A tumor microenvironment-responsive nano-assembly, its preparation method and application

Abstract

The present invention relates to a tumor microenvironment-responsive nano-assembly and its preparation method and application, belonging to the technical fields of combined drug therapy, new excipients and new dosage forms. A tumor microenvironment-responsive nano-assembly is co-assembled by DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizers through intermolecular forces, and is modified with a PEG modifier; wherein, the mass ratio of DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizers is 1:0.1-10:0.1-15, and the mass ratio of the sum of DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizers to the PEG modifier is 1:0.1-9. The nano-assembly described in the present invention has sensitive 1 O2 self-reporting ability, the ability to improve the tumor hypoxic microenvironment and the ability to deplete GSH in tumor cells, and thus has good anti-tumor effects and high safety, providing a new strategy for the synergistic combination of singlet oxygen detection probes and other drugs.

Description

Technical Field

[0001] The invention relates to a tumor microenvironment responsive nano assembly and a preparation method and application thereof, belonging to the technical field of drug preparation combined therapy and new auxiliary materials and new dosage forms. Background Art

[0002] Photodynamic therapy (PDT) is an emerging cancer treatment method that has been widely used in clinical treatment in recent years due to its low invasiveness and high therapeutic selectivity. The mechanism of PDT is that under the irradiation of laser of a specific wavelength, photosensitizers can convert oxygen into singlet oxygen ( 1 O2), 1 O2 can directly act on tumor cells, inducing cell apoptosis or necrosis. Although PDT has made remarkable progress in the field of cancer treatment, it still faces challenges in clinical application. 1 The O2 content is difficult to monitor in real time and accurately, which directly leads to the difficulty in accurately controlling the therapeutic effect of PDT in clinical applications. 1 If the O2 content is insufficient, the expected therapeutic effect may not be achieved; on the contrary, if 1 Excessive O2 content may cause excessive damage to normal tissues. This lack of treatment precision limits the widespread application and in-depth development of PDT in clinical practice. 1 The generation and detection of O2 often adopt a divide-and-conquer strategy, that is, using independent systems to monitor and evaluate them separately. However, this separate application model has obvious disadvantages: it cannot reflect the dynamic changes in the PDT treatment process in real time. 1 The actual change of O2 content leads to the lack of accurate basis for clinicians to formulate treatment plans, making it difficult to achieve personalized precision treatment. Therefore, building a new nanosystem that integrates diagnosis and treatment has become a hot topic and urgent need in the current PDT research field.

[0003] In addition, the complexity of the tumor microenvironment has become a consensus generally accepted by the medical community. Among them, the hypoxic microenvironment unique to solid tumors is an obstacle to the clinical application of oxygen-dependent PDT. The oxygen partial pressure in normal tissues is usually maintained at a steady-state level of about 30 mmHg, which is essential for the normal physiological function of cells. However, due to abnormal vascular structure and dysfunction and the high metabolic rate of tumor cells, many tumors have a serious lack of oxygen supply. The oxygen partial pressure in some tumor tissues even drops below 2.5 mmHg. This extreme hypoxic state greatly limits the photodynamic process. 1The generation of O2 directly reduces the oxidative killing ability of PDT against tumor cells. To break through this limitation, various strategies have been widely studied to address tumor hypoxia. Among them, the design of oxygen delivery systems aims to precisely deliver external oxygen into tumors to increase the local oxygen partial pressure; in addition, by inhibiting the oxygen consumption mechanism of tumor cells, such as regulating the expression of related genes or blocking specific metabolic pathways, the oxygenation status of the tumor microenvironment can be indirectly improved. However, these strategies all face challenges in practical applications, such as oxygen leakage that may occur during the transportation of oxygen-carrying materials and the rapid consumption of the supplemented oxygen in the tumor site, etc.

[0004] In addition to the hypoxic microenvironment, tumor cells also maintain the redox balance of their microenvironment through a series of self-protection mechanisms, and the high concentration of glutathione (GSH) is a key link among them. As an important antioxidant within cells, GSH can effectively scavenge various harmful free radicals including reactive oxygen species (ROS), thereby protecting tumor cells from apoptosis induced by oxidative stress. During the PDT treatment process, this role of GSH undoubtedly weakens 1 the killing effect of O2 on tumor cells and further reduces the treatment efficiency of PDT. In view of this, it is particularly important to develop new nanotechnologies that can simultaneously relieve tumor hypoxia and cause GSH depletion in tumor cells. Summary of the Invention

[0005] To solve the problems existing in the prior art during PDT treatment, such as hypoxia at the tumor site, poor treatment effect in a high-GSH environment, and difficult control of the singlet oxygen 1 O2 content, etc., the present invention provides a tumor microenvironment-responsive nanoassembly and its preparation method and application. The present invention designs a nanoassembly (DMCP nanoparticle) jointly formed by DPA-MOF nanoparticles with the function of real-time monitoring of 1 O2, manganese dioxide (MnO2) with the ability to consume GSH and generate oxygen, and porphyrin-based photosensitizers, and promotes the treatment effect of PDT by self-reporting the 1 O2 during the PDT treatment process and regulating the tumor microenvironment. MnO2 can catalyze the overexpressed H2O2 at the tumor site to generate oxygen, and at the same time consume the GSH at the tumor site. Under light irradiation, the photosensitizer converts oxygen into a large amount of 1 O2, effectively killing tumor cells. At the same time, the real-time monitoring of 1 O2 by DPA-MOF nanoparticles in this nano-system can achieve precise PDT treatment, effectively inducing tumor death without causing superimposed toxicity to normal cells. This nanoassembly with self-reporting and self-enhancing functions provides a new drug delivery mode for multi-modal, highly efficient and low-toxic tumor treatment using precise PDT.

[0006] The present invention achieves the above object through the following technical solutions:

[0007] The present invention provides a tumor microenvironment-responsive nano-assembly, which is co-assembled by DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers through intermolecular forces and modified with a PEG modifier. Among them,

[0008] The mass ratio of the DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers is 1:0.1-10:0.1-15, and the mass ratio of the sum of the DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers to the PEG modifier is 1:0.1-9.

