An ultrasonically responsive nanoreactor, its preparation method and application

Abstract

This invention relates to the pharmaceutical field, specifically to an ultrasound-responsive nanoreactor, its preparation method, and its applications. The ultrasound-responsive nanoreactor of this invention comprises a manganese porphyrin-based metal-organic framework (MOF) and a lipid membrane. The MOF serves as a carrier, and the lipid membrane coats the outer surface of the MOF. Maleimide-modified hydroxychloroquine is loaded onto the MOF. This application is the first to apply a clever click chemistry-based design to enhance the efficacy of autophagy inhibition, pioneering a novel concept of autophagy capture using the Mal-SH reaction in situ within tumors. By combining a novel nanoreactor with powerful autophagy inhibition and the SDT effect, the inventors significantly improve the in vitro and in vivo antitumor effects, providing a forward-looking approach and research foundation for click chemistry-based biomedical applications and offering a new research direction for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to an ultrasonic-responsive nanoreactor, its preparation method, and its application. Background Technology

[0002] Autophagy, an evolutionarily conserved cellular protection process, protects cells from oxidative damage and cellular stress, maintaining intracellular homeostasis. However, autophagy may increase the resistance of tumor cells to therapeutic stimuli because autophagic enzymes degrade drugs or reactive oxygen species (ROS)-inactivated proteins and organelles, maintaining tumor cell viability. Sounddynamic therapy (SDT) is a novel anti-tumor treatment with minimal invasiveness and high penetration; however, many studies have found that SDT-mediated apoptosis can be repaired and reversed through autophagy, leading to incomplete cell death. To overcome this treatment resistance, synergistic use of autophagy inhibitors with SDT is considered a promising strategy to resensitize cancer cells to treatment.

[0003] Currently, various drugs have been developed to block the early or late stages of autophagy. Hydroxychloroquine (HCQ) is one of the most widely used chemicals. It inhibits the final stage of autophagy flux by blocking the fusion of autophagosomes and lysosomes and preventing the degradation of substrates in autophagosomes. This action cuts off the nutrient supply from apoptotic cells to avoid energy cycling in tumors. Hydroxychloroquine can disrupt the complete autophagy process; therefore, the more hydroxychloroquine accumulates in autophagosomes or lysosomes, the higher the autophagy blocking efficiency. However, there are relatively few studies on increasing the hydroxychloroquine content in autophagosomes or lysosomes. Although hydroxychloroquine has been found to induce autophagy and inhibit the development of certain diseases, its specific mechanism of action and targets are still not fully understood. Research on increasing the hydroxychloroquine content in autophagosomes or lysosomes is still in the exploratory and research stage, and the effect of inhibiting autophagy is still limited by the rapid metabolism and low selectivity of small molecule inhibitors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an ultrasound-responsive nanoreactor that can enhance autophagy blocking and synergize with SDT, and can be used as a drug for tumor treatment.

[0005] The basic solution provided by this invention is: an ultrasonic-responsive nanoreactor, comprising a manganese porphyrin-based metal-organic framework and a lipid membrane, wherein the manganese porphyrin-based metal-organic framework is a carrier, the lipid membrane is coated on the outer surface of the manganese porphyrin-based metal-organic framework, and maleimide-modified hydroxychloroquine is loaded on the manganese porphyrin-based metal-organic framework.

[0006] The working principle and advantages of this invention are as follows:

[0007] The inventors discovered that the classical click reaction between maleimide (Mal) and thiol (-SH) is highly efficient and specific. Tumor-derived proteins are released from cellular debris upon SDT stimulation, and the thiol groups exposed on damaged proteins can bind with high sensitivity to maleimide groups. Therefore, this application innovatively proposes a novel concept: utilizing the in-situ Mal-SH reaction in tumors to achieve autophagic capture. Specifically, the inventors synthesized for the first time Mal-modified hydroxychloroquine (Mal-SH). M When SDT (Solution-Driven Therapy) is performed, tumor cells are killed, producing cellular or protein fragments. Maleimide-modified hydroxychloroquine then covalently binds to the -SH groups of the damaged proteins via a Mal-SH reaction, anchoring itself to the protein fragments. Subsequently, due to the inherent self-protective mechanism of cancer cells, autophagy is rapidly activated. Autophagosomes then engulf the damaged proteins and simultaneously phagocytose the protein-anchored hydroxychloroquine, allowing them to actively access the anti-autophagy hydroxychloroquine. Finally, hydroxychloroquine consumes hydrogen ions, altering the internal pH of proteolytic enzymes during the fusion of autophagosomes and lysosomes, thereby blocking autophagy flux and significantly enhancing autophagy blocking efficiency.

