, indicating that CPZI-Ms produced significant ·OH free radicals; by 8 h, the fluorescence signal of ROS was no longer confined to lysosomes, but distributed throughout the cytoplasm, which may be due to the damage of Cu after lysosome damage. 2+ due to the escape.

[0077] It can be seen from the above that the CPZI-Ms of the present invention can be preferentially internalized into lysosomes and can be degraded to release copper ions and hydrogen peroxide, thereby spontaneously promoting the generation of a large number of hydroxyl radicals in the lysosomes, leading to lysosomal damage.

[0078] Example 5

[0079] [CPZIs and CPZI-Ms inhibit autophagy and macropinocytosis]

[0080] Panc02 cells were seeded in 24-well plates and allowed to grow in an incubator for 24 h. On the next day, the medium was replaced with serum-free medium and cultured in serum-free medium for another 24 h, during which different treatments (ML385, CPZIs, CPZI-Ms) or untreated controls were performed. After 24 h of treatment, the cells were washed with PBS.

[0081] Western blot and fluorescence staining were used to evaluate the levels of autophagy markers in one part of the cells, and 1 mg mL -1 The cells were then cultured in serum-free medium containing TMR-dextran and continued to be cultured at 37°C for 1 hour. Afterwards, the cells were thoroughly washed and fixed with 4% formaldehyde solution. Finally, they were stained with DAPI and the intracellular TMR-dextran uptake was captured using a fluorescence microscope, and the ATP content was measured using an ATP detection kit.

[0082] Figure 8 The results showed that after 24 h of incubation with the nanoreactors CPZIs and CPZI-Ms, the expression levels of the autophagy markers LC3B and P62 increased significantly, indicating that the autophagic flux in the cells was initiated and aggravated due to the enhanced oxidative stress. However, P62 (aggregate 1 / P62) failed to be degraded but accumulated instead, indicating that the fusion of lysosomes and autophagosomes failed, resulting in the restricted formation of autophagosomes and thus blocking the autophagic flux.

[0083] Fig. 9 A shows that CPZIs treatment significantly upregulated the expression of NRF2, indicating that the macropinocytosis response was activated; in contrast, CPZI-Ms effectively reversed the upregulation of NRF2 stimulated by CPZIs, keeping NRF2 at a low level, which was comparable to the effect of ML385 alone; Fig. 9 B Immunofluorescence staining shows the same results; the macropinocytosis process was then reflected by tracking the uptake of TMR fluorescently labeled dextran (TMR-dextran). Fig. 9 As shown in C and 9D, TMR-dextran uptake was significantly increased after CPZIs treatment, but was greatly reduced in the CPZI-Ms group; in addition, Fig. 9 E shows that the levels of key macropinocytosis-related proteins SDC1 and NHE1 were upregulated in Panc02 cells treated with CPZIs compared with the control group, while they were downregulated in the CPZI-Ms group. These results indicate that CPZI-mediated autophagy blockade significantly activated macropinocytosis, and CPZI-Ms effectively inhibited this process by inhibiting NRF2. Next, the effect of CPZI-Ms on the energy metabolism of tumor cells was evaluated. Fig. 9As shown in F, CPZI-Ms significantly reduced the ATP level in Panc02 cells, indicating that the energy supply of tumor cells was disrupted.

[0084] Example 6

[0085] [The therapeutic effect of CPZI-Ms on tumors]

[0086] Cell level: Panc02 cells were cultured in 96-well plates at 37°C for 24 h; then, the cells were treated with different concentrations of different groups (control group, ML385, CPZIs, CPZI-Ms) for 24 h; then, the cell viability was determined according to the CCK-8 method.

[0087] Animal level: Panc02 cells were suspended in 100 μL of saline, and then the mixture was subcutaneously injected into the right back of the mouse to establish a subcutaneous tumor model. When the tumor size reached approximately 90 cubic millimeters, the mice were randomly divided into four groups (n=5 in each group) and treated with saline, ML385 (15 mg / kg), CPZIs (15 mg / kg), and CPZI-Ms (15 mg / kg). The drug was injected intravenously once a day for a total of three injections, and the weight and tumor volume of the mice were recorded every two days. The calculation of tumor volume refers to the formula V=L×W ^2 ×1 / 2 (V, volume; L, tumor length; W, tumor width). In addition, after treatment, the weight of the mouse tumor was recorded.