[0009] In the method of the present invention, the DPA-MOF nanoparticles can be prepared by reacting singlet oxygen detection probe DPA, zirconium oxychloride octahydrate, and benzoic acid in a dimethylformamide (DMF) solution through ultrasonic treatment and then stirring reaction in an oil bath.

[0010] In the above technical solution, the mass ratio of the detection probe DPA, zirconium oxychloride octahydrate, and benzoic acid during the preparation of the DPA-MOF nanoparticles is 1:0.5:5-9.

[0011] In the above technical solution, the oil bath temperature is 100°C-105°C.

[0012] In the above technical solution, the stirring reaction conditions are stirring reaction at 800-1200 rpm for 5-6 h.

[0013] In the method of the present invention, the DPA-MOF nanoparticles are used as 1 an O2 detection probe, and the manganese dioxide is a functional material for improving the tumor hypoxic microenvironment.

[0014] In the method of the present invention, the manganese dioxide adheres to the surface of the DPA-MOF nanoparticles to form a shell structure.

[0015] Furthermore, the porphyrin photosensitizers are one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, tetracarboxyphenyl porphyrin, and chlorin e6.

[0016] Preferably, the porphyrin photosensitizer is chlorin e6 (Ce6).

[0017] Furthermore, the intermolecular force is an electrostatic force.

[0018] Furthermore, the PEG modifier is one or more of PCL-PEG, DSPE-PEG, PLGA-PEG, and PE-PEG, and the molecular weight of PEG is 200-20000.

[0019] Preferably, the PEG modifier is DSPE-PEG, and the molecular weight of PEG is 2000.

[0020] The present invention also provides a preparation method of a tumor microenvironment-responsive nano-assembly, comprising the following steps:

[0021] Disperse a certain amount of DPA-MOF nanoparticles in deionized water to obtain a DPA-MOF nanoparticle solution, then add a poly(allylamine hydrochloride) solution, and stir and react at 800 - 1200 rpm for 1 - 2 h to obtain a mixed solution; dropwise add a potassium permanganate solution into the above mixed solution, continue to stir for 1 - 2 h to obtain DM nanoparticles; subsequently disperse the above DM nanoparticles in a DMF solution, dropwise add a porphyrin photosensitizer solution to modify it, and stir and react at 800 - 1200 rpm for 10 - 14 h to obtain DMC nanoparticles; disperse the above DMC nanoparticles in water to obtain a DMC nanoparticle solution, and then dropwise add an organic solvent containing a PEG modifier to the DMC nanoparticle solution under stirring at 800 - 1200 rpm, react for 12 h, and remove the organic solvent after completion to obtain DMCP nanoparticles, which are the tumor microenvironment-responsive nano-assembly.

[0022] In the above technical solution, the concentration of the DPA-MOF nanoparticles is 0.1 - 1 mg / mL, the concentration of the DMC nanoparticles is 2 - 3 mg / mL, and the concentration of the PEG modifier is 2 - 3 mg / mL.

[0023] In the above technical solution, the porphyrin photosensitizer solution is prepared by dissolving a photosensitizer in an organic solvent, and the concentration is 800 - 1000 nM.

[0024] In the above technical solution, the poly(allylamine hydrochloride) solution is prepared by dissolving poly(allylamine hydrochloride) in deionized water, and the concentration is 12 - 15 mg / mL.

[0025] In the above technical solution, the potassium permanganate solution is prepared by dissolving potassium permanganate in deionized water, and the concentration is 8 - 10 mg / mL.

[0026] In the above technical solution, the DMC nanoparticles are nanoparticles not modified by a PEG modifier, and the DMCP nanoparticles are nanoparticles modified by a PEG modifier.

[0027] Further, the organic solvent is one or more of N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

[0028] Further, the method for removing the organic solvent is centrifugation.

[0029] The operations of the present invention related to stirring are all carried out in a stirring device provided by the prior art, preferably a magnetic stirrer.

[0030] The preparation method of the tumor microenvironment-responsive nano-assembly of the present invention involves process steps that are all carried out at room temperature.

[0031] The present invention also provides an application of a tumor microenvironment-responsive nano-assembly in the preparation of a drug delivery system.

[0032] The present invention also provides an application of a tumor microenvironment-responsive nano-assembly in the preparation of an anti-tumor drug.

[0033] The present invention also provides an application of a tumor microenvironment-responsive nano-assembly in the preparation of an injection administration, oral administration or topical administration system.

[0034] The beneficial effects of the present invention compared with the prior art are as follows:

[0035] 1. The nano-metal-organic framework (Nano-MOF) adopted in the present invention is a class of novel materials with great potential, having unique physical and chemical properties, including an ultra-large specific surface area, high porosity, highly adjustable size, and excellent biocompatibility. These characteristics endow Nano-MOF with a powerful gas adsorption capacity, enabling it to efficiently capture 1 O2 during the PDT treatment process, significantly enhancing the detection sensitivity of 1 O2, and also providing the possibility for real-time monitoring of 1 O2 during the treatment process. More importantly, Nano-MOF in the present invention has easy modifiability. Through a carefully designed modification strategy, it is endowed with the function of enhancing the PDT treatment effect, such as alleviating tumor hypoxia and depleting GSH in tumor cells. The present invention uses Nano-MOF as a carrier to integrally integrate a photosensitizer, a tumor hypoxia alleviator, and a GSH depleting agent, realizing the co-delivery of multiple components. It can not only achieve real-time monitoring and precise control of 1 O2 during the PDT process, but also improve the tumor microenvironment and enhance the sensitivity of tumor cells to PDT, ultimately achieving precise tumor treatment.

[0036] 2. A tumor microenvironment-responsive nano-assembly (DMCP nanoparticles) prepared by the present invention can be used for singlet oxygen detection and tumor microenvironment regulation to enhance the photodynamic therapy effect. DMCP nanoparticles reflect the PDT treatment process by timely capturing and detecting 1 O2, providing precise PDT treatment. MnO2 can produce more 1 O2 under light illumination conditions by increasing the generation of oxygen at the tumor site and depleting GSH, while reducing the consumption of 1Consumption of O2 enhances PDT-induced tumor death.