[0008] To further enhance drug efficacy, this application designs a multifunctional nanoreactor, Lip@M- M H, a strategy to enhance autophagy blockade and synergize with SDT through in-situ click reaction to improve tumor treatment efficacy. This application prepares a structurally tunable nanoscale manganese porphyrin-based metal-organic framework (MnTCPP MOF) for delivery. M HCQ, MnTCPP MOF can achieve [the following]: M The efficient loading of HCQ and its timed, targeted release of hydroxychloroquine enable targeted drug delivery, thereby improving therapeutic efficacy. Furthermore, the MnTCPP MOF can act as a sonosensitive agent to generate ROS under ultrasound irradiation, inducing apoptosis in cancer cells. Due to the excellent near-infrared absorption and paramagnetism of MnTCPP, this nanoreactor has the potential to serve as a contrast agent for magnetic resonance imaging (MRI) and photoacoustic imaging (PAI), providing visualization guidance during treatment. (The Lip@M- described in this application...) M H is lipidmembrane@MnTCPP MOF- M HCQ is an abbreviation for lipid membrane, Lip is an abbreviation for lipid membrane, and MnTCPP MOF is an abbreviation for manganese porphyrin-based metal-organic framework. M H is hydroxychloroquine modified with Mal. M (HCQ is an abbreviation)

[0009] In addition, to preventM The inventors addressed the non-specific binding of HCQ to non-target proteins by coating a PEGylated lipid membrane onto a nanoreactor. M The Mal group in HCQ is shielded before the nanoreactor is exposed to tumor-derived proteins. PEGylation generates a hydration cloud around the nanoreactor, reducing interactions between the nanoreactor and blood components. The inventors' use of a PEGylated lipid membrane to protect the Mal group is also previously unreported. Simultaneously, the PEGylated lipid membrane prevents drug leakage, further improving drug bioavailability. Furthermore, the PEGylated lipid membrane enhances the biocompatibility of the nanoreactor, prolonging blood circulation time. When the nanoreactor reaches the tumor region and is subjected to ultrasound irradiation, it not only initiates the SDT effect of the nanoreactor but also decomposes the lipid membrane and promotes the release of hydroxychloroquine. More importantly, in the presence of damaged proteins released in situ from cell debris, autophagosomes actively contact these proteins and the hydroxychloroquine anchored on them, significantly promoting the accumulation of anti-autophagy drugs within the autophagosomes, thereby improving the efficiency of autophagy blockade.

[0010] In summary, the inventors of this application have, for the first time, applied an ingenious design based on click chemistry to enhance the efficacy of autophagy inhibition, pioneering a novel concept of autophagy capture using the Mal-SH reaction in situ within tumors, and for the first time synthesized Mal-modified hydroxychloroquine. M HCQ, none of these studies have been reported before. The inventors have significantly improved the in vitro and in vivo anti-tumor effects by combining a novel nanoreactor with powerful autophagy inhibition and SDT effects, providing a forward-looking approach and a solid research foundation for click chemistry-based biomedical applications, and also offering a completely new research direction for tumor treatment.

[0011] Furthermore, the structure of the maleimide-modified hydroxychloroquine is as follows:

[0012] .

[0013] Furthermore, the pore size of the manganese porphyrin-based metal-organic framework (MOF) is 1.27 nm. MnTCPP MOFs possess a mesoporous structure; excessively small pore sizes hinder drug loading, while excessively large pore sizes reduce the material's structural stability and chemical properties. The MnTCPP MOF material with the aforementioned pore size enables highly efficient loading of hydroxychloroquine, effectively protecting the drug molecule while also facilitating drug release and control.

[0014] This invention also provides a method for preparing an ultrasonically responsive nanoreactor, including... M Preparation of HCQ, preparation of MnTCPPMOF and Lip@M- MPreparation of H, in which M The preparation of HCQ includes the following steps:

[0015] Hydroxychloroquine was dissolved in N,N-dimethylformamide, and then the solution was mixed with 3-maleimide propionic acid, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, and carbodiimide. The mixture was stirred and evaporated to dryness to obtain maleimide-modified hydroxychloroquine. M HCQ is then purified.

[0016] Furthermore, Lip@M- M The preparation of H includes the following steps:

[0017] S1: Dispalmitoylphosphatidylcholine, polydiethanol-distearate phosphatidylethanolamine and cholesterol are dissolved in chloroform, and the mixed solution is then rotary evaporated in a water bath to form a lipid membrane, which is then dissolved.

[0018] S2: Dissolve manganese porphyrin-based metal-organic frameworks in a mixed solution of N,N-dimethylformamide and ultrapure water, then disperse maleimide-modified hydroxychloroquine in the mixture and stir at room temperature;

[0019] S3: The lipid membrane prepared in S1 is added to the mixture in S2, stirred again, rinsed with deionized water, and centrifuged to finally obtain the nanoreactor Lip@M-MH.

[0020] Furthermore, in S1, the mass ratio of dipalmitoylphosphatidylcholine, polydiethanol-distearate phosphatidylethanolamine, and cholesterol is 3:1:1.

[0021] Furthermore, in S1, the water bath temperature is 50-55℃.

[0022] Furthermore, in step S1, deionized water is used to dissolve the lipid membrane. Conventional methods typically involve using phosphate buffer to dissolve the lipid membrane, and the inventors had also attempted to dissolve the formed lipid membrane in phosphate buffer. However, they found that the lipid membrane dissolved in phosphate buffer could not encapsulate MnTCPP. Through extensive experimentation and analysis, the inventors discovered that dissolving the lipid membrane in deionized water allowed for successful encapsulation of MnTCPP.