[0088] Fig.10 It was shown that ML385 itself did not show obvious killing effect at the cellular level, while the nanoreactor CPZI-Ms inhibited the proliferation of tumor cells and showed concentration dependence, and the killing effect of CPZI-Ms on cells was better than that of the CPZI group.

[0089] Fig.11 A and 11B show that ML385 alone could not inhibit the rapid growth of tumors at the in vivo level, while CPZIs and CPZI-Ms treatments showed better therapeutic effects, and the therapeutic effect of the CPZI-Ms group was significantly better than that of the CPZI group.

[0090] Then, immunofluorescence staining of autophagy and macropinocytosis-related biomarkers was performed on tumor tissues from different groups. Fig.11As shown in C, the levels of LC3B and P62 increased in mice treated with CPZIs and CPZI-Ms, indicating that cellular autophagy was initiated but failed to complete due to lysosomal dysfunction caused by the nanoreactor; in addition, CPZIs-induced autophagy blockade triggered the activation of compensatory macropinocytosis, manifested by increased levels of NRF2, NHE1, and SDC1, however, CPZI-Ms loaded with ML385 effectively inhibited the upregulation of NRF2, thereby reducing the expression of NHE1 and SDC1 and blocking the response of macropinocytosis.

[0091] Therefore, it can be confirmed that the CPZI-Ms of the present invention can cause damage to the autophagic clearance function of tumor cells, leading to the accumulation of harmful substances in the cells, and prevent the cells from absorbing extracellular nutrients by inhibiting macropinocytosis, thereby cutting off the key survival pathway of tumor cells, and ultimately causing tumor cells to irreversible death.

[0092] The present invention significantly improves the killing power against tumor cells by simultaneously inhibiting autophagy and macropinocytosis, thereby improving the effect of tumor treatment.

[0093] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A nanoreactor for jointly blocking autophagy and macropinocytosis, characterized in that: The nanoreactor consists of a nanoreactor that integrates copper peroxide (CP), Zn 2+ The first nanoparticle CPZIs is synthesized by ions and 2-methylimidazole (2-MIM), and the NRF2 inhibitor is loaded in the pores of the CPZIs to constitute the second nanoparticle CPZI-Ms.

2. The nanoreactor for jointly blocking autophagy and macropinocytosis according to claim 1, characterized in that: The NRF2 inhibitors include ML385.

3. The nanoreactor for jointly blocking autophagy and macropinocytosis according to claim 1, characterized in that: The loading rate of the NRF2 inhibitor is greater than 50%.

4. The nanoreactor for jointly blocking autophagy and macropinocytosis according to claim 1, characterized in that: The particle size of the second nanoparticle CPZI-Ms is 170 nm to 200 nm, and the Zeta potential is +5 mV to +10 mV.

5. A method for preparing a nanoreactor for jointly blocking autophagy and macropinocytosis according to any one of claims 1 to 4, characterized in that: The following steps are involved: dissolving PVP in an aqueous solution containing CuCl2·2H2O to form a first solution; NaOH, 2-MIM, H2O2, and zinc acetate are sequentially added to the first solution to form a second solution. The second solution is stirred at room temperature to react under strong alkaline conditions. After the reaction is completed, the solution is centrifuged and washed with water to obtain the first nanoparticle CPZIs. CPZIs and NRF2 inhibitors are mixed, stirred, and centrifuged in methanol to load the NRF2 inhibitors into the pores of CPZIs to obtain the second nanoparticle CPZI-Ms.

6. The preparation method according to claim 5, characterized in that The concentration of the CuCl2·2H2O aqueous solution is 0.005M~0.015M.

7. The preparation method according to claim 5, characterized in that In the first solution, the mass ratio of PVP to CuCl2·2H2O is (0.04-0.06):

1.

8. The preparation method according to claim 5, characterized in that The pH of the second solution is greater than 11, and in the second solution, the mass ratio of 2-MIM, H2O2, zinc acetate and CuCl2·2H2O is 1:(0.12-0.15):(0.02-0.04):(0.02-0.03).

9. The preparation method according to claim 5, characterized in that The mass ratio of CPZIs to NRF2 inhibitor is 1:(0.5-2), and the NRF2 inhibitor includes ML385.

10. Use of the nanoreactor for combined blocking of autophagy and macropinocytosis according to any one of claims 1 to 4 in the preparation of autophagy blocking tumor drugs.

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