[0037] 3. The tumor microenvironment-responsive nano-assembly prepared by the present invention achieves technical effects such as high sensitivity, good stability, and low toxicity and side effects, meets the urgent needs for highly efficient and low-toxic preparations in clinical practice, provides a new strategy for the assembly of singlet oxygen detection probes in synergistic combination with other drugs, and promotes the conceptual progress of precision PDT for enhancing the anti-cancer mode. Description of the Drawings

[0038] Figure 1 Scanning electron micrographs of the DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1.

[0039] Figure 2 Transmission electron micrographs of the DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1.

[0040] Figure 3 Energy-dispersive X-ray spectroscopy of the DMCP nanoparticles obtained in Example 1.

[0041] Figure 4 X-ray photoelectron spectroscopy of the DMCP nanoparticles obtained in Example 1 (a) and local high-resolution X-ray photon energy spectrum of Mn 2p in the DMCP nanoparticles.

[0042] Figure 5 X-ray diffraction pattern of the DMCP nanoparticles obtained in Example 1.

[0043] Figure 6 Colloidal stability diagram of the DMCP nanoparticles obtained in Example 1.

[0044] Figure 7 Ultraviolet absorption spectrum of the DMCP nanoparticles obtained in Example 1.

[0045] Figure 8 Fluorescence absorption spectrum of the DMCP nanoparticles obtained in Example 1.

[0046] Figure 9 GSH consumption determination diagram of the DMCP nanoparticles obtained in Example 1.

[0047] Figure 10 O2 generation of the DMCP nanoparticles obtained in Example 1 under illumination (a) and non-illumination (b) conditions 1 O2 generation.

[0048] Figure 11 O2 generation of the DMCP nanoparticles obtained in Example 1 under illumination (a) and non-illumination (b) conditions after adding MnO2 1 O2 generation.

[0049] Figure 12 Fluorescence change study of DPA-MOF nanoparticles obtained in Example 1 under conditions with GSH (a) or without GSH (b), and detection study of 1 O2 by DMCP nanoparticles under non-irradiation conditions (c).

[0050] Figure 13 Fluorescence change graph (a) and detection linear range graph (b) of the detection sensitivity of O2 by DMCP nanoparticles obtained in Example 1 under irradiation conditions. 1

[0051] Figure 14 Selectivity study graph of O2 by DMCP nanoparticles obtained in Example 1. 1

[0052] Figure 15 Safety investigation graphs of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 against 4T1 cells.

[0053] Figure 16 Safety investigation graphs of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 against MCF-7 cells.

[0054] Figure 17 Safety investigation graphs of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 against 293T cells.

[0055] Figure 18 Uptake and quantification graphs of DMCP nanoparticles obtained in Example 1 in 4T1 cells.

[0056] Figure 19 Uptake and quantification graphs of DMCP nanoparticles obtained in Example 1 in MCF-7 cells.

[0057] Figure 20 Cytotoxicity investigation of DMCP nanoparticles obtained in Example 1 against 4T1 cells.

[0058] Figure 21 Cytotoxicity investigation of DMCP nanoparticles obtained in Example 1 against MCF-7 cells.

[0059] Figure 22 Investigation of the consumption of GSH in 4T1 cells by DMCP nanoparticles obtained in Example 1.

[0060] Figure 23 Investigation of the consumption of GSH in MCF-7 cells by DMCP nanoparticles obtained in Example 1.

[0061] ​​Figure 24 The DMCP nanoparticles obtained in Example 1 were grown in 4T1 cells 1 O2 production situation.

[0062] Figure 25 The DMCP nanoparticles obtained in Example 1 were grown in MCF-7 cells. 1 O2 production situation.

[0063] Figure 26 The DMCP nanoparticles obtained in Example 1 acted on 4T1 cells. 1 Research on the detection of O2.

[0064] Figure 27 The DMCP nanoparticles obtained in Example 1 acted on MCF-7 cells. 1 Research on the detection of O2. DETAILED DESCRIPTION

[0065] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0066] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0067] Example 1

[0068] Preparation method of non-PEGylated DMC nanoparticles (DPA-MOF@MnO2@Ce6): 1The O2 detection probe DPA (20.9 mg, 0.05 mmol, Adamas Reagent Co., Ltd., Shanghai), zirconium oxychloride octahydrate (ZrOCl2·8H2O, 10 mg, 0.03 mmol), and benzoic acid (BA, 100 mg, 0.82 mmol) were dispersed in 50 mL of N,N-dimethylformamide (DMF) solution. After ultrasonic dispersion to make it uniform, it was placed in an oil bath at 100 °C and stirred for 5 h, and then the DMF solution was removed by centrifugation to obtain DPA-MOF nanoparticles. Subsequently, a certain amount of DPA-MOF nanoparticles (5 mg) was dispersed in 30 mL of deionized water solution to obtain a DPA-MOF nanoparticle solution. Then, poly(allylamine hydrochloride) (PAH) solution (1.25 mL, 14.4 mg / mL, solvent is deionized water) was added, and it was stirred and mixed at room temperature (stirring rate is 800 rpm) for 2 h. After that, potassium permanganate (KMnO4) solution (1.25 mL, 8 mg / mL, solvent is deionized water) was added dropwise to the above mixed solution, and stirring was continued for 1 h. The solvent was removed by centrifugation to obtain DM nanoparticles. The above DM nanoparticles (4 mg) were dispersed in DMF solution (2 mL). Subsequently, Ce6 solution (100 μL, 8 mM, solvent is DMF solution) was added dropwise for modification. After stirring and reacting at 1000 rpm for 10 h, it was washed with water multiple times, and the organic solvent was removed by centrifugation to obtain non-PEGylated DMC nanoparticles.