[0023] The ultrasound-responsive nanoreactor described in this invention can be used to prepare drugs for treating tumors. The ultrasound-responsive nanoreactor of this invention exhibits significantly enhanced autophagy blocking efficiency, which can further enhance SDT-induced apoptosis, thereby inhibiting tumor cell growth and making it suitable for use as a tumor therapeutic drug.

[0024] The present invention relates to the application of any of the ultrasound-responsive nanoreactors in the preparation of contrast agents. Because the ultrasound-responsive nanoreactor of this invention possesses paramagnetism and good near-infrared absorption, it is a dual-modal (magnetic resonance / photoacoustic) enhancement contrast agent with imaging potential, which can provide visual guidance during treatment. Attached Figure Description

[0025] Figure 1 for M The characterization of HCQ, where (a) is M The mass spectrometry results of HCQ; (b) is M HPLC results of HCQ; (c) shows HCQ and M The UV-Vis spectrum of HCQ; (d) is M HCQ proton nuclear magnetic resonance hydrogen spectrum ( Figure 1 The top left corner of d is M (Chemical structural formula of HCQ).

[0026] Figure 2 Characterization of MnTCPP MOF, where (a) is transmission electron microscopy (TEM) of MnTCPP MOF; (b) is scanning electron microscopy (SEM) of MnTCPP MOF; (c) is the specific surface area of ​​MnTCPP MOF; (d) is the pore size of MnTCPP MOF; and (e) is... M Standard concentration curve of HCQ.

[0027] Figure 3 For Lip@M- M The characterization of H, where (a) is Lip@M- M (a) Transmission electron microscopy of H; (b) Elemental analysis; (c) XRD; (d) UV-Vis spectrum; (e) Particle size on the left, potential on the right; (f) Particle size stability; (g) Lip@M- M H and Lip@M- M The drug release behavior of H+US; (h) is the transmission electron microscope after drug release; (i) is the scanning electron microscope after dissolving the lipid membrane with phosphate buffer and mixing with MnTCPP.

[0028] Figure 4 The acoustic-dynamic performance and phagocytic reaction of the nanoreactor were measured. (a) shows the ESR spectra of PBS, MnTCPP, and Lip@M with and without sonication; (b) shows the UV-Vis absorption spectra of MnTCPP and Lip@M-mediated methylene blue degradation with and without sonication; (c) and (d) show the concentration- and time-dependent SOSG probe results of Lip@M phagocytosis. 1 O2; (e) and (f) are respectively Lip@M- MThe images and flow cytometry results of intracellular confocal fluorescence microscopy images of H and 4T1 cells after co-incubation for 0.5, 1, 2, and 4 hours; (g) and (h) are the confocal fluorescence microscopy images and flow cytometry results of 4T1 cells stained with DCFH-DA after different treatments.

[0029] Figure 5 To detect the enhancement of autophagy inhibition by in situ click reaction, (a) is a schematic diagram of in situ protein capture mediated by the Mal-SH click reaction; (b) is a schematic diagram of cell debris preparation and transwell chamber-simulated protein capture; (c) is... M Zeta potentials of HCQ and OVA, and their relationship with HCQ and M Zeta potential of OVA after HCQ incubation; (d) is M Zeta potentials of HCQ and OVA, and Lip@M- M H and Lip@M- M (e) Zeta potential of OVA after H+ US incubation; (f) Protein quantification of the upper transwell chamber after different treatments; (g) Protein quantification of the lower transwell chamber after different treatments; (h) Western blot detection of LC3B-II / I, p62, and Beclin-1 expression in different groups; (h), (i), and (j) Quantitative analysis of relative gray values ​​of LC3B-II / I, p62, and Beclin-1, respectively; (k) Immunofluorescence images of LC3B in 4T1 cells after different treatments; (l) Lip@M- M Biological transmission electron microscopy images of 4T1 cells processed with H+US.

[0030] Figure 6 For the biosafety and cytotoxicity testing of different nanoreactors, the figure shows (a) Lip@M- containing different concentrations of MnTCPP. M Cell viability after co-incubation of H with 4T1 and HUVEC for 24 hours; Figure (b) shows the cell viability after co-incubation of MnTCPP containing different concentrations with 4T1 and HUVEC for 24 hours; (c) shows the relative cell viability after co-incubation of 4T1 cells with different concentrations in different reactors; (d) and (e) show the level of apoptosis and corresponding quantitative analysis after different treatments by flow cytometry; (f) shows the fluorescence images of 4T1 cells after different treatments stained with PI and calcein AM respectively.

[0031] Figure 7 The in vitro and in vivo fluorescence, photoacoustic, and magnetic resonance imaging of the nanoreactor were used for detection. Figure (a) shows the intravenous injection of Lip@M- M(a) Fluorescence images of tumor sites in 4T1 tumor-bearing mice after H44 hours; (b) In vitro fluorescence images of tumors and major organs 48 hours after injection of the nanoreactor; (c) Fluorescence images of different concentrations of Lip@M- M In vitro photoacoustic signal values ​​and photoacoustic images of H (inset); (d) Lip@M- administered intravenously at different time intervals. M Photoacoustic image of the tumor region after H; (e) is Lip@M- M (f) shows the hysteresis curve of H; (g) shows the in vitro t1-weighted relaxation coefficient and magnetic resonance images of nanoreactors at different concentrations (inset); (g) shows the in vivo t1-weighted imaging of 4T1 tumor-bearing mice before and after intravenous administration of the nanoreactor; (h) shows the corresponding t1-weighted MRI signal intensity of the tumor area before and after injection.