[0069] Preparation method of PEGylated DMCP nanoparticles (DPA-MOF@MnO2@Ce6@PEG): The above non-PEGylated DMC nanoparticles (4 mg) were dispersed in water (2 mL). The DMF solution containing DSPE-PEG (1 mL, 2 mg / mL, molecular weight is 2000) was added dropwise to the above solution under stirring (stirring rate is 1000 rpm), and the reaction was carried out for 12 h. Finally, it was washed with water multiple times, and the DMF solution was removed by centrifugation to obtain DMCP nanoparticles. The prepared DMCP nanoparticles are different from any other Zr-based MOF and have unique 1 O2 detection and self-reporting ability.

[0070] The scanning electron microscopy of DPA-MOF nanoparticles and DMCP nanoparticles is shown in Figure 1 , and it can be seen that: The prepared DPA-MOF nanoparticles have uniform particle size, are spherical, have good dispersibility, and the particle diameter is about 120 nm; after the DMCP nanoparticles are modified layer by layer, the surface of the nanoparticles becomes slightly rough, and the morphology of the nanoparticles does not change significantly, still being a spherical structure, having good dispersibility, and the particle diameter is about 130 nm. The transmission electron microscopy of DPA-MOF nanoparticles and DMCP nanoparticles is shown in Figure 2, it can be seen that: DPA-MOF nanoparticles exhibit a spherical structure with uniform size, smooth surface, good dispersibility, and the size is about 160 nm; after layer-by-layer modification of DMCP nanoparticles, a wrinkled structure appears on the surface, which can preliminarily prove the successful synthesis of DMCP nanoparticles.

[0071] The average hydrodynamic diameter of DPA-MOF and DMCP nanoparticles was measured by dynamic light scattering (DLS) technology, and the surface charge differences of each nanoparticle (NPs) were studied by measuring the Zeta potential of different nanoparticles. The results are shown in Table 1.

[0072] Table 1 Particle size and Zeta potential of nanoassemblies

[0073]

[0074] It can be seen from Table 1 that: the hydrodynamic diameter of DPA-MOF nanoparticles is about 234 nm, and after modification, the hydrodynamic diameter of DMCP nanoparticles slightly increases to about 256 nm; the Zeta potentials of DPA-MOF and DMCP nanoparticles are -20.93 and -5.13 mV, respectively.

[0075] Energy dispersive X-ray spectroscopy (EDS) was used to analyze the elemental composition, content and distribution of DMCP nanoparticles. The results are shown in Figure 3 , it can be seen that: C, O, Zr, Mn, N and P elements are evenly distributed on the surface of DMCP nanoparticles. Among them, C, O and Zr elements are the characteristic elements contained in the DPA-MOF framework, while Mn, N and P elements intuitively prove the successful modification of MnO2, Ce6 and PEG.

[0076] X-ray photoelectron spectroscopy (XPS) was used to analyze DMCP nanoparticles. In order to more accurately measure the elemental composition and chemical state of elements of DMCP nanoparticles, the results are shown in Figure 4 , it can be seen that: C, N, O, Mn, N and P elements exist in DMCP nanoparticles, which is consistent with the above EDS results, further proving the successful modification of MnO2, Ce6 and PEG; in the local high-resolution spectrum ( Figure 4 -b), the binding energies of 642.2 eV and 652.6 eV correspond to the spin orbits of Mn(IV)2p 3 / 2 and Mn(IV)2p 1 / 2 , which further indicates that MnO2 is successfully coated on the surface of DPA-MOF.

[0077] X-ray powder diffraction (XRD) was used to analyze the crystal form characteristics of DPA-MOF and DMCP nanoparticles. The results are shown in Figure 5, it can be seen that the positions and intensities of the XRD diffraction peaks of the DPA-MOF nanoparticles are basically consistent with the simulated diffraction peaks, indicating that the DPA-MOF nanoparticles have been successfully prepared and have good crystallinity; in addition, the XRD diffraction peaks of the DMCP nanoparticles are basically the same as those of the DPA-MOF nanoparticles, indicating that the layer-by-layer modified MnO2, Ce6, and PEG have no obvious effect on the crystal form of the DPA-MOF nanoparticles.

[0078] Colloidal stability test of DMCP nanoparticles:

[0079] In in vitro and in vivo biological studies, the stability of nanomaterials is crucial for their expected functions. Therefore, the stability of the obtained DMCP nanoparticles in different physiological solutions was evaluated. The DMCP nanoparticles were immersed in H2O, PBS buffer, and complete DMEM medium for different times, and the hydrodynamic diameter of the DMCP nanoparticles was measured at 0, 12, 24, 48, and 72 h. The results are shown in Figure 6 , it can be seen that after the DMCP nanoparticles were treated with different physiological solutions for 72 h, their hydrodynamic diameter did not change significantly, indicating that the DMCP nanoparticles have good colloidal stability, providing an experimental basis for their application in the biological field.

[0080] Spectral performance analysis of DMCP nanoparticles:

[0081] The ultraviolet absorption performance of the DMCP nanoparticles was investigated. The results are shown in Figure 7 , it can be seen that the characteristic absorption peaks of the photosensitizer Ce6 appear at 404 nm, 503 nm, 533 nm, 607 nm, and 663 nm, which are all caused by the π-electron transition in the Ce6 structure; the DPA-MOF shows characteristic absorption peaks at 343 nm, 360 nm, 380 nm, and 400 nm; in the ultraviolet-visible absorption spectrum of the DMCP nanoparticles, the characteristic absorption peaks of both DPA-MOF and Ce6 are present, which further confirms the successful preparation of the nanoparticles; compared with free Ce6, the characteristic absorption peak of Ce6 in the DMCP nanoparticles is red-shifted from 664 nm to 674 nm, which may be caused by the π-π stacking interaction of Ce6 molecules; in addition, after the DPA-MOF is loaded with MnO2, its ultraviolet-visible absorption spectrum does not change significantly. Similarly, the addition of PEG has no obvious effect on the ultraviolet-visible absorption spectrum of the nanoparticles.

[0082] The excitation spectrum and emission spectrum of the DMCP nanoparticles were measured by a fluorescence spectrophotometer. The results are shown in Figure 8First, with an excitation wavelength of 400 nm, the emission spectrum was collected. It can be seen that the DMCP nanoparticles showed a strong emission peak at 439 nm. Then, with an emission wavelength of 439 nm, the excitation spectrum was collected, and it can be seen that the maximum excitation wavelength was 407 nm. From the excitation spectrum and emission spectrum of the DMCP nanoparticles, it can be determined that the maximum excitation wavelength and the corresponding emission wavelength of DPA were 407 nm and 439 nm, respectively.