[0032] Figure 8 (a) is the fluorescence signal in the tumor region during in vivo fluorescence; Figure 8 (b) Fluorescence signals of various organs and tumors after ex vivo; Figure 8 (c) represents the full wavelength of photoacoustic imaging; Figure 8 (d) is in vivo photoacoustic imaging.

[0033] Figure 9 For the nanoreactor Lip@M- M The in vivo biocompatibility of H, where (a) includes PBS, deionized water, MnTCPP, M-MH, and Lip@M- M Assessment of hemolytic effect after H treatment of cells; (b)-(d) shows intravenous administration of Lip@M- M Blood biochemical results of Kunming mice sacrificed at different time intervals after H; (e)-(m) represent intravenous administration of Lip@M- M Blood routine examination results of mice at different time intervals after H, n = 6; (n) represents the results of intravenous administration of Lip@M- M Images of H&E staining of the heart, liver, spleen, lungs, and kidneys of mice sacrificed at different time intervals after H. Detailed Implementation

[0034] The following detailed explanation illustrates the specific implementation methods:

[0035] Example 1: Lip@M- M H

[0036] This embodiment provides an ultrasonic-responsive nanoreactor, abbreviated as Lip@M- M H comprises a manganese porphyrin-based metal-organic framework and a lipid membrane, wherein the manganese porphyrin-based metal-organic framework serves as a carrier, the lipid membrane coats the outer surface of the manganese porphyrin-based metal-organic framework, and maleimide-modified hydroxychloroquine is loaded on the manganese porphyrin-based metal-organic framework.

[0037] Lip@M- M The method for preparing H includes the following steps:

[0038] Step 1: M Preparation of HCQ

[0039] 0.8 g of hydroxychloroquine was dissolved in 40 ml of N,N-dimethylformamide. This solution was then mixed with 481 g of 3-maleimide propionic acid, 170 g of 4-dimethylaminopyridine, 270 g of 1-hydroxybenzotriazole, and 5 ml of carbodiimide. The mixture was magnetically stirred at room temperature for 6-8 hours (6 hours in this example). After evaporation to dryness, orange-red crystals of maleimide-modified hydroxychloroquine were obtained. M HCQ was finally purified using high performance liquid chromatography.

[0040] Step 2: Preparation of MnTCPP MOF

[0041] 30 mg zirconium chloride hexahydrate, 0.28 g benzoic acid, and 10.98 mg manganese porphyrin were dissolved in 14 ml N,N-dimethylformamide and magnetically stirred at 90 °C for 5 hours. After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 15,000 rpm for 15 minutes. The product was washed three times with N,N-dimethylformamide and deionized water and then resuspended in deionized water to obtain the manganese porphyrin metal-organic framework MnTCPP MOF.

[0042] Step 3: Lip@M- M Preparation of H

[0043] S1: Dipalmitoylphosphatidylcholine, poly(diethanolamine)-distearate phosphatidylethanolamine, and cholesterol (the mass ratio of dipalmitoylphosphatidylcholine, poly(diethanolamine)-distearate phosphatidylethanolamine, and cholesterol is 3:1:1; in this embodiment, the masses of dipalmitoylphosphatidylcholine, poly(diethanolamine)-distearate phosphatidylethanolamine, and cholesterol are 6 mg, 2 mg, and 2 mg, respectively) are dissolved in 5 ml of chloroform. The mixed solution is then transferred to a 100 ml round-bottom flask and rotary evaporated in a water bath at 50-55°C (the water bath temperature in this embodiment is 55°C) to form a lipid film. Then, 10 ml of deionized water is added to a beaker to dissolve the lipid film.

[0044] S2: Dissolve 1 mg MnTCPP MOF in a mixed solution of 200 μl N,N-dimethylformamide and 800 μl ultrapure water, then add 0.2 mg... M HCQ was dispersed in the mixture and magnetically stirred at room temperature for 12 hours;

[0045] S3: Then, the 2.5 ml lipid membrane prepared in S1 was added to the mixture in S2. After stirring for 3 hours, it was rinsed with deionized water and centrifuged at 15000 rpm for 20 minutes to finally obtain the nanoreactor Lip@M- M H.

[0046] Example 2

[0047] The difference between Example 2 and Example 1 is that step 3, Lip@M- M In the preparation process of H, the deionized water in S1 is replaced with phosphate buffer.