[0083] The ability of DMCP nanoparticles to consume GSH in solution:

[0084] 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) was used to detect the content of GSH in the reaction system to study the ability of DMCP nanoparticles to regulate the simulated tumor microenvironment (TME). DTNB, also known as Ellamn's reagent, can interact with the sulfhydryl group (-SH) in GSH to generate the yellow product 2-nitro-5-thiobenzoic acid (NTB). In the ultraviolet-visible absorption spectrum, DTNB has a maximum absorption peak at 323 nm, while the maximum absorption peak of NTB appears at 412 nm. Therefore, the content of GSH in the reaction system can be indirectly judged by the change of the characteristic absorption peak in the ultraviolet-visible absorption spectrum. The content of NTB is proportional to the content of GSH, and the content of DTNB is inversely proportional to the content of GSH. A decrease in the content of NTB or an increase in the content of DTNB means the depletion of GSH. The results are shown in Figure 9 , and it can be seen that: with the increase in the concentration of DMCP nanoparticles, the absorbance at 412 nm decreased significantly, and the absorption peak at 323 nm increased continuously, both indicating an increase in the consumption of GSH.

[0085] The ability of DMCP nanoparticles to generate 1 O2 in solution:

[0086] 1,3-Diphenylisobenzofuran (DPBF) is a commonly used commercial 1 O2 detection probe with a characteristic absorption peak at 410 nm. After this probe undergoes an irreversible oxidation reaction with 1 O2 to generate the oxidation product DBB, the intensity of the ultraviolet-visible absorption peak at 410 nm decreases. The ethanol solution of DPBF (5 mM) was mixed with the DMCP nanoparticle dispersion. Under NIR laser irradiation, the mixed solution was aspirated at regular intervals, filtered through a 0.22 μm filter membrane, and the change in the absorbance of DPBF at the maximum absorption wavelength was measured. At the same time, the ultraviolet-visible spectrum measured under the same time under non-irradiation conditions was used as a control. The results are shown in Figure 10 , and it can be seen that: under irradiation conditions, the absorbance value of DPBF at λ 410nm showed an obvious downward trend, indicating that under NIR laser excitation, the Ce6 loaded in the DMCP nanoparticles continuously generated1 O2; In comparison, under non-illuminated conditions, the absorption peak of DPBF hardly changed. This can prove that under the excitation of light with appropriate wavelengths, DMCP nanoparticles have good 1 O2 generation ability.

[0087] After adding MnO2, the 1 O2 generation ability of DMCP nanoparticles can be enhanced. Add the aqueous dispersion of DMCP nanoparticles to the TME simulation solution (composed of PBS buffer solution with pH 6.5 and 10 mM GSH solution), and observe the changes in the UV-visible absorption spectrum of DMCP nanoparticles after different illumination times to determine whether DMCP nanoparticles have the ability to generate and detect 1 O2. At the same time, use the UV-visible absorption spectrum of this system at the same time intervals under non-illuminated conditions as a control. The results are shown in Figure 11 , and it can be seen that: as the illumination time increases, the intensity of the characteristic absorption peak of anthracene ring in DMCP nanoparticles is continuously decreasing, indicating that under the excitation of NIR laser, Ce6 in the nanoparticles generates 1 O2, which is captured by the probe DPA-MOF, causing its absorbance to decrease; while under non-illuminated conditions, the intensity of the characteristic absorption peak of anthracene ring has no obvious change, indicating that no 1 O2 is generated in the system under light-shielded conditions, and it also reflects that DMCP nanoparticles have good stability. Through the above results, it can be preliminarily judged that DMCP nanoparticles have good 1 O2 generation and detection ability.

[0088] In addition, in order to investigate the tumor microenvironment regulation ability of DMCP nanoparticles, the system containing DMCP nanoparticles was compared with the system without MnO2 (that is, the system only contains free Ce6 as the 1 O2 source, and DPA-MOF is the 1 O2 detection probe) in the simulated tumor microenvironment for generating 1 O2. The Ce6 content in the two systems was controlled to be the same. The results are shown in Figure 12 -a, b, and it can be seen that: in the system containing DMCP nanoparticles, as the illumination time increases, the characteristic absorption peak of anthracene ring gradually decreases, indicating that 1 O2 is continuously generated in this system; while in the system without MnO2, the UV absorption peak of anthracene ring decreases to a certain extent within 15 minutes of illumination, but within the subsequent illumination time, the absorbance of DPA has no obvious change. The changes in the intensity of the anthracene ring absorption peak in the two systems also represent that there are significant differences in the 1 O2 content in the two systems. DMCP nanoparticles generate more 1There is more O2 because MnO2 in the DMCP nanoparticles plays an important role in the simulated TME. MnO2 consumes GSH, reducing the consumption of O2 by GSH, thus indirectly increasing the 1 content of O2 in the system. 1 content of O2.

[0089] Detection of O2 production ability of DMCP nanoparticles in solution 1 ability:

[0090] DPA-MOF can emit blue fluorescence under excitation at an appropriate wavelength. When it captures 1 O2, corresponding endoperoxides will be generated, resulting in quenching of the blue fluorescence. Using this fluorescence response mechanism, the detection of 1 O2 can be achieved through the change of the fluorescence of DPA-MOF itself. Therefore, using fluorescence spectroscopy technology, the fluorescence emission spectra of the system containing DMCP nanoparticles and the system without MnO2 were obtained, and the ability of DMCP nanoparticles to enhance 1 O2 production and detection 1 ability of O2 was investigated.