[0048] In step three Lip@M- M In the preparation process S1 of H, the inventors also attempted to dissolve the formed lipid membrane in phosphate buffer, such as... Figure 3 As shown in Figure i, scanning electron microscopy revealed that after dissolving the lipid membrane in phosphate buffer and then mixing it with MnTCPP, no thin film was formed around the MnTCPP, indicating that the lipid membrane failed to successfully coat the MnTCPP. Through extensive experimentation and analysis, the inventors discovered that dissolving the lipid membrane in deionized water allowed for successful coating of MnTCPP. Figure 3 As shown in Figure a, a light-colored thin film can be seen on the periphery of MnTCPP in transmission electron microscopy, indicating that the lipid membrane has been successfully coated on the surface of MnTCPP MOF. (In this application, MnTCPP is an abbreviation for MnTCPP MOF)

[0049] Comparative Example 1: Lip@M (Lip@M in this application is an abbreviation for lipid membrane@MnTCPP MOF, where lipid membrane is the English name for lipid membrane, Lip is an abbreviation for lipid membrane, and MnTCPPMOF is an abbreviation for manganese porphyrin metal-organic framework)

[0050] The difference between Comparative Example 1 and Example 1 is that the preparation step in Step 1 is omitted, and the step "add 0.2 mg" in Step 3S2 is also omitted. M The operation of "dispersing HCQ in the mixture" is performed.

[0051] Comparative Example 2: Lip@MH (Lip@MH in this application is an abbreviation for lipid membrane@MnTCPP MOF-HCQ)

[0052] The difference between Comparative Example 2 and Example 1 is that the preparation step in step 1 is omitted, and in step 3S2, "another 0.2 mg" is added. M In the operation of "dispersing HCQ in the mixture" M Replace HCQ with HCQ.

[0053] Comparative Example 3: MnTCPP

[0054] The difference between Comparative Example 3 and Example 1 is that the preparation steps of Step 1 and Step 3 are omitted.

[0055] I. Physicochemical Properties Characterization

[0056] (one) M Characterization of HCQ

[0057] This application successfully prepared hydroxychloroquine modified with a maleimide group. M HCQ. (e.g.) Figure 1 As shown in figure a, the mass spectrometry results indicate that the molecular weight of the final product obtained is 487.7 Da, which is close to the theoretical molecular weight of the product. Figure 1 As shown in b, the high-performance liquid chromatography (HPLC) results indicate that the purity of the final product obtained is 95.31%, all of which demonstrate that the maleimide-modified hydroxychloroquine has high purity. Furthermore, as... Figure 1 As shown in c and 1d, the UV-Vis spectrum and proton NMR spectrum confirm that... M The chemical structure and maximum absorption value of HCQ indicate that the Mal group was successfully coupled with HCQ. M The chemical structural formula of HCQ is as follows: Figure 1 As shown in the upper left corner of d.

[0058] (II) Characterization of MnTCPP MOF

[0059] like Figure 2 As shown in Figures a and 2b, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) results indicate that the MnTCPP MOFs before lipid membrane coating are spindle-shaped, uniformly dispersed, and exhibit good homogeneity in solution. Figure 2 As shown in c and 2d, nitrogen adsorption analysis indicates that the specific surface area and average pore size of the MnTCPP MOF are 1257 m². 2 / g and 1.27 nm indicate that the MnTCPP MOF possesses a mesoporous structure and reliable loading efficiency. Figure 2 As shown in e, the HPLC results show that MnTCPP... M The encapsulation efficiency of HCQ is 72.2±7.5%.

[0060] (III) Example 1 Lip@M- M Characterization of H

[0061] The inventors successfully coated a polyethylene glycol-modified lipid membrane onto MnTCPP- M The final nanoreactor Lip@M- is formed on HCQ. M H, to temporarily shield the activity of the nanoreactor. After encapsulation with a lipid membrane, as... Figure 3As shown in Figure a, a light-colored thin film can be seen on the periphery of MnTCPP in transmission electron microscopy, indicating that the lipid membrane has been successfully coated on the surface of the MnTCPP MOF. Figure 3 As shown in b, elemental analysis reveals that oxygen (O), phosphorus (P), zirconium (Zr), and manganese (Mn) are present in Lip@M- M The distribution of H further verifies the successful synthesis of the nanoreactor. Furthermore, as... Figure 3 As shown in c and 3d, X-ray diffraction (XRD) and UV-Vis spectral data indicate that MnTCPP and M The properties of HCQ were well preserved in the nanoreactor. For example... Figure 3 As shown in Figure e, the Malvern particle size potentiometer measured the particle size of the nanoreactor covered with the lipid membrane to be approximately 192.93 ± 20.91 nm, and the potential to be approximately -16.16 ± 1.34 mV. Furthermore, as... Figure 3 As shown in f, Lip@M- M When H was resuspended in various physiological solutions, including deionized water, phosphate-buffered saline (PBS), culture medium, and physiological saline, the particle size of the nanoreactor remained largely unchanged, demonstrating good stability. Furthermore, the inventors investigated the release of H from the nanoreactor under ultrasonic irradiation (US) and non-irradiation conditions. M HCQ behavior. For example... Figure 3 As shown in g, approximately 81% of the drug was released 48 hours after ultrasonic irradiation, significantly higher than the 48% release rate of nanoparticles without ultrasonic irradiation. Meanwhile, as... Figure 3 As shown in h, transmission electron microscopy also confirmed the disruption of the lipid membrane, indicating that ultrasonic irradiation of the nanoreactor can decompose the liposome membrane and promote the release of hydroxychloroquine. (The "+US" markings in the data images of this application indicate that the nanoreactor has undergone ultrasonic irradiation.)