[0091] The DMCP nanoparticle aqueous dispersion was added to the TME simulation solution (25 μg·mL -1 ), and after incubation for 20 min, it was irradiated with 655 nm NIR laser, and the fluorescence emission spectra of DPA-MOF at different time points were collected (λex = 407 nm, λem = 420 - 600 nm). The results are shown in Figure 12 -c. It can be seen that in the DMCP nanoparticle system containing MnO2, as the irradiation time prolongs, the fluorescence intensity of DPA-MOF gradually decreases, indicating that DPA-MOF successfully captures the 1 O2 generated in the system, forming corresponding endoperoxides, resulting in quenching of its own fluorescence; in the system without MnO2, when irradiated with the laser for the same time, the fluorescence intensity of DPA-MOF only decreases to a small extent, indicating that the 1 content of O2 in this system is low. This is because there is a high concentration of GSH in the system, and the reducing GSH will consume the 1 O2 generated in the system; under non-irradiation conditions, the fluorescence emission spectrum of DPA-MOF hardly changes, indicating that there is no 1 O2 generation in this system. It can also be proved from this that the change of the fluorescence intensity of DPA-MOF has nothing to do with laser irradiation. Only when it captures 1 O2, the emission peak will decrease significantly.

[0092] Study on the detection sensitivity of DMCP nanoparticles to 1 O2 in solution:

[0093] Investigate the detection sensitivity of DMCP nanoparticles as a nano-fluorescent probe for 1 O2. Using the NaClO + H2O2 system to generate exogenous 1 O2, DMCP nanoparticles, NaClO (10 mM), and different concentrations of H2O2 (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 5 mM) were successively added to the reaction system. After the reaction system reacted for 15 min at room temperature under non-illuminated conditions, the changes in the fluorescence emission spectrum of DPA-MOF were measured. The content of 1 O2 was quantitatively analyzed using the fluorescence emission intensity ratio (F0 / F) of DPA-MOF to determine the linear range and calculate the limit of detection (LOD). Among them, F0 represents the initial emission intensity of DPA under the condition of no H2O2, and F represents the fluorescence emission intensity of DPA after adding different concentrations of H2O2. The results are shown in Figure 13 , and it can be seen that: as 1 the concentration of 1 O2 continuously increases, the fluorescence emission intensity of DPA-MOF continuously decreases. When the concentration of 1 O2 increases from 0 to 5 mM, the F0 / F value increases from 1 to 2.63. In the range of 0.2 - 3 mM, 1 there is a good linear relationship between the concentration of

[0094] O2 and the F0 / F value (r = 0.996); the detection limit of DMCP nanoparticles for 1 O2 is about 79 μM.

[0095] ROS are natural by-products during the normal metabolism of oxygen in the body, mainly including superoxide anion radical (·O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (·OH), and 1 O2 and other free radicals and non-free radical substances. To investigate the specificity of DMCP nanoparticles for the detection of 1 O2, we measured the selectivity of DMCP nanoparticles for 1 O2 in the presence of other interfering substances such as ROS or ions, and at the same time set a blank group (DMCP nanoparticle dispersion without ROS substances) as a control to control the consistent concentration of ROS. After incubating DMCP nanoparticles with different types of ROS for 15 min, the changes in the fluorescence emission spectrum of DMCP nanoparticles were measured. The concentrations of ROS and interfering ions used were 5 mM, and the preparation methods are as follows:

[0096] 1 O2: 5 mM NaClO + 5 mM H2O2;

[0097] ·O2 - : 5 mM xanthine + 500 mU xanthine oxidase;

[0098] H2O2: Dilute 30% H2O2 aqueous solution to 5 mM;

[0099] ·OH: 5 mM FeSO4·7H2O + 5 mM H2O2;

[0100] ClO - : Dilute 100 mM NaClO to 5 mM.

[0101] Measure the fluorescence intensity ratio (F / F0) of DMCP nanoparticles after reacting with various ROS substances. The results are shown in Figure 14 , and it can be seen that: in the presence of 1 O2, the F / F0 of DMCP nanoparticles decreases significantly; while in the presence of other interfering substances, the F / F0 of DMCP nanoparticles does not change significantly, proving that DMCP nanoparticles have good selectivity for the detection of 1 O2.

[0102] Safety study of DMCP nanoparticles on 4T1 cells, MCF-7 cells and 293T cells:

[0103] Dilute 4T1 cells, MCF-7 cells and 293T cells with good growth status with culture medium to make cell suspensions, and inoculate them into 96-well plates at a cell density of 3×10 3 / well. Add 200 μL of cell suspension to each well, set 4 replicate wells for each group of samples, and culture them in a cell incubator for 12 h to allow the cells to adhere and grow. Replace the original culture medium in the 96-well plate with a suspension containing different concentrations of Ce6, DMCP nanoparticles or DPA-MOF. Among them, in the Ce6 and DMCP nanoparticle groups, control the same Ce6 content, and set the Ce6 content to 0, 1, 2, 4, 8, 16 and 20 μg·mL -1 , and set the concentration of DPA-MOF to 0, 5, 10, 20, 40, 80 and 100 μg·mL -1 . To exclude the interference of the background, a blank control group containing only PBS buffer solution was set. After culturing for 12 h, remove the culture medium containing Ce6 solution, DPA-MOF and DMCP nanoparticles, wash with PBS buffer solution, add 100 μL of culture medium to each well, and at the same time add 20 μL of MTT solution (5 mg·mL -1 ), continue to culture for 4 h, then pour out the culture medium, add 150 μL of DMSO to each well, and gently shake with a plate shaker for 5 min to fully dissolve the generated formazan. Finally, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at 490 nm and calculate the cell survival rate. The results are shown inFigures 15 - 17 , it can be seen that: when there is no NIR laser irradiation, as the concentrations of Ce6 solution, DPA-MOF, and DMCP nanoparticles increase, the survival rates of the three types of cells can all be maintained above 70%. This indicates that free Ce6 and the prepared nanofluorescent probe DPA-MOF and DMCP nanoparticles have low toxicity under non-irradiation conditions, have little impact on the growth and proliferation of normal cells and tumor cells, and have good safety.

[0104] Study on the uptake behavior of DMCP nanoparticles:

[0105] Digest the 4T1 cells in good growth state to make a cell suspension, and inoculate it into a 24-well plate with coverslips. The cell density is 5×10 4 / well, and culture for 12 h to allow the cells to adhere and grow on the coverslips. Then, replace the original culture medium with a culture medium containing DMCP nanoparticles and continue to incubate for 0, 4, 8, 12, and 16 h. Then, remove the culture medium and wash three times with PBS buffer solution to remove the excess DMCP nanoparticles. Next, fix the cells with 4% tissue cell fixative in the dark for 30 min, and then wash three times with PBS buffer solution to remove the excess fixative. Finally, add the nuclear stain Hoechst33324, stain in the dark for 10 min, then take out the coverslips, and fix the coverslips on the glass slides with a small amount of anti-fluorescence quenching mounting medium. Observe the uptake of DMCP nanoparticles by 4T1 cells by observing the blue fluorescence and red fluorescence through CLSM. Among them, the blue fluorescence emitted by Hoechst33324 can be used to determine the position of the cell nucleus. The Ce6 loaded in the DMCP nanoparticles can emit red fluorescence under the excitation of a laser with an appropriate wavelength. According to the change in the intensity of this red fluorescence, the uptake of DMCP nanoparticles by cells can be directly determined. In addition, for the uptake study of MCF-7 cells, except that the MCF-7 cells are incubated for 24 h and then the culture medium containing DMCP nanoparticles is added, the remaining steps are the same as those of the uptake experiment of 4T1 cells. Use Image J software to analyze the average fluorescence intensity of the obtained fluorescence images, and the results are shown in Figure 18 and Figure 19 .

[0106] From the CLSM imaging diagrams of DMCP nanoparticles incubated with 4T1 cells for 0, 4, 8, 12, and 16 h ( Figure 18 -a), it can be seen that: the cell nucleus is stained blue, and the red fluorescence comes from DMCP nanoparticles. The red fluorescence surrounds the cell nucleus, from which it can be determined that DMCP nanoparticles are successfully taken up by 4T1 cells. From the average fluorescence intensity analysis diagram ( Figure 18-b) It can be seen that compared with the blank control group (cells not treated with DMCP nanoparticles), after incubation with DMCP nanoparticles for 4, 8, 12, and 16 h, the intracellular red fluorescence intensity gradually increased with the prolongation of the incubation time and reached an equilibrium state at 12 h. Thus, it is concluded that the optimal uptake time of 4T1 cells for DMCP nanoparticles is 12 h.

[0107] From the CLSM imaging diagrams of the incubation of DMCP nanoparticles with MCF-7 cells for 0, 4, 8, 12, and 16 h ( Figure 19 -a), it can be determined that DMCP nanoparticles have been effectively taken up by MCF-7 cells. From the average fluorescence intensity analysis diagram ( Figure 19 -b), it can be seen by comparison that after MCF-7 cells were co-incubated with DMCP nanoparticles for 12 h, the red fluorescence presented in the cells was the strongest, indicating that the optimal uptake time of MCF-7 cells for DMCP nanoparticles is 12 h.

[0108] Study on the cytotoxicity of DMCP nanoparticles to 4T1 cells and MCF-7 cells:

[0109] The MTT assay was used to determine the PDT anti-tumor effect of the DMCP nanoparticles under 655 nm NIR laser irradiation. 4T1 cells and MCF-7 cells with good cell states were digested to prepare cell suspensions, which were inoculated into 96-well plates at a cell density of about 3×10 3 / well. After culturing for 12 h, the original culture medium was discarded, and suspensions of different concentrations of Ce6 and DMCP nanoparticles were added. After laser irradiation for different times (0, 5, 10, and 20 min), incubation was continued for 24 h, and then the PDT treatment effects of each experimental group were compared by the MTT method. At the same time, the cytotoxicity of DMCP nanoparticles to 4T1 cells and MCF-7 cells was evaluated, and the results are shown in Figure 20 and Figure 21 . Figure 20 For the change in the survival rate of 4T1 cells after treatment with different concentrations of Ce6 and DMCP nanoparticles and laser irradiation, Figure 21 For the change in the survival rate of MCF-7 cells after the same treatment, it can be seen that when the concentrations of Ce6 or DMCP nanoparticles are the same, with the increase in the irradiation time, the cell viability gradually decreases; after 20 min of irradiation, the inhibition rate of the group containing DMCP nanoparticles on tumor cells is the highest, and there is no obvious damage to the cells in the group without DMCP nanoparticles, indicating that the PDT killing effect of the cells treated with DMCP nanoparticles is more obvious. This is because the MnO2 loaded in the DMCP nanoparticles can consume the overexpressed GSH in the TME, reduce the consumption of GSH for 1 O2, increase the content of 1 O2 in the cells, and thus amplify the PDT treatment effect.

[0110] Study on the depletion of GSH in 4T1 cells and MCF-7 cells by DMCP nanoparticles:

[0111] A GSH detection kit was used to further study the depletion of intracellular GSH by DMCP nanoparticles. The well-conditioned 4T1 cells and MCF-7 cells were digested into cell suspensions and seeded in 6-well plates at a density of 2×10 5 / well. After incubation for 12 h, the original medium was replaced with a medium containing different concentrations of Ce6 or DMCP nanoparticles (controlling the same Ce6 content in both). After continued incubation for 12 h, the medium was removed, and the cells were washed 3 times with PBS buffer solution. Trypsin was added to digest the cells, and the cell suspension was collected and centrifuged. The cells were resuspended with 1 mL of Reagent I (GSH extraction solution), and repeatedly frozen and thawed 2 - 3 times (frozen in liquid nitrogen and dissolved in a 37 °C water bath). The supernatant was collected after centrifugation at 10000 rpm for 10 min. According to the ratio provided in the kit instructions, 20 μL of the sample, 140 μL of Reagent II (GSH analysis buffer), and 40 μL of Reagent III (chromogenic agent DTNB) were added to the 96-well plate in sequence. After standing for 2 min for the reaction, the absorbance value at 405 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The measurement process of the GSH content in MCF-7 cells was the same as that of 4T1 cells, except that the medium containing DMCP nanoparticles was added after 24 h of culture. The above experimental results are shown in Figure 22 and Figure 23 , and it can be seen that different concentrations of Ce6 had no obvious effect on the GSH level in 4T1 cells. However, after the cells were incubated with DMCP nanoparticles, the intracellular GSH content gradually decreased with the increase in the concentrations of the Ce6 solution and DMCP nanoparticles. When the concentrations of the Ce6 solution and DMCP nanoparticles were 1.09 μg·mL -1 (where the Ce6 concentration was 200 ng·mL -1 ), the intracellular GSH content in 4T1 cells could be reduced to about 48%; after treatment with different concentrations of Ce6 or DMCP nanoparticles, the change in the GSH level in MCF-7 cells was similar to that in 4T1 cells. After incubation with Ce6, there was no obvious change in the intracellular GSH level, while for the cells treated with DMCP nanoparticles, the GSH level decreased significantly. The above experimental results indicate that DMCP nanoparticles can effectively scavenge the overexpressed GSH in tumor cells and reduce the GSH content.