[0062] II. Experiments demonstrating the effectiveness of nanoreactors

[0063] Experiment 1: The ability of nanoreactors to generate ROS

[0064] To evaluate the effect of the nanosystem on SDT (Spin-Derived Transformation), the ability of MnTCPP to generate ROS was verified by electron spin resonance. Figure 4 As shown in Figure a, after ultrasonic irradiation of MnTCPP and Lip@M, a typical 1:2:2:1 signal of 5,5-dimethyl-1-pyrrolline-n-oxide was observed, demonstrating the capture of hydroxyl radicals (•OH). However, this characteristic signal was not detected in MnTCPP and Lip@M samples that had not undergone ultrasonic irradiation, nor in ultrasonically irradiated PBS samples. Furthermore, •OH can gradually degrade methylene blue, changing its color from blue to colorless, exhibiting obvious photodegradation characteristics. Figure 4As shown in b, this application found that the characteristic peak intensity at approximately 664 nm was significantly reduced only in the comparative example 3 MnTCPP + US and the comparative example 1 Lip@M + US sample solutions, indicating that ultrasound can trigger the generation of •OH from MnTCPP. Furthermore, using a singlet oxygen sensor (SOSG) as a probe, the ultrasound-induced singlet oxygen (•OH) was observed. 1 The generation of O2. For example... Figure 4 As shown in c and 4d, the fluorescence intensity of the SOSG peak gradually increased with increasing MnTCPP concentration and prolonged ultrasonic irradiation time. These results indicate that MnTCPP and MnTCPP-containing nanosystems are acoustic sensitizers capable of generating acoustic dynamic effects.

[0065] Furthermore, the Lip@M- of Example 1 was determined using confocal laser scanning microscopy (CLSM) and flow cytometry. M Cellular uptake behavior of H. For example Figure 4 As shown in e, 4T1 cells and Lip@M- from Example 1 are compared. M After incubation with H for 0.5, 1, 2, and 4 hours, red fluorescent signals gradually appeared around the cancer cells. Figure 4 As shown in f, the quantitative analysis results by flow cytometry were consistent with the CLSM image data, indicating that the phagocytosis of the nanoreactors was time-dependent. Finally, intracellular ROS generated by the nanoreactors were further detected using 2,7-dichlorodihydrofluorescein. Figure 4 As shown in g and 4h, in the control group without ultrasound irradiation (the control group in this application refers to the cells co-incubated with serum-free 1640 medium without nanoparticles) and the comparative example of non-ultrasound irradiation, namely, MnTCPP and Lip@M- M In group H, only slight fluorescence was observed, while in comparative example three (MnTCPP + US) and example one (Lip@M- M In the H + US group, a significant intensity of ROS was produced. Both CLSM and flow cytometry results showed that, in Example 1, Lip@M- M The cells treated with H+US showed the strongest fluorescence signal because the anti-autophagy drug prevented the metabolism of ROS-inactivating proteins.

[0066] Experiment 2: Autophagy Inhibition Efficiency of Nanoreactors

[0067] To further verify the click chemistry-based mechanism of enhanced autophagy blocking, and to evaluate... M The anti-autophagy efficiency of HCQ was verified through a series of experiments in this invention. The exposed Mal group can capture tumor proteins through reaction with -SH, therefore, as... Figure 5 As shown in figure a, ovalbumin (OVA) containing free SH groups was used to mimic damaged tumor-derived proteins in vitro. Figure 5As shown in c, the inventor discovered that, with M The zeta potential of OVA co-incubated with HCQ increased from -23.0 mV to -12.4 mV, while the zeta potential of OVA co-incubated with HCQ did not change significantly, indicating that after incubation... M HCQ has been anchored to the protein. Furthermore, this application also investigated the reaction of the nanoreactor with OVA before and after sonication, such as... Figure 5 As shown in d, Example 1 Lip@M- was treated with ultrasound. M The zeta potential of H-incubated oocytes and M The trend of HCQ is similar; while Example 1 Lip@M- M No significant changes were observed in the H group without ultrasound. This is because the polyethylene glycol-modified coating inhibits the reaction to some extent, but ultrasound can remove the coating to restore the reaction. M The reactivity of HCQ.