[0112] Study on the generation of 1 O2 by DMCP nanoparticles in vitro:

[0113] The well-conditioned 4T1 or MCF-7 cells were seeded at 5×10 4The cells with a cell density of 4 / well were seeded in a 24-well plate. After the cells adhered, Ce6 or DMCP nanoparticle suspension was added (the Ce6 content in the two suspensions was controlled to be the same). After incubation for 12 h, the culture medium was aspirated, and the cells were washed 3 times with PBS buffer solution to remove the un-uptaken Ce6 solution and DMCP nanoparticles. Then, DCFH-DA solution (20 μM) was added, and after incubation at 37 °C for 30 min, the cells were irradiated with 655 nm laser for 20 min. Finally, fluorescence imaging of the cells after different irradiation times was performed using an inverted fluorescence microscope, and the results are shown in Figure 24 and Figure 25 , and it can be seen that: compared with the control group, in the Ce6 and DMCP nanoparticle groups of 4T1 cells and MCF-7 cells, green fluorescence appeared after laser irradiation, indicating that Ce6 solution or DMCP nanoparticles produced 1 O 2； in cells under the excitation of NIR laser. However, the green fluorescence intensity of the DMCP nanoparticle group was significantly stronger than that of the Ce6 group, indicating that compared with the single Ce6 group, DMCP nanoparticles can produce more 1 O2 in cells. This is because the MnO2 contained in DMCP nanoparticles consumed GSH and reduced the consumption of 1 O2 by GSH. Thus, it was further proved that DMCP nanoparticles have the potential to enhance the efficacy of PDT.

[0114] Detection of 1 O2 by DMCP nanoparticles in vitro:

[0115] The ability of DMCP nanoparticles to detect 1 O2 was studied in vitro. The well-conditioned 4T1 or MCF-7 cells were seeded in a 24-well plate at a cell density of 5×10 4 / well. After the cells adhered, 1 mL of DMCP nanoparticle (2 μg·mL -1 ) suspension was added to each well. After incubation for 12 h, the cells were irradiated with 655 nm laser for different times (0, 5, 10, and 20 min). Then, the culture medium was aspirated, and the cells were washed 3 times with PBS buffer solution. Finally, fluorescence imaging of the cells after different irradiation times was performed using an inverted fluorescence microscope. The generation of 1 O2 during PDT was reflected by the change in the fluorescence intensity of DPA itself, and the results are shown in Figure 26 and Figure 27 . It can be seen from the change in the fluorescence intensity of DPA in cells at different irradiation times that in the two types of tumor cells treated with DMCP nanoparticles, as the irradiation time increased, the blue fluorescence of DPA gradually weakened, indicating that the DMCP nanoparticles can detect the 1 O2 generated during PDT through the change in the fluorescence of the probe itself.​​​

Claims

1. A tumor microenvironment-responsive nano-assembly, characterized in that: The nano-assembly is co-assembled by DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers through electrostatic forces and modified with a PEG modifier; wherein, the mass ratio of the DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers is 1:0.1-10:0.1-15, and the mass ratio of the sum of the DPA-MOF nanoparticles, manganese dioxide, and porphyrin photosensitizers to the PEG modifier is 1:0.1-9; the porphyrin photosensitizer is chlorin e6; the PEG modifier is one or more of DSPE-PEG and PE-PEG, and the molecular weight of PEG is 2000-20000; the DPA-MOF nanoparticles are prepared by reacting singlet oxygen detection probe DPA, zirconium oxychloride octahydrate, and benzoic acid in a dimethylformamide (DMF) solution through ultrasonic treatment and then stirring reaction in an oil bath. In the preparation process of the DPA-MOF nanoparticles, the mass ratio of the detection probe DPA, zirconium oxychloride octahydrate, and benzoic acid is 1:0.5:5-9, the oil bath temperature is 100°C-105°C, and the stirring reaction conditions are stirring reaction at 800-1200 rpm for 5-6 h; The tumor microenvironment-responsive nano-assembly is prepared as follows: Disperse a certain amount of DPA-MOF nanoparticles in deionized water to obtain a DPA-MOF nanoparticle solution, then add a poly(allylamine hydrochloride) solution, and stir and react at 800-1200 rpm for 1-2 h to obtain a mixed solution; Dropwise add a potassium permanganate solution to the above mixed solution, and continue stirring for 1-2 h to obtain DM nanoparticles; Disperse the above DM nanoparticles in a DMF solution, and dropwise add a porphyrin photosensitizer solution to modify them, and stir and react at 800-1200 rpm for 10-14 h to obtain DMC nanoparticles; Disperse the above DMC nanoparticles in water to obtain a DMC nanoparticle solution, and then dropwise add an organic solvent containing a PEG modifier to the DMC nanoparticle solution while stirring at 800-1200 rpm, react for 12 h, and remove the organic solvent after completion to obtain DMCP nanoparticles, which are the tumor microenvironment-responsive nano-assembly.

2. The nano-assembly according to claim 1, characterized in that: The organic solvent is one or more of N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

3. Use of the tumor microenvironment-responsive nano-assembly according to claim 1 or 2 in the preparation of an anti-tumor drug.

4. The application according to claim 3, characterized in that: The drug is administered by injection, oral administration, or topical administration.

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