[0068] In addition, this application provides cell debris and a transwell device to further verify the above theory. For example... Figure 5 As shown in b, different drug formulations are placed in the lower transwell chamber, and cell debris is placed in the upper chamber. For example... Figure 5 As shown in e and 5f, after 24 hours of incubation... M HCQ group and Example 1 Lip@M- M The H + US group had a higher amount of inferior luminal protein than other groups, while the amount of superior luminal protein residue was relatively lower. These results indicate that, via in situ Mal-SH reaction, maleimide-functionalized HCQ binds more cell debris than HCQ. Based on this, the present invention subsequently evaluated it in vitro. M The autophagy inhibition efficiency of HCQ. The LC3B spot is associated with autophagosome formation; immunofluorescence images of the LC3B spot are shown using CLSM. Figure 5 As shown in k, compared with the control group, Comparative Example 3 MnTCPP + US group, Comparative Example 2 Lip@MH + US group, and Example 1 Lip@M- M More green LC3B punctate spots appeared in the H + US group, indicating a significant increase in the number of autophagosomes in cancer cells due to the blockage of autophagic flux. Furthermore, in Example 1, Lip@M- M The highest fluorescence intensity was observed in the H+US group because more autophagosomes were blocked. Figure 5 As shown in gj, Western blotting also revealed that autophagy-related proteins LC3B-II / I and beclin-1 were present in Comparative Example 3 (MnTCPP + US), Comparative Example 2 (Lip@MH + US), and Example 1 (Lip@M- MUpregulation in the H+US group indicates that autophagy begins after SDT activation. More importantly, p62 levels were higher in Example 1 Lip@M- M The H+US group showed a sharp increase, with p62, an autophagy substrate, being recruited by and degraded within autophagosomes. This indicates that even after autophagy has been initiated, nanoreactors can interfere with the integrated autophagy process, leading to the accumulation of autophagy substrates. Bio-TEM was used to directly observe intracellular autophagic vesicles, such as... Figure 5 As shown in i, Example 1 Lip@M- M Following H+ US treatment, numerous autolysosomes encapsulating nanoreactors were observed in 4T1 cells. In summary, these results confirm that, due to click chemistry-mediated autophagy capture, [the following is a continuation of the previous sentence]. M HCQ can effectively inhibit SDT-induced autophagy.

[0069] Experiment 3: In vitro cytotoxicity study

[0070] Phospholipids have a composition similar to that of biological cell membranes, thus exhibiting ideal biocompatibility. Therefore, this application evaluates the biosafety of lipid membranes before and after coating with nanomaterials. Figure 6 As shown in a, different concentrations of Lip@M- M After incubation with H, the cell survival rate was higher than 80%; however, as Figure 6 As shown in b, increasing MnTCPP concentration partially inhibits the growth of 4T1 cells and human umbilical vein endothelial cells (HUVECs). This indicates that the polyethylene glycol lipid membrane not only prevents premature exposure of the Mal group but also enhances the biocompatibility of the nanoreactor.

[0071] Further investigation into the combined efficacy of SDT and autophagy inhibitors will be conducted using cell viability assessment. Figure 6 As shown in c, Comparative Example 1, Lip@M + US, only showed moderate antitumor effects, which is related to the single treatment modality and cell-protective autophagy; Comparative Example 2, Lip@MH + US activation therapy, showed better cytotoxicity, indicating a synergistic effect between SDT and autophagy blocking strategies; Example 1, Lip@M- M The viability of cells in the H+US group decreased sharply to below 20%, indicating that enhanced autophagy inhibition efficiency can further enhance SDT-induced apoptosis, thereby inhibiting cancer cell growth. Furthermore, flow cytometry-based quantitative analysis of apoptosis also confirmed these results, such as... Figure 6 As shown in d and 6e, similar to the results of the cytotoxicity experiment, Example 1 Lip@M- M The highest apoptosis rate was observed in tumor cells after H+US treatment. This invention also uses calcein-am and propidium iodide (PI) fluorescent dyes for visual observation of dead and live cells. For example... Figure 6 As shown in f, the experimental results show that, in Example 1, Lip@M- M The H+US group emitted the strongest red fluorescence, indicating that the Lip@M- cells in Example 1 emitted the strongest red fluorescence. M H+ US causes the most damage to cells.

[0072] Experiment 4: Study on biological distribution

[0073] Because polydiethanolation can prolong the blood circulation time of the nanoreactor and increase drug accumulation, this application employs real-time fluorescence imaging technology to monitor the biodistribution of the nanosystem after intravenous injection. For example... Figure 7 As shown in Figures a and 8a, at 24 and 48 hours post-injection, mice intravenously injected with the unpolydiethanolated nanoreactor exhibited weak intratumoral fluorescence intensity, indicating rapid clearance from the bloodstream. However, mice intravenously injected with the PEG-modified nanoreactor showed enhanced fluorescence over time, reaching a maximum at 24 hours and maintaining a relatively high level over 48 hours. These results suggest that PEG-modified nanosystems can persist in the tumor region for a longer period. Correspondingly, in vitro fluorescence imaging was performed on mouse tumors and major organs (heart, liver, spleen, lung, and kidney). Figure 7 As shown in b and 8b, due to the rapid phagocytosis of the reticuloendothelial system, the fluorescence intensity of the liver and spleen of the non-PEGylated mice was relatively high. However, the PEGylated nanoreactors accumulated less in the spleen, and the tumor accumulation was relatively large.

[0074] Experiment 5: Imaging Effect Study

[0075] Non-invasive imaging techniques can provide real-time treatment guidance and visual monitoring for solid tumors. Due to its excellent near-infrared absorption and magnetism, MnTCPP nanoreactors show promise as a contrast enhancer for PAI and MRI. Figure 8 As shown in c, the Lip@M- in Example 1 of photoacoustic imaging was initially determined through full-spectrum scanning. M The imaging window for H was determined, and an excitation wavelength of 720 nm was selected as the optimal wavelength for subsequent experiments. Figure 7 As shown in Figure c, in the comparative example, increasing the concentration of 3MnTCPP from 12.5 µg / mL to 400 µg / mL enhanced the PAI signal intensity of the nanoreactor. Then, at predetermined time points after injection, [the following data was obtained]. Figure 7 The photoacoustic image of the body shown in d. Figure 8 As shown in d, the corresponding quantitative analysis shows that Example 1 Lip@M- M The photoacoustic signal at the H tumor site was significantly enhanced and time-dependent, peaking at approximately 24 hours and gradually weakening after 48 hours. To confirm the magnetic behavior of the nanoreactor, such as... Figure 7As shown in figure e, the hysteresis loop is described and exhibits paramagnetism, possibly originating from Mn. 2+ Then, the present invention systematically investigated the T1-weighted MRI performance of the nanoreactor in vitro and in vivo. For example... Figure 7 As shown in f, the MRI signal intensity depends on the concentration of Mn. Based on the slope of the fitted line, the Lip@M- in Example 1 is determined. M The longitudinal relaxation coefficient (r1) of H is 1.8704 mM. -1 s -1 In vivo applications, such as Figure 7 As shown at g and 7h, the tumor site showed significant enhancement on t1-weighted MRI after intravenous administration, as indicated by Lip@M in Example 1 of drug administration. -M The signal intensity after injection was 1.52 times higher than before injection.

[0076] Experiment 6: Biosafety Testing

[0077] To determine the short-term and long-term biosafety of the nanoreactor, a hemolysis test was performed in this application. Figure 9 As shown in Figure a, the inventors found that the hemolysis rate of the comparative example, 3MnTCPP, was slightly higher, but after encapsulation with a lipid membrane, the hemolysis rate decreased from over 5% to 2.82%, indicating improved biocompatibility. Subsequently, this invention systematically evaluated the effects of injecting Example 1, Lip@M- into Kunming mice. M Complete blood count and blood biochemical parameters at 1, 3, 7, 14, and 28 days post-H. (e.g.) Figure 9 As shown in the BM, no significant abnormalities were found compared to the control group, indicating no significant systemic toxicity. Figure 9 As shown in Figure n, H&E staining of major organs revealed no abnormal inflammatory responses or histopathological changes, either in the short or long term. These results clearly demonstrate that the multifunctional nanoreactor exhibits good biocompatibility in vivo, paving the way for future clinical translation.

[0078] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An ultrasonically responsive nanoreactor, characterized in that, It includes a manganese porphyrin-based metal-organic framework and a lipid membrane, wherein the manganese porphyrin-based metal-organic framework is a carrier, and the lipid membrane is a polyethylene glycol-based lipid membrane coated on the outer surface of the manganese porphyrin-based metal-organic framework. Maleimide-modified hydroxychloroquine is loaded on the manganese porphyrin-based metal-organic framework.

2. The ultrasonically responsive nanoreactor as described in claim 1, characterized in that, The structure of the maleimide-modified hydroxychloroquine is as follows: 。 3. The ultrasonically responsive nanoreactor as described in claim 2, characterized in that, The manganese porphyrin-based metal-organic framework has a pore size of 1.27 nm.

4. The method for preparing the ultrasonically responsive nanoreactor according to claim 1, comprising: M Preparation of HCQ, preparation of MnTCPP MOF and nanoreactor Lip@M- M The preparation of H is characterized by, M The preparation of HCQ includes the following steps: Hydroxychloroquine was dissolved in N,N-dimethylformamide, and then the solution was mixed with 3-maleimide propionic acid, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, and carbodiimide. The mixture was stirred and evaporated to dryness to obtain maleimide-modified hydroxychloroquine. M HCQ is then purified.

5. The method for preparing the ultrasonically responsive nanoreactor as described in claim 4, characterized in that, Lip@M- M The preparation of H includes the following steps: S1: Dispalmitoylphosphatidylcholine, polydiethanol-distearate phosphatidylethanolamine and cholesterol are dissolved in chloroform, and the mixed solution is then rotary evaporated in a water bath to form a lipid membrane. The lipid membrane is then dissolved in deionized water. S2: Dissolve manganese porphyrin-based metal-organic frameworks in a mixed solution of N,N-dimethylformamide and ultrapure water, then disperse maleimide-modified hydroxychloroquine in the mixture and stir at room temperature; S3: The lipid membrane prepared in S1 was added to the mixture in S2, stirred again, rinsed with deionized water, and centrifuged to finally obtain the nanoreactor Lip@M- M H.

6. The method for preparing the ultrasonically responsive nanoreactor as described in claim 5, characterized in that, In S1, the mass ratio of dipalmitoylphosphatidylcholine, polydiethanol-distearate phosphatidylethanolamine, and cholesterol is 3:1:

1.

7. The method for preparing the ultrasonically responsive nanoreactor as described in claim 6, characterized in that, In S1, the water bath temperature is 50-55℃.

8. The method for preparing the ultrasonically responsive nanoreactor as described in claim 7, characterized in that, In S1, deionized water is used to dissolve the lipid membrane.

9. The use of any one of the ultrasonic-responsive nanoreactors according to claims 1-3 in the preparation of drugs for treating tumors.

10. The application of any one of the ultrasonically responsive nanoreactors according to claims 1-3 in the preparation of contrast agents.